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Occurrence and treatment of plant sterols in pulp and paper mill effluents Mahmood-Khan, Zahid 2005

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Occurrence and Treatment of Plant Sterols in and Paper Mill Effluents by ZAHID MAHMOOD-KHAN B. Sc. Agricultural Engineering The University of Agriculture Faisalabad, Pakistan, 1991 M. Sc. Agricultural Engineering (Environmental Eng. Program) The University of Agriculture Faisalabad, Pakistan, 1995 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES CML ENGINEERING The University of British Columbia Vancouver, BC, Canada, March 2005 © Zahid Mahmood Khan, 2005 Abstract Abstract Pulp and paper mill effluents (PPMEs) contain plant sterols or phytosterols that may exert adverse effects on growth, physiology and reproduction of aquatic life by acting as endocrine disrupting chemicals (EDCs) or hormonally active agents (HAAs). Phytosterols form a part of wood extractives and structurally resemble steroid hormones. A suggested procedure was modified for isolation and analysis of PPME sterols. The technique involved liquid-liquid extractions with methyl-t-butyl ether (MTBE), trimethylsilylation by BSTFA (N, O-Bis tri-methylsilyl-triflouroacetamide) and gas-chromatography coupled with mass-spectrometry. Emulsion formation, incomplete silylation and chromatographic peak overlapping were the major problems encountered. The emulsion problems were resolved by additional amounts of solvents (MTBE and ethanol) and centrifugation. The silylation was improved by increasing the reaction temperature from 20 to 70°C. This shortened the incubation time from 12 to 4 h. The chromatographic separation was achieved through carrier gas flow and oven temperature adjustments. The modifications improved reproducibility and method sensitivity, and reduced the total time required for analysis. The modified technique was successfully used for analyzing sterols in PPMEs collected from two British Columbia pulp and paper mills. Six different sterols were quantitatively analyzed. B-sitosterol (B-Sito), B-sitostanol (B-Sitosta) and campesterol (Campe) were the major phytosterols that accounted for about 70% of the total sterols in PPMEs. Cholesterol (Chole), stigmasterol (Stigma) and ergosterol (Ergo) collectively accounted for about 30% or less. Total sterol concentrations were about 800-4,000 u,g/L in primary effluents, 250-1,200 p.g/L in final effluents and 12,000-40,000 pg/L in recycle and waste activated sludges. A more detailed survey, at two mills, revealed a general removal of sterols from PPMEs across the UNOX-AST (pure oxygen-activated sludge treatment) systems. The sterols removal efficiencies were variable. About 72% of the sterols were removed at Mill A and about 66% at Mill B. Bio-adsorption and selective biodegradation were the suggested mechanisms of sterols removal during secondary treatment. Sterol mass balance calculations across the UNOX-AST systems, revealed that about 30% of the incoming sterols were being discharged to the receiving waters with final effluents. Another 40% or more of the sterols left the treatment systems with excess sludge, indicating that 70% or more of the sterols may leave the treatment systems without biodegradation. Thus, a typical pulp mill (producing 1000 air dried tones /d) may discharge about 20 kg/day of sterols in treated effluents only. Two lab-scale suspended growth bioreactors, treating PPMEs, were used to investigate the fate and behavior of sterols during secondary treatment. A removal efficiency of about 90% was achieved and maintained for major sterols: B-Sito, B-Sitosta and Campe, with a solids retention time of 11-13 d and a hydraulic retention time of 10-12 h. The biological treatment was sensitive to process pH, solids detention time and hydraulic retention time. Under suitable process conditions, biodegradation/transformation appeared to be the major mechanism of sterols removal. During the conditions sub-optimal for sterols biodegradation, bio-adsorption became the major mechanism of removal and a larger portion of the influent sterols was discharged with waste sludge as well as final effluent. The investigation of sterols adsorption to inactivated sludge, revealed comparable adsorption kinetics for all three major sterols tested: B-Sito, B-Sitosta and Campe. Isotherms were generated by fitting a linear Freundlich adsorption model that suggested two adsorption regions for each sterol. In the high adsorption region, the inactivated biomass appeared to have the highest capacity for Campe, the lowest for B-Sito, and intermediate for B-Sitosta. The adsorption capacity of the inactivated biomass showed a biphasic behavior and increased with increasing equilibrium concentration of sterols, suggesting that bio-adsorption ii Abstract may be effectively used for sterols removal from PPMEs at higher concentrations. The results of sterols biodegradation and bio-adsorption studies presented, can be used in secondary treatment process modeling and design to incorporate specific organic pollutant removal. Key words: pulp mill effluents, endocrine disrupting chemicals, hormonally active agents, biological removal and mass flow of phytosterols, bio-adsorption of plant sterols, fi-sitosterol, (J-sitostanol, campesterol, stigmasterol, wood extractives. iii Table of Contents Table of Contents Abstract ii Table of Contents iv List of Tables x List of Figures xi List of Abbreviations xiv Acknowledgements xvi Dedications xviii 1. General Introduction 1 2. Literature Review 3 2.1 Introduction 3 2.2 Biochemical Effects Related to PPMEs 4 2.2.1 Dioxins/Difurans and Chlorinated Organics 4 2.2.2 Phytoestrogens and Plant Sterols 8 2.3 Nature of Steroids and Sterols 14 2.3.1 Steroids 14 2.3.2 Sterols and Stands 15 2.4 Sources of Plant Sterols 17 2.4.1 Occurrence in PPMEs 18 2.4.2 Occurrence in Sewage Effluents 19 2.5 Degradation and Transformation of Sterols 20 2.6 Secondary Treatment of PPMEs and Sterols Removal 22 2.7 Adsorption of Plant Sterols 25 2.8 Adsorption of Other Organics 26 2.9 Summary 32 2.10 Research Question and Hypothesis 33 3. Quantification of Plant Sterols 35 3.1 Introduction 35 3.1.1 Research Question 37 iv Table of Contents 3.2 The Selected Technique 37 3.2.1 Analyte Selection 38 3.2.2 Hydrogen Ion Concentration (pH) 38 3.2.3 Sterol Extraction Mode 39 3.2.4 Liquid/Liquid Extraction 39 3.2.5 Silylation Derivatization 40 3.2.6 Internal Standard 40 3.2.7 Surrogate 41 3.2.8 Gas Chromatography/Mass Spectrometry (GC/MS) Analysis 41 3.3 Plant Sterol Standards 41 3.4 PPME Sampling 42 3.4.1 Preliminary Sampling ; 43 3.4.2 On-Site Sampling 43 3.5 Results and Discussions 43 3.5.1 Silylation of Sterols 43 3.5.2 Calibration and Quantification 49 3.5.3 QA/QC 50 3.5.4 Distribution of Plant Sterols in Local PPMEs 54 3.5.5 Plant Sterols and Secondary Wastewater Treatment 57 3.6 Conclusions 59 4. Survey of Plant Sterols in PPMEs..!:. 60 4.1 Introduction 60 4.1.1 Research Questions 61 4.2 Sterols Analysis 61 4.2.1 Extraction from PPMEs 61 4.2.2 Silylation, Analysis and Quantification 62 4.3 PPMEs Sampling 62 4.3.1 Sampling Program I 63 4.3.2 Sampling Program II 64 4.3.3 Sampling Program III 64 4.4 Results and Discussions 66 4.4.1 A Snapshot of Plant Sterols in PPMEs (Sampling Program I) 66 4.4.2 Sampling Program II 73 4.4.3 Behavior of Plant Sterols during Secondary Treatment of PPMEs (Sampling Program III) 77 4.4.4 Sterols in Recycle and Waste Activated Sludge (Sampling Program III) 87 4.4.5 Sterols Mass Balance (Sampling Program III) 90 4.5 Conclusions 94 v Table of Contents 5. Removal of Plant Sterols in Lab-Scale Biological Reactors 96 5.1 Introduction 96 5.1.1 Research Questions 97 5.2 Methods and Materials 98 5.2.1 Lab-Scale Suspended Growth Activated Sludge Systems 98 5.2.2 Nutrients, pH Control and Process Temperature 100 5.2.3 Sterols Spiking 101 5.2.4 Chemical Analyses 103 5.2.5 Sterols Removal Experiments 104 5.3 Results and Discussions 106 5.3.1 Plant Sterols in Influent to the Lab-Scale Bioreactors 106 5.3.2 Plant Sterols in Treated Effluents 108 5.3.3 Effect of HRT and SRT on the Lab-Scale Secondary Treatment of Sterols 112 5.3.4 Sterol Mass Flows Entering and Leaving the Lab-Scale Bioreactors 117 5.3.5 Cumulative Mass Flows and Contribution of Biodegradation 121 5.4 Conclusions 126 6. The Bio-Adsorption of Phytosterols on Inactivated Biomass „ 128 6.1 Introduction 128 6.1.1 Research Questions 129 6.2 Experimental 130 6.2.1 Adsorbent (biomass) 130 6.2.2 Biomass Inactivation 130 6.2.3 Sterols Stock Solution 132 6.2.4 Adsorption Experiments 132 6.2.5 Chemical Analysis 133 6.2.6 Characteristics of Sterol Spiked PPME 134 6.3 Results and Discussions 135 6.3.1 Adsorption Kinetics of Phytosterols 135 6.3.2 Adsorption Equilibria of Plant Sterols 143 6.3.3 Isotherm Modeling 148 6.3.4 Sterols Bio-adsorption and Removal during Secondary Treatment 155 6.4 Conclusions 157 7. Overall Conclusions & Recommendations 159 7.1 Overall Conclusions 159 vi Table of Contents 7.1.1 Can plant sterols be successfully quantified in PPMEs? 159 7.1.2 Which sterols are present in (local) PPMEs? Are they removed during secondary treatment? 160 7.1.3 To what extent can biodegradation contribute to the overall removal of plant sterols during the biological secondary treatment of PPMEs? 160 7.1.4 What are the kinetics of sterols bio-adsorption and what is the adsorptive behavior of plant sterols with secondary solids? 161 7.2 New Knowledge & Engineering Significance 163 7.2.1 Contaminant Quantification 163 7.2.2 Pollutant Removal through Secondary Biological Treatment 163 7.2.3 Pollutant Bio-adsorption and Water/Wastewater reuse 164 7.3 Recommendations 166 7.3.1 Biological Treatment of Semi-Hydrophobic Organic Pollutants 166 7.3.2 Organic Pollutant Bio-adsorption Studies 166 7.4 Thesis Writing 167 8. References 168 9. Appendices 181 9.1 Appendix A. Chemical Analysis of Plant Sterols 181 A 1. Target plant sterol standards, surrogate and internal standard 181 A 2. Peak area and silylation time for B-Sito, B-Sitosta and Chole at 65 and 70°C 182 A 3. Unsilylated normalized peak area of B-Sito & Camp 183 A 4. Normalized peak area of B-Sito & Camp (Silylation at 70°C) 184 A 5. Peak area of plant sterols at 50 ug/L (Silylation at 70°C) 185 A 6. Peak area of plant sterols at 100 ug/L (Silylation at 70°C) 186 A 7. Peak area of plant sterols at 200 ug/L (Silylation at 70°C) 187 A 8. Peak area of plant sterols at 500 |jg/L (Silylation at 70°C) 188 A 9. Variation in normalized peak area of plant sterols with silylation incubation time (200ug/L; Silylation at 70°C) 189 A 10. Average normalized peak area of five plant sterols and derivatization incubation time (250ug/L; Silylation at 70°C) 189 A 11. Standard curve for plant sterols (Silylation 4 h at 70°C) 190 A 12. Chromatogram of different plant sterols: STD-25 192 A 13. Plant sterols detected in PPMEs before biological treatment 193 A 14. Plant sterols detected in PPMEs after biological treatment 194 A 15. Plant sterols or phytosterols found in PPMEs 195 A 16. Plant Sterols MDL and QA/QC 196 A 17. Regression for line of fit and residual plots for S-Sito 201 9.2 Appendix B. Plant Sterols in PPMEs 203 B 1. Plant sterols (p.g/L) in primary treated PPMEs at Mill B 203 vii Table of Contents B 2. Summary of 8-week monitoring results* for primary effluent at Mill B 204 B 3. Plant sterols (ug/L) in secondary treated PPMEs at Mill B 205 B 4. Summary of 8-week monitoring results* for final effluent at Mill B 206 B 5. Removal of plant sterols from PPMEs through secondary treatment at Mill B 207 B 6. PPMEs and sludge sampling locations around the UNOX-AST secondary treatment system at Mill A and Mill B - On-site Sampling Program I 208 B 7. Sampling stations around UNOX-AST secondary treatment system at Mill B - On-site Sampling Program II 209 B 8. Plant sterols variation during secondary treatment 210 B 9. Variation of PPME plant sterols around UNOX-AST system 213 B 10. Average sterols at the UNOX-AST system at Mill B 217 B 11. Total sterols variation at different sampling stations around the UNOX-AST system at Mill B 218 B 12. Total sterols variation across the UNOX-AST system 219 B 13. Total sterols variation in recycle and waste secondary sludge from the UNOX-AST bioreactor 221 9.3 Appendix C. Biological Removal of Plant Sterols 222 C 1. Schematic of small-scale suspended growth AST system: C pH control, D diffuser aerator, E final effluent, F influent feed, H heated water, M stirrer motor, N Nutrients, P air supply pump, R recycle, T temperature control, S mechanical stirrer, V control valve 222 C 2. Lab-scale Reactor 1 & 2 influent sterol concentrations (ug /L) 223 C 3. Reactor 1 effluent sterols (ug /L), removal efficiency (%), flow rate (l/d), HRT (h) and pH : 225 C 4. Reactor 2 effluent sterols (ug /L), removal efficiency (%), flow rate (l/d), HRT (h) and pH 228 C 5. Reactor 1 mixed liquor sterols (ug/L), SRT (d), and WAS flow (mL/d) 230 C 6. Reactor 2 mixed liquor sterols (Mg/L), SRT (d), and WAS flow (mL/d) 232 C 7. Reactor 1 daily mass flow of total sterols (mg/d), removed (%), biodegraded (%), and non-degraded (%) 233 C 8. Reactor 2 daily mass flow of total sterols (mg/d), removed (%), biodegraded (%), and non-degraded (%) 236 C 9. Reactor 1 cumulative mass flows of total sterols influent (mg), effluent (mg), WAS (mg), removed (%), retained (%), accumulated (mg), ML sterols (mg/L), biodegraded (%)238 C 10. Reactor 2 cumulative mass flows of total sterols influent (mg), effluent (mg), WAS (mg), removed (%), retained (%), accumulated (mg), ML sterols (mg/L), biodegraded (%)240 9.4 Appendix D. Inactivation of Biomass 243 D 1. Inactivation of Biomass and Head Space Monitoring for C02 243 D 2. Head space C02 for active (1,2 & fresh) and inactive biomass (T1 & T3) ...246 D 3. Head space C02 for active (3 & 4) and inactive biomass (T2 & T3) 247 9.5 Appendix E. Adsorption of Plant Sterols 248 E 1. Set 1: Batch 1-7 data for sterols adsorption to the inactivated secondary sludge 248 viii Table of Contents E 2. Set 1: Batch 1-7 figures showing sterols adsorption to the inactivated secondary sludge 250 E 3. Set 1: Liquid phase sterol concentration variation with inactivated M L S S dose 253 E 4. Set 1: Sterol equilibrium concentrations, adsorption capacities of the inactivated secondary sludge and adsorption isotherms 255 E 5. Set 2: Batch 8-14 data for sterols adsorption to the inactivated secondary sludge ...257 E 6. Set 2: Batch 8-14 figures showing sterols adsorption to the inactivated secondary sludge 259 E 7. Set 2: Liquid phase sterol concentration variation with inactivated M L S S dose 263 E 8. Set 2: Sterol equilibrium concentrations, adsorption capacities of the inactivated secondary sludge and adsorption isotherms 264 E 9. Set 3: Batch 15-22 data for sterols adsorption to the inactivated secondary sludge .266 E 10. Set 3: Batch 15-22 figures showing sterols adsorption to the inactivated secondary sludge 269 E 11. Set 2: Liquid phase sterol concentration variation with inactivated M L S S dose 274 E 12. Set 2: Sterol equilibrium concentrations, adsorption capacities of the inactivated secondary sludge and adsorption isotherms 275 9.6 Appendix F. Thesis Writing 277 F 1. Action Strategies 277 F 2. Further Motivation and Support 279 F 3. References & Resources 280 IX List of Tables List of Tables Table 2-1. Percent removal and degradation of wood resin components in activated sludge (AST) treatment process (Kaplin et al. 1997) 25 Table 3-1. Target plant sterols, surrogate and internal standard 42 Table 3-2. Plant sterols (ug/L) found in primary treated PPMEs from Mill A 54 Table 3-3. Plant sterols (ug/L) found in biologically treated PPMEs from Mill B 56 Table 3-4. Comparison of plant sterols (ug/L) found in primary and secondary treated PPMEs ...57 Table 4-1. Plant sterols (ug/L) in PPMEs at Mill A (Sampling Program I) 67 Table 4-2. Plant sterols (jig/L) in PPMEs at Mill B (Sampling Program I) 68 Table 4-3. Phytosterols (Mg/L) in recycle secondary sludge (Sampling Program I) 70 Table 4-4. Estimated mass flow rate (kg/day) of plant sterols (Sampling Program-I) 72 Table 4-5. Plant sterols in primary and secondary effluents at Mill B during 8-week monitoring of PPMEs (Sampling Program II) 74 Table 4-6. Sterols (ug/L) in the influent to the UNOX-AST system at Mill B each sampling cycle (Program III) 78 Table 4-7. Sterols (ug/L) in final effluent from UNOX-AST system at Mill B for each sampling cycle (Program II) 83 Table 4-8. Relative proportion (%) of individual sterols in different UNOX-AST streams at Mill B (Program III) 84 Table 4-9. Removal of plant sterols (%) through the UNOX-AST system at Mill B (Program III) ..85 Table 4-10. Average mass flow (kg/day) of sterols across the UNOX-AST system at Mill B 91 Table 5-1. Solubility of solid sterols in different solvents 102 Table 5-2. Characteristics of Primary Effluent from Mill B 103 Table 5-3. Different phases of lab-scale bioreactor operation 105 Table 5-4. Dissolved and particulate plant sterols in primary-treated effluents 107 Table 6-1. MLSS and sampling times for sterols adsorption experiments 133 Table 6-2. Characteristics of sterol-spiked PPME wastewater used for sterol bio-adsorption studies 134 Table 6-3. Low and high adsorption capacity regions for different sterols (Set 1-3) 147 Table 6-4. Isotherm model coefficients for different sterols (Set 1) 150 Table 6-5. Isotherm model coefficients for different sterols (Set 2) 151 Table 6-6. Isotherm model coefficients for different sterols (Set 3) 152 x List of Figures List of Figures Figure 2-1. Structure of steroid nucleus (Perhydrocyclopentanophenanthrene) and a general steroid 14 Figure 2-2. A common zoosterol 16 Figure 2-3. Chemical structure of some plant sterols 17 Figure 3-1. Increase in peak areas of sterol derivatives with incubation time at 65°C 44 Figure 3-2. Increase in peak areas of sterol derivatives with incubation time at 70°C 45 Figure 3-3. Optimization of cholesterol and B-sitosterol peak areas (silylated at 70°C) 46 Figure 3-4. Change in cholesterol and stigmasterol peak areas with incubation time (silylation at 70°C) 47 Figure 3-5. Average normalized peak area of five sterols (optimization of silylated sterol peak areas at 70°C) 48 Figure 3-6. QA/QC analysis for B-Sito (silylation 4 h at 70°C) 50 Figure 3-7. QA/QC Analysis for Chole (silylation 4 h at 70°C) 51 Figure 3-8. Chromatogram for a primary effluent sample showing six plant sterols found in PPMEs (silylation 4 h at 70°C) 52 Figure 3-9. Chromatogram for a biologically treated final effluent sample showing five different plant sterols present in PPMEs (silylation 4 hours at 70°C) 53 Figure 4-1. Sampling Program I locations, schematic of UNOX-AST system and mass balance system boundaries (outside dotted line) 63 Figure 4-2. UNOX-AST system sampling station location details of P1 to P8 at Mill B (On-site Sampling Program II) 65 Figure 4-3. Plant sterols in PPMEs and secondary sludge (Sampling Program I) 71 Figure 4-4. Time series plot of plant sterols in primary effluent at Mill B 75 Figure 4-5. Time series plot of plant sterols in final secondary effluent at Mill B ...76 Figure 4-6. Sterols in primary-treated influent to UNOX-AST-Sampling Station P1, Mill B. Vertical bars indicate standard error estimates 79 Figure 4-7. Sterols in biobasin Cell-A, UNOX-AST-Sampling Station P2, Mill B. Vertical bars indicate standard error estimates 80 Figure 4-8. Sterols in biobasin Cell-B, UNOX-AST-Sampling Station P3, Mill B. Vertical bars indicate standard error estimates 81 Figure 4-9. Sterols in biobasin Cell-C, UNOX-AST-Sampling Station P4, Mill B. Vertical bars indicate standard error estimates 81 Figure 4-10. Sterols in mixed liquor at biobasin outlet, UNOX-AST-Sampling Station P5, Mill B. Vertical bars indicate standard error estimates 82 Figure 4-11. Sterols in final effluent, UNOX-AST-Sampling Station P6, Mill B. Vertical bars indicate standard error estimates 84 XI List of Figures Figure 4-12. PPME total sterols during various sampling cycles around the UNOX-AST system Mill B (Sampling Program III). Vertical bars indicate 95% confidence interval estimates. P E refers to primary-treated effluent and FE-secondary-treated final effluent 86 Figure 4-13. Sterols in recycle activated sludge, UNOX-AST-Sampling Station P7, Mill B. Vertical bars indicate standard error estimates 87 Figure 4-14. Sterols in waste activated sludge, UNOX-AST-Sampling Station P8, Mill B. Vertical bars indicate standard error estimates 88 Figure 4-15. A comparison of sterols in recycle and waste activated sludge (RAS and WAS), UNOX-AST-Sampling Stations P7 & P8, Mill B. Vertical bars indicate standard error estimates 89 Figure 4-16. Total sterols mass flows around the UNOX-AST system at Mill B ...92 Figure 5-1. Schematic of lab-scale suspended growth AST system: C pH control, D diffuser aerator, E final effluent, F influent feed, H heated water, M stirrer motor, N Nutrients, P air supply pump, R recycle, T temperature control, S mechanical stirrer, V control valve 98 Figure 5-2. Dimensions (in cm) of the lab-scale bioreactors and secondary clarifiers 99 Figure 5-3. Plant sterols in the influent to the lab-scale bioreactors 107 Figure 5-4. Total-sterols removal efficiency Reactor 1 109 Figure 5-5. Total-sterols removal efficiency Reactor 2 109 Figure 5-6. Process pH and total-sterols removal performance of Reactor 1 (_1) and Reactor 2 (_2) 111 Figure 5-7. Reactor 1 hydraulic and solids retention times and total-sterols removal 112 Figure 5-8. Reactor 2 hydraulic and solids retention times and total-sterols removal 113 Figure 5-9. Variation in mixed liquor plant sterols with SRT in Reactor 1 115 Figure 5-10. Variation in mixed liquor plant sterols with SRT in Reactor 2 116 Figure 5-11. Daily mass flows of total sterols entering and leaving Reactor 1 System 118 Figure 5-12. Daily mass flows of total sterols entering and leaving Reactor 2 System 119 Figure 5-13. Reactor 1 System: Cumulative mass flows, average removal, retention, biodegradation, and ML (mixed liquor) total sterols 122 Figure 5-14. Reactor 2 System: Cumulative mass flows, average removal, retention biodegradation, and ML (mixed liquor) total sterols 123 Figure 6-1. Phytosterols remaining in solution- 0 mg/L MLSS batch 136 Figure 6-2. Phytosterols remaining in solution- 20 mg/L MLSS batch 136 Figure 6-3. Phytosterols remaining in solution-100 mg/L MLSS batch 137 Figure 6-4. Phytosterols remaining in solution- 200 mg/L MLSS batch 137 Figure 6-5. Phytosterols remaining in solution-1000 mg/L MLSS batch 138 Figure 6-6. Phytosterols remaining in solution- 2000 mg/L MLSS batch 138 Figure 6-7. Phytosterols remaining in solution- 40 mg/L MLSS batch (Set 2) 139 Figure 6-8. Phytosterols remaining in solution- 2000 mg/L MLSS batch (Set 2) 140 Figure 6-9. Percentage removal of B-Sito with time and MLSS. C is the concentration remaining in solution at any time t and Ci is the initial concentration 141 xu List of Figures Figure 6-10. Dimensionless liquid phase concentration of B-Sito and MLSS. C is the concentration in solution at time t and Ci is initial concentration 141 Figure 6-11. Sterol adsorption capacities of inactivated MLSS (Set 1) 144 Figure 6-12. Sterol adsorption capacities of inactivated MLSS (Set 2) 145 Figure 6-13. Sterol adsorption capacities of inactivated MLSS (Set 3) 146 Figure 6-14. Set 1: Freundlich isotherms for Campe, B-Sitosta and B-Sito. Legend suffix _a denotes high adsorption Region A, and _b denotes low adsorption Region B 150 Figure 6-15. Set 2: Freundlich isotherms for Campe, B-Sitosta and B-Sito. Legend suffix _a denotes high adsorption Region A and _b denotes low adsorption Region B 151 Figure 6-16. Set 3: Freundlich isotherms for Campe, B-Sitosta and B-Sito. Legend suffix _a denotes high adsorption Region A and _b denotes low adsorption Region B 152 xiii List of Abbreviations List of Abbreviations AC Adsorptive Capacity Adt Air Dried Tonne AHH (Hepatic) Aryl Hydrocarbon Hydroxylase AhR Aryl Hydrogen Receptor AOX Adsorbable Organic Halides AR Androgen Receptor ASBs Aerated Stabilization Basins ASE Accelerated Solvent Extraction AST Activated Sludge Treatment BKMEs Bleached Kraft Mill Effluents BMEs Bleached Mill Effluents BOD Biochemical Oxygen Demand BODs 5-Day Biochemical Oxygen Demand BSTFA N, O-Bis Tri-Methylsilyl-Triflouroacetamide Campe Campesterol Chole Cholesterol COD Chemical Oxygen Demand DMP Dimethylphenol DOC Dissolved Organic Carbon DOM Dissolved Organic Matter B2 17 B-Estradiol ECF Elemental Chlorine Free EDCs Endocrine Disrupting Chemicals ER Estrogen Receptor Ergo Ergosterol EROD Hepatic Ethoxyresorufin-O-Deethylase GAC Granular Activated Carbon GC-FID Gas Chromatograph - Flame Ionization Detector GC-MS Gas Chromatograph - Mass Spectrometer HAAs Hormonally Active Agents HCR High-Efficiency Compact Reactor HDCs Hormone Disrupting Chemicals HOCs/HOPs Hydrophobic Organic Contaminants/Hydrophobic Organic Pollutants HRT Hydraulic Retention Time xiv 1 ist of Abbreviations LH (Plasma) Luteinizing Hormone (Concentrations) MFO (Hepatic) Mixed-Function Oxygenase or Oxidase MLSS Mixed Liquor Suspended Solids MTBE Methyl-T-Butyl Ether NCASI National Council for Air and Stream Improvement NP Nonylphenol NSPs Non Settling Particles or colloids OM Organic Matter PAHs Poly-Nuclear Aromatic Hydrocarbons PCB Polychlorinated Biphenyl PCDDs Polychlorinated Dibenzo-p-Dioxins PCDFs Polychlorinated Dibenzofurans POP Pentachlorophenol PHAHs Polyhaloginated Aromatic Hydrocarbons PPMEs Pulp and Paper Mill Effluents RNA Ribonucleic Acid RAS Recycle Secondary Sludge SD Standard Deviation SPE Solid-Phase Extraction SRT Solids Retention Time SSBP Sex Steroid Binding Protein (I-Sito B-Sitosterol or Sitosterol B-Sitosta B-Sitostanol or Stigmastanol Stigma Stigmasterol T2 Testosterone TCF Total Chlorine Free TMP Thermo-Mechanical Pulp TMS Trimethylsilyl Group TOC Total Organic Carbon TSS Total Suspended Solids Vtg Vitellogenin WAS Waste Secondary Sludge WWTP Wastewater Treatment Plant XOCs Xenobiotic Organic Compounds X V Acknowledgements Acknowledgements After getting through the stages of qualifying exam, preliminary proposal & experiments, proposal defense and "the actual research" that usually involves a long-period of hard work, patience, determination & extreme persistence, a Ph.D. Candidate, with a large amount of experimental results & associated data in hand, may think that "the toughest part" of the Ph.D. process is probably over. Therefore the Candidate may expect a down-hill journey, but that is not the case. After all the hard & demanding research work, "thesis writing" may particularly demand the most concentrated effort. The huge amount of additional work required as well as the attached high expectations can easily make the writing task overwhelming. The thesis is the formal presentation of all the work and efforts done during the whole program and it should meet the technical requirements of a high professional standard. This means a lot of suffering and difficulty may still have to be faced by the Candidate. The situation can particularly become tense, if the Candidate takes up a full-time or even a part-time work commitment for financial survival before finishing "The Writing." It is not uncommon that the writing takes much longer time than expected, and 'a delay of 1 or 2 years is warned.' The initial high motivation of the Ph.D. Candidate can drop to extremely low levels more than once during the whole process. I went through those easier-said-than-done stages & situations the severity of which was too challenging to continue. Has it not been a great mercy of God, I could probably never finish this relentless undertaking. I have suggested some action strategies in Appendix F for the benefit & support of the enthusiastic Ph.D. candidates of the future, who may read this thesis for a variety of reasons. The story would be incomplete without the mention of invaluable help & support from God, the family here in Canada and in Pakistan, the friends & class-mates, colleagues at PPC, UBC and GVRD. Many others including Susan Harper & Paula Parkinson at the Environmental Engineering Labs., and Peter Tyler & Tim Patterson at PPC who helped whenever required. I am grateful to my Supervisor Dr. Eric R Hall for his understanding, cooperation and guidance during the whole Ph.D. process. Dr. Hall's style of informative questioning, thoughtfulness to the situation, readiness to help and situation handling need to be especially mentioned. I am also grateful to my Supervisory Committee that included Dr. Ken J Hall, xvi Acknowledgements Dr. Sheldon J B Duff, and Dr. Colette Breuil for their time to time guidance and help in the whole program. Dr. Bill Ramey, Dr. Don Mavinic and Professor Jim Atwater also need to be mentioned for their kind support and guidance whenever needed. I was fortunate to get financial help for bearing the costs of advanced studies, research and professional development from a number of sources that include Government of Pakistan, Natural Sciences & Engineering Research of Canada (NSERC), my research supervisor Dr. Eric R Hall, and my part-time as well as full-time work commitments. xvii Dedications Dedications I would like to dedicate this dissertation humbly to Allah SWT (God), His messenger Mohammed (may peace and blessings of Allah SWT be upon him), my parents Roshan Deen Khan, Noor Bibi, the extended family and all the friends. For the luxury of all the blessings, provisions, a high-level of education and for the gift of life itself, I would like to be grateful for ever. Zahid Mahmood-Khan xviii 1 General Introduction 1 . General Introduction Certain chemicals, present in our wastewaters and receiving water bodies, have been reported to mimic natural hormones. Such chemicals may interfere in the normal function of endocrine systems of aquatic and other wildlife (McLachlan 1985; Harries et al. 1997). The presence of hormone mimicking or endocrine disrupting chemicals (EDCs), also called by some as hormonally active agents (HAAs), has recently gained considerable environmental interest. Ecological integrity concerns are particularly related to aquatic environments, which serve as sinks for the products of industrialized societies. Although a direct relationship between EDCs and their effects in humans has not been established (Sharpe and Skakkebaek 1993; Safe 1995; Ashby 1997) a large number of synthetic and naturally occurring chemicals have been identified as potential EDCs or HAAs. Many of them are widely used for domestic and industrial purposes and have varied routes to the environment. For example: PVC (poly vinyl chloride) plastics are a major source of phthalates (dicarboxlic acids) and dioxins/difurans. Other main categories of EDCs/HAAs include persistent organohalogens, pesticides, penta- to nonyl-phenols, bisphenol-A, styrene dimers and trimers, and heavy metals. Pulp and paper mill effluents (PPMEs) are suspected to contain some chemicals like plant sterols that can be potential EDCs or HAAs for fish and aquatic wildlife (Howell et al. 1980; Andersson et al. 1988; Davis and Bortone 1992; Maclatchy and Van Der Kraak 1995). The observed biochemical responses in fish exposed to PPMEs include masculinization of female fish resulting in the development of male-like secondary sex organs and behavior, delayed sexual maturity, decreased gonad growth, reduced body length, enlarged liver, altered steroid profiles, induction of mixed-function oxygenase (MFO) and vitellogenin secretion activities as well as metabolic disorders (Andersson et al. 1988; Krotzer 1990; Pelissero et al. 1991; Munkittrick et al. 1994; Cody and Bortone 1997). Phytosterols and other chemicals like polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) were the major candidates thought to be responsible for the observed responses (Munkittrick et al. 1994; Servos et al. 1994; Zacharewski et al. 1995; Zacharewski 1997). However, biochemical responses including MFO inductions in fish exposed to PPMEs from thermo-mechanical pulp mills, modernized bleached kraft mills, 1 1 General Introduction 1994; Lehtinen et al. 1999) point to the EDCs or HAAs as being non-halogenated organic compounds. A possible source of EDCs in P P M E s is wood itself (Servos et al. 1994). Plant sterols, or phytosterols that originate from wood, can be present in P P M E s that are suspected to induce adverse endocrine effects (Sandstrom et al. 1988; Zacharewski et al. 1995; Stromberg et al. 1996; Cook et al. 1997). Some EDCs present in P P M E s , like li-sitosterol o r its microbial transformation byproducts, were thought to act as androgenic hormones (Rosa-Molinar and Williams 1984) that affected an entire population of mosquitofish, {Gambusia affinis holbrooki) in a 20 km length of a creek downstream of the discharge point of P P M E s (Howell et al. 1980). Arrhenoidal effects were noted by Denton et al. (1985) in fish exposed to plant sterols (primarily B-sitosterol and stigmastanol) in the presence of myco-bacteria. Plant sterols or their transformation byproducts are suspected as an important source of androstane steroids that may be acting as EDCs to produce the observed aquatic biological effects (Denton et al. 1985; Hunsinger et al. 1988; Krotzer 1990; Pelissero et al. 1991; Zacharewski et al. 1995; Cody and Bortone 1997). Phytosterols have been detected in varying amounts in P P M E s obtained from different Finnish and United States mills (Stromberg et al. 1996; Cook et al. 1997). Hence, it is of interest to examine the presence level of plant sterols in P P M E s from Canadian sources, to assess the discharge of sterols to our receiving waters. The accumulating evidence suggesting that plant sterols may be an important source of E D C s , has provided motivation for research into the control and treatment of sterols in P P M E s . Although removal of plant sterols from P P M E s through secondary wastewater treatment has been reported (Verta et al. 1996; Magnus et al. 2000b), many details about the fate and the behavior of plant sterols in secondary wastewater treatment systems still remain unknown. Therefore, it is necessary to investigate not only the occurrence of plant sterols in P P M E s from local sources but also the effectiveness of the secondary wastewater treatment provided at the mills. 2 2 Literature Review 2.1 Introduction 2. Literature Review 2.1 Introduction Some evidence has accumulated suggesting that PPMEs may contain a single chemical or a combination of chemicals acting as EDCs or HAAs. These chemicals may exert endocrine disrupting effects on aquatic wildlife and upset the ecological integrity of the receiving environments. PPMEs contain a complex mixture of a number of different organic chemicals including methanol, acetone, organic acids, cellulose, lignin, sulphides and mercaptans, adsorbable organic halides (AOXs), sterols, steryl esters, triglycerides, and other lipophilic extractives (Orsa and Holmbom 1994; Mimms et al. 1989). The assessment of individual PPME components that may be EDCs is difficult, and many of these chemicals are unidentified as well as unknown (Fenner-Crisp and Fisher 1997). Parks et al. (2001) also reported some unidentified substances whose presence in PPMEs was considered to be associated with masculinization of female fish. However, plant sterols and chlorinated organics like dibenzo-p-dioxins/difurans (PCDDs/PCDFs) have been speculated to be the potential causative agents for the observed biochemical effects in fish exposed to PPMEs (Zacharewski et al. 1995). The endocrine system is a combination of glands and hormones that affect biological growth, development and reproduction in higher forms of life. EDCs or HAAs are compounds that can block, mimic, stimulate, or inhibit the action and/or production of natural hormones, thereby disrupting the endocrine system's ability to function properly (McGovern and McDonald 2003). Hence, endocrine disruption is not necessarily a toxicological end point but a functional change leading to abnormal and adverse effects. McCann (2004) defined an endocrine disrupter (or disruptor) as an exogenous substance that causes health effects in an intact organism, or its progeny, secondary to changes in endocrine function. The potential EDCs or HAAs found in PPMEs, have been reported to induce a variety of estrogenic or androgenic effects accompanied with behavioral modifications in fish and other 3 2 Literature Review 2.2 Biochemical Effects Related to PPMEs aquatic life. The following sections provide more details about these chemicals and the observed biochemical implications. 2.2 Biochemical Effects Related to PPMEs 2.2.1 Dioxins/Difurans and Chlorinated Organics Chronic toxicity of low-levels of dioxins/difurans and other hydrophobic chlorinated organic compounds in PPMEs relates to bioaccumulation. Chlorinated phenolics and resin acids in bleaching effluents accumulate in fish tissues, particularly in liver (Holmbom and Lehtinen 1980; Landner et al. 1977). This may eventually lead to carcinogenesis and endocrine disruptions. The chlorinated organic compounds formed during pulp bleaching, have generally been considered to be important mutagenic agents in PPMEs. Exposure to bleached kraft mill effluents (BKMEs) caused a complex pattern of effects on both primary (MFO-enzyme) and secondary adaptatory (hematological and osmoregulational) mechanisms in exposed rainbow trout fish. The strongest physiological effects were observed when the fish were exposed to traditional chlorine bleaching effluents without secondary treatment (Lehtinen et al. 1990). However, the response of specific parameters such as the enzyme activities was not manifested in a dose-response manner probably due to the presence of other EDCs or HAAs -like substances that may either stimulate or inhibit the biochemical responses. Strong physiological and biochemical responses were reported in white sucker in the St. Maurice River, Quebec, receiving BKMEs (Hodson et al. 1992). Ten-fold higher activity of hepatic (relating to liver) aryl hydrocarbon hydroxylase (AHH) was noted in fish immediately after, and five-fold higher AHH activity was observed up to 95 km downstream of a BKME discharge point, as compared to the reference fish. Tetrachlorodibenzo-p-dioxin (TCDD) and tetrachlorodibenzofuran (TCDF) were detected in gutted fish samples. The strong correlation observed between the AHH induction and the tissue contamination, denoted severe effects of the exposure of TCDD/TCDF containing BKMEs. Van Der Kraak et al. (1992) demonstrated that pre-spawning exposure of BKMEs affected reproduction in white sucker by acting at multiple cites in the pituitary-gonadal axis. The exposed fish exhibited delayed 4 2 Literature Review 2.2 Biochemical Effects Related to PPMEs sexual maturity, reduced gonadal size, reduced secondary sexual characteristics and depressed circulating steroid levels relative to those of reference populations. European carp, inhabiting a shallow-water lake receiving treated PPMEs, showed significantly elevated hepatic microsomal EROD (ethoxyresorufin-O-deethylase in cellular structures relating to liver) levels relative to the reference fish living in a nearby unexposed water body (Ahokas et al. 1994). The increased EROD levels indicated higher activity of cytochrome P-450 (an intra-cellular electron transport hemoprotein) linked MFO (mixed function oxidase) detoxification system. The MFO activity correlated with site adsorbable organic halide (AOX) concentrations and poly chlorinated dioxins/difurans (PCDD/PCDFs) measured in carp muscle. Induction of cytochrome P-450-linked MFO, an important detoxification enzyme system in fish and other vertebrates, indicated possible exposure of carp to EDC-like environmental contaminants of lipophilic nature. TCDD and related polychlorinated aromatic hydrocarbons modulate the levels of several hormones causing a spectrum of morphological and functional developmental deficits (Bimbaum 1995). The alteration of hormones through exposure to environmental contaminants like TCDD and related dioxins, can initiate a cascade of biochemical effects. White sucker collected from the Terrace Bay mill effluent receiving area, Jackfish Bay, Lake Superior, Ontario, Canada, two years following the installation of secondary treatment facilities at the site, exhibited decreased gonadal size, depressed plasma sex steroid levels, increased liver size and induced EROD activity (Munkittrick et al. 1994). It was also observed that the absence of chlorine free bleaching or the presence of secondary treatment did not eliminate the responses in fish downstream of several pulp mills. PPME exposure studies on freshwater fish, in a state of late vitellogenesis, showed relatively lower concentrations of plasma steroids: 17 fc-estradiol and testosterone (Karels et al. 2001). The lower plasma steroid concentrations in late vitellogenic female perch, 1 km downstream of PPME discharge, coincided with lower gonad size and fecundity and higher concentrations of resin acids and chlorophenolics in the bile. The exposed perch population also showed a different size and age distribution compared with the reference fish. The observed responses seemed to be restricted to less than a 6 km range, due mainly to hydrology and dilution of effluents. Nonetheless, the evidence suggested that the elemental chlorine-free PPMEs also contained some potential EDCs that were capable of causing the 5 2 Literature Review 2.2 Biochemical Effects Related to PPMEs reproductive and population changes in perch. However, plasma steroid levels were not affected in roach, another freshwater fish tested in the same study, revealing the differences in immuno-competence of different species of aquatic life. Thus, roach appeared to be a more adapted species for chronic low-level exposure to PPMEs. Bioaccumulation of poly chlorinated organics depends upon site and species specific parameters including the food-chain choice from the filter feeding insects by a specific fish species. Mountain whitefish (Prosopium williamsoni) showed relatively higher levels of TCDD and TCDF concentrations as compared to another bottom feeding species, longnose sucker (Catostomus catostomos). Longnose sucker TCDD/TCDF levels were at least an order of magnitude lower than those of the whitefish (Owens et al. 1994). Polychlorinated dioxins/furans (PCDD/PCDFs) and contaminants with comparable hydrophobicity (log > 5) accounted for hepatic MFO activity inductions in male white sucker population exposed to PPMEs from a bleached kraft mill. The fish appeared to have accumulated PCDD/PCDFs during their entire life time. Other non-dioxin contaminant AhR (aryl hydrocarbon receptor) ligands of log Kow 2 to 5 in livers of exposed fish exhibited significant competition for AR (androgen receptor), ER (estrogen receptor) and sex steroid binding protein (SSBP) indicating potential effects on hormone signaling and transport (Hewitt et al. 2000). Most of the ligands for AhR and sex steroid receptors AR, ER, and SSBP were cleared from tissues of pre-exposed fish after a short term removal from PPMEs. Some BKME constituents bind to both white sucker and goldfish SSBP, and possible modification in the properties of their SSBP of the native species residing downstream of the discharge of PPMEs. The changes in SSBP characteristics, in terms of their affinity and capacity, may differ from one species to another and can be more pronounced in females undergoing vitellogenesis and final oocyte maturation rather than in non-reproductive stage (Pryce-Hobby et al. 2003). The effects of the exposure to PPMEs containing EDCs, can be more detrimental to fish in the juvenile stage. PCDDs/PCDFs however, may not be the only chemicals involved in causing the physiological responses observed in fish near pulp mills. The presence of non-chlorinated EDCs in PPMEs released by pulp mills producing unbleached pulp and paperboard, was indicated through enzyme-based bio-monitoring of rainbow trout and whitefish. The fish exposed to the unbleached PPMEs, which contained no chlorinated compounds, demonstrated significant effects on cytochrome P-4501A enzyme activities (Lindstrom-Seppa 6 2 Literature Review 2.2 Biochemical Effects Related to PPMEs et al. 1992). Moreover, biochemical responses including elevated hepatic MFO inductions, depressed sex plasma steroid levels, decreased gonad size and increased liver size have been observed in fish exposed to effluents from thermo-mechanical pulp mills, modernized bleached kraft mills, sulfite mills, unbleached kraft mills, with or without secondary treatment facilities, all emitting negligible amounts of PCDDs and PCDFs (Lehtinen et al. 1990; Munkittrick et al. 1994). Although white sucker collected near bleached kraft mills exhibited the highest EROD inductions and dioxin levels, elevated enzyme activity was also observed in fish from sites that did not use chlorine. The absence of chlorine bleaching or the presence of secondary treatment did not eliminate the observed responses in fish. This evidence strongly suggested that one or more chemicals other than halogenated organic compounds, present in PPMEs, were also acting as EDCs. The failure of the presence of PCDDs/PCDFs to correlate with several biochemical responses, confirmed that PCDDs/PCDFs might not be the only chemicals involved in causing the physiological responses observed in fish near pulp mills (Servos et al. 1994). Fish exposed to a wide variety of PPMEs indicated that the pulping process itself, rather than the bleaching process, might be the major source of EDC-like environmental contaminants that were capable of inducing the EROD based hepatic MFO activity. These enzymatic inducers may have originated from the wood, or from the byproducts of pulping processes. Hence, the chlorinated compounds formed during pulp bleaching, may not be the only compounds responsible for the inducing capacity of the PPMEs. Although there were no chlorinated compounds present in the unbleached effluents of the studied mills, induced cytochrome P-470 mono-oxygenase activities were observed. It is however, possible that the inducers originated in the wood material (Lindstrom-Seppa et al. 1992). Further, internal and external process improvements have led to minimization or even elimination of a number of chlorinated organic pollutants from PPMEs. For example, conversion of a kraft mill bleaching process to 100% chlorine dioxide essentially eliminated the discharge of PCDDs and PCDFs (Owens et al. 1994). Therefore, the presence of other chemicals that are potential EDCs or environmental estrogens like phytoestrogens and plant sterols needs to be investigated. 7 2 Literature Review ? 2 Biochemical Effects Related tn PPMF* 2.2.2 P h y t o e s t r o g e n s a n d P lan t S t e r o l s Some naturally-occurring compounds like phytoestrogens and flavonoids, may compromise reproductive capacity and elicit toxic effects in livestock, rodents and fish (Pelissero et al. 1991). Such compounds modulate activities of different enzymes, and affect metabolism and biological activities of body estrogens and fatty acids, inducing estrogenic responses (Miksick 1994). Therefore, naturally occurring compounds released from wood during the pulping processes and discharged in PPMEs may induce receptor-mediated responses in exposed fish (Munkittrick et al. 1994; Servos et al. 1994). The estrogenicity of phytoestrogens is highly dependent on their stereo-chemical structure, particularly the distance between the two hydroxylated groups carried by the two aromatic nuclei and the angle of the twist of the two hydroxylated rings with respect to each other (Pelissero et al. 1991). Some phytosterols have a stereo-chemical structure close to that of the natural hormone estradiol- 17-p, and are, therefore, likely to be potent EDCs. Isoflavones, and coumestans are other phytoestrogens that may induce higher vitellogenin (a precursor of major egg yolk proteins) levels in fish exposed to them (Pelissero et al. 1991). The presence of phytoestrogens in plants used for animal feed and the deleterious effects of excessive exposure have been recognized for several decades (Kaldas and Hughes 1989). However, the observed impacts of phyto-origin EDCs or HAAs include both androgenic as well as estrogenic effects. Plant sterols can also be a potential source of androstane steroids that are capable of stimulating abnormal growth in fish (Rosa-Molinar and Williams 1984). Masculinized female mosquitofish (Gambusia affinis holbrooki) were found up to 6 km downstream from the discharge point of PPMEs (Howell et al. 1980; Rosa-Molinar and Williams 1984), while no masculinized fish were found upstream. Phytoestrogens like plant sterols and flavonoids were suspected to modulate the normal function of the fish endocrine system, probably through receptor-mediated responses. Such reactions can induce other biochemical effects resulting in defective growth and reproduction of fish in the receiving waters. Phytosterols in tall oils, present in kraft mill effluents, are a possible source of environmental contaminants causing masculinization. Cody and Bortone (1997) associated tall oil reclamation efforts with an overall reduction in the observed masculinization in the 8 2 Literature Review 2.2 Biochemical Effects Related to PPMEs receiving water fish under study. The degree of arrhenoidity (masculinization) or phenotypic sex change in fish can be influenced by the timing and duration of the exposure to androgenic agents, and environmental conditions like water temperature, stream flow, effluent treatment and other factors that affect the metabolic activity. Of all the plant/wood-derived potential EDCs, plant sterols are of most concern, and they have been reported to occur in PPMEs. Due to structural similarity plant sterols may probably act as precursors in the formation of different hormones or hormone-like steroids. The effects of phytosterols exposure may depend upon a variety of environmental conditions including season, tree species being pulped, degree of effluent treatment, effluent dilution, developmental stage and the species of the organism under exposure. The observed response may further depend upon the nature of the activated androgen-estrogen receptor complex rather than that of the hormone bound to it (Le Menn et al: 1980). Arrhenoidy or masculinization of both G. affinis mosquitofish and Heterandria formosa least killifish (Poeciidae family) was observed after exposure to transformed phytosterols that included sitosterol and stigmasterol. In stream side-pool experiments after three or more weeks of exposure to PPME, a group of G. affinis emerged that was statistically intermediate between masculinized and non-masculinized reference samples (Davis and Bortone 1992). Arrhenoidy of H. formosa was observed to be equivalent or more pronounced than that of G. affinis. Microbially-degraded phytosterols and other halogenated organics like dioxins/furans were the suspected EDCs for the androgenic responses. The degree of arrhenoidy may be affected by the seasonal levels of phytosterol precursors in the processed trees (Casey 1980) and environmental parameters such as dissolved oxygen, temperature, microbial activity, and other chemicals in PPMEs. The likelihood that a female anal fin may become modified in the direction of a typical male gonopod appears to be high in juvenile specimens of fish, it decreases with age and becomes limited in older specimens because of a fixation of the fin in a female pattern and the decreased growth characteristics (Turner 1941). However, additional structure may be formed within the six differentiation areas to the extent to which it is possible, in a fin already fixed in a female pattern. Davis and Bortone (1992) also described permanent androgenic effects in female fish of poeciliid species, which related to the concentration and duration of exposure as well as the life stage of the fish. 9 2 Literature Review 2.2 Biochemical Effects Related to PPMEs A survey of the aquatic environmental impacts associated with PPME discharges from 12 Canadian pulp mills showed different types of hormonal effects in fish, including decreased levels of circulating sex steroids, decreased gonad size and increased liver size. Induction of EROD (hepatic ethoxyresorufin-O-deethylase) enzymes and depressions of plasma sex steroid levels during gonadal growth were found in fish downstream of pulp mill sites. These changes were seen at some mills without chlorine bleaching and at mills that had secondary treatment, indicating the presence of non-chlorinated EDCs (Munkittrick et al. 1994). Although the effects could be seen at low concentrations of a few ug/L, the dilution level was the dominant factor for determining the presence or absence of these responses. Strong androgenic effects were observed in female mosquitofish (Gambusia affinis holbrooki) exposed to (i-sitosterol and stigmastanol, in the presence of the microorganism Mycobacteria smegmatis, suggesting that sterols may be transformed to active hormones (Denton et al. 1985). The most conspicuous feature of the masculinized female fish was the development of male-like gonopodia within six days, followed by anal fin ray changes a few days later. It was thought that androgenic-type substances might be formed during the degradation process of stigmastanol, (J-sitosterol and campesterol. Hunsinger et al. (1988) confirmed morphological changes in female fish, but detected no gross histological changes in ovaries. Degraded products of phytosterols (65% stigmasterol-30% fi-sitosterol) were linked to the masculinized female fish and the induced gonopodial development (Krotzer 1990). The masculinized female fish also exhibited male-like reproductive behavior. Lehtinen et al. (1990) reported altered primary and secondary adaptory mechanisms in a complex physiological pattern by stimulating or inhibiting detoxification of enzymes in the exposed fish. Intraperitoneal (within the area containing abdominal organs) injection of a phytosterol, R-sitosterol, into goldfish at doses 10, 20 or 100 p.g/g caused a dose-dependent decrease in plasma levels of testosterone and 11-ketotestosterone in males and testosterone and 17fi-estradiol in females. Similar effects were noted when goldfish were exposed to R-sitosterol in water at 75-1200 pg/L concentrations (Van Der Kraak and MacLatchy 1994). No effects on the levels of gonadotropin (a gonadotropic plasma hormone acting on or stimulating the gonads) were detected indicating that the pituitary gland rather than the gonad, was the primary target of B-sitosterol action. The waterborne exposure experiments suggested that &-10 2 Literature Review 2.2 Biochemical Effects Related to PPMEs sitosterol levels found in the vicinity of pulp mills (Ontario, Canada) were capable of producing reproductive effects in goldfish. Other biochemical responses include estrogenic effects seen through vitellogenin expressions in bioassays. Abnormal production of yolk protein vitellogenin (Vtg) and inhibition of testicular growth in adult male rainbow trout cock-fish was observed within three weeks of exposure to treated sewage discharges containing alkylphenols or lipophilic sterols (Harries et al. 1997). Wood-derived fi-sitosterol and abietic acid mixtures were found to be estrogenic to juvenile and methyltestosterone-treated fish (Mellanen et al. 1996). Both preparations induced expressions of the vitellogenin gene in the liver of rainbow trout, indicating possible reproductive dysfunction in the exposed fish. Plant sterols such as fi-sitosterol are common in pines (Browning 1963), and can be found in the vicinity of pulp and paper mills. These compounds were considered potentially estrogenic to fish (Van Der Kraak et al. 1994; Zacharewski et al. 1995). Both fi-sitosterol and abietic acid were found in PPMEs (Oikari and Holmbom 1996) and their presence was linked to the weak estrogenicity observed in debarking effluent (Mellanen et al. 1996). Pelissero et al. (1991) reported that phytoestrogens were estrogen agonists in nature, and confirmed the estrogenic potency of phytoestrogens in fish. PPMEs have been shown to affect reproductive and endocrine functions in fish. Tremblay and Van Der Kraak (1998) investigated the potential of fi-sitosterol through a series of in vitro and in vivo assays based on different levels of cellular organization in rainbow trout using the environmental estrogen nonylphenol (NP) and 17 fi-estradiol (E2) as references. In a receptor binding assay, fi-sitosterol was found to have a lower affinity for the rainbow trout hepatic estrogen receptor (ER) than NP and E 2. In a whole cell assay using primary cultures of hepatocyes, fi-sitosterol induced the production of vitellogenin (Vtg), which is an estrogen-dependent process. The estrogenic actions of fi-sitosterol and NP were confirmed in a whole animal assay, where Vtg production was induced in sexually immature rainbow trout exposed to waterborne fi-sitosterol, NP, and E 2 for 21 days. However, the Vtg production induced by fi-sitosterol and NP was much lower than E2. Plasma testosterone, pregnenolone and total cholesterol levels were reduced by fi-sitosterol but not by E 2 and NP, implying that this effect was independent of its estrogenic activity. Nonetheless, all the three assays: receptor binding, hepatocyte Vtg production, and in vivo Vtg production confirmed the estrogenicity of fi-sitosterol in trout. The in vivo assay demonstrated other effects not related to fi-sitosterol, 11 2 Literature Review 2.2 Biochemical Effects Related to PPMEs suggesting that there were multiple mechanisms through which fi-sitosterol can potentially modulate the endocrine system of trout. Both sexes of lake trout, in the maturing stage, were exposed to 10 and 20 ug/L phytosterols, mainly fi-sitosterol, for 4.5 months prior to spawning by Lehtinen et al. (1999). The results indicated a dose-dependent egg mortality, smaller egg size, lower weight of the yolk sac stage larvae, and a higher prevalence of deformed or otherwise diseased larvae, especially at the higher phytosterol doses. Several physiological parameters including higher plasma estradiol and EROD activity implied slower maturation of the exposed female fish. Indications of accelerated maturation of male fish were obtained from the same groups. The results indicated that naturally occurring wood-derived compounds in PPMEs were probably responsible for the reproductive impacts previously observed in fish, both in laboratory and in the water bodies receiving PPMEs. Vtg does not usually occur in juvenile or male fish. However, the liver of juvenile or male fish can be induced by estrogen to synthesize and secrete considerable amounts of this protein. Vtg levels can thus be used as biomarkers for estrogenic ecotoxicological contamination. Juvenile whitefish were exposed to diluted PPMEs from three operating pulp, paper, and paperboard mills in Southern lake Saimaa, Finland. All three mills used activated sludge secondary treatment systems for their effluents. Expressions of Vtg gene, a biomarker of estrogenic contamination of effluents, and increased mRNA (messenger ribonucleic acid) levels were found in fish caged in the vicinity of one of the three mills (Mellanen et al. 1999). The mill was found to discharge wood-derived compounds, such as plant sterols and resin acids, into the lake in amounts considerably exceeding those from the other two mills. The increased Vtg gene expressions and the mRNA levels, a more specific and reliable evidence for estrogen receptor mediated actins in vivo, suggested that PPMEs were a source of estrogenic contamination. Sexually immature rainbow trout were subjected to a 21 day in vivo exposure to two sources of BMEs (bleached mill effluents), two preparations of fi-sitosterol, and E 2 (17 fi-estradiol). Plasma vitellogenin (Vtg) levels were significantly elevated in all exposures, establishing the presence of estrogenic EDCs in the PPMEs and confirming the estrogenicity of a plant sterol: fi-sitosterol (Tremblay and Van Der Kraak 1999). Exposure to BMEs significantly reduced plasma pregnenolone levels and induced MFO activity but cholesterol levels were unchanged, fi-sitosterol exposure induced hepatic MFO activity and reduced both 12 2 Literature Review 2,2 Biochemical Effects Related to PPMEs the sex steroid precursor pregnenolone and cholesterol in a dose-dependent manner. This suggested that, in addition to fi-sitosterol, other compounds of an EDC nature existed in PPMEs that affected the fish physiology and reproductive end-points through more than one mechanism of action. The pulp and paper industry is a major user of non-ionic surfactants like alkylphenol polyethoxylates. Nonylphenol, a well known E 2 agonist, is a major degradation product of these surfactants and could have contributed to the observed Vtg stimulations. The exposure of marine fish, Viviparous blenny, to phytosterols during different phases of the life cycle of the parent fish (from oogenesis to parturition or giving birth and from breeding to parturition) and their offspring by rearing either in clean or phytosterol contaminated brackish water was studied by Mattsson et al. (2001a). The results implied that blenny offspring were affected by phytosterols through the exposure of the parental generation. Phytosterols affected the embryological development of the larvae before hatching as well as the levels of circulating hormones of the parent fish. In an other investigation by Mattsson et al. (2001b) juvenile female rainbow trout were exposed for 4.5 months to two dilutions of untreated and activated sludge treated-effluents from a pulp mill producing ECF (elemental chlorine-free) pulp. The results indicated that exposure to treated PPMEs elevated the metabolic turnover rate in fish followed by an increase in energy demand that was suspected as a result of interference with hormonal regulation. The endocrinological responses also indicated the biochemical transformation processes may occur in wastewater treatment systems. Plant sterols appeared to act as endocrine disruptors in farmbred juvenile European polecat (Nieminen et al. 2002), however, the induced effects were different from those reported in fish exposed to plant sterols. The estradiol levels of polecats of both sexes increased significantly with an increase in plant sterol doses. A positive correlation existed between the increasing plant sterols dose and the plasma testosterone, a phenomenon previously observed in the polecat with another endocrine disruptor, bisphenol A. The carbohydrate and lipid metabolism of the polecat were significantly changed including changes in the thyroid axis, and liver EROD activities (Nieminen et al. 2002). The induced effects resulting from sub-acute exposure of plant sterols: B-sitosterol and sitostanol, that 13 2 Literature Review 2.3 Nature of Steroids and Sterols revealed several metabolic changes, may become more intense in the presence of other EDCs that may be acting synergistically. 2.3 Nature of Steroids and Sterols 2.3.1 S t e r o i d s Steroids are a widely occurring group of colorless and often saturated natural products possessing the tetra-cyclic carbon skeleton that consists of three six carbon cyclohexane rings (A, B, and C) in the nonlinear or phenanthrene arrangement and one five carbon ring (D) with the structure as shown in Figure 2-1. This fused and reduced ring system, "perhydrocyclopentanophenantherene" is called "the steroid nucleus". Steroids contain the steroid nucleus and bear structural resemblance to terpenes, where terpenes are lipids having structures that are dissectionable to isoprene-like fragments (White et al. 1964). General structure of a Steroid nucleus steroid Figure 2-1. Structure of steroid nucleus (Perhydrocyclopentanophenanthrene) and a general steroid Steroids comprise a large variety of chemicals and a number of the steroids are natural hormones. Minor variations occur in different steroids due to nuclear substitutions, and degree of unsaturation, however, the diversity of the steroid compounds arises mainly from 14 2 Literature Review 2.3 Nature of Steroids and Sterols variations in the attached side-chains. Letters a or describe the orientation of attached hydrogen atoms or constituent groups (R1t R2) at C-18 and C-19, below or above the general plane of the ring system respectively. Usually, and R2 are methyl groups and R3 is a side chain. In some steroids and/or R3 may be absent. Sterols form a sub-category of the large group of steroid chemicals. The chemical structure of sterols is similar to hormones and some sterols serve as parent steroids in the formation of some important hormones, which are involved in growth and reproduction processes. More details about sterols are given in the succeeding sections. 2.3.2 Sterols and Stanols Sterols are unsaponifiable steroid alcohols with an aliphatic side chain. Sterols are widely found in nature both as free sterols and as esters of higher aliphatic acids. Depending upon their source, they can be: zoosterols (from animals) phytosterols (from plants) mycosterols (from yeasts and fungi) marine sterols (from sponges) Saturated derivatives of sterols are referred to as stanols. Sterols are isolated from the unsaponifiable fraction of fats, and are able to crystallize rapidly, usually forming a mixture of closely related compounds (Shoppee 1964). During the latter half of the eighteenth century, cholesterol (Figure 2-2) was discovered as a major constituent of human gallstones, and it is now known that it also accounts for about 17% of the solid matter of the human brain (Shoppee 1964). Cholesterol is a white crystalline substance soluble in alcohol and ether. 15 2 Literature Review 2.3 Nature of Steroids and Sterols CH 3 CH3* C H 3 C H 3 i T : H H HO H Cholesterol Figure 2-2. A common zoosterol Cholesterol was initially called cholestrine, but the name was changed to cholesterol when Berthelot (1859) showed it to be an alcohol (Myant 1981). Cholesterol is a characteristic sterol of higher animals that arises from acetic acid units and degrades in the organisms to cholic acid and other steroid hormones for which it serves as the parent steroid. Cholesterol is a major zoosterol while G-sitosterol, campesterol and stigmasterol are phytosterols. Ergosterol is the most common of the mycosterols that are found in yeast, and chalinasterol and stellasterol are examples of marine sterols (Shoppee 1964). The structures of some typical phytosterols are shown in Figure 2-3, as they are the focus of this investigation. 16 C2H 5 Stigmasterol Campesterol Figure 2-3. Chemical structure of some plant sterols 2.4 Sources of Plant Sterols Plants and wood constitute a major source of sterols in our water environment. Due to their chemical nature, sterols may persist in receiving waters for relatively long periods of time. As stated earlier, pine trees and other plants are natural sources of the phytosterols that may be present in varying amounts in the wood pulping effluents (Browning 1963; Conner et al. 1976). Phytosterols occur in both softwood and hardwood species and mostly form the unsaponifiable neutral fraction of wood extractives (Rydholm 1965; Pollak and Kritchevsky 1981; Sandstrom etal. 1996). 17 2 Literature Review 2.4 Sources of Plant Sterols 2.4.1 O c c u r r e n c e in P P M E s Wood derived sterols can make their way into pulping effluents as digestion by-products (Conner et al. 1976). Fatty alcohols and phytosterols like fi-sitosterol and stigmasterol mainly constitute the unsaponifiable neutral portion of wood extractives in approximate proportions of 30:63:7 (Rydholm 1965; Sandstrom et al. 1996). Sitosterols are lipophilic and are slightly soluble in water at all pH levels. During the chemical or semi-chemical pulping processes, lipids, including sterols, are released from wood. The released sterols mainly contribute to pitch deposits or wood resins (Peng et al. 1999) while some dissolved and suspended sterols make their way into the pulping effluents that are released into settling ponds or lagoons where phytosterols can accumulate and in the presence of certain microorganisms, sitosterols may be converted into androgenic substances (Denton et al. 1985) and finally enter the receiving waters where fish and other aquatic vertebrates are exposed to them. Chemical characterization of effluents from several Swedish bleached kraft mills using either conventional or modified cooking in combination with oxygen delignification and elemental chlorine-free (ECF) or totally chlorine-free (TCF) bleaching, indicated the presence of plant sterols in the effluents (Stromberg et al. 1996). A considerable variation in sterol content was observed among the BKMEs (bleached kraft mill effluents) studied and fi-sitosterol was the dominating sterol in the PPMEs investigated. The total sterol fraction appeared to be relatively higher in the case of mills using hardwood. The analysis of PPMEs from 22 US mills revealed the presence of plant sterols in varying amounts. li-Sitosterol was a major sterols fraction. The total sterol concentrations ranged from 71 u.g/L to 535 u.g/L (Cook et al. 1997). fi-Sitosterol was the only sterol quantified in all the effluents tested. The mills using recycled fibers generally had lower sterol discharges, although no relationships were observed between discharge rate and treatment system, location, or bleached and unbleached pulp production. While investigating the fate of extractives during secondary treatment of PPMEs from Norwegian mills, Magnus et al. (2000b) identified fi-sitosterol, campesterol, campestanol and fi-sitostanol in MTBE extractable material. Magnus et al. (2000b) quantified only one plant 18 2 Literature Review 2.4 Sources of Plant Sterols sterol: fi-sitosterol that was found up to 14.2 mg/L in MTBE-extractable material from the total mill effluents, 5.0 mg/L in primary treated effluents and 0.2 mg/L after secondary clarification. Total mill effluents carried (^ -sitosterol in both particulate as well as dissolved phases. However, about 36% of the extractable material could not be identified. McKague and Reeve (2003) analyzed PPMEs and sludges for plant sterols and reported that sitosterol concentrations ranged from 78-200 g/L in the unfiltered lagoon effluents. The sitosterol concentrations ranged up to 4.8 mg/g in the sludges and were reported to be about 7,000-70,000 times higher than those in the effluents, indicating plant sterols partitioning and binding to the microbial biosolids and sludges. Hence, there exists a considerable difference in the reported concentrations of sterols in PPMEs and often the individual sterols concentrations have not been quantified or mentioned. It is not clear what is a typical concentration range of sterols and which particular sterols other than li-Sitosterol can be expected in PPMEs from a typical pulp mill. Most of the research work related to sterols in PPMEs seem to have been done in U.S. and Scandinavian countries. Therefore, it is of interest to analyze PPMEs from Canadian mills to identify as well as quantify particular sterols present, to estimate their discharge to our receiving waters and any potential ecological concerns associated with them. 2.4.2 O c c u r r e n c e in S e w a g e Eff luents Domestic wastewaters contain large amounts of organic matter, about 20-25% of which is composed of lipids mainly from kitchen wastes (Bowerman and Dryden 1962; Loehr and de Navarra 1969). R-Sitosterol and cholesterol are main components of these lipids and are derived from vegetable oils and animal fats (Gunstone 1967). Moreover, sterols are also present in fecal wastes. Nearly 50% of these sterols are 5-R-stanol or coprostanols (Martin et al. 1973). Coprostanols are produced by bacterial hydrogenation of cholesterol, campesterol and S-sitosterol in mammalian guts (Eneroth et al. 1964). Raw sewage analysis shows that about 80% of the fatty acids and sterols are in particulate form, and about 80% of the particulate sterols are from fecal sources. The effluents from a number of sewage treatment works in the United Kingdom were estrogenic to fish caged downstream of their discharge points at distances of up to several 19 2 Literature Review 2.5 Degradation and Transformation of Sterols kilometers. The responses included abnormal production of yolk protein vitellogenin (Vtg) and inhibition of testicular growth in adult male rainbow trout within three weeks (Harries et al. 1997). Sumpter (1995) also found that the effluents from domestic wastewater treatment plants entering British rivers contained estrogenic EDCs that stimulated vitellogenin synthesis in male fish. Poorly treated alkylphenolic compounds or lipids like sterols were suspected EDCs or HAAs. Coprostanol, a reduced metabolite of cholesterol, is produced by micro-organisms and is present in urban effluents. Coprostanol is accumulated by organisms living in the vicinity of municipal outfalls. Freshwater mussels exposed in situ to the downstream waters of a municipal effluent plume for two months had accumulated coprostanol in their soft tissue, as compared with the mussels in the reference lake and upstream sites. The exposed mussels had significantly higher levels of vitelline like (resembling egg yolk) proteins. The addition of coprostanol to cytosol resulted in the increase of unbound labeled estradiol, which suggested that coprostanol prevented the binding of the labeled hormone to the cytosolic proteins. The weak binding affinity of coprostanol that elicited vitellogenic responses confirmed that coprostanol is estrogenic to freshwater mussels (Gagne et al. 2001). The formation of two intermediate compounds by the incubation of coprostanol in gonad homogenate extracts, further implied that coprostanol or other sterols may be biologically transformed into more potent estrogenic metabolites. 2.5 Degradation and Transformation of Sterols The ability of aerobic microorganisms of the soil and the gastrointestinal tract to degrade and transform plant sterols like fi-sitosterol has been reported (Charney and Herzog 1967; Arima et al. 1969; Nagasawa et al. 1969; Marsheck et al. 1972). However, some degradation byproducts of these sterols may closely resemble steroid hormones. Aerobic and/or anaerobic sedimentary microorganisms may transform li-sitosterol and other sterols into androgenic hormones like 5-fi-androstan3, 17-dione and androst-4-en-3, 17-dion (Rosa-Molinar and Williams 1984; Taylor et al. 1981). Such androstane derivatives of sterols may 20 2 Literature Review 2.5 Degradation and Transformation of Sterols ultimately interfere with endocrine systems and produce hermaphroditism* or other morphological effects. Plant sterols are presumably utilized by some groups of mycoplasmic" bacteria that possess sterols in their membranes (Brock and Madigan 1991). Degraded sterols can be androgenic to female mosquito fish, however, the exact identity and chemical nature of these compounds is not clear (Denton et al. 1985; Hunsinger et al. 1988). A side chain cleavage may be responsible for the conversion of C-29 parent phytosterols into common androgens, a mechanism similar to the conversion of cholesterol to pregnanolone (Hunsinger et al. 1988). Such transformations and additional metabolic reactions by microflora like hydroxylations and conjugations would increase polarity and would certainly favor the distribution of the androgenic metabolites in water. This would decrease adsorption inside fish because of their highly lipoidal gill apparatus, reducing internal effects. However, anal fin rays remain in contact with water, and would receive continuous exposure resulting in apparent responses. Although it has been documented that sterols are biodegradable, the complex structure of phytosterols present in the wood pulping effluents as digestion by-products (Conner et al. 1976) results in an inherent resistance to rapid breakdown into humic and fulvic acids. Further, effluent treatment systems do not appear to destroy phytosterols completely. Some of these phytosterols and their transformation products that are released to the environment may be acting as androgenic factors responsible for fish masculinization, as observed for poeciliid fish under lab conditions (Bortone and Davis 1994). Nonetheless the complexity of paper mill effluents presents a considerable challenge to specific identification of androgenic factors. As described before, some groups of bacteria can transform sterols to steroidal hormones, under a certain set of conditions. Marshek et al. (1972) reported such conversions of cholesterol, Is-sitosterol and stigmasterol. While Conner et al. (1976) demonstrated efficient conversion of sterols (85% (i-sitosterol and campesterol) present in tall oil obtained from kraft pulping of pine wood chips, into C19 steroids using Mycobacterium sp. NRRL B-3638. The obtained C19 could be used in several types of hormonal drugs. an animal or plant having both male and female reproductive organs ** polymorphic gram-negative bacteria, chiefly non-motile and parasitic prokaryotes, usually found in mammals 21 2 Literature Review ?.6 Secondary Treatment of P P M E s and Sterols Remova l While studying the physiological and phylogenic diversity of bacteria growing on resin acids, Mohn et al. (1999) reported at least two pure aerobic cultures Rodococcus strains DhA-55 and lpA-13 that could use B-sitosterol as a sole source of substrate. Therefore, biodegradation of sterols seems possible, but whether these potential EDCs are biodegraded during secondary wastewater treatment or not is explored in the succeeding section. 2.6 Secondary Treatment of PPMEs and Sterols Removal Studies conducted in 1988 and 1989 on Jackfish Bay, Lake Superior, Canada, showed several impacts of primary treated BKME discharges in both white sucker and lake whitefish (Munkittrick et al. 1991; McMaster et al. 1991 and 1992). The impacts included increased MFO activity and an altered suite of responses related to reproduction: depressed plasma levels of gonadal sex steroids, delayed age to maturation, altered fecundity, and reduced secondary sexual characteristics. The mill discharging into Jackfish Bay installed a secondary biological treatment system incorporating an aerated stabilization basin in September 1989 and made some in-plant process changes to reduce daily effluent discharge volumes, biochemical oxygen demand (BOD), and pollutant loading. These changes reduced BOD by 95%, total suspended solids by 20-30%, eliminated acute toxicity, improved water clarity in the bay, and substantially reduced the temperature of treated PPMEs. However, the persistence of hepatic MFO activity indicated that secondary treatment was not successful in removing the sub-lethal or chronic biological responses for the fish in the studied lake (Munkittrick et al. 1992). A rapid disappearance of MFO induction during a maintenance shutdown at the mill suggested that the MFO induction was not related to historical sediment contamination and that the metabolic clearance of the suspected inducers was rapid, at least in the species studied. Moreover, the effects of steroids may be indirectly related to MFO induction. Stromberg et al. (1996) reported variable removal of resin acids, fatty acids and sterols through different secondary treatment plants treating effluents from Swedish pulp and paper mills using soft and hard wood species. Study of whole mill effluents from 15 Finnish pulp mills revealed that the biological treatment of PPMEs generally removed lethal and sub-lethal toxicity to test organisms (Priha 1996). However, treated PPMEs from three Finnish mills 22 2 Literature Review 2.6 Secondary Treatment of PPMEs and Sterols Removal were clearly toxic to bacteria. Cook et al. (1997) surveyed 22 US pulp and paper mills, incorporating mainly activated sludge treatment (AST) and aerated stabilization basin (ASB) secondary treatment systems. The reported sterol removal efficiencies ranged from as low as 13% to as high as 95% (average 54%) for the surveyed secondary treatment systems (NCASI 1997). The estimated discharge level of plant sterols over a 13 months period was up to 28.6 g/adt (grams of sterols per air dried tonne of pulp) in treated PPMEs from the surveyed U.S. mills. In general, AST systems appeared to be relatively better than ASBs for removing plant sterols. Further, in the case of ASBs, a general trend for stigmasterol was an increase across the treatment system, while in AST systems all the tested sterols were generally removed. Comparison of treated effluents from different secondary wastewater treatment systems revealed that biological treatment was superior to physico-chemical treatment in removing the lipid contents of sewage including sterols (Quemeneur and Marty 1994). However, an increase was observed in fi-sitosterol content in the effluent that could be a result of biological input of cholesterol and fi-sitosterol during the complex degradation process, much of which remains unknown. A full scale high-efficiency compact reactor (HCR) activated sludge plant for treating PPMEs from an integrated newsprint mill was studied for the removal of organic compounds by Magnus et al. (2000a). The full scale HCR plant process removal efficiencies were 79% for COD, 77% for TOC and 98% for BOD5. The organics removal data revealed that almost all (100%) of the acetates, 93% of the carbohydrates, and 85% of the MTBE-extractable material was removed. However, the lignin-like material that was removed only to about 45% and the chelating agents like diethylenetriamine penta-acetic acid (DTPA) were not removed significantly. The lignin-like material was the main residual organics fraction in the wastewater after biological treatment, contributing 75% of the calculated COD. Hence, even a highly efficient treatment process that removes as much as 98% of the total BOD, may not remove some organic contaminants successfully. The investigation of the removal of wood extractives during the full-scale operation of an (HCR) activated sludge plant revealed that the identified lipophilic extractives (193 mg/L free/bound fatty acids, sterols, and resin acids) were mostly (95%) attached to particles and 70% were removed through primary clarification (Magnus et al. 2000b). In the bioreactors, 99% of lignans and resin and fatty acids were removed, fc-sitosterol was the only sterol 23 2 Literature Review 2.6 Secondary Treatment of PPMEs and Sterols Removal quantified and it was reduced by 96%. The removal mechanisms for B-sitosterol were both biosorption (46%) and biodegradation/transformation (50%). However, the unidentified MTBE-extractable material (36%) was only 37% removed. The unidentified extractive residuals in the biologically treated effluents were mainly found in the membrane filtrate showing that this non-biodegradable material was both extractable by MTBE and in the dissolved phase. If part of the unidentified lipophilic portion was composed of sterols, then the removal efficiency may be less than reported. However, the results indicate that biosorption and biodegradation may be equally responsible for sterols removal. Removal of resin acids and sterols from PPMEs, through activated sludge treatment, was investigated by Kostamo and Kukkonen (2003) in eastern Finland, during a study of the fate of wood extractives and toxicity. Secondary wastewater treatment appeared to remove most (~ 97%) of the wood extractives. The activated sludge treatment successfully removed PPME toxicity to luminescence bacteria and reduced the resin acids by over 94%. However, the degradation or biotransformation of sterols into other compounds was only about 41%. Furthermore, in general, less than 5% of the resin acids, but over 41% of the sterols, were removed in bio-sludge sent to the sludge thickener. Most of the discharged fraction of the extractives was attached to particles. Process disturbances, due to a sludge thickener overflow and a 1-day mill shutdown that increased the load of wood extractives, did not affect the operational efficiency of the secondary treatment system, probably due to the efficient adsorption of the extractives onto the bio-sludge. The overall performance as well as the operational stability of the activated sludge treatment plant were good. However, the concentrations of the wood extractives in the discharged water were still at a level that can have sub-chronic effects on organisms living in the receiving waters. Biological secondary treatment may be relatively effective in removing wood extractives including sterols from PPMEs. The reported literature supports the idea that some biodegradation of sterols is possible, but there are no correlations reported to associate the removal efficiency to the treatment process variables or the operating conditions of the secondary wastewater treatment plants. 24 2 Literature Review 2.7 Adsorption of Plant Sterols 2.7 Adsorption of Plant Sterols The removal and degradation of wood resin components in an activated sludge treatment (AST) plant treating PPMEs from Finnish mills was studied by Kaplin et al. (1997). The results in percent removal of all the three components of wood resins are reproduced in Table 2-1. Maximum removal and degradation/transformation were reported for resin acids. However, the observed overall removal was the lowest for plant sterols as compared with other wood resin components. Additionally, the poorest degradation/transformation was also reported for plant sterols. Sterols also showed great variations between the two different sampling series. Wood resin components, especially the free fatty acids, were mostly ( > 60%) partitioned to suspended solids. More than 74% of fatty acids were sorbed to particles. However, the resin acids and plant sterols were relatively less sorbed to suspended particles and the reported partitioning was not the same at all sampling points around the activated sludge treatment plant (Kaplin et al. 1997). The character and amount of suspended solids in PPMEs and the pH were among the suggested factors affecting the partitioning of wood resin components. Table 2-1. Percent removal and degradation of wood resin components in activated sludge (AST) treatment process (Kaplin et al. 1997) Wood Resin Component Removal from PPMEs (%) Degraded or transformed (%) 86 54-78 31-66 Adsorbed to sludge (%) 12 21 -58 44 -95 Discharged with final effluent (%) 2 5 - 7 6 - 9 Resin Acids 98 Fatty Acids 93 - 95 Sterols 91 - 94 About 46% of incoming fi-sitosterol was removed with excess sludge during secondary biological treatment that mostly removed the major phytosterol, fi-sitosterol, from PPMEs 25 2 Literature Review 2.8 Adsorption of Other Oraanics (Magnus et al. 2000b). The identification of fi-sitosterol at a concentration of 2.2 mg/g in secondary sludge suggested partitioning adsorption of sterols to bio-sludge. Analysis of PPMEs and sludges by McKague and Reeve (2003), showed sitosterol concentrations of up to 4.8 mg/g of secondary sludge. They further reported that the sterols were strongly bound to microbial biosolids with sitosterol concentrations in the sludges up to 70,000 times higher than those in the effluents. Sterols also appeared to be extracted relatively more effectively through accelerated solvent extraction (ASE) using toluene refluxing at 100°C (McKague and Reeve 2003 and 2001). The strong bonding of sterols to the biosolids suggested that the bio-availability of such compounds for biodegradation may be significantly reduced. At a Finnish elemental chlorine free bleached kraft pulp mill, activated sludge treatment reduced resin acids by more than 94% and sterols by over 41% (Kostamo and Kukkonen 2003). Furthermore, less than 5% of the resin acids and over 41% of the sterols were removed in the secondary sludge transferred to the sludge thickener. Most of the extractives were discharged attached to particles that hinted at efficient adsorption of wood extractives onto the bio-sludge. More insight into the adsorption process of plant sterols needs to be developed in order to understand and utilize the phenomenon of sterols bio-adsorption for reactor design and secondary treatment configuration optimization, to treat such organic pollutants up to a required standard. 2.8 Adsorption of Other Organics Hydrophobic organic contaminants (HOCs) have a natural tendency for biosorption that has been studied by many researchers as described in this section. Stringfellow and Alvarez-Cohen (1999) investigated the removal mechanisms of poly-nuclear aromatic hydrocarbons (PAHs) from refinery wastewaters typically treated with biological processes, in order to examine the relationship between PAH biosorption to bacterial biomass and PAH biodegradation. Biosorption decreased the rate of biodegradation in the short term. However, biosorption enhanced PAH removal from the wastewater and PAH retention in the treatment system where PAHs were ultimately degraded. PAH biosorption was not correlated with PAH 26 2 Literature Review 2.8 Adsorption of Other Organics degradation capabilities but the mean cell hydrophobicity was slightly correlated with PAH sorption. Different bacterial strains and species had different PAH sorption capacities (or partitioning Coefficient Kp). Non-degraders with high K p had significant impact on PAH biodegradation in vitro. The biosorption was identified as a bacterial surface adsorption phenomenon rather than a non-specific partitioning into the bacterial cell. The rate and affinity of HOCs biosorption to cyanobacteria" and algal detritus increased with a decrease in the adsorbent's average polarity, which was influenced by the nature and the degree of mineralization of the adsorbent (Koelmans et al. 1995). The dynamics of organic micro-pollutant biosorption to cyanobacteria and detritus further demonstrated that the rate of HOC desorption decreased with increasing hydrophobicity and with decreasing surface area to volume ratio. Hence slower desorption kinetics can be expected for HOCs with log Kow (octanol-water partitioning coefficient) values greater than 6, for example polychlorinated biphenyls (PCBs) and plant sterols. Wastewater organics adsorption equilibria and kinetics, that are important to determine the effectiveness of any adsorption system, were investigated by Chaudhary et al. (2003) using GAC (granular activated carbon). Freundlich isotherm and association theory were used to describe the adsorption behavior. The association theory was found to describe and predict the overall adsorption equilibrium of the wastewater system only slightly better than a Freundlich isotherm. However, the successful description and prediction of the wastewater. organics adsorption pattern demonstrated the usefulness of both of the adsorption theories. The mass transfer coefficient (MT) was observed to increase with an increase in the adsorbent dose and mixing intensity in the batch experiments. Adsorption is one of the most widely used treatment processes in the separation and purifying industry and the use of an adsorption process with GAC to purify and treat both natural water and wastewater provides a means to achieve almost complete removal of contaminants during advanced treatment processes. GAC is one of the most effective and widely used materials for these purposes. However, the phenomenon of adsorption to microorganisms i.e. biosorption, is neither well understood nor established properly in any thorough way as yet. The organics that adsorb to the biomass can be biodegraded simultaneously, and the resulting growth of biomass generates more adsorbent surface that chiefly aquatic chlorophyll-containing nonvascular polyphyletic prokaryotes: also called blue-green algae 27 2 Literature Review 2.8 Adsorption of Other Oraanics is available for further bio-adsorption. Klimenko et al. (2002) assessed the contribution of physical adsorption and biodegradation in a cumulative biosorption process using non-ionic and ionic surfactants as well as phenol as sorbates. The contribution of biodegradation to the biosorption process was estimated by comparing the equilibrium and dynamic bio-adsorptive capacities of the adsorbent, microorganisms attached to GAC. The dynamic adsorptive capacity (DAC) was always lower than the equilibrium adsorptive capacity (EAC), due to the absence of the kinetic factors, when there were no accompanying processes of biodegradation. Hence, the general efficiency of the bio-adsorption process depended upon the contribution of the biodegradation component. The impact of physico-chemical factors of the adsorption process was indicated as the change in the Gibbs free energy (-dG). The higher the change -dG, the lower the contribution of the biological oxidation to the biosorption process. A higher energy of adsorptive binding between the adsorbed component and the adsorbent surface, hinders the back diffusion of the adsorbed molecules. An essential decrease in the DAC is expected as the biodegradation products have predominantly lower molecular mass and higher solubility. The higher solubility reduces the adsorbability of these compounds, and the biodegradation degree so reached inhibits further biodegradation of the original compounds. The contribution of bio-adsorption is affected by the biodegradation processes and the EACs for the biodegraded or transformed products can be reduced significantly. Such a trend confirms the varying roles of biodegradation/transformation and adsorption mechanisms, which may result in fluctuating removal of compounds such as sterols during secondary treatment of PPMEs. The adsorption behavior of pentachlorophenol (PCP) from aqueous solution to activated sludge biomass was quantitatively characterized by Jianlong et al. (2000). The biosorption of PCP was described using a Freundlich adsorption isotherm. The experimental results indicated that the initial pH value and biomass concentration were important parameters affecting the adsorption capacity that increased with decreasing biomass concentration in the range less than 5000 mg/L and pH between 6 and 8. Both the biomass concentration and pH only affected the capacity constant KF of the Freundlich equation while the intensity constant n remained constant. The hydrogen ion concentration (pH) primarily affects the degree of ionization of the sorbate (PCP) and the surface properties of the sorbent (biomass). With an increase in pH, the overall surface charge on the cells may become negative, lowering the electrostatic attraction between the negatively charged PCP 28 2 Literature Review 2.8 Adsorption of Other Organics molecules and the binding sites of the biomass surface. These changes may lead to a shift in the EAC. Interestingly, the percentage of PCP removal increased with the increasing activated sludge amount, but conversely, the adsorbed amount of PCP per unit biomass quantity decreased with the increasing biomass concentration. The loss of adsorption capacity was thought to be due to the biomass binding sites remaining unsaturated during the adsorption reaction. Behavior of the sorption of HOPs onto particulates in an aquatic environment is significantly altered by the presence of DOM and its characteristics. Lee and Kuo (1999) quantified the overall effect of dissolved organic matter (DOM) on the sorption of HOPs. A mechanistic sorption model was used that combined different equilibrium relationships, including adsorption of DOM on particulates, the binding between the organic pollutants and DOM, and the sorption of organic pollutants on particulates with or without the presence of the DOM. In general, sorption equilibrium coefficients or EAC initially increased with the concentrations of DOM and then decreased after reaching a maximum value. An initial increase in DOM increases the adsorption of DOM to the adsorbent. The DOM adsorption, in turn, increases the organic carbon fraction of the adsorbent, thereby increasing the sorption of HOPs. After the adsorption of the DOM reaches a maximum, the sorptive capacity of HOP on DOM-coated sediment also tends to approach its maximum. As the concentration of DOM further increases, the surplus DOM in the aqueous phase increases the solubility of the HOP and, subsequently, reduces the apparent sorptive coefficient of the pollutant. This phenomenon of the reduced overall partition coefficient is termed the third phase effect. Finlayson et al. (1999) studied sorption of organic contaminants to fresh biomass in a minimally perturbed state. Significant removal of organic contaminants was observed through adsorption to biomass. The results showed that 40% of halogenated hydrocarbons and 75% of PAHs were removed through biosorption. Further, the addition of chromium ion (Cr+ 3), at low concentrations of 0.02-0.08 mg/mg VSS, was observed to change the biomass floe structure leading to increased sorption of organics to the biomass floes. Simultaneous biosorption of phenol and nickel (Ni+2) ions to dried activated sludge from binary mixtures was studied by Aksu et al. (1999), and compared with single phenol or N i + 2 in a batch system. The phenol and N i + 2 EAC of microorganisms was shown to be a function of initial pH and single or dual pollutants concentrations. Synergistic or antagonistic interactions occurring between the two or more components in a solution seemed to affect the individual 29 2 Literature Review 2.8 Adsorption of Other Oroanics component uptake by the microorganisms. Although the dried activated sludge had a higher adsorption capacity for phenol and nickel in a single component situation, the adsorption uptake of phenol and nickel decreased in binary mixtures because of the antagonistic interactions between the components/pollutants. The initial adsorption pH and the concentration levels of individual pollutants significantly affected the pollutant uptake. The reason for this behavior was claimed to be the competition for different kinds of adsorption sites on the cells and/or the screening effect by the second pollutant. Although the second component addition affected the adsorption uptake and rate negatively, dried activated sludge offered a practical approach to multi-component removal of pollutants from wastewaters. Gulyas et al. (1999) found that the sorption capacity of activated sludge for 2,6-dimethylphenol (DMP) was significantly affected by the duration of sludge aeration. The maximum sorption capacity was reached in 43 days of aeration (20.9 mg DMP/g MLSS at an initial concentration of 50 mg/L and a biomass concentration of 1 g MLSS/L). Aeration for longer periods resulted in a decrease in the sorption capacity of the sludge. The influence of aeration time on sorption capacity was thought to be due to changes in the sludge biocenosis and related changes in biomass surface structures. The sorption capacity of activated sludge was significantly lower than that of activated carbon. Nevertheless, activated sludge, which has been aerated for a period that leads to the maximum or optimum sorption capacity, can be used as a non-expensive adsorbent and some refractory organic pollutants of medium polarity may also be eliminated in a multistage biosorption process. The organics removal performance of an activated sludge process is influenced by the effect of biological adsorption or bio-adsorption of both the soluble and the particulate organic substances in the wastewater. The bio-adsorption capacity of activated sludge and the rate of organics degradation were found to be functions of COD concentration and temperature (Fujie et al. 1997). The biosorption capacity of activated sludge increased with an increase in COD concentration in the mixed liquor and temperature. The pollutant concentration fluctuations in raw municipal influent were considerably damped due to bio-adsorption of organics by the activated sludge. Resin acids, common organic pollutants in PPMEs, were found to be removed through a two phase partitioning process. The majority of resin acids were first removed rapidly to non-acclimated or inactivated anaerobic biomass, followed by a slower removal phase 30 2 Literature Review 2.8 Adsorption of Other Oraanics requiring about 5 days to reach equilibrium (Hall and Liver 1996). Lower biomass concentrations required longer equilibrium times. The adsorption of resin acids did not appear to be a reversible process. However, equilibrium was achieved after 12 h when non-acclimated inactivated aerobic biomass was used. The partitioning of resin acids onto inactivated aerobic biomass also followed a two-phase process that could be adequately described by a linear partitioning model. Sorption of HOCs like pentachlorophenol (PCP) is a dynamic process during the secondary wastewater treatment. Jacobsen et al. (1991) showed that PCP sorption may account for up to 50% removal of the influent load at low SRT (< 3 days) in the activated sludge process. No biodegradation was detected at the low SRT. On the other hand, sorption accounted for less than 10 % removal of PCP at high SRT ( > 14 days). Sorption isotherms indicated linearity up to 100 ug/L of dissolved PCP. Biodegradation of PCP was reported to have increased with increasing SRT. This indicated that biodegradation was probably occurring through catabolic growth of a specific fraction of biomass. At SRT > 8 days the 1 s t order biodegradation rate constant was approximately 2.5E-3 (mg MLSS-day) at 15°C. Melcer and Bedford (1988) also studied the fate of PCP in bench-scale continuous flow activated sludge reactors, and reported that relatively long SRTs were required for high PCP removals. In 10-20 day SRT systems, PCP concentrations of 0.1 to 12 mg/L degraded to less than 10 ug/L. Incomplete removal of PCP was observed in the 5 day SRT systems. However, the 15 day SRT activated sludge units achieved almost a complete removal of PCP at an influent level of 350 pg/L. Jacobsen et al. (1996) showed that the bio-adsorption of lipophilic HOCs like PCP is an important removal process in the biological secondary treatment process. The adsorption of PCP to activated sludge biomass was shown to follow linear sorption isotherm trends at low concentrations of up to 100 pg/L PCP in dissolved phase. The pH had a major impact on linear sorption coefficient, Kp, but the concentration of dissolved organic matter and added pH buffer also showed some influence. The linear sorption coefficient Kp increased significantly with a decrease in pH and decreased with an increase in pH within the tested range of pH from 5.9 to 8.0. 31 2 Literature Review 2.9 Summary The investigations related to the fate of HOCs and some other toxic and inhibitory organic contaminants like PCP, suggest that bio-adsorption is an important removal process in secondary wastewater treatment. The removal of organic pollutants, especially HOCs, during secondary wastewater treatment is a complex and dynamic phenomenon. It involves partitioning and adsorption of the organics to biomass and other suspended matter, which may or may not be accompanied by a simultaneous biodegradation process. The complexities involved indicate a considerable need for further research, particularly with respect to phytosterols removal from PPMEs. 2.9 Summary Plant sterols are suspected to induce physiological and biochemical responses in fish and other aquatic life by acting as hormone-like substances or as precursors of androgenic or estrogenic steroids. The chemical structure of plant sterols is similar to that of steroid hormones of vertebrate organisms. The transformation products of sterols may particularly resemble the steroid hormones more closely. Consequently, the discharge and control of plant sterols in PPMEs is important in the context of preserving and sustaining the ecological integrity of our receiving waters. Such contaminants have been termed endocrine disruption chemicals (EDCs) or hormonally active agents (HAAs) and may be poorly removed by the best conventional technologies available for wastewater treatment at present. The exact chemical nature of EDCs in PPMEs is not known, due to the complex nature of PPMEs. Chlorinated chemicals including dioxins and difurans were initially thought to be the primary EDCs responsible for the abnormal biochemical effects observed in fish. However, the evidence of abnormal physiological and reproductive responses in fish exposed to PPMEs from mills that are chlorine-free or effluents that contain negligible amounts of chlorinated compounds, suggests that there are other chemicals that are also EDCs. Plant sterols such as fi-sitosterol and/or their biotransformation products are suspected to cause fish masculinization or feminization by acting either as EDCs or precursors of EDCs. Plant sterols are naturally occurring compounds that originate from wood itself and are found in pulping effluents in varying concentrations as pulping by-products. Sterols are also present in the lipid portion of domestic wastewaters, with fi-sitosterol and cholesterol as main 32 2 Literature Review 2.10 Research Question and Hypothesis sterols originating from vegetable oils and animal fats. Sterols may be relatively resistant to biodegradation; however, some microorganisms have been reported to be capable of degrading sterols. Some studies have indicated removal of plant sterols from PPMEs by secondary biological wastewater treatment. A review of the published literature suggests that activated sludge treatment may be better suited for the removal of organic pollutants like plant sterols, as compared with the other major treatment system used by the pulp and paper industry, aerated stabilization basins. The chemical nature of plant sterols may lead to a natural tendency for bio-adsorption, which may be affected by a number of pertinent factors during secondary wastewater treatment. Secondary systems treating PPMEs may contain acclimatized sterols-degrading biomass, thereby making biodegradation of sterols an important process in the adsorption and removal of plant sterols from PPMEs. Nonetheless, a considerable knowledge gap exists regarding the fate of these compounds, the contribution of removal mechanisms involved and the factors that influence the treatment and control of such compounds during secondary treatment of PPMEs. 2.10 Research Question and Hypothesis The following research question was proposed: "Can phytosterols be removed in secondary wastewater treatment systems treating PPMEs?" The attempt to answer the above question further lead to finding the answers of the following sub-questions. How can plant sterols be analyzed in a complex mixture of P P M E s ? (Developing, adopting and testing proper chemical analysis protocols) 33 2 Literature Review 2.10 Research Question and Hypothesis Which plant sterols exist in local PPMEs? (Survey of effluents from local pulp and paper mills) (Types of major phytosterols present in PPMEs) (Sterol concentration range in local PPMEs) What happens to these sterols in a wastewater treatment plant? (Mass flow of sterols across a secondary biological system) If the phytosterols can be generally removed through secondary wastewater treatment of PPMEs (based on the results of the studies described in Chapter 3 and Chapter 4) the major research question could be modified to: To what extent can the sterols be removed in biological treatment systems? (Investigation of sterols' removal in lab-scale biological reactors) Answering the above question further raised the following sub-questions: To what extent does biodegradation of sterols contribute to the overall removal of sterols during the secondary treatment of PPMEs? (Investigation of sterols removal and biodegradation in lab-scale biological reactors treating PPMEs) To what extent does bio-adsorption of sterols contribute to the overall removal of sterols during the secondary treatment of PPMEs? (Investigation of sterol adsorption on to inactivated biosolids obtained from biological reactors treating PPMEs) 34 3 Quantification of Plant Sterols 3.1 Introduction 3. Quantification of Plant Sterols 3.1 Introduction Sterols occur in softwood as well as hardwood species (Gao et al. 1995; Holl and Goller 1982), usually with fi-sitosterol as the major component and they are often found in detectable concentrations in receiving waters in the vicinity of pulp and paper mills (Cook et al. 1997). Plant sterols, along with other compounds, have been suspected as potential EDCs or HAAs that may be estrogenic or androgenic to fish (Lehtinen et al. 1999; Zacharewski et al. 1995; Van Der Kraak and MacLatchy 1994). fi-Sitosterol and abietic acid present in PPMEs (Mellanen et al. 1999; Oikari and Holmbom 1996) may be directly or indirectly responsible for estrogenicity in debarking effluents (Mellanen et al. 1996 and 1999) assessed by vitellogenin expression bioassays. The presence of such phytosterols has also been noted by Stromberg et al. (1996) in acetone extracts of elemental chlorine free (ECF) and totally chlorine free (TCF) oxygen bleached modern pulp mill effluents. Consequently, it is important to isolate and analyze phytosterols occurring in PPMEs (pulp and paper mill effluents). Quantitative analysis of plant sterol from PPMEs is complex because PPMEs contain a mixture of chemicals including dissolved wood extractives, methanol, acetone, organic acids, cellulose and lignin products, sulphides and mercaptans, adsorbable organic halides (AOXs), sterols, steryle esters, triglycerides and other lipophilic compounds (Orsa and Holmbom 1994; Mimms et al. 1989). Although, standard analytical protocols have not been published for isolation and analysis of sterols, several analytical methods have been proposed for the analysis of wood extractives from PPMEs (Leach and Thakore 1976; Holmbom 1980; Ekman and Holmbom 1989; Alvarado et al. 1992), and from wood pulps (Rowe 1965; Sithole 1993; Gao et al. 1995; Peng et al. 1999). In general, these procedures focused on lipophilic fractions collectively and analyzed wood extractives as a whole. Plant sterols were analyzed indirectly. None of these methods was developed exclusively for sterols and most of the analytical procedures appeared to be laborious as well as expensive for analyzing large numbers of effluent samples in a reasonable amount of time. Therefore, an inherent difficulty 35 3 Quantification of Plant Sterols 3.1 Introduction in the present study was the development of analytical protocols to extract, isolate and quantify plant sterols present in PPMEs, with adequate levels of certainty and reproducibility. Thus, having a reliable technique for chemical analysis and quantification of plant sterols became the first objective of this research project. Sterols analysis has historically been complex and different researchers have used several approaches. However, extractions using solvents like acetone, chloroform-methanol mixtures or diethyl ether along with gas-liquid chromatography (GLC or GC) combined with mass spectrometry (MS) can be used for analyzing neutral steroids in complex mixtures (Abramovich et al. 1963; Eneroth et al. 1964; Monroe 1971; Holmbolm 1980). Other published literature also confirms the effectiveness of solvent extractions and chromatography for sterols analysis (Holl and Goller 1982; Ekman and Holmbom 1989; Suckling et al. 1990; Sithole 1992; Cocito and Delpheni 1994; Jansson et al. 1994; Gao et al. 1995; Hillinger et al. 1996; Elhmmali et al. 2000). Orsa and Holmbom (1994) proposed a relatively convenient analytical procedure for quantitative determination of lipophilic wood extractives, lignins and sterols, from papermaking process waters. After experiments with several solvents, Orsa and Holmbom (1994) chose methyl-t-butyl ether (MTBE) because it gave relatively high yields for lipophilic extractive groups and extracted lignans to a higher degree. The method comprised of MTBE extractions, silylation derivatization and gas chromatography. The National Council for Air and Stream Improvement (NCASI) further developed this technique utilizing a previously proposed Method 85.02 (NCASI 1986) for analyzing resin and fatty acids from PPMEs, and recommended a similar procedure for analyzing plant sterols (Cook et al. 1997). Other attempts at sterol isolation using adsorption/desorption on XAD-2 as described by Junk et al. (1974) were not successful (NCASI 1997), due to the semi-polar nature of plant sterols. Steam distillation did not work effectively because of the non-volatile nature of phytosterols. Solid phase extraction methods using C i 8 Empore™ disks, as well as liquid-liquid extraction employing NCASI Method 85.02 (NCASI 1986) for combined analysis of resin and fatty acids including sterols, resulted in poor recoveries of ethylated phytosterols and poor analytical reproducibilities (NCASI 1997). Chen et al. (1994) described a solid-phase extraction (SPE) method for separating lipid classes from acetone extracts of wood and pulps. However, the method was used only for qualitative separation of sterols. Mosbye et al. (2000) also suggested an SPE procedure for the combined analysis of fatty acids, resin 36 3 Quantification of Plant Sterols 3.2 The Selected Technique acids and sterols in white water from the mechanical pulp and paper industry, in which quantification was based on gas chromatography of the silylated compounds. The procedure was developed for application to a white water matrix, and did not appear to be more suitable than the liquid-liquid extraction procedure similar to that suggested by Orsa and Holmbolm (1994) and Cook et al. (1997). McKague and Reeve (2001 and 2003) investigated other methods of extraction and analysis of plant sterols, and reported that aggressive extraction procedures, like accelerated solvent extraction (ASE) with toluene refluxing at 100°C, were required for satisfactory analysis of aqueous samples and pulp mill sludges containing considerable amount of solids. The extraction of phytosterols with MTBE appeared to be a relatively more effective technique for isolating phytosterols from the complex mixture of PPME (Magnus et al. 2000b). Subsequent application of gas-liquid chromatography (GC) coupled with mass spectrometry (MS) seemed to be a promising technique for quantitative analysis plant sterols extracted from PPMEs. Therefore, the NCASI procedure described by Cook et al. (1997) that was similar to the method proposed by Orsa and Holmbom (1994) was selected for analyzing plant sterols in PPMEs for the present investigation. However, certain analytical problems were encountered before the procedure could be used successfully. Therefore, a considerable effort was invested to develop a reliable analytical technique in order to monitor sterols in PPMEs from different mills. 3.1.1 Research Question How can plant sterols be analyzed in a complex mixture of PPMEs? 3.2 The Selected Technique A second version of the analytical method STER-97 for the determination of phytosterols in PPMEs was published by NCASI in October 1997 with more details (NCASI 1997). This procedure was selected for the present study and it involved liquid-liquid lipophilic extractions using MTBE and silylation derivatization of the extracted sterols followed by 37 3 Quantification of Plant Sterols 3 2 The Selected Technique GC/FID (Gas Chromatography/Flame ionization Detection) analysis. The procedure was adopted with modifications and used for analyzing plant sterols in PPMEs obtained from local Canadian mills. Mass spectrometry (MS) was used instead of FID. 3.2.1 Analyte Selection As stated earlier, sterols are found in the plant kingdom, particularly in pine trees that are used in the pulping industry. Sitosterols are also expected to be present with other plant sterols (Browning 1963; Pollak and Kritchevsky 1981; Rydholm 1965). In light of the published literature (Cook et al. 1997; Verta et al. 1996; Cocito and Delfini 1994; Suckling et al 1990; Hafizoglu 1989; Ekman et al. 1989; Abramovitch et al. 1963; Rowe 1965; Monroe 1971) and the results of a preliminary screening of PPMEs from local mills, the following plant sterols were considered most likely to be found in PPMEs: • fi-sitosterol • fi-sitostanol • stigmastanol • stigmasterol • ergosterol and • campesterol These plant sterols were, therefore, selected as target analytes in the present study. Figure 2-3 on page 17 shows the chemical structure of some of these phytosterols. 3.2.2 Hydrogen Ion Concentration (pH) Near neutral pH (6-7) was selected for extraction in the present study, to minimize the effect of any changes in the state of extraneous organic materials with a change in pH and thus avoid potential for varying baselines and possible interferences. 38 3 Quantification of Plant Sterols 3.2 The Selected Technique Extraction experiments conducted by NCASI (1997) confirmed effective recovery of phytosterols from reagent grade water at pH values of 5 to 7. Orsa and Holmbom (1994) also reported that the extractive fractions (resin and fatty acids, and sterols) were extracted almost equally well at all pH levels (3.5 to 8) studied. 3.2.3 S t e r o l E x t r a c t i o n M o d e Whole mill effluents were used for isolation and quantification of plant sterols in PPMEs. Filtered solids and filtrate (filtered liquid) were also analyzed when required. Preliminary studies done in this study demonstrated that liquid/liquid extraction of the whole effluent gave comparable or even better results than Soxhlet extraction of filtered solids in combination with liquid/liquid extraction of the filtrate. 3.2.4 L i q u i d / L i q u i d E x t r a c t i o n A 25-100 mL aliquot of effluent was taken (and adjusted to pH 6-7 if required). The sample was then extracted with 15-40 mL of MTBE. The extractions were done in pre-fired clean Pyrex® glass centrifuge tubes fitted with Teflon lined screw caps. For effluents, the mixture of PPME and MTBE was shaken vigorously by hand and then with the help of a mechanical shaker (Burrell ™ Wrist-Action Shaker E-23, Burrell Corp., PA, USA) at a speed of about 140 cycles/min. for 15-20 min. The contents were allowed to settle for 10 minutes and then centrifuged for 10 minutes at an average relative centrifugal force of approximately 1600xg. The settled solvent extract was then collected as supernatant. The procedure was repeated two or three times for each sample to recover most (~95%) of the sterols. For sludge samples, 2-5 mL sludge was extracted three times with a combination of 4 mL of MTBE and 1 mL methanol by shaking with a wrist-action mechanical shaker for 15-20 minutes before centrifuging. Alternatively for sludge samples, freeze-drying and 12 h Soxhlet extractions with MTBE was also tried without better results. The solvent extracts from each sample were collected and combined. Then water was removed from the extracts by adding anhydrous magnesium sulfate in an Erlenmeyer flask. 39 3 Quantification of Plant Sterols 3.2 The Selected Technique The magnesium sulfate was then removed by filtering the extract using two rinses of MTBE. The extracts were concentrated to about 0.5 mL by placing them in a rotavaporator, transferred to a 5 mL glass reaction vial, and concentrated to dryness using nitrogen blowdown. 3.2.5 S i l y l a t i o n Der iva t i za t ion The extracted sterols previously concentrated to dryness were re-dissolved in 100 u l of hexane and 50-100 pL of MTBE, and suspended in excess silylating derivatization agent using approximately 10-15 p.L of BSTFA (N, O-Bis tri-methylsilyl-triflouroacetamide) per u.g of the estimated amount of sterols. The extracts were well mixed with silylation agent BSTFA using a vortex mixer and were incubated at a given temperature for a given amount of time as described in subsequent sections. Silylation is substitution of a TSM [trimethylsilyl - Si (CH3)3] group in a molecule in place of an active hydrogen (or a metal component of a salt). Silylation reduces the polarity of the compound and thus decreases the possibility of hydrogen bonding and increases stability and volatility of silylated derivatives during chromatographic analyses. It also improves the resolution and symmetry of chromatographic peaks. The effect of small differences between parent compounds is increased and peak overlapping and tailing effects are reduced after silylation. The analysis of the extracted sterols indicated that the silylated sterols were better suited for gas chromatography as compared with unsilylated sterols. 3.2.6 Internal S t a n d a r d Dotriacontane was used as an internal standard (IS) as recommended by Cook et al. (1997). It accounted for gas chromatograph instrument performance and response. Dotriacontane is not a chemical constituent of PPMEs and does not interfere with sterol peaks. It has a molecular weight of 450 g/mole that is similar to the weights of derivatized phytosterols that range from 466 to 482 g/mole. 40 3 Quantification of Plant Sterols 3.3 Plant Sterol Standards 3.2.7 S u r r o g a t e Cholesterol (Chole) was selected as a surrogate to monitor the extraction efficiencies and other losses to the glassware during the sample extraction and preparation. A number of chemicals that were initially thought to be suitable surrogates were employed, but they did not prove successful. Cholesterol is not always present in PPMEs. However, traces of cholesterol can be found in pine bark (Rowe 1965). Therefore, the presence of background levels of cholesterol was also assessed in PPME samples. 3.2.8 G a s C h r o m a t o g r a p h y / M a s s S p e c t r o m e t r y ( G C / M S ) A n a l y s i s The silylated sterol extracts were analyzed quantitatively using a Hewlett-Packard Model HP-6890 Series Gas Chromatograph/HP-5973 Mass Spectrometer Detector (GC/MS) System with a J&W DB-5 MS capillary column 29 m long, 0.25 pm coating, 0.25 mm internal diameter. The GC oven conditions were: 130°C, 1 min. hold; (Ramp 1) 130°C to 285°C @ 15°C/min. 3 min. hold; (Ramp 2) 285°C to 310°C @ 2°C/min. 1 min hold; post run 315°C 3 min hold; inlet at 290°C, carrier gas helium at 11.2 PSI and 53.6 mL/min; mass selective detector at 280°C. The conditions were modified to obtain better peaks, higher sensitivity and reduced peak overlapping. The HP 5973 Mass Spectrometer Detector coupled with the GC system provided satisfactory detection and identification of phytosterols and other test chemicals. 3.3 Plant Sterol Standards Authentic standards were obtained for each plant sterol. Table 3-1 presents the details of sterol standards, internal standard and surrogate along with their name codes, approximate retention times, and molecular weights with and without silylation derivatization. 41 3 Quantification of Plant Sterols 3 4 PPME Sampling Table 3-1. Target plant sterols, surrogate and internal standard Name Code Chemical Name (Company, purity) Formula MW MW (d) RT (min.) Chole Cholesterol (Sigma, 98%) C27H46O 386.70 458.70 17.83 Ergo Ergosterol (Sigma, 90%) C28H44O 396.33 468.33 19.03 Campe Campesterol (Sigma, 98%) C28H48O 400.70 472.70 19.37 Stigma Stigmasterol (Fluka, 98%) C29H48O 412.67 484.67 19.73 fi-Sito fJ-Sitosterol (Sigma, 98%) C29H50O 414.70 486.70 20.68 fi-Sitosta B-Sitostanol (Sigma, 98%) C29H52O 416.71 488.71 20.89 IS-3 Dotriacontane (Aldridge, 97%) C32H66 450.90 - 18.65 {MW=Molecular Weight; MW (d)=Silylated Molecular Weight; IS=lnternal Standard; RT= Approximate Retention Time} 3.4 PPME Sampling Two pulp and paper mills, Mill A and Mill B, located on the West Coast of British Columbia were selected for PPME sampling. Both of the mills use state-of-the-art UNOX (Union Carbide ™ pure oxygen) activated sludge treatment (AST) bioreactors to treat their wastewaters. UNOX-AST systems use pure oxygen to maintain high-levels of dissolved oxygen and thus offer a relatively high degree of wastewater treatment in terms of biochemical oxygen demand (BOD), suspended solids and acute toxicity removal. During the period of sampling, Mill A was producing kraft and newsprint pulp, using 100% hemlock for kraft pulp and a mix of spruce, pine and fir for newsprint with a bleaching sequence of ODEopDED. Mill B was producing kraft pulp, with a wood furnish of about 70% hemlock, 15% cedar, 15% fir, with DEoEopD bleaching sequence. 42 3 Quantification of Plant Sterols 3.4.1 P r e l i m i n a r y S a m p l i n g 3.5 Results and Discussions During the testing and development phase of the adopted analytical procedure, PPME samples were shipped to UBC on a weekly basis from the pulp and paper mills for a period of about three months. The PPME samples were shipped in plastic containers and included primary effluent (secondary influent to the UNOX-AST biobasins) and final effluent (overflow from the secondary clarifiers) from Mill A and Mill B. 3.4.2 O n - S l t e S a m p l i n g After the chemical analysis protocols were tested and modified to give reliable and consistent results for phytosterols present in PPMEs, on-site sampling was conducted. The details of the on-site sampling procedures and their results are discussed in Chapter 4 which exclusively deals with the occurrence of plant sterols in PPMEs. The focus of this chapter is the chemical analysis of plant sterols and the application of the selected analytical technique. 3.5 Results and Discussions 3.5.1 S i l y l a t i o n o f S t e r o l s The initial problem with the recommended procedure was inefficient silylation derivatization of plant sterols as recognized by the presence of underivatized peaks in the chromatograms and confirmed by mass spectrometry. This resulted in poor calibration curves without much linearity and trend. Soon it was discovered that effective silylation of the extracted sterols was not attained by leaving the mixture of sterol extracts in contact with excess of BSTFA overnight either at room temperature, or at an elevated reaction temperature of about 40°C. BSTFA is a highly reactive silylation agent that can be used for silylation (derivatization) of the active hydrogen(s) of non-hindered hydroxyl group(s) in the study of steroid profiles by 4 3 3 Quantification of Plant Sterols 3.5 Results and Discussions gas-liquid chromatography or GLC (Gleispach 1974). Cook et al. (1997) suggested an overnight incubation at room temperature, whereas others have suggested relatively short incubation times at elevated reaction temperatures i.e. Orsa and Holmbom (1994) recommended 20 minutes for silylation at 70°C and NCASI (1997) suggested about 1 hour of incubation at room temperature. Therefore, in the present study, the extracted sterols and the silylation agent, BSTFA, were incubated at different reaction conditions, starting from an overnight incubation at room temperature, and from 1 to 24 hours at 40, 65 and 70°C. The results are shown in Figure 3-1 to Figure 3-5. More detailed results are given in Appendix A. CO 9> co CD 0_ 40 000 35 000 30 000 25 000 < 20 000 15 000 10 000 5 000 • 1 h a 3 h 40 000 10 50 100 250 B-Sitosterol (pg/L) 35 000 4 30 000 25 000 co f 20 000 co 0) Q_ 15 000 10 000 5 000 -\ a 1 h s 3 h I::1 10 50 100 250 Cholesterol (ug/L) Figure 3-1. Increase in peak areas of sterol derivatives with incubation time at 65°C Most of the underivatized sterol peaks disappeared, especially at lower sterol concentrations, when the incubation temperature was increased from 40 to 65°C. However, at higher sterol concentrations, small underivatized sterol peaks could still be detected at the 65°C incubation temperature. The probable reason for the absence of underivatized peaks is 44 ( 3 Quantification of Plant Sterols 3.5 Results and Discussions that the underivatized sterols have a tendency to stick in the GC column and at low concentrations this would have reduced analytical recoveries greatly. Moreover, the peak areas of silylated sterols were found to increase when incubation time was increased from 1 to 3 hours, hence, the absence of underivatized peaks in the chromatograms did not guarantee the completion of the silylation reaction at the stated conditions (Figure 3-1). The silylation reaction was then allowed to proceed at 70°C for 1 and 3 hours. No underivatized peaks could be detected at any concentration tested at this incubation temperature (70°C). However, peak areas of silylated sterols showed an increasing trend with an increase in incubation time at this temperature as well. Figure 3-2 shows silylated sterol peak areas for different concentrations of S-sitosterol (fi-Sito) and cholesterol (Chole). When the incubation time was increased from 1 to 3 hours, the peak area increased as much as 100% at lower concentrations for each of the sterols tested. This demonstrated that complete derivatization of extracted sterols could not be accomplished in 1 hour at either 65°C or 70°C. 90 000 80 000 -I 70 000 60 000 -I g 50 000 -I < | 40 000 Q_ 30 000 20 000 10 000 0 El 1 h i 3h PS 10 50 100 250 B-Sitosterol (ug/L) 90 000 80 000 70 000 60 000 | 50 000 < | 40 000 30 000 20 000 10 000 0 4 • 1 h n 3 h 4 10 50 100 250 Cholesterol (ug/L) Figure 3-2. Increase in peak areas of sterol derivatives with incubation time at 70°C 45 3 Quantification of Plant Sterols 3.5 Results and Discussions An increase in silylation time caused relatively higher gains in peak areas at lower concentrations for the tested sterols. This effect may be due to lower reaction velocities at lower sterol (reactant) concentrations, assuming other conditions were the same. Similarly, at higher sterol concentrations, a shorter reaction time was required to achieve the same peak area in a chromatogram. In general, incubation at 70°C produced better silylation results with good chromatographic peaks and high peak areas. (0 CO a> Q_ "8 N O z 2.0 1.0 •a--. Chole-I - A - - - B-Sito-I •a -Cho l - l l - A - •- fi-Sito-ll a—Chol-Ave •A— B-Sito-Ave 0.0 -I • i ' i ' i ' i • i 1 i • h 0 1 2 3 4 5 6 7 Silylation Time (h) Figure 3-3. Optimization of cholesterol and B-sitosterol peak areas (silylated at 70°C) In subsequent experiments, the silylation reaction was allowed to proceed for up to 24 hours. Samples were taken from the same reaction vial (or batch reactor) at different times and analyzed for silylated sterols. In order to reduce the analytical instrument variability, sterol peak areas were normalized by dividing them with the peak area of the internal standard. The results indicated that silylation approached completion after about 3 hours, 46 3 Quantification of Plant Sterols 3.5 Results and Discussions causing the peak areas of the silylated sterols to reach their maximum values that declined to relatively steady values after 5 hours of incubation (Figure 3-3). Other important observations included the fact that at a given reaction temperature, different individual plant sterols seemed to follow a similar trend with silylation time. Therefore, it was decided to use only two individual phytosterols in further experiments to indicate the reaction trends for the rest of the sterols. The moderately reversible nature of the silylation reaction was also indicated by some decline in peak areas of silylated sterols, Chole and fi-Sito, after 5 hours of incubation at 70°C (Figure 3-4). co to Q_ N "55 CD E I Silylation Time (h) Figure 3-4. Change in cholesterol and stigmasterol peak areas with incubation time (silylation at 70°C) It was further confirmed that peak areas of silylated sterols Chole (cholesterol) and Stigma (stigmasterol) approached near maximum values at about 3.5-4 hours of incubation at 47 3 Quantification of Plant Sterols 3.5 Results and Discussions 70°C (Figure 3-4). Hence, silylation conditions of 3.5-4 hours of incubation at 70°C were selected for optimum silylation of sterols during the analyses. This modified set of silylation conditions reduced the silylation duration from 12 hours to 3.5-4 hours for each batch of samples, and resulted in increased peak areas of sterols. When the silylation reaction was allowed to proceed for 24 hours for five different plant sterols, the normalized peak areas of silylated plant sterols did not improve significantly (Figure 3-5). The prolonged incubation of sterols at a given reaction temperature (70°C), did not prove to be detrimental for the silylation derivatization, as the normalized peak area of silylated sterols slightly increased after 24 hours. It was further confirmed that all the tested sterols followed similar trends, as anticipated (Figure 3-5). 0.0 -I i i i i i i i i i i i 1 0 2 4 6 8 10 12 14 16 18 20 22 24 Silylation Time (h) Figure 3-5. Average normalized peak area of five sterols (optimization of silylated sterol peak areas at 70°C) 48 3 Quantification of Plant Sterols 3.5 Results and Discussions The identification of inefficient silylation derivatization is important because it can cause analytical problems and systematic error in the pollutant measurements. Method sensitivity will be reduced and low concentrations may not be detected due to the poor chromatography of the underivatized sterols. 3.5.2 C a l i b r a t i o n a n d Quan t i f i ca t i on For the purpose of calibration and quantification, six different sterols standards including Chole were used and it was found that each sterol would require its own individual calibration curve for its analysis (Figure 3-5). Another reason to include Chole in the sterol standards was the possibility of its presence in some PPME samples. As stated above, 3.5-4 hours of incubation at 70°C were considered optimal for silylation derivatization of phytosterols. Hence, derivatization time could be reduced by 66% (from 12 to 4 hours). Better calibration curves were generated with acceptable linearity and a correlation coefficient of about 0.98 (Appendix A1). Further reduction in the total analytical time was possible by modifying GC oven temperature conditions and holding times, but this had a negative effect on the standard calibration curves and overall linearity. Instrument gas flow and temperature program parameters were also adjusted for achieving reproducible non-overlapping peaks with reduced tails. In other experiments, it was observed that phytosterol standards could be derivatized effectively in 3-3.5 hours, but the same was not true for some effluent and sludge sample extracts. Some underivatized sterol peaks were encountered when PPME and sludge samples were incubated for less than 3.5 hours. These problems were addressed by increasing the reaction time to 4 hours at the same incubation temperature (70°C), making the sterol extracts free of water and keeping the extracted sterols dissolved in a mixed solvent composed of the silylation agent, hexane and a small amount (50-100 ul_)of MTBE. The average method detection limit (MDL) for the method itself was estimated to be 5 ug/L for individual phytosterols. However, 8-10 ug/L was taken as the practical quantification limit (PQL) for the sterols extracted from PPMEs. Sterol extracts from treated PPMEs were relatively cleaner as compared with the extracts from secondary sludge. Chromatograms presented in Figure 3-8, Figure 3-9, and 49 3 Quantification of Plant Sterols 3.5 Results and Discussions Appendix A 6 show chromatographic peaks for silylated plant sterols analyzed in a sterol standard, primary treated effluent (biobasin influent) and biologically treated or final effluent. 3.5.3 QA/QC Field and laboratory blanks were tested and the sterol extracts were analyzed in duplicate for QA/QC (quality assurance and quality control). In addition, some effluent samples were also extracted and analyzed in triplicate. The observed instrument variation was about 5-8%, while different extracts from the same effluent sample varied about 15%. For effluent samples, surrogate recoveries were relatively high with values of about 92% and above. However, the recoveries from sludge samples were relatively low with values of about 65% on average. More QA/QC data are given at the end in Appendix A 8. Results for reproducibility and precision for two plant sterols S-Sito and Chole are shown in Figure 3-6 and Figure 3-7. Gas chromatograms for primary effluent and biologically treated secondary final effluent are also shown in Figure 3-8 and Figure 3-9, respectively. Figure 3-6. Q A / Q C analysis for B-Sito (silylation 4 h at 70°C) 50 3 Quantification of Plant Sterols 3.5 Results and Discussions Cholesterol (Chole) Concentration (x10 ug/L) Figure 3-7. Q A / Q C Analysis for Chole (silylation 4 h at 70°C) Phytosterol calibration curves were prepared using eight or more standard points, and a separate calibration curve was used for each phytosterol. The calibration was checked as often as required and the instrument was re-calibrated when there was a change in sample source or some alteration in the effluent matrix was expected. The identification of each analyzed plant sterol was confirmed by its mass spectrogram for Q A / Q C purposes. Standard blanks and duplicate samples were also used to reduce analytical variability and to check for sample contamination. 51 3 Quantification of Plant Sterols 3.5 Results and Discussions c 3 o "5 o X CO a> a. «aOOO<»j *30QQG0-j I 1 mm*! • • ••{ ssoeoaao I: 1 j o i : i to. Retention Time (min.) Figure 3-8. Chromatogram for a primary effluent sample showing six plant sterols found in PPMEs (silylation 4 h at 70°C) 52 3 Quantification of Plant Sterols 3.5 Results and Discussions C 4530GC o. S2 g <i> X j«: co co 0_ mm :'CS; I 1 J h A i i I I n n. ,s n i M J:kA / I A i:i /i III: a-ca. i t 85 2 S. t t7.5.8 .49 -,r,«cj «r.«K> <$.oo ts'zo i*.eo «.«o 15.00 I€JC ?848 vt.m nj?n<s.m WMWASZO.®; se.8» 2 t e o 21.20 *t«c< Retention Time (min.) Figure 3-9. Chromatogram for a biologically treated final effluent sample showing five different plant sterols present in PPMEs (silylation 4 hours at 70°C) 53 3 Quantification of Plant Sterols 3.5 Results and Discussions 3.5.4 D i s t r i b u t i o n of P lan t S t e r o l s in L o c a l P P M E s After obtaining calibration curves with good linearity and reproducibility for each individual sterol, the modified analytical technique was applied for assessing the presence of phytosterols in pulp and paper mill effluents. For this purpose, primary treated and secondary treated PPMEs were obtained from two British Columbia coastal pulp and paper mills, Mill A and Mill B. Six different sterols were found in PPMEs from the two pulp and paper mills tested. The modified procedure was successfully applied to both primary as well as secondary treated effluents. Primary treated PPME (secondary influent) samples were mainly received from Mill A, and Table 3-2 presents the results for five weeks of sampling. Table 3-2. Plant sterols (pg/L) found in primary treated PPMEs from Mill A Sample Chole Ergo Campe Stigma S-SitO fi-Sitosta Total Mill A Weekl 35 ND 106 23 225 155 544 Mill A Week 2 47 30 152 34 324 222 809 Mill A Week 3 47 23 192 36 355 232 884 Mill A Week 4 39 ND 159 30 303 188 719 Mill A Week 5 55 ND 184 33 391 261 924 Average Value 45 27 159 31 320 211 792 Standard Deviation 8 5 34 5 63 41 152 Relative Standard Deviation % 18 19 21 16 20 19 19 Confidence Interval (95%) ± 10 ±6 ±42 ±6 ±76 ±51 ± 188 {Chole=Cholesterol; Ergo=Ergosterol; Campe=Campesterol; Stigma=Stigmasterol; B-Sito=IS-Sitosterol; B-Siiosta =R-Sitostanol; ND=Not Detected} 54 3 Quantification of Plant Sterols 3.5 Results and Discussions On average, primary treated PPMEs contained about 800 ± 190 pg/L total sterols in terms of the six plant sterols tested, including cholesterol (Chole), ergosterol (Ergo), campesterol (Campe), stigmasterol (Stigma), fi-sitosterol (fi-Sito) and fi-sitostanol (fi-Sitosta) or stigmastanol. The most abundant plant sterol was fi-Sito while Ergo appeared to be the least abundant of the sterols tested. Phytosterols make up about 6.0% of the wood extractives and about 10.6% of the bark extractives of pines (Hafizoglu 1989) and fi-Sito is a main phytosterol that is found in wood pulps and other plant materials (Browning 1963). This plant sterol (fi-Sito) has also been identified in the receiving waters within the vicinity of pulp and paper mills (Cook et al. 1997). fi-Sito alone was detected at up to 390 u.g/L for some samples, accounting for about 48% of the total sterols. On average, fi-Sito represented about 39%, while fi-Sitosta and comprised about 26% and 20%, of the total sterols, respectively (Table 3-2). Hence, fi-Sito, fi-Sitosta and Campe collectively composed more than 70% of the total sterols found in primary treated PPMEs from Mill A. Hence, it was confirmed that plant sterols were present in easily detectable concentrations in PPMEs from local sources. The plant sterols that originate from wood, make their way through the pulping processes and finally show up in PPMEs. The primary treatment of PPMEs may partially remove the incoming sterols but a significant portion of the plant sterols was still present in the primary treated effluents. In general, these results appear to be in agreement with the reported literature (Magnus et al. 2000b). Secondary treated PPME samples were mainly received from Mill B, and the results are presented in Table 3-3. Almost all of the six plant sterols that were present in primary treated PPMEs, were also detected in biologically treated PPMEs but in lower quantities. Chole was mostly absent from the biologically treated PPMEs or final secondary effluents. Chole and Ergo were the least abundant plant sterols in secondary treated effluents. On average, the total plant sterol concentration, in terms of the six analyzed sterols, was approximately 211 ±90 pg/L (Table 3-3). fi-Sitosta was the major plant sterol found in secondary treated PPMEs, instead of fi-Sito, which was the most abundant sterol in primary treated PPMEs. fi-Sitosta accounted for about 43% of the total plant sterols in terms of the six sterols tested in this study. fi-Sito and Campe followed fi-Sitosta quantitatively. Stigma, 55 3.5 Results and Discussions 3 Quantification of Plant Sterols Ergo and Chole were not present in detectable quantities in all the biologically treated PPMEs, however, some samples did contain these plant sterols in relatively low quantities. Table 3-3. Plant sterols (ug/L) found in biologically treated PPMEs from Mill B Sample Number Chole Ergo Campe Stigma fi-Sito fi-Sitosta Total Mill B Week 1 ND ND 35 11 33 118 197 Mill B Week 2 9 16 36.5 10 33 122 225 MilIB Week 3 ND 23 15.5 ND 43 50 132 Mill B Week 4 ND ND ND ND 50 47 98 MilIB Week5 ND 18 18.3 ND 116 118 271 Average Value 9 19 26 10 55 91 211 ±90 Standard Deviation 4 11 1 35 39 70 Relative Standard 21 42 6 64 43 33 Deviation % Confidence Interval ±5 ±14 ±43 ±48 ±87 (95%) {Chole=Cholesterol; Ergo=Ergosterol; Campe=Campesterol; Stigma=Stigmasterol; B-Sito=R-Sitosterol; B-Sitosta =B-Sitostanol; ND=Not Detected} A possible reason for the difference in the distribution of the plant sterols in primary and secondary treated PPMEs, is the different source of each type of PPME received. Primary treated effluents came from Mill A while the secondary treated effluents came from Mill B. A preferential removal of fi-Sito or any other individual sterol during the secondary wastewater treatment can affect the general distribution of the plant sterols in secondary treated final effluents. Cook et al. (1997) presented similar results obtained through a survey of 22 pulp and paper mills in the United States. However, Cook et al. (1997) did not find any correlation with the type of plant sterols present in PPMEs and their sources. 56 3 Quantification of Plant Sterols 3.5.5 P lan t S t e r o l s a n d S e c o n d a r y W a s t e w a t e r Trea tment 3.5 Results and Discussions The primary and secondary treated PPME samples discussed above, came from different mills. However, if the levels of plant sterols in primary treated PPMEs from Mill B are assumed to be similar to those found in primary effluents from Mill A, relatively lower sterol concentrations in secondary effluents from Mill A would suggest that the sterols were probably removed from PPMEs through the secondary wastewater treatment provided at the mills. The removal efficiencies for individual sterols could not be calculated because the samples came from different mills, but average plant sterol concentrations are shown in Table 3-4 for comparison purposes. The lower concentrations of phytosterols in secondary treated PPMEs as compared to the phytosterols in primary treated PPMEs, suggested removal of the plant sterols in secondary biological treatment systems. However, the extent and mechanism of plant sterols removal were not clear from the data. Additionally, it was also not clear whether the removed sterols were biodegraded or they were just removed from the liquid phase of PPMEs. Table 3-4. Compar i son of plant sterols (pg/L) found in primary and secondary treated P P M E s Effluent Stream Chole Ergo Campe Stigma fi-Sito fi-Sitosta Total Primary Effluents 45 27 159 31 320 211 792 . (Mill A) Secondary Effluents 9 19 26 10 55 91 211 (Mill B) {Chole=Cholesterol; Ergo=Ergosterol; Campe=Campesterol; Stigma=Stigmasterol; B-Sito=l2-Sitosterol; B-Sitosta =B-Sitostanol; ND=Not Detected} Some investigators have shown effective removal of organics and other contaminants from PPMEs through modern activated sludge treatment (Oikari and Holmbom 1996) and others have shown biodegradation of wood resins through biological secondary treatment (Werker 1998). Wood resins have a resemblance to plant sterols in their chemical structure, 57 3 Quantification of Plant Sterols 3.5 Results and Discussions and some microbial transformations of plant sterols have been reported (Marsheck et al. 1972) that may suggest some removal of sterols through biodegradation. Other evidence suggests sorption of organic contaminants like phytosterols onto suspended solids and biomass in the aeration basin (Sundin et al. 1999; Kaplin et al. 1997). However, more investigation seemed necessary to establish and evaluate the removal of plant sterols during secondary treatment of PPMEs. The complexities involved in the chemical analysis of sterols and the lack of a standardized analytical method may be two of the reasons why many researchers have not studied the behavior of plant sterols during secondary treatment of PPMEs. The need for a reliable method for analyzing a particular environmental contaminant such as plant sterols cannot be overemphasized. The achievements of this investigation include: identification of the ineffective silylation derivatization of the extracted plant sterols, modification of the suggested procedure for reliable analysis of plant sterols and successful application of the developed technique to detect and quantify plant sterols in primary and secondary treated PPMEs from local sources. Based upon the results obtained so far, several research questions arise that have not been fully answered at this stage. Among them the following questions appear to be more important because of their engineering significance. • What are the expected mass flow rates of plant sterols in secondary treated and untreated PPMEs, and waste secondary sludges? • Are the plant sterols effectively removed from PPMEs through secondary treatment? Such information is useful for subsequent environmental research including risk assessment, contaminant discharge limit considerations, wastewater treatment effectiveness assessments or the decisions about the treatment level requirements, the management and disposal considerations and programs for secondary sludge that may be contaminant rich. An attempt has been made to answer these questions within the scope of this thesis, in the next chapter that deals with the level of phytosterols in PPMEs and their secondary treatment. 58 3 Quantification of Plant Sterols 3.6 Conclusions 3.6 Conclusions A gas-liquid chromatographic analytical technique was modified for effective isolation, extraction and chemical analysis of plant sterols, suspected EDCs or HAAs, from a complex mixture of PPMEs. The procedure involved liquid-liquid extractions with MTBE and optimum silylation derivatization followed by GC/MS analysis. The modified technique was successfully used for plant sterols detection and quantification in primary and secondary treated PPMEs. Optimum silylation derivatization is required for effective chromatographic determination of the sterols extracted from PPMEs. Silylation appeared to improve the quality of gas chromatograms, analytical reproducibility and detection. Optimum silylation derivatization of sterols was achieved by incubating the extracted sterols with BSTFA at 70°C for about 4 hours. The silylation derivatization of sterols was also found to be unstable and reversible to some extent. The modified set of silylation conditions reduced the silylation duration from 12 to 4 hours, thereby reducing the total analytical time required for sterol analysis and improving analytical recoveries and method sensitivity for the low concentrations found in secondary treated PPMEs. Six different plant sterols were detected and quantified in PPMEs collected from two mills. fi-Sito, B-Sitosta and Campe were the major plant sterols measured in these samples. Total sterols content was about 800 ± 190 pg/L in primary treated PPMEs and about 211 ± 90 pg/L in biologically treated PPMEs or final secondary effluents. Chole, Ergo and Stigma were also present in PPMEs, but in relatively lower quantities and these sterols were not present in all of the secondary treated PPMEs. A general comparison of plant sterols found in primary and secondary treated PPMEs suggests sterols removal across the biological secondary wastewater treatment systems sampled. 59 4 Survey of Plant Sterols in PPMEs 4.1 Introduction 4. Survey of Plant Sterols in PPMEs 4.1 Introduction Specific data regarding phytosterol concentrations in mill effluents are not readily available, and a considerable difference exists in the reported levels of phytosterols in PPMEs. Cook et al. (1997) reported total sterols removal efficiencies for nine U.S. mills utilizing activated sludge treatment (AST) or aerated stabilization basin (ASB) secondary treatment systems. The results, which varied from negative values to about 90%, indicated a high variability in the overall removal of sterols entering secondary wastewater treatment systems. Moreover, the discharge of sterols at various mills varied from 7 to 29 g/adt (grams per air dried ton of pulp) in treated PPMEs. Similarly, the measured removal of sterols varied from 53 to 99% across secondary effluent treatment systems at Swedish mills pulping softwood and hardwood species (Stromberg et al. 1996). Assuming an average mill production rate of 1000 adt/day, the total sterol discharge from an average mill may be as high as 29 kg/day, however, the fluctuations in sterol levels and their removal efficiencies make the interpretation of the results difficult. Hence, it is of interest to investigate the occurrence, treatment and discharge of phytosterols in PPMEs. Preliminary analysis confirmed the presence of several plant sterols in primary effluents from two coastal British Columbia mills and suggested some removal of phytosterols during secondary treatment (Chapter 3). Wastewater treatment at an elemental chlorine-free (ECF) bleached kraft pulp mill in Finland also indicated an overall high removal of wood extractives but the estimated transformation or biodegradation of sterols was only about 41% (Kostamo and Kukkonen 2003). Similarly, Magnus et al. (2000b) reported about 50% biodegradation of sterols across a high-efficiency compact reactor (HCR) treating PPMEs from an integrated pulp and paper mill in Norway. Cook et al (1997) observed that activated sludge treatment (AST) systems were relatively better in removing phytosterols as compared to aerated stabilization basins (ASBs). However, the concentration of stigmasterol, a plant sterol, appeared to increase during the secondary treatment in ASBs (Cook et al. 1997). The considerable variation reported in the removal of phytosterols during wastewater treatment, 60 4 Survey of Plant Sterols in PPMEs 4.2 Sterols Analysis makes it interesting as well as necessary to investigate the removal of plant sterols during secondary treatment, to develop understanding about the assimilation of such environmental contaminants and improve there control. Not much information is available regarding the occurrence, removal and behavior of plant sterols entering biological secondary wastewater treatment systems at Canadian mills. To address the existing knowledge gaps, two state-of-the-art UNOX-AST (Union Carbide pure oxygen-activated sludge treatment) systems at British Columbia coastal mills were sampled for the present investigation. The objective of the study was to expand the knowledge of phytosterols presence in PPMEs and the fate of phytosterols entering the biological wastewater treatment systems. Such information is useful in developing strategies for effective treatment of such compounds and a knowledge of their environmental discharge and presence is necessary for any ecological risk assessment studies. 4.1.1 R e s e a r c h Q u e s t i o n s What is the concentration range of phytosterols that may exist in PPMEs? What is the fate of plant sterols during the secondary wastewater treatment? 4.2 Sterols Analysis 4.2.1 E x t r a c t i o n f r o m P P M E s Depending upon the type of the PPMEs (primary or secondary), 25-100 mL of effluent was extracted three times with 15-40 mL of methyl-t-butyl ether (MTBE) using pre-fired, clean Pyrex® centrifuge glass tubes. The detailed procedure for plant sterols extraction is given in Section 3.3.3. 61 4 Survey of Plant Sterols in PPMEs 4.2.2 S i l y l a t i o n , A n a l y s i s a n d Quan t i f i ca t ion 4.3 PPMEs Sampling The isolated plant sterols were then derivatized to trimethylsilyl ethers for improved gas chromatographic analysis. Water-free sterol extracts were incubated at 70°C for 4 hours in excess of silylation agent, BSTFA, according to the modified silylation conditions (Chapter 3). The silylated sterols were analyzed using a Gas Chromatograph/Mass Spectrometer (GC/MS) System (Section 3.3.8). Authentic standards of target phytosterols were purchased from reliable sources as indicated in Table 3-1. More details of sterols quantification procedure are given in Chapter 3. 4.3 PPMEs Sampling Two British Columbia coastal pulp and paper mills, designated as Mill A and Mill B, were selected for sampling. Both of the mills use state-of-the-art UNOX®-AST (Union Carbide pure oxygen-Activated Sludge Treatment) systems for wastewater treatment. UNOX systems utilize high-purity oxygen to achieve and maintain higher levels of dissolved oxygen aimed at providing better oxygen transfer for relatively higher levels of BOD (biochemical oxygen demand), COD (chemical oxygen demand), and acute toxicity removal from PPMEs. Compared to conventional AST systems, UNOX-AST systems potentially result in shorter detention times, more concentrated secondary sludge, smaller footprints and odor-control due to enclosed bio-basins. During the course of sampling, both mills were operating under typical production conditions. Mill A was producing kraft and newsprint pulp, using 100% hemlock wood furnish for the kraft pulp mill and a mix of spruce, pine and fir for the newsprint pulp mill. The bleaching sequence at Mill A was ODEopDED. Mill B was producing kraft pulp with a wood furnish of about 70% hemlock, 15% cedar, 15% fir. The bleaching sequence at Mill B was DEoEopD. The average combined mill effluent flow rates were 55 m3/adt at Mill A and 76 m3/adt at Mill B. 62 4 Survey of Plant Sterols in PPMEs 4.3.1 S a m p l i n g P r o g r a m I 4.3 PPMEs Sampling At each of the two mills, three sampling locations were chosen (Figure 4-1), for obtaining samples of influent to the bioreactors (Primary Effluent or PE), overflow from the secondary clarifiers (Final Effluent or FE), and recycle secondary sludge (RAS) from the secondary clarifiers. For acquiring representative samples, a set of four grab samples was collected at each of the three sampling locations using the sampling nozzles/valves provided in the delivery pipes carrying secondary influent, final effluent and RAS (Figure 4-1), both at Mill A and Mill B. Each sampling nozzle was allowed to flow for 1-2 min. before a sample was taken. An approximate interval of half an hour was maintained between consecutive samples at each sampling point. •— I Primary j / j Influent j I I 7 j Primary j L / j Effluent j Final Effluent Primary Clarifier — • Primary Sludge <8> Inlet Box UNOX Bio-Reactor — • Secondary Recycle Sludge Outlet Box Secondary Clarifier • ...VVasfe Sludge ® = Sampling Point Figure 4-1. Sampling Program I locations, schematic of UNOX-AST system and mass balance system boundaries (outside dotted line) For minimizing possible adsorption losses to the inner walls of the containers, all samples were collected in pre-fired clean amber glass containers with Teflon lined screw 63 4 Survey of Plant Sterols in PPMEs 4.3 PPMEs Sampling caps. A volume of 300 mL was collected for each sample of primary effluent, 600 mL each for final effluent samples, and 100 mL each for secondary sludge samples. For sample preservation, the collected effluent samples were stored in ice-packed coolers to keep the samples at a temperature of 4°C or less. The PPME samples were then brought back to the university and stored in a refrigerator at about 4°C for 1-3 d before analyses . For QA/QC (quality assurance and quality control) additional samples were collected in 1 L and 20 L containers for duplicate analyses along with field blanks. The samples were taken after rinsing the containers with the sample. On-site sampling, using clean glass containers, was aimed at having more representative results as compared with those shipped to the university, in plastic containers (Chapter 3). Moreover, the storage time prior to analysis was also reduced from 3 to 1 d. 4.3.2 S a m p l i n g P r o g r a m II In a separate sampling program, the variation in effluent concentrations of phytosterols was monitored in PPMEs before and after secondary treatment, on a weekly basis for a period of eight weeks. Grab samples of primary effluents and final secondary effluents from UNOX-AST system at Mill B were shipped in 25 L Nalgene ™ polyethylene containers to the university where they were refrigerated at 4°C before analysis. 4.3.3 S a m p l i n g P r o g r a m III In Sampling Program III, a more detailed sampling was completed to produce a better understanding of sterol concentration profiles in the UNOX-AST biological treatment system at Mill B. The UNOX-bioreactor consisted of two separate AST trains. Each train was further divided into three cells A, B, and C, which were connected in series. During the normal operation of the wastewater treatment system, one out of the two AST trains was kept on-line while the other AST train was kept off-line as a stand-by for emergency use and/or for regular maintenance shutdowns. 64 4 Survey of Plant Sterols in PPMEs 4.3 PPMEs Sampling Sampling Program III consisted of eight sampling cycles around the UNOX-AST system. In each sampling cycle, samples were collected at eight different locations from P1 to P8 as shown in Figure 4-2. Primary effluent samples were collected at sampling point P1; bioreactor mixed liquor (ML) samples from Cell A, Cell B and Cell C at sampling points P2, P3 and P4; biobasin outflow samples from the outlet box (P5); final effluent samples at sampling point P6; recycle and waste activated sludge (RAS and WAS) samples at sampling points P7 and P8 respectively (Figure 4-2). Eight sampling cycles were completed maintaining a time interval of about 1.15 h (i.e. 1 h and 9 min.) between each cycle. This interval was approximately the time required to complete a full cycle of sampling around the UNOX-AST system. The total time required for completing all eight cycles of sample collection at each of the eight sampling stations (P1 to P8), also covered the nominal hydraulic retention time (HRT) of about 7.7 hours that allowed the influent to pass through the treatment system. Primary Sludge Final Effluent Figure 4-2. U N O X - A S T system sampling station location details of P1 to P8 at Mill B (On-site Sampling Program II) 65 4 Survey of Plant Sterols in PPMEs 4,4 Results and Discussions All samples were collected in pre-fired clean amber glass containers with Teflon lined screw caps, containing MTBE solvent in different amounts that were considered appropriate for each type of sample (25-40 mL for primary effluent, mixed liquor, and biobasin outflow samples; 60-70 mL for final effluent; 5-7 mL for RAS and WAS samples). The solvent-containing sampling bottles were used for arresting any biodegradation, transformation or other changes like adsorption of plant sterols by starting the process of sterols extraction on-site. A volume of 50-60 mL each was collected for primary effluent samples (P1); 15-30 mL each for ML samples from each of the three biobasin cells (A, B, and C) and the biobasin outflow samples at the outlet box (P2, P3, P4 and P5); 70-100 mL each for final effluent samples (P6); and 2-5 mL each for RAS and WAS samples (P7 and P8; Figure 4-2). The collected samples were mixed thoroughly with MTBE and cooled down immediately after collection by storing them in ice-packed coolers where the storage temperature was kept at 4°C or less. The collected samples were brought back to the university (Environmental Engineering Laboratories) to complete the process of sterol extraction from PPMEs and subsequent chemical analyses of plant sterols. 4.4 Results and Discussions 4.4.1 A S n a p s h o t o f P lan t S t e r o l s in P P M E s ( S a m p l i n g P r o g r a m I) To assess the concentrations of plant sterols in PPMEs from the two selected pulp and paper mills, four grab samples of primary effluent, final effluent and recycle or return secondary sludge were collected from Mill A and Mill B, over a 90 minute sampling period. The results present a snapshot of the levels of the target plant sterols, during a period of normal operation of the mills. Individual sterols, as well as total sterols in primary and final effluents from both of the mills are presented in Table 4-1 and Table 4-2. The results for sterols in secondary sludge are presented in Table 4-3 and Figure 4-3. Estimated mass flows for total sterols are given in Table 4-4. 66 4 Survey of Plant Sterols in PPMEs 4.4 Results and Discussions Campesterol (Campe), fi-sitosterol (li-Sito), and li-sitostanol (li-Sitosta) were the major contributors to the total sterol content of PPMEs at both mills. Cholesterol (Chole) and stigmasterol (Stigma) were also present generally in lower quantities, while ergosterol (Ergo) represented the smallest fraction. Total sterol concentrations in the primary treated (biologically untreated) PPMEs ranged from 350 to 950 u.g/L (Table 4-1 and Table 4-2). fi-Sito was detected up to 500 pg/L and accounted for 30 to 50% of the total sterols. li-Sito, fi-Sitosta and Campe collectively comprised about 70% of the total sterol concentrations present in primary as well as secondary treated PPMEs. The total sterols detected in primary treated PPMEs from Mill A were slightly lower in concentration than those of Mill B. Table 4-1. Plant sterols (pg/L) in PPMEs at Mill A (Sampling Program I) Sample Chole Ergo Campe Stigma (3-Sito P-Sitosta Total Primary Effluent Sample 1 76 17 71 29 112 65 370 Sample 2 160 17 112 39 143 70 540 Sample 3 64 56 99 55 178 247 700 Sample 4 131 17 59 29 69 65 372 Average Value 108 27 85 38 126 112 496 Standard Deviation 45 20 24 12 46 90 158 Range (95% C.I.) (276-799) Secondary Effluent* Sample 1 15 13 46 20 15 18 126 Sample 2 17 11 53 19 18 22 141 Sample 3 18 16 57 16 18 24 149 Average Value 17 13 52 18 17 21 139 Standard Deviation 2 2 5 2 2 3 32 Range (95% C.I.) (43-234) Removal % 85 50 39 53 87 81 72% {* Sample 4 data did not pass QA/QC} 67 4 Survey of Plant Sterols in PPMEs 4.4 Results and Discussions On average, total sterols in the secondary treated PPMEs were around 150 pg/L. fi-Sito, fi-Sitosta and Campe were also the major contributors to the total sterol concentration in treated PPMEs. The sterol concentrations in secondary treated effluents from both mills were similar (Table 4-1 and Table 4-2). However, Campe comprised the largest fraction of sterols in secondary effluents, rather than fi-Sito, which was the major contributor to total sterols in primary effluents. This suggests that individual sterol fractions present in primary effluents may not be removed to the same extent during the secondary treatment. Table 4-2. Plant sterols (u.g/L) in PPMEs at Mill B (Sampling Program I) Sample Chole Ergo Campe Stigma p-Sito (3-Sitosta Total Primary Effluent* Sample 1 96 15 37 36 115 50 349 Sample 2 48 17 21 22 78 44 229 Sample 3 30 20 143 32 528 201 954 Average Value 58 17 67 30 240 98 511 Standard Deviation 34 2 66 7 250 89 389 Range (95% C.I.) (200-1400) Secondary Effluent* Sample 1 26 16 40 20 33 42 177 Sample 2 20 18 32 21 41 32 163 Sample 3 22 19 37 20 38 40 176 Average Value 23 18 36 20 37 38 172 Standard Deviation 3 2 4 1 4 6 32 Range (95% C.I.) (77-268) Removal % 61 -1 46 34 84 61 66% C Sample 4 data did not pass QA/QC} The percentage distributions of individual sterols were similar to those obtained in the preliminary analysis of PPMEs (Chapter 3), but the individual sterol concentrations were 68 4 Survey of Plant Sterols in PPMEs 4.4 Results and Discussions different. At both mills, the on-site sampling confirmed the presence of the same six sterols that were detected during the preliminary testing of PPMEs. Compared to the previous analysis (Table 3-2 and Table 3-3), on-site sampling Program I indicated a relatively wide range of phytosterol concentrations in PPMEs. The effluent samples, taken before and after the secondary biological treatment, also allowed the estimation of sterol removal efficiencies. Detected plant sterols appeared to be generally removed from PPMEs across the secondary treatment systems, but the removal efficiencies varied from approximately 34 to 84% (Table 4-1 and Table 4-2). The nominal sterols removal efficiency for total sterols was 72% at Mill A and 66% at Mill B. The secondary biological treatment systems at both mills produced comparable sterols removal efficiencies, but the removal efficiencies of individual plant sterols were not similar. The removal of the major plant sterols, B-Sito and B-Sitosta, was between 60-85% and the removal of other plant sterols, like Stigma and Campe, was less than 50%. Ergo was the least abundant sterol found in PPMEs at both mills, and no removal of Ergo was observed across the secondary wastewater treatment system at Mill B. The observed removal of plant sterols should be regarded as approximate, because the timing of secondary effluent and sludge sampling did not completely allow for the hydraulic retention times of the treatment systems. Magnus et al. (2000b) reported relatively high (96%) removal of wood extractives and sterols (B-Sito was the only plant sterol quantified) across a high-efficiency compact biological reactor treating TMP wastewater from an integrated newsprint mill. The estimated biodegradation for B-Sito was about 50%. Kostamo and Kukkonen (2003) reported 97% removal of wood extractives including sterols, but only about 41% of the sterols were degraded or transformed in an AST plant treating ECF effluents. The details of individual plant sterol removals were not available. Therefore, the observed removal of sterols from PPMEs across secondary treatment at two mills surveyed, may or may not suggest significant biodegradation of plant sterols. The results for plant sterols in secondary sludge are shown in Table 4-3, Figure 4-3 and Table 4-4. The secondary sludge sampled from Mill A contained an average total sterol content around 24,000 pg/L, while the secondary sludge from Mill B contained approximately 13,000 pg/L. On average, the individual sterols in RAS (recycle activated sludge) from Mill A were higher than that from Mill B and the concentration of Ergo was markedly high in RAS from Mill A. However, the concentrations of B-Sito and Campe were similar in both cases. 69 4 Survey of Plant Sterols in PPMEs 4.4 Results and Discussions The overall range of total sterols found in secondary sludge (RAS) at both mills varied from 10,000 to 32,000 ug/L. Table 4-3. Phytosterols (ug/L.) in recycle secondary sludge (Sampling Program I) Sample Chole Ergo Campe Stigma p-Sito p-Sitosta Total Mill A * Sample 1 1611 3656 2685 2205 6250 2347 18754 Sample 2 1900 4490 2838 2192 6626 2562 20607 Sample 3 1137 13273 2747 986 7688 6815 32646 Average Value 1549 7140 2757 1794 6855 3908 24002 Standard Deviation 385 5328 77 700 745 2520 7542 Range (95% C.I.) (5200-42000) Mill B * Sample 1 162 1405 3086 607 8108 1217 14585 Sample 2 197 1342 2800 482 7404 748 12974 Sample 3 180 998 2173 421 5723 830 10324 Average Value 180 1248 2686 504 7078 932 12628 Standard Deviation 18 219 467 95 1225 250 2151 Range (95% C.I.) (7200-18000) f* Sample 4 data did not pass QA/QC} The RAS samples from each of the UNOX-AST systems contained sterols at much higher concentrations than the influents and the effluents at these treatment systems (Table 4-3, Figure 4-3 and Table 4-4), suggesting an accumulation of plant sterols within the secondary sludge. This accumulation of sterols, may however, be the main cause of the observed sterols removal across the UNOX-AST systems. The probable reason for sterols accumulation in secondary sludge may be the moderately non-polar nature of the sterol molecules. The moderate hydrophobicity may result in partitioning and sorption of sterols onto the colloidal as well as the particulate suspended material including secondary biosolids (McKague and Reeve 2003). 70 4 Survey of Plant Sterols in PPMEs 44 Results and Discussions 600 500 4 E3 Primary Effluent • Final Effluent • Recycle Sludge 30 000 4 25 000 Mill A Mill B Figure 4-3. Plant sterols in PPMEs and secondary sludge (Sampling Program I) The RAS samples from Mill A indicated higher sterols concentrations than the RAS collected from Mill B (Figure 4-3). This indicated lower quantities of sterols being accumulated in secondary sludge at Mill B as compared to that of Mill A. More accumulation of sterols in secondary sludge at Mill A, may mean less biodegradation or more adsorption taking place at Mill A. Similar concentrations of B-Sito and Campe were measured in RAS from both mills, however, the concentrations of Chole, Ergo and B-Sitosta were orders of magnitude higher in RAS from Mill A than those in RAS from Mill B (Table 4-3). Differences in individual sterols present in secondary sludges from these mills may arise from the differences in the biodegradation/transformation as well as the adsorption behavior of sterols towards the secondary sludge present in each wastewater treatment systems. Additionally, Mill A was treating a small portion of sewage flow (~ 5%) from a nearby residential colony along with the PPME from the mill while the WWTP at Mill B treated PPME exclusively. Municipal 71 4 Survey of Plant Sterols in PPMEs 4.4 Results and Discussions wastewaters are reported to contain trace amounts of sterols like Chole and fi-Sito (Gunstone 1967). The contribution of such a small sewage input may not account for the higher concentration of Chole and other sterols found in RAS from Mill A, but this may have lead to the growth of dissimilar activated sludge microbial communities that have different adsorptive and degradation capacities. A transformation of concentration data into mass flow rates revealed that on average each mill was discharging approximately 12 kg/day sterols in its treated effluents (Table 4-4). These sterols that were discharged with secondary effluents accounted for about 30% of the sterols incoming with the primary effluents at each mill. Table 4-4. Estimated mass flow rate (kg/day) of plant sterols (Sampling Program-I) Sample Flow Stream Total Sterols (ug/L) Flow Rate Mass Flow Percentage (m3/day) (kg/day) (%) Mill A Primary Effluent Secondary Effluent 496 (SD* 158) 86400 139 (SD32) 85104 43 12 (100%) 28 Waste Activated Sludge* Mill B Primary Effluent Secondary Effluent 24002 (SD7542) 1296 511 (SD* 389) 68256 172 (SD32) 67392 31 35 12 72 100% (100%) 34 Waste Activated Sludge** 12628 (SD2151) 864 11 31 65% {*SD = Standard Deviation; **Recycle and Waste Activated Sludge sterol concentrations were assumed to be the same} Assuming that waste secondary sludge (WAS) contained similar amounts of sterols as that of RAS, about 70% of the incoming sterols were estimated to be discharged with WAS at Mill A and about 30 % of the incoming sterols appeared to be discharged with WAS at Mill B. 72 4 Survey of Plant Sterols in PPMEs 4.4 Results and Discussions Hence, the sterols mass balance provided no evidence of biodegradation of phytosterols during the secondary treatment at Mill A and only about 35% at Mill B (Table 4-4). The short term sampling results indicated that most of the incoming phytosterols passed through the secondary wastewater treatment systems with little biodegradation. Accumulation of phytosterols in the mixed liquor and secondary sludges appeared to be mostly responsible for the observed removal of sterols from PPMEs. Poor biodegradation of sterols in treatment systems (Quemeneur and Marty 1994) and strong partitioning of fi-Sito and fi-Sitosta onto biosolids has been reported (McKague and Reeve 2001). Therefore, if the biodegradation rate of phytosterols in activated sludge is lower than the sterols mass loading rate, sterol accumulation in the sludge is expected until the sorptive capacity of the sludge is reached, resulting in higher sterol concentrations in secondary clarifier underflow and mixed liquor suspended solids (MLSS). More investigation was required to clarify the mechanisms of sterols removal and degradation for developing effective control strategies and to improve the design of wastewater treatment systems treating PPMEs. 4.4.2 Sampling Program II The fluctuations associated with the measured phytosterol concentrations at both mills indicated a need for analysis of PPMEs over a longer period of time. Mill B was selected for such monitoring, and phytosterols were analyzed weekly in PPME grab samples of primary and secondary effluents collected over a period of eight weeks. The observed sterol concentrations are shown in Table 4-5, the time series plots of individual and total sterols are shown in Figure 4-4 and Figure 4-5. The associated data for individual sterols are shown in tabulated and box plot formats in Appendix B. Average total sterol concentrations in primary effluents ranged from 900 to 1200 pg/L and the total sterol content of biologically treated and clarified final effluent varied from 130 to 230 pg/L (Table 4-5). Chole was not detected in treated or untreated PPMEs at Mill B. Individual and total sterol concentrations in primary and final effluents were relatively consistent between week 2 and week 5 (Figure 4-4 and Figure 4-5). However, higher 73 4 Survey of Plant Sterols in PPMEs 4,4 Results and Discussions variability in the other samples indicated a non-steady mass flow of sterols in and out of UNOX bioreactors. Table 4-5. Plant sterols in primary and secondary effluents at Mill B during 8-week monitoring of PPMEs (Sampling Program II) Sample Primary Effluent Final Effluent Removal (M9/L) (M9/L) (%) Week 1 843.9 196.4 76.7 Week 2 1109.1 179.3 83.8 Week 3 1212.6 199.6 83.5 Week 4 1186.9 196.2 83.5 Week 5 1180.8 190.4 83.9 Week 6 857.7 280.1 67.3 Week 7 1222.9 94.4 92.3 Week 8 837.1 101.9 87.8 Average 1056.4 179.8 82.4 SD 177.3 59.1 7.5 RSD (%) 16.8 32.9 9.1 SE= SD/nA0.5 62.7 20.9 95% t * SE = ± ± 148.3 ±49.4 95% C.I. Low Limit 908.1 130.3 74.6 95% C.I. High Limit 1204.7 229.2 89.1 {SD=Standard Deviation; SE=Standard Error of the Average; C.I.=Confidence Interval} Time series monitoring of PPMEs exhibited relatively narrow sterol concentration ranges for both primary and secondary effluents (Table 4-5), as compared with those observed during the short term sampling Program I at Mill B (Table 4-2). The monitoring samples were shipped to the laboratory in 20 L plastic containers, and had to be stored for 2-3 days before analysis. Some reduction in sterol concentrations, due to the adsorption of 74 4 Survey of Plant Sterols in PPMEs 4.4 Results and Discussions lipophilic wood extractives including sterols, to the container walls was possible. However, biodegradation losses due to the presence of microorganisms were expected to be negligible, as the sterols mass balance calculations suggested limited biodegradation of sterols during the secondary treatment. Additionally, total sterol concentrations in some of the monitoring samples were higher than those analyzed in on-site sampling Program I. During the time series monitoring, the sterols removal efficiency was about 80%, based on average concentrations of the sum of the analyzed sterols entering the UNOX-AST system, for a period of three to four weeks (Figure 4-4 and Figure 4-5). This is higher than the 66% removal estimated during the screening sampling at Mill B (Program I). The discrepancy cannot be adequately explained but one contributing factor may be that the 8-week monitoring did not compensate for the retention time of the UNOX-AST system. Figure 4-4. Time series plot of plant sterols in primary effluent at Mill B 75 4 Survey of Plant Sterols in PPMEs 4.4 Results and Discussions Based on average concentrations, the removal efficiencies for individual sterols varied between 45% for Ergo to 87% for Campe. fi-Sito, fi-Sitosta, and Stigma showed average removal of 83, 84 and 79% that almost matched the calculated removal of total sterols (83%). Ambrus et al. (1995) reported microbial transformations of some plant sterols (B-Sito and Stigma) by genetically modified strains of Mycobacterium sp. BCS 396. Marsheck et al. (1972) also documented bacterial transformations of Chole and Stigma without appreciable degradation of the steroid nucleus, during attempts to demonstrate microbial conversion of these sterols to androstane steroids. Marsheck et al. (1972) also hinted at the significance of the side chain associated with these sterols in their susceptibility to microbial degradation. Therefore, selective removal of individual sterols through biodegradation is possible during the secondary treatment. 300 Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 Figure 4-5. Time series plot of plant sterols in final secondary effluent at Mill B 76 4 Survey of Plant Sterols in PPMEs 4.4 Results and Discussions In general, secondary biological treatment appeared to remove plant sterols from PPME at Mill B. Nonetheless, other related questions regarding the extent of bio-adsorption of sterols to the secondary solids, the degree of sterol biodegradation or transformation in the UNOX-AST bioreactors and the removal of sorbed sterols through waste secondary sludge, could not be answered fully. This provided motive for further investigations about the behavior of phytosterols and the involved removal mechanisms during secondary biological treatment of PPMEs. 4.4.3 B e h a v i o r o f P lan t S t e r o l s d u r i n g S e c o n d a r y T r e a t m e n t o f P P M E s ( S a m p l i n g P r o g r a m III) A subsequent, more detailed on-site Sampling Program III focused on the remaining questions about sterols behavior during secondary treatment. The program allowed for the treatment system detention time, and additionally included the sampling of MLSS, biobasin outflow, RAS and WAS streams at Mill B (Figure 4-2) to resolve some of the questions raised in previous sampling programs. Biodegradation of the target plant sterols was arrested immediately by starting the sterols extraction process on-site. Eight sampling stations or sampling points (P1 to P8) were selected around the UNOX-AST system. Sampling was done at these stations (Figure 4-2) for a period of about nine hours, a period slightly longer than the nominal hydraulic detention time of about 8 hours for the UNOX-AST bioreactor. The results for individual and total sterols in secondary influent (primary-treated effluent) are presented in Table 4-6 and Figure 4-6, for mixed liquor flow from biobasin Cell-A, Cell-B, Cell-C and outlet-box (biobasin outflow before the secondary clarifiers) in Figure 4-7, Figure 4-8, Figure 4-9, and Figure 4-10, respectively. Table 4-7 and Figure 4-11 show the results for overflow from the secondary clarifiers (final effluent). Vertical bars on the total sterol curves in these figures show the standard error estimates*. The standard error bars are shown to indicates the variability associated with the average values (Siegel 1988). Table 4-8 shows the relative concentrations of individual sterols in different streams at the treatment system. Sterol removal efficiencies for overall average of total sterols as well as individual sterols are given in Table 4-9. Total sterols removal and UNOX-AST influent as well as * Standard error of the average = (standard deviation)/(square root of the number of data values) 77 4 Survey of Plant Sterols in PPMEs 4.4 Results and Discussions effluent sterol concentrations are shown in Figure 4-12. The vertical bars in Figure 4-12 illustrate the (two-sided) 95% confidence interval" for average total sterol concentrations and the removal efficiencies. The associated data are reported in Appendix B 8 to B 12. Table 4-6. Sterols (pg/L) in the influent to the UNOX-AST system at Mill B each sampling cycle (Program III) S Cyc le 9 C h 0 l e E r 9 ° C a m P e Stigma fi-Sito fi-Sitosta Total Sterols 1 0.0 0.0 241.0 86.5 328.5 177.9 833.9 2 0.0 39.8 294.4 158.9 345.1 259.9 1098.1 3 0.0 25.9 326.9 157.7 378.7 300.1 1189.3 4 0.0 11.8 493.6 234.1 548.7 321.7 1610.0 5 0.0 33.6 446.4 505.8 564.1 420.3 1970.2 6 0.0 11.2 675.9 583.6 848.4 493.7 2612.8 7 0.0 51.7 308.8 165.8 380.6 289.6 1196.6 8 0.0 0.0 611.3 262.1 816.8 466.2 2156.3 Average 0.0 21.8 424.8 269.3 526.4 341.2 1583.4 SD 0.0 19.0 159.0 179.2 208.7 109.0 614.2 SE 0.0 6.7 56.2 63.4 73.8 38.5 217.1 t(n-1)xSE ±0.0 ±15.9 ±132.9 ±149.8 ±174.5 ±91.1 ±513.6 95% C.I. Low 0.0 5.8 291.9 119.5 351.8 250.1 1069.8 95% C.I. High 0.0 37.7 557.7 419.1 700.9 432.3 2096.9 {SD=Standard Deviation; SE=Standard Error of the Average; C.I.=Confidence Interval} The total sterols content varied approximately from 1000 to 2500 pg/L in primary-treated effluent - secondary influent (Figure 4-6), from 2000 to 7000 pg/L in the UNOX-AST biobasin mixed liquor (Figure 4-7 to Figure 4-9), from 2000 to 4000 pg/L in outflow from the Confidence interval = average ± [ t(n-l) x standard error] where t(n-l) is the critical value 78 4 Survey of Plant Sterols in PPMEs 4.4 Results and Discussions biobasin (Figure 4-10) and from 100 to 700 ug/L in final effluent from secondary clarifiers (Figure 4-11). In primary-treated PPME, fi-Sito was the major plant sterol and Campe was 2 n d most abundant sterols followed by fi-Sitosta, Stigma and Ergo, respectively. UNOX-AST Influent 3 000 -, 1.15 2.30 3.45 4.60 5.75 6.90 8.05 9.20 Sampling Time (h) —•—Ergo A Campe - X - Stigma —»—R-Sito — B — fi-Sitosta —o— Total Sterols Figure 4-6. Sterols in primary-treated influent to UNOX-AST-Sampling Station P1, Mill B. Vertical bars indicate standard error estimates The plant sterols concentrations in mixed liquor inside the three biobasin cells and in the biobasin outflow before clarification were higher than those in primary-treated secondary influent (Figure 4-7 to Figure 4-10). This confirmed a trend of sterols accumulation within the UNOX-AST mixed liquor and secondary sludge. The accumulation of plant sterols was probably happening through the mechanism of adsorption to the microbial biomass and suspended secondary solids. Moderately non-polar plant sterols would have a natural tendency to adsorb to microbial biomass and secondary solids. Plants sterols have been observed to be removed from PPMEs (Mahmood-Khan and Hall 2003) and one of the main mechanisms suggested for sterols removal was through their partitioning and adsorption to secondary solids and sludge (McKague and Reeve 2003). 79 4 Survey of Plant Sterols in PPMEs 4.4 Results and Discussions UNOXBioreactor Cell A 6 000 -i CO 1.15 2.30 3.45 4.60 5.75 6.90 8.05 9.20 Sampling Time (h) —-—Ergo A Campe —X--Stigma -«—S-Sito —a—B-Sitosta —o--Total Sterols Figure 4-7. Sterols in biobasin Cell-A, UNOX-AST-Sampling Station P2, Mill B. Vertical bars indicate standard error estimates The concentration of plant sterols in mixed liquor and biobasin outflow was also expected to increase due to the combination of primary-treated influent and return activated sludge (RAS) entering the UNOX-AST biobasin. RAS contained about 16 times higher sterol concentrations than primary-treated influent (Figure 4-6 and Figure 4-15). The measured sterol concentrations in mixed liquor were roughly three (2.86) times higher (Figure 4-7 to Figure 4-10) than the theoretical mixed liquor sterol concentrations that would result from mass balance calculations. The probable reason for this may be the fact that secondary solids spend more time in the treatment system than liquid effluents as the solids retention time (SRT) is usually greater than the hydraulic retention time (HRT). Hence mixed liquor sterols would tend to increase until the sorptive capacity of the solids is exhausted or reaches an equilibrium. However, a portion of the incoming plant sterols may be degraded or transformed in the biobasin even if biodegradation of sterols was proceeding at a low rate. 80 4 Survey of Plant Sterols in PPMEs 4.4 Results and Discussions UNOXBioreactor Cell B 1.15 2.30 3.45 4.60 5.75 6.90 8.05 9.20 Sampling Cycle Figure 4-8. Sterols in biobasin Cell-B, UNOX-AST-Sampling Station P3, Mill B. Vertical bars indicate standard error estimates UNOXBioreactor Cell C 6 000 5 000 4 w 3 000 2 CO 2 000 1 000 4 —«—Ergo —A—Campe X Stigma —a—B-Sito —a— 6-Sitosta —o—Total Sterols 1.15 2.30 3.45 4.60 5.75 6.90 8.05 9.20 Sampling Cycle Figure 4-9. Sterols in biobasin Cell-C, UNOX-AST-Sampling Station P4, Mill B. Vertical bars indicate standard error estimates 81 4 Survey of Plant Sterols in PPMEs 4.4 Results and Discussions UNOX-AST Biobasin Outflow 4 500 - i 1.15 2.30 3.45 4.60 5.75 6.90 8.05 9.20 Sampling Time (h) —«— Ergo —A—Campe —H— Stigma -•—B-Sito —o— S-Sitosta —o—Total Sterols Figure 4-10. Sterols in mixed liquor at biobasin outlet, UNOX-AST-Sampling Station P5, Mill B. Vertical bars indicate standard error estimates The relatively high mixed liquor sterol concentrations were reduced sharply by about 90% after secondary clarification and final effluent contained total sterols between 110 to 670 pg/L (Figure 4-10, Table 4-7 and Figure 4-11). Secondary clarification is a gravity-assisted sedimentation process and the reduction in sterol concentrations through secondary clarification indicates that the removed sterols were either associated with secondary solids or present in particulate form. The general composition of individual sterols inside the UNOX-AST biobasin and in secondary effluent appeared to be different than that in primary-treated influent entering the biobasin. The relative concentrations of fi-Sito and fi-Sitosta increased from 33 to 40% and 21 to 28% in mixed liquor and those of Campe and Stigma decreased from 27 to 20% and 17 to 11% respectively (Table 4-8). The causes of such variations are not known. However, a decrease in the relative proportions of Campe and Stigma suggested that these sterols were removed to a higher degree through secondary treatment as compared with fi-Sito and Ergo, 82 4 Survey of Plant Sterols in PPMEs 4.4 Results and Discussions the relative proportions of which increased while passing through the UNOX-AST (Table 4-8). Nonetheless, a general decrease in total sterol concentrations was observed (Table 4-9) and this has also been reported previously by some (Mahmood-Khan and Hall 2003, Kostamo et al. 2004). The changes in the relative concentrations of the individual sterols passing through secondary treatment have not been reported before. Table 4-7. Sterols (pg/L) in final effluent from UNOX-AST system at Mill B for each sampling cycle (Program II) Sampling Cycle Chole Ergo Campe Stigma S-Sito fi-Sitosta Total Sterols 1 0.0 6.2 29.6 15.2 42.5 28.2 121.7 2 0.0 9.6 26.3 13.5 37.7 24.3 111.5 3 0.0 14.4 28.5 15.2 38.9 25.2 122.1 4 0.0 42.6 94.7 38.9 132.3 90.2 398.7 5 0.0 0.0 137.7 111.2 276.2 141.5 666.5 6 0.0 0.0 74.3 55.7 150.3 72.9 353.2 7 0.0 0.0 59.1 32.7 163.3 56.8 311.9 u 8 0.0 0.0 45.3 30.8 115.4 46.3 237.8 Average 0.0 9.1 61.9 39.1 119.6 60.7 290.4 SD 0.0 14.6 39.1 32.5 81.7 40.4 188.7 SE 0.0 5.2 13.8 11.5 28.9 14.3 66.7 t*SE = ± 0.0 12.2 32.7 27.2 68.3 33.8 157.7 95% C.I. Low 0.0 0.0 29.3 12.0 51.3 26.9 132.7 95% C.I. High 0.0 21.3 94.6 66.3 187.9 94.4 448.2 {SD=Standard Deviation; SE=Standard Error of the Average; Cd.=Confidence Interval} 83 4 Survey of Plant Sterols in PPMEs 4.4 Results and Discussions Figure 4-11.Sterols in final effluent, UNOX-AST-Sampling Station P6, Mill B. Vertical bars indicate standard error estimates Table 4-8. Relative proportion (%) of individual sterols in different UNOX-AST streams at Mill B (Program III) Description Chole Ergo Campe Stigma fi-Sito fi-Sitosta Total Sterols Primary Effluent 0.0 1.4 26.8 17.0 33.2 21.5 100 Biobasin Cell-A 0.0 0.3 19.7 12.8 42.6 24.7 100 Biobasin Cell-B 0.0 0.6 19.5 12.4 41.3 26.2 100 Biobasin Cell-C 0.0 0.1 19.6 10.8 43.9 25.6 100 Biobasin Outflow 0.0 1.3 19.9 11.4 39.3 28.2 100 Final Effluent 0.0 3.1 21.3 13.5 41.2 20.9 100 84 4 Survey of Plant Sterols in PPMEs 4.4 Results and Discussions In final effluent, the differences in relative concentrations were noted only for B-Sito and Campe, the former increased to about 43% and the latter decreased to about 20% as compared with those in primary effluents(Table 4-8 and Figure 4-11). This indicated a relatively greater dissolved fraction of B-Sito present in secondary-treated effluent and hinted at lower removal of fi-Sito through secondary treatment. The relative proportions of other individual sterols in final effluent and mixed liquor appeared to be similar, in that the major plant sterol was B-Sito, the minor sterol was Ergo, and Chole was not detected (Table 4-7 and Table 4-8). Similar sterol proportions in final effluent and mixed liquor can also be expected because the most of the biological activity takes place in aeration basin where the required levels of oxygen, substrate and nutrients are maintained. Additionally, most of the adsorption of plant sterols on the secondary solids is also expected to occur within the biobasin, due to the continuous mixing and agitation provided there. Table 4-9. Removal of plant sterols (%) through the UNOX-AST system at Mill B (Program III) Description Chole Ergo Campe Stigma B-Sito S-Sitosta Total Sterols Cycle 1 & 8 ND ND 81.2 64.4 64.9 74.0 71.5 Overall Average ND 58.1 85.4 85.5 77.3 82.2 81.7 Estimated High - 108.2 94.8 97.1 92.7 93.8 93.7 Estimate Low - -265.3 67.6 44.5 46.6 62.2 58.1 {ND=Not Detected; High & Low estimates from 2-sided 95% Confidence Interval of the mean} Considering the overall average of total sterols, secondary-treated effluent appeared to contain about 20% of the sterols that initially entered the secondary treatment system with primary-treated influent (Table 4-9 and Figure 4-12). The overall average removal was around 81% and removal was observed for most of the phytosterols as the PPME passed through the UNOX-AST system. The treatment system hydraulic retention time (HRT) considerations required that the plant sterols entering the UNOX-AST system during Sampling Cycle 1 be checked against the plant sterols leaving the UNOX-AST system during Sampling Cycle 8. This showed that the sterols removal was approximately 71% instead of 81% (Table 4-9). It is also interesting to note that individual sterols showed different levels of 85 4 Survey of Plant Sterols in PPMEs 4.4 Results and Discussions removal. fi-Sito and Stigma were removed to about 64% with a confidence interval from about 45 to 95%. Relative to other sterols, Campe showed the highest removal efficiencies. fi-Sito showed relatively less removal and was the least removed sterols as indicated by the overall average sterol removal estimates. 1.15 2.30 3.45 4.60 5.75 6.90 8.05 9.20 Ave Sampling Time (h) •a—PE —A—FE - o — % Removal - Low x Average - High Figure 4-12. PPME total sterols during various sampling cycles around the UNOX-AST system Mill B (Sampling Program III). Vertical bars indicate 95% confidence interval estimates. PE refers to primary-treated effluent and FE-secondary-treated final effluent It appears that fi-Sito may be the most persistent phytosterol in secondary-treated PPMEs. This was confirmed by the presence of fi-Sito as a major sterol in almost all the secondary-treated PPMEs. Comparatively high concentrations of fi-Sito in primary-treated influents and lower removal efficiencies for fi-Sito may be the reasons why fi-Sito has been 86 4 Survey of Plant Sterols in PPMEs 4.4 Results and Discussions detected in most of the secondary treated and untreated PPMEs (Cook et al. 1997; Magnus et al 2000b; Mahmood-Khan and Hall 2003). Relatively high removal efficiencies were observed for other sterols: Campe, Stigma and B-Sitosta. However, the removal efficiencies for Chole and Ergo could not be measured satisfactorily, due to their absence or presence at non-detectable levels in the samples tested in this investigation. 4.4.4 Sterols in Recycle and Waste Activated Sludge (Sampling Program III) The results for individual and total sterols in recycle and waste activated sludge (RAS and WAS) from the UNOX-AST system at Mill B are shown in Figure 4-13 to Figure 4-15. The vertical bars indicate standard error estimates of the average, and associated data for RAS and WAS are given in Appendix B 11. RAS 40 000 -, , 1.15 2.30 3.45 4.60 5.75 6.90 8.05 9.20 Sampling Time (h) —*—Ergo A Campe - K - Stigma -»—S-Sito -a— B-Sitosta —o— Total Sterols Figure 4-13. Sterols in recycle activated sludge, UNOX-AST-Sampling Station P7, Mill B. Vertical bars indicate standard error estimates 87 4 Survey of Plant Sterols in PPMEs 4.4 Results and Discussions W A S 70 000 -, 1.15 2.30 3.45 4.60 5.75 6.90 8.05 9.20 Sampling Time (h) —*— Ergo —A—Campe - X — Stigma —«—B-Sito — B — S-Sitosta —«—Total Sterols Figure 4-14. Sterols in waste activated sludge, UNOX-AST-Sampling Station P8, Mill B. Vertical bars indicate standard error estimates Total sterols in RAS were around 20,000-35,000 pg/L with an average of about 25,000 pg/L (Figure 4-13). WAS contained 20,000-50,000 pg/L and an average of about 35,000 pg/L total sterols, however, only two measurements exceeded 40,000 pg/L (Figure 4-14). The individual sterol proportions were similar to those found in mixed liquor. After fi-Sito, fi-Sitosta was the most abundant sterol found in both RAS and WAS, instead of Campe which was the 2 n d most abundant sterol entering the treatment system with primary-treated effluents probably due to the reasons discussed in the preceding section. The secondary sludge (RAS and WAS) from UNOX-AST contained approximately 20 to 30 times higher sterol concentrations than primary-treated effluents (Table 4-6), confirming the sorptive tendency of plant sterols to secondary biosolids and sludges. Therefore, a significant portion of the sterols may potentially leave the treatment system with WAS. 88 4 Survey of Plant Sterols in PPMEs 4.4 Results and Discussions McKague and Reeve (2003) reported that B-Sito concentrations in sludges can be up to 70,000 times higher than those in pulp mill effluents. However, such a high concentration of sterols in secondary sludges was not detected in the present study. 1.15 2.30 3.45 4.60 5.75 6.90 8.05 9.20 Ave Sampling Time (h) O RAS • WAS Figure 4-15. A comparison of sterols in recycle and waste activated s ludge (RAS and WAS) , UNOX-AST-Sampl ing Stations P7 & P8, Mill B . Vert ical bars indicate standard error estimates On average, total sterols concentrations in WAS were slightly higher that those in RAS (Figure 4-15). The difference in sterols content of RAS and WAS may be due to the fact that the two streams are not drawn from the same point in the secondary clarifiers. As a result, RAS and WAS do not have the same solids content. The WAS is pumped from the bottom of the secondary clarifiers (Figure 4-2), below the point of RAS withdrawal, in order to produce a more concentrated WAS. During the period of sampling, the suspended solids concentration was about 12,000 mg/L for the return sludge line delivering RAS to the bioreactor and about 24,000 mg/L for the waste sludge line that earned WAS. The higher concentration of 89 4 Survey of Plant Sterols in PPMEs 4.4 Results and Discussions suspended solids in WAS is the probable reason for higher sterols concentrations measured in WAS. However, WAS sterols did not appear to increase in the same proportion as the suspended solids which were twice as much in WAS as compared to that in RAS. This further suggests that sterols were probably attached to the biosolids but not all of the sterols were attached to the biosolids. A conversion of sterols data in per unit sludge mass concentrations yields 1.6 to 2.91 mg/g total sterols in RAS and 0.83 to 2.08 mg/g total sterols in WAS. The only similar reported values are those from McKague and Reeve (2003) who measured fi-Sito as high as 4.8 mg/g in sludges. Such concentrations could not be confirmed in this study. However, differences in type of sludge used and experimental conditions may result in different sludge-loading of sterols. Nonetheless, in sterol mass balance calculations, the higher sterols content for WAS may further increase the estimates of untreated sterols discharged from treatment system with waste sludge. This will have the effect of reducing the estimated biodegradation of sterols and making the role of sterols bio-adsorption more significant under these conditions. 4.4.5 Sterols Mass Balance (Sampling Program III) Sterol mass flows, based on the average concentration of total sterols measured in all the sampling cycles at each of the sampling locations around the UNOX-AST system, are given in Table 4-10 and shown in Figure 4-16. More details for total sterols at these locations in each sampling cycle are shown in Appendices B 12 to B 15. The secondary treatment system appeared to receive an average daily load of 108 kg/day total sterols (Table 4-10 and Figure 4-16), out of which an average of 20 kg/day (19%) were leaving the system with final effluents. Another 31 kg/day (29% of the incoming sterols) left the secondary treatment system with WAS on average. This indicated that about 51% of the incoming sterols left the system without undergoing biodegradation. The remaining portion, approximately 49% of the daily sterols load, appeared to be biodegraded or transformed (Figure 4-16). Additionally, the mixed liquor sterol mass flows (Cell-A, Cell-B, Cell-C and biobasin outflow) and RAS appeared to contain 5-10 times more sterols than the daily load of 108 kg/day. 90 4 Survey of Plant Sterols in PPMEs 4.4 Results and Discussions Table 4-10. Average mass flow (kg/day) of sterols across the U N O X - A S T system a t Mill B Stream Sample Sterols Flow* Flow* Mass Flow % Load M9/L L/Sec MLD Kg/day Primary Effluent Average 1583.4 786.6 67.96 107.6 100.0 Lower 95% C. I. 1069.8 72.7 67.6 Upper 95% C. I. 2096.9 142.5 132.4 Cel l -A Average 3015.1 1164.6 100.62 303.4 281.9 Lower 95% C. I. 1962.9 197.5 183.5 Upper 95% C. I. 4067.2 409.2 380.3 Cel l -B Average 3639.7 1164.6 100.62 366.2 340.3 Lower 95% C. I. 2177.1 219.1 203.6 Upper 95% C. I. 5102.2 513.4 477.1 Cel l -C Average 2643.5 1164.6 100.62 266.0 247.2 Lower 95% C. I. 1908.5 192.0 178.5 Upper 95% c . i. : 3378.5 339.9 315.9 Biobas in Outflow Average** 2841.8 1164.6 100.62 285.9 265.7 Lower 95% C. I. 2300.7 231.5 215.1 Upper 95% C. I. 3382.8 340.4 316.3 Final Effluent Average 290.4 786.6 67.96 19.7 18.3 Lower 95% C. I. 132.7 9.0 8.4 Upper 95% C. I. 448.2 30.5 28.3 R A S Average 25422.0 378.0 32.66 830.3 771.5 Lower 95% C. I. 19308.5 630.6 586.0 Upper 95% C. I. 31535.4 1029.9 957.1 W A S Average 35846.7 10.0 0.86 31.0 28.8 Lower 95% C. I. 25008.3 21.6 20.1 Upper 95% C. I. 46685.1 40.3 37.5 {C. I. Confidence Interval; *Mill data; **The secondary clarifier did not appear to be in a steady state} 91 4 Survey of Plant Sterols in PPMEs 4.4 Results and Discussions Primary Effluent 73-143 kg/day 108 kg/day (100%) UNOX Biobasin 300-633 kg/day (3-6 times) RAS ^ Secondary Clarifier / Final Effluent 20 kg/day i (19%) Figure 4-16. Total sterols mass flows around the UNOX-AST system at Mill B Considering 95% confidence interval, secondary-treated effluent may contain from 8 to 28% (9-31 kg/day), and WAS may contain from 20 to 38% (22-40 kg/day) of the incoming daily sterols load (Table 4-10). This indicated that the amount of non-degraded sterols leaving the UNOX-AST system may vary from 28 to 66%. Hence the data suggest a relatively wide range for the estimated biodegradation/transformation that may vary from about 72 to 34% (Table 4-10). Such a variation may considerably influence and compromise the 92 4 Survey of Plant Sterols in PPMEs 4.4 Results and Discussions effectiveness of a treatment system. Why or how such a variation may take place during the normal operation of the treatment system is an important question that may have implications for operation as well as design of the treatment system. It has been shown that the plant sterols removed from PPMEs, tended to accumulate in secondary sludge where a part of these sterols was probably biodegraded or transformed and the remaining sterols left the treatment system with WAS. Accumulation of plant sterols due to their sorptive affinities for secondary solids can be expected to result in high concentrations of sterols in mixed liquor and RAS (McKague and Reeve 2003; Mahmood-Khan and Hall 2003) and some biodegradation/transformation of sterols can also be expected (Kostamo and Kukkonen 2003; Conner et al 1976). However, what factors caused the sterols removal efficiency and/or biodegradation fluctuations were not clear. Nonetheless, the role of microbial biodegradation of plant sterols had become more important in the light of these results that indicate that the sterols were captured and removed quickly from PPMEs and a portion was probably biodegraded during secondary treatment. Overall, this investigation provided new information about the occurrence levels of plant sterols in primary-treated effluents, mixed liquor and biobasin outflow, secondary-treated effluents, return and waste activated sludge, and the behavior of plant sterols in biological treatment systems. It also provided sterols removal as well as biodegradation/transformation estimates that increased the insight of the process of sterols removal during the secondary treatment of PPMEs. The investigation answered some of the research questions relating to the occurrence and treatment of plant sterols in PPMEs, and supported the main hypothesis of the research that 'plant sterols can be removed in secondary treatment systems treating PPMEs'. However, the investigation also generated more questions that evolved more interest and provided the motivation for further research to find the important factors that control the removal and biodegradation of organic contaminants like plant sterols during secondary treatment of PPMEs. Therefore, the removal of plant sterols in biological systems was further studied in lab-scale biological reactors in order to improve the operation and design of wastewater treatment systems, as described in Chapter 5. 93 4 Survey of Plant Sterols in PPMEs 4.5 Conclusions 4.5 Conclusions The presence of fi-Sito, fi-Sitosta, Campe, Stigma, Ergo and Chole was confirmed through different sampling programs at two British Columbia costal mills. Chole and Ergo appeared to be the least abundant sterols found in PPMEs. fi-Sito was the most abundant sterol analyzed in PPMEs. Campe was the 2 n d most abundant sterols in primary-treated effluents and fi-Sitosta was the 2 n d most abundant sterol in secondary-treated effluents. fi-Sito, Campe and fi-Sitosta comprised the dominant fraction that accounted for about 70% of the total sterols present in PPMEs, RAS as well as WAS. Stigma, Ergo and Chole make the minor fraction of plant sterols present in PPMEs, accounting for up to 30% or less. Total sterol concentrations varied approximately from 250 to 2500 pg/L in PEs, 100 to 700 pg/L in FEs, 2000 to 4000 pg/L in biobasin outflows, 20,000 to 30,000 pg/L (average 25,000 pg/L) in RAS and 20,000 to 50,000 pg/L (average 35,000 pg/L) in WAS samples. Phytosterols appeared to be removed from PPMEs through secondary treatment at both mills. Total sterol removal efficiencies ranged from 58 to 93% with average estimates between 70 and 80%. Activated sludge (RAS and WAS) from UNOX-AST systems contained about 15-30 times higher sterol concentrations than primary-treated PPMEs, suggesting sorption and accumulation of plant sterols in biobasin mixed liquor and secondary sludges. Sterols mass balance considerations showed that a significant amount, from 22-55% to 70% or more, of the incoming sterols may leave the treatment system with final effluents and WAS without undergoing biodegradation. Sampling Program III provided estimates of biodegradation or transformation of plant sterols that ranged from 34 to 72%. The factors contributing to the observed variation are not clear. Individual sterols exhibited different removal efficiencies. Among the dominant fraction of sterols analyzed in PPMEs, relatively poor removal (64%) for fi-Sito and relatively high removal (81%) for Campe was observed. The removal efficiencies for Ergo and Chole could not be measured satisfactorily because of their low level presence. Sorption and limited biodegradation were probable mechanisms contributing to the observed removal of phytosterols across the secondary treatment systems. The general composition of individual sterols in primary and secondary treated PPMEs was similar except the relative proportions 94 4 Survey of Plant Sterols in PPMEs 4.5 Conclusions of Campe and B-Sito. The former decreased and the latter increased in secondary-treated effluents and biobasin mixed liquor as compared with those in primary-treated effluents. On average, 18-30% or more of the incoming sterols may leave the treatment system with final effluents into the receiving waters and another 20-38% or more may leave the treatment system with WAS. A single pulp mill may discharge about 20 kg/day of sterols with its treated effluents without including the sterols leaving the system with WAS. 95 5 Removal of Plant Sterols in Lab-Scale Biological Reactors 5.1 Introduction 5. Removal of Plant Sterols in Lab-Scale Biological Reactors 5.1 Introduction Currently, there are concerns over the observed estrogenic, and in some cases, androgenic effects observed in fish and aquatic life in pulp and paper mill effluent (PPME) receiving waters. These effects have been attributed to certain chemicals, including plant sterols (Chapter 2). If not treated and controlled property, plant sterols contained in PPMEs (Chapter 3) can be introduced to the receiving waters (Chapter 4), where they may potentially act as endocrine disrupting chemicals (EDCs) or hormonally active agents (HAAs). Activated sludge treatment (AST) is a common method of effluent treatment used by the pulp and paper industry. The technique of suspended growth wastewater treatment was developed in the beginning of 20 th century and AST is the most common form of the suspended growth systems. Most of the AST systems in use by the pulp and paper industry are effectively removing the majority of the .organic pollutants and thus reducing the biochemical oxygen demand (BOD) and acute toxicity (Springer 1993). All activated sludge processes depend on the natural selection and growth of microorganisms contained in the sludge floes. The activated sludge process has been proven to be both cost effective and reliable. However, AST systems have shown instability in reducing some other organic pollutants including plant sterols in PPMEs (Cook et al. 1997; Van Ginkel et al. 1999b; Mahmood-Khan and Hall 2003). Some studies have shown plant sterols removal in AST systems treating PPMEs but the behavior and fate of the sterols entering biological wastewater systems is not clear (Magnus et al. 2000b; Mahmood-Khan and Hall 2003). The previous investigations detailed in Chapters 3 and 4 confirmed that, in general, plant sterols do occur in PPMEs and that they can be removed during secondary wastewater treatment. However, it has also been observed by others that plant sterols have a tendency to adsorb on to secondary sludge (McKague and Reeve 2003). This raises questions about the mechanisms of removal of plant sterols during wastewater treatment. The reported removal of plant sterols from PPMEs may or may not relate to their biodegradation (Chapter 4). Selective microbial degradation or 96 5 Removal of Plant Sterols in Lab-Scale Biological Reactors 5.2 Methods and Materials 5.2 Methods and Materials 5.2.1 L a b - S c a l e S u s p e n d e d G r o w t h A c t i v a t e d S l u d g e S y s t e m s Activated sludge treatment (AST) is one of the most common systems used for treating PPMEs. It involves the production of an activated mass of microorganisms capable of stabilizing a waste aerobically. AST systems have shown superior sterol removal efficiencies relative to other common treatment systems like ASBs - aerated stabilization basins (NCASI 1997). In the present study, AST was used in the form of two identical laboratory-scale biological reactors as shown below (Figure 5-1). Heated Water Air Supply Air Supply M P >• R Heated Water Figure 5-1. Schematic of lab-scale suspended growth A S T system: C pH control, D diffuser aerator, E final effluent, F influent feed, H heated water, M stirrer motor, N Nutrients, P air supply pump, R recycle, T temperature control , S mechanical stirrer, V control valve 98 S Removal of Plant Sterols in Lab-Scale Biological Reactors 5.1 Introduction transformation of sterols like Chole and fi-Sito has been reported by Arima et al. (1969), Ambrus et al. (1995), Niven et al. (2001); and Kostamo and Kukkonen (2003). A recent survey of Finnish wastewater treatment plants at an ECF (elemental chlorine free) kraft pulp mill, a paper mill, and an integrated ECF kraft pulp and paper mill, by Kostamo et al. (2004) have indicated that AST removed most of the wood extractives and sterols from PPMEs at these mills. The degradation/transformation estimates for wood extractives varied from 35 to 99%. Hence, biodegradation/transformation of sterols can probably play an important role in sterols removal during secondary biological treatment such as AST. However, it is not clear why and how the degradation/transformation of wood extractives and sterols varied over such a wide range during secondary treatment. Other studies have indicated adsorption of sterols to secondary solids, to be a major mechanism of removal (Magnus et al. 200b; Mahmood-Khan and Hall 2003; McKague and Reeve 2003) Whether adsorption is the major mechanism involved in sterols removal or whether biodegradation plays the major role during secondary treatment of PPMEs. These are important questions to understand and improve the treatment and control of such organic contaminants in wastewaters. The aim of the present study was, therefore, to examine the extent of sterols removal and the role of biodegradation during laboratory scale biological secondary treatment of PPMEs. Phytosterol-containing PPMEs were treated using two lab-scale biological reactors equipped with secondary clarifiers, and the removal of sterols was monitored at various influent sterol concentrations under different treatment conditions. 5.1.1 R e s e a r c h Q u e s t i o n s To what extent can the sterols be removed in biological treatment sys tems? C a n the biological treatment systems sustain an effective removal of plant s terols? To what extent does biodegradation of sterols contribute to the overall removal of sterols during the secondary treatment of P P M E s ? 97 5 Removal of Plant Sterols in Lab-Scale Biological Reactors 5.2 Methods and Materials Each laboratory bioreactor was made of Plexiglas ™, with a 4 L aeration basin working capacity. The bioreactors were fitted with double-jacketed walls to provide the passage of circulating hot water for process temperature control. The dimensions of the bioreactors are shown in Figure 5-2. A double-jacketed secondary clarifier made with Plexiglas ™ was also provided for each bioreactor (Figure 5-1 and Figure 5-2), so that the two systems could be operated separately. Other mechanisms included influent and nutrients feeding through peristaltic pumps, pH controllers, mechanical mixing, air supply diffusers, and arrangements for return secondary sludge and sludge wasting (directly from the reactors), to allow the desired operational changes. Hot Water -4 29.2 t 21.6 15.24 O o o 4 L o o k 8 Air 21.6 Inflow Hot Water Figure 5-2. Dimensions (in cm) of the lab-scale bioreactors and secondary clarifiers 99 5 Removal of Plant Sterols in Lab-Scale Biological Reactors 5.2 Methods and Materials The influent to the lab-scale bioreactors consisted of primary treated PPME that was either collected from Mill B or was shipped from the mill in 25 L Nalgene ™ high density polyethylene containers that were stored in a refrigerator at 4°C before use. The bioreactors were inoculated with biomass obtained from the full-scale UNOX-AST bioreactors at Mill B, to grow acclimated biosolids for the treatment of sterol-containing PPMEs. A desired concentration of sterols was achieved by spiking the primary-treated effluent with sterols previously recovered from wood waste, when required. The start-up process took almost two months. Air was supplied using Aqua Fizzzz ™ (A-962) fine bubble diffusers. Two diffusers were used in each bioreactor. Sufficient air was supplied to keep the DO (dissolved oxygen) level around 5-6 mg/L. The high DO levels (5-6 mg/L) were maintained for achieving oxygen transfer conditions similar to those of the full-scale UNOX-AST biobasin. Additional mixing was provided using variable speed mechanical mixers Electric Arrow ™ (Model # 1750) stirring at about 160-180 RPM. 5.2.2 Nut r i en t s , p H C o n t r o l a n d P r o c e s s Tempera tu r e Nutrients were supplied in an approximate proportion of 100:3.5:1 for COD:N:P. NH4CI was used for nitrogen, and a mixture of K 2 HP0 4 and KH 2 P0 4 was used for P. The pH control solutions were 0.2 N H 2 S0 4 and 0.2 N NaOH. After the start-up phase, 0.2 N NaOH was mostly required to keep the reactor pH at the desired level. There were three phases of the biological sterol treatment study and Reactor 1 was operated at a pH of 6.6-6.8 throughout the three phases, as it is typical to operate AST systems at near neutral pH conditions. Reactor 2 was started at a pH of about 7.0 ± 0.2 in the 1s t phase, but later in the 2 n d and 3 r d phase its pH was increased to slightly alkaline conditions of about 7.6-7.8, to cover the normal range of process pH at the full-scale UNOX-AST Mill B system. Further, it has been indicated that slightly alkaline conditions may favor the treatment of organic pollutants in PPMEs for a variety of reasons (Van Ginkel et al. 1999a; Werker and Hall 1999). The process pH was measured using pH/mV ChemCadet Cole Parmer ™ (Model 100 S Removal of Plant Sterols in Lab-Scale Biological Reactors 5.2 Methods and Materials # 5984-50) meter and maintained by using Cole Parmer ™ pH/pump systems (Model # 7142-60). Process temperature was maintained at 38-39°C by using a VWR ™ Constant Temperature Circulator (Model #1130 A) water bath for both bioreactors through out the whole period of study. This is the temperature at which the full-scale UNOX-AST plant was typically operated at Mill B. 5.2.3 S t e r o l s S p i k i n g The primary-treated effluent was screened through a coarse strainer to remove fiber fines, into a 60 L holding tank and used as influent to the bioreactors. The primary-treated influent was spiked when desired, with plant sterols supplied by BC Chemicals, 2711 Pulp Mill Road, Prince George, BC, Canada, V2N 2K3, and were received in a fluffy powdered solid state. The dissolution of the sterols in PPMEs was brought about in a stepwise procedure. The sterols were first dissolved in iso-propanol (2-propanol) after examining the solubility of the sterols for different solvents that included methanolf; ethanol, acetone, hexane and 2-propanol (Table 5-1). Each of these five solvents were separately added to conical-bottom penny-head stopper Pyrex ™ glass centrifuge tubes of 15, or 10 mL capacity, graduated to 0.1 mL and calibrated to contain a specific volume (Corning 8084 15). The centrifuge tubes contained a given mass of sterols to which solvent was added slowly, mixed with a vortex mixer and heated to about 40°C for 5-6 minutes. The solvent was added until the solid-state sterols were completely dissolved and the solvent volume was noted after the addition of last drop. This volume was considered to represent the solubility of the sterols in a specific solvent under consideration. Table 5-1 shows the results obtained. Relative to other solvents, the least amount of 2-propanol was required to dissolve a given amount of sterols (Table 5-1). Therefore, 2-propanol was selected to pre-dissolve the semi-crystalline solid sterols, to keep the resulting change in the PPME matrix to a minimum. The 2-propanol sterol solution was then introduced in to a small volume of primary-treated effluent that was heated to a temperature of about 40°C. Then this solution was introduced to larger volumes of primary-treated effluent. 101 S Removal of Plant Sterols in Lab-Scale Biological Reactors 5.2 Methods and Materials Table 5-1. Solubility of solid sterols in different solvents Solvent Solubility at 40 °C (mg/mL) Methanol 1.8-2 Ethanol 6 - 7 Hexane 13.6-15 Acetone 28 - 30 Iso-propanol 32-36 The whole process was completed in the following three steps: I. A sterols stock-solution was prepared by dissolving 200 mg of sterols in about 5.5 mL of iso-propahol (2-propanol) at about 40°C. II. The 5.5 mL sterols stock-solution was then introduced to approximately 4 L of primary-treated effluent at about 55°C and continuously mixed on a stirrer plate heater for 4-6 hours to get an intermediate PPME solution. III. The intermediate sterol solution was then introduced to a given volume (40-60 L, as required) of bioreactor influent and mixed for 2 hours at room temperature to produce the desired concentration of plant sterols in the laboratory bioreactor influent. The spiked influent was stored at about 4-6°C in a refrigerator, where it was continuously stirred and fed to the laboratory bioreactors. One batch of the bioreactor feed was usually consumed within one week. The general characteristics of the bioreactor influent are given in Table 5-2. The fraction of PPME sterols that passed through Whatman ™ AH-934 fiberglass filters was considered 102 S Removal of Plant Sterols in Lab-Scale Biological Reactors 5.2 Methods and Materials to be the dissolved fraction in this study. The particulate fraction was determined by the difference in the analyzed amount of sterols in the whole sample and the filtrate. Typically, 70-130 mg/L of COD was added as 2-propanol to the spiked primary-treated influent. Table 5-2. Characteristics of Primary Effluent from Mill B Characteristic Low High Typical COD 1100 mg/L 1500 mg/L 1300 mg/L BOD 300 mg/L 380 mg/L 340 mg/L TSS 70 mg/L 120 mg/L 80 mg/L PH 6.8 7.8 7.2 Temperature 37 °C 41 °C 39 °C Native Sterols 600 pg/L 2500 pg/L 1400 pg/L Spiked Sterols 1000 pg/L 4500 pg/L 2500 p/L Dissolved Sterols 30% 70% 50% 5.2.4 C h e m i c a l A n a l y s e s Plant sterols were analyzed in the bioreactor influent (primary-treated PPME), secondary-treated effluent and mixed liquor or secondary sludge, according to the analytical procedure described in detail in Chapter 3. Total solids suspended solids (TSS), and volatile suspended solids (VSS) solids were analyzed in primary-treated influent, secondary-treated effluent and mixed liquor and secondary sludge, according to the Standard Methods for the Examination of Water and Wastewater 2540 D and 2540 E (Clesceri et al. 1989). 103 S Removal of Plant Sterols in Lab-Scale Biological Reactors 5.2 Methods and Materials Chemical oxygen demand (COD) was measured through a closed reflux colorimetric Standard Method 5220 D (Clesceri et al. 1989), using Hach ™ DR/2000 Spectrophotometer by reading absorbance values at 600 nm. Total organic carbon (TOC) measurements were made through combustion-infrared Standard Method 5310 B (Clesceri et al. 1989), using a Shimadzu ™ TOC-500 (Shimadzu Corporation Kyoto, Japan) analyzer. The instrument was calibrated for each batch of samples. 5.2.5 S t e r o l s R e m o v a l E x p e r i m e n t s Both lab-scale bioreactors were started by inoculation with about 300-400 mL of return activated sludge to a final MLSS concentration of about 1500 mg/L. The return sludge was freshly received from Mill B in 1 L plastic containers that allowed for some headspace air for biomass respiration during travel. Additional return activated sludge was also added to the bioreactors for re-seeding after the 2 n d and the 3 r d week. Real primary-treated effluent was fed to the lab-scale AST systems at a relatively low flow rate with HRTs (hydraulic retention time) of about 24 h. % The removal of sterols from PPMEs was assessed across the continuous-flow lab-scale biological systems in three different phases, of about 2 to 3 months each. Samples were taken after 2-3 HRT intervals, so that the effluent sterol concentration had stabilized. Each study phase was continued for approximately 3 times the average SRT (solids retention time) of the system in that phase. SRT was controlled by wasting mixed liquor directly from the reactors. The influent phytosterols concentration range and the process control variables (HRT, SRT and pH) are given in Table 5-3. 104 5 Removal of Plant Sterols in Lab-Scale Biological Reactors 5.2 Methods and Materials Table 5-3. Different phases of lab-scale bioreactor operation Experimental Phas e (Duration) Sterol Concentration (M9/L) HRT (h) SRT (d) PH Reactor 1 I (3 months) II (3 months) III (2.5 months) 1000-1500 2000-3500 3500-4500 28-20 20-13 13-8 27-23 23-10 10-7 6.6 ±0.2 6.7 ±0.2 6.7 ±0.2 Reactor 2 I (3 months)* II (3 months) III (2 months) 1000-1500 2000-3500 3500-4500 24-20 20-11 11-9 25-22 22-12 12-8 7.0 ±0.2 7.6 ± 0.2 7.6 ± 0.2 * Results up to day 40 could not be reported because of reactor failures and sterol spiking problems 105 S Removal of Plant Sterols in Lab-Scale Biological Reactors 5.3 Results and Discussions 5.3 Results and Discussions 5.3.1 P l an t S t e r o l s in Influent to the L a b - S c a l e B i o r e a c t o r s During the previous surveys of two full-scale secondary treatment systems treating PPMEs, it was concluded that plant sterols were removed during the biological treatment of PPMEs (Chapter 4), but the mechanism was not determined. In the current phase of study, more detailed investigation was performed using two lab-scale biological reactors, Reactor 1 and Reactor 2, equipped with secondary clarifiers (Figure 5-1). Primary-treated effluents from Mill B were spiked with plant sterols and used as influent for the lab-scale bioreactors. Plant sterols previously recovered from wood waste were used for spiking the primary-treated PPME. The process of re-dissolving the sterols in primary-treated PPME was complex and it was found that if the solid sterols were first dissolved in a solvent, the dissolution of plant sterols was relatively easier and quicker. Different water miscible solvents like methanol, ethanol and acetone were used for this purpose. Iso-propanol (2-propanol) was found to be the solvent with relatively high solubility for phytosterols and hence required the least Solvent volume to dissolve a given amount of plant sterols (Table 5-1). Therefore, 2-propanol was chosen to pre-dissolve the plant sterols that were introduced to primary-treated PPME in a step-wise process as explained in the Section 5.2.3. The dissolved portion of sterols in spiked PPME was approximately 10% higher as compared to that in the un-spiked PPME (Table 5-4). This was probably due to the higher concentrations of sterols present in spiked PEs. Plant sterol concentrations in the influent to the lab-scale bioreactors are shown in Figure 5-3. In the 1s t phase (day 40-90), the lab-scale bioreactor influent contained plant sterols at a concentration of about 1000 pg/L, corresponding to the lower end of the range of sterols concentrations observed in PPMEs during mill surveys and effluent monitoring (Chapter 4). The bioreactors, seeded with RAS obtained from the full-scale UNOX-AST system at Mill B, were started with a relatively low flow rate of about 3.3-3.5 L/d. The influent flow rate and plant sterols concentrations were gradually increased after about 45 days of operation. By then the bioreactors had stabilized and a steady concentration of sterols was obtained in treated effluents. 106 S Removal of Plant Sterols in Lab-Scale Biological Reactors 5.3 Results and Discussions Table 5-4. Dissolved and particulate plant sterols in primary-treated effluents Sample Dissolved Particulate Total Sterols (pg/L) Un-spiked 38 -65% 35-62 % 1000-2500 Spiked 30 -60% 40-70 % 1000-4500 In the 2 n d phase (day 90-180) of the biological sterol removal study, the influent sterol concentrations were increased to an intermediate range of 2000-3500 pg/L. By the end of the 3 r d phase (day 180-255) of the study, the influent sterol concentrations had been increased to around 4500 pg/L, which corresponded to the higher end of the anticipated range of the plant sterols concentration in PEs (Figure 5-3). Figure 5-3. Plant sterols in the influent to the lab-scale bioreactors 107 5 Removal of Plant Sterols in Lab-Scale Biological Reactors 5.3 Results and Discussions The influent total sterols composition remained more or less similar, in terms of the individual sterol fractions, throughout the three phases of the experiment. fi-Sito, B-Sitosta and Campe were the dominant plant sterols entering the bioreactors. The overall average composition of influent plant sterols was: B-Sito 46%, B-Sitosta 31%, Campe 19% and Stigma 4% (Appendix C1). This is in agreement with the results obtained earlier. B-Sito was the major plant sterol in the bioreactor influent and Stigma was the least abundant plant sterol. B-Sito has been described to be the major plant sterol contained in wood extracts (Rydholm 1965; Shoppee 1964) and in PPMEs (Mahmood-Khan and Hall 2003; Magnus et al. 2000b; Cook et al. 1997). Ergo and Chole remained mostly non-detectable. 5.3.2 P l an t S t e r o l s in Trea ted Eff luents The overall removal of plant sterols in all the three phases is shown in Figure 5-4 and Figure 5-5 for Reactor 1 and Reactor 2 (indicated by the legend suffix _1 and _2) respectively. The results for the Is' and 2 n d phases (up to day 180) show a consistent removal of about 90% of the sterols entering Reactor 1, indicating that a high removal of plant sterols can be attained through secondary treatment of PPMEs. However, in the 3 r d phase, the sterols removal decreased to about 76% when Reactor 1 was operated at high-rate conditions (HRTs 13-8 h; SRT 10-7d) similar to that of the full-scale UNOX-AST systems at the mill (Figure 5-4). In the 1s t phase (up to day 90), Reactor 2 exhibited lower sterols removal efficiencies relative to Reactor 1. Reactor 2 also took more time than Reactor 1 to achieve more than 90% removal of sterols (day 90 to 130). However as the influent sterols concentrations were increased during the 2 n d phase of study (day 90 to 180), the total sterols removal efficiency dropped well below 70% after about day 150 (Figure 5-5). The total-sterol removal efficiency of Reactor 2 appeared to improve after the 210th day of operation in the 3 r d phase (day 180 to 240) and approached about 90% removal by the end of study. 108 5 Removal of Plant Sterols in Lab-Scale Biological Reactors 5.3 Results and Discussions 2 co To 5 o Influent —5K—Effluent_1 • % RemovaM Figure 5-4. Total-sterols removal efficiency Reactor 1 r 5 000 4 500 3 3 000 2 2 500 55 o Influent -)lt-Effluent_2 — a — % Removal 2 Figure 5-5. Total-sterols removal efficiency Reactor 2 109 5 Removal of Plant Sterols in Lab-Scale Biological Reactors 5.3 Results and Discussions Phytosterols are physically in a crystalline solid state at room temperature, the melting points being in the range of 100 to 180°C. Sitosterol has a melting point of 136°C (Chapman and Hall 1996), and campesterol melts at about 157°C (Budavari et al. 1996). Sterols are chemically stable, non-volatile and moderately non-polar in nature. Therefore, loss or removal of plant sterols by volatilization, stripping and chemical degradation may be neglected because these processes are not primary mechanisms of sterols removal, under typical environmental conditions existing at secondary wastewater treatment systems. Therefore, the observed removal of sterols was thought to be a combined result of adsorption to the suspended solids, as well as biodegradation of sterols in the mixed culture environment of activated sludge treatment. Sterols removal from PPMEs was expected based upon the earlier investigations (Chapter 4). However, the factors involved that control the extent of sterols removal through adsorption or biodegradation were not clear from these results of removal efficiencies. It is to be noted that, both bioreactors received the same influent and were operated at the same temperatures with similar loadings, but the results show different performance of each unit for sterols removal (Figure 5-6). The main difference between the operation of the two reactors was the operating pH. Reactor 2 was operated at a pH slightly higher than that of Reactor 1 (Figure 5-6). A possible explanation of the difference in behavior may relate to a spectrum of interacting factors. Firstly, the pH differences within the typical range used for biological treatment may cause variations in the biobasin mixed cultures (Werker 1998). Secondly, pH-associated changes in the solubility of plant sterols and/or other organics present in PPMEs may alter their availability for microbial degradation (Werker and Hall 1999). For example, the solubility of PPME organics like resin acids and kraft lignin increases with increasing pH (Marton 1964; Werker 1998). Higher solubility tends to reduce the sorptive behavior of such compounds (Klimenko et al. 2002; Shaw 1992). A reduction in sterols sorption and/or biodegradation will cause the sterol removal performance to decline, until such capacity is re-gained. Therefore, the differences in the performance of the two reactors may be attributable to the operating pH of each reactor. 110 K Removal of P ' » " t Sterols in I ah.Sr.alfi Biolonical Reactors a Results and Discussions Figure 5-6. P roces s pH and total-sterols removal performance of Reactor 1 (_D and Reactor 2 (_2) With some differences, both of the lab-scale reactors were able to remove plant sterols from PPMEs. However, the overall operation of Reactor 1 appeared to be more successful relative to that of Reactor 2. These results suggest that a high (-95%) removal of sterols can be achieved through secondary treatment of PPMEs, and near neutral pH may be more favorable for phytosterols removal across biological systems. Whether such removal of sterols from PPMEs and the associated risk of chronic toxicity are acceptable for the ecological sustainability of a receiving environment will depend upon the specific conditions of a particular receiving environment. However, the quantitative estimates of sterols biodegradation are presented in the subsequent sections. 111 S Removal of Plant Sterols in Lab-Scale Biological Reactors 5.3 Results and Discussions 5.3.3 Effect o f H R T a n d S R T o n the L a b - S c a l e S e c o n d a r y T r e a t m e n t o f S t e r o l s The lab-scale biological reactors were started with relatively long hydraulic and solids retention times (HRTs and SRTs in the 1st phase). However, once the biological process was stabilized at about 80% removal of sterols, the influent sterols concentration was increased and the retention times were decreased gradually to match the high-rate mill scale conditions of about 10 h HRT and 10 day SRT by the end of the 3 r d phase of study. The results are shown in Figure 5-7 and Figure 5-8. The plant sterols associated with the biomass lab-scale AST systems are also presented in Figure 5-9 and Figure 5-10 for the 2 n d and 3 r d phases of the study for the assessment of sterols behavior in secondary wastewater treatment systems. As the HRT and SRT were reduced and sterol loads increased, Reactor 1 continued to show about 90% removal efficiency for phytosterols, until the system approached 8 day SRT and 12 h HRT in the 3 r d phase of study (day 180 to 250). Further reduction in SRT and HRT reduced the removal efficiency (Figure 5-7). However, the loss in removal efficiency was recovered when the SRT was increased to about 10 days and the HRT to about 11 h. -e—HRT 1 - A — S R T 1 - * ^ % RemovaM Figure 5-7. Reactor 1 hydraulic and solids retention times and total-sterols removal 112 S Removal of Plant Sterols in I ah-Scate Biolonical Reactors 3 Rftsults and Discussions Total-sterols removal efficiency for Reactor 2 started declining in the 2 n d phase (day 90 to180), well before Reactor 1, and by the end of the 2 n d phase (day180) the removal decreased to about 60% when system SRT and HRT were reduced to below 14 day and 13 h respectively (Figure 5-8). The loss in Reactor 2-sterols removal performance was also recovered when SRT was increased back to about 12 days and HRT to about 11 h. or c/) or x 120 150 Day HRT 2 -A—SRT_2 % Removal_2 Figure 5-8. Reactor 2 hydraulic and sol ids retention times and total-sterols removal These results suggest that an SRT of 11-12 days and an HRT of about 11 h were critical for effective removal of sterols during secondary treatment under the conditions of this experiment. The sterol removal efficiencies generally declined when the detention times (SRT and HRT) were below the critical values. SRT is a variable of fundamental importance that is functionally related to the steady-state specific growth rate of biomass in a completely-mixed stirred tank biological reactor or CSTR (Grady et al. 1999; Metcalf and Eddy 2003). The growth of biomass results in the removal of pollutant that is used as substrate or biodegraded. It is known that a decrease in 113 5 Removal of Plant Sterols in Lab-Scale Biological Reactors 5.3 Results and Discussions SRT, within the operating range of values, increases the microbial specific growth rate as well as the observed yield. However, for a fixed reactor volume, the amount of biomass in the reactor also decreases (Grady et al. 1999). Hence, if the plant sterols were biodegraded in the lab-scale suspended growth reactors, the overall sterol removal through biodegradation will decrease with a reduction in SRT. Under these conditions a reduction in HRT will further reduce the biomass available for pollutant degradation (Grady et al. 1999), resulting in an additional loss of reactor performance in regard to sterols (or a biodegradable pollutant) removal. The observed loss in sterols removal was recovered through a small incremental increase in system SRT and HRT for both reactors. This was probably the result of improved biodegradation of sterols through the re-establishment of sterol-degrading microorganisms. It shows the importance of retention times in regard to the removal of plant sterols or organic pollutants of similar nature, during the operation of biological reactors treating PPMEs. Although use of HRT has diminished in importance for sizing suspended growth reactors (WEF and ASCE 1998), the results of this study suggest that HRT can be an important consideration for design as well as operation of biological reactors or treatment systems treating sterol-containing PPMEs. Therefore, both retention times: SRT and HRT can impact the performance of biological reactors treating PPMEs within the normal range usually observed in full-scale operation. This may also be a possible cause of the reported fluctuations in sterols removal at full-scale treatment plants at different mills (Kostamo et al. 2004; Mahmood-Khan and Hall 2003). However, as discussed previously, biodegradation is not the only major mechanism of sterols removal, and the fluctuations in the amount of sterol removed through adsorption to secondary solids will also impact the overall sterols removal. At this stage, it seems important to examine the effect of increased loading and reduced retention times especially SRT, on the concentration of mixed liquor sterols. The variations in mixed liquor sterol concentrations during the 2 n d and 3 r d phases of the study are shown in Figure 5-9 and Figure 5-10 for Reactor 1 and Reactor 2, respectively. The results reveal that plant sterols associated with secondary solids were not affected by the increased loadings and reduction in the retention times until approximately day 170 for Reactor 1 and day 180 for Reactor 2. However, after day 180 (in the 3 r d phase) the mixed liquor sterol concentrations increased substantially with decreasing SRTs (Figure 5-9 and 114 S Removal of Plant Sterols in Lab-Scale Biological Reactors 5.3 Results and Discussions Figure 5-10). The mixed liquor sterol concentrations increased quickly after the SRT was decreased to less than 12 days for Reactor 1 and less than 11 days for Reactor 2. A similar trend was observed in both bioreactors treating PPMEs. Under these conditions, decreasing SRTs increased the amount of sterols discharged with waste activated sludge (WAS). This probably suggests a switching between the two mechanisms of sterols removal where the role of biodegradation/transformation appears to have decreased and that of bio-adsorption appears to have increased. Nonetheless, once the process retention times were increased, the mixed liquor sterol concentrations decreased again indicating an increased biodegradation/transformation capacity probably through the re-establishment of sterol-degrading biomass depending upon the process conditions suitable for their growth. 80 110 140 170 200 230 260 Day campe - o — Stigma - A — B - S i t o -x— S-Sitosta - * - M L _ 1 Sterols - o — S R T J Figure 5-9. Variation in mixed liquor plant sterols with S R T in Reactor 1 Another contributing factor may be the influent sterol concentrations that can affect reactor biomass growth and hence biomass concentrations present in the biological reactors. However, the influent sterol concentrations were relatively constant past day 190 of operation 115 35 000 30 S Removal of Plant Sterols in Lab-Sr.ale Biological Reactors fi 3 Results and Discussions in the 3 r d phase of the study (Figure 5-3). The effect of SRT is probably much greater than that of HRT in regard to the sterol concentrations associated with reactor biomass for two reasons. First, the HRT was changed in a relatively narrow range of operating values. Second, the SRT effectively controls the amount of biomass present in the biobasin as well as the pollutant concentration in the biobasin during the operation of suspended growth secondary treatment systems (Grady et al. 1999; Metcalf and Eddy 2003). 35 000 £ 10 000 80 100 120 140 160 180 200 220 240 260 Day .Campe -a—Stigma - A - B - S i t o B-Sitosta - * - M L _ 2 Sterols - © - S R T 2 Figure 5-10. Variation in mixed liquor plant sterols with S R T in Reactor 2 At this stage, it seemed necessary to examine the mass flows and system accumulation of plant sterols to have conclusive evidence regarding the shifting role of sterols biodegradation/transformation and bioadsorption during the normal operation of biological reactors. This is the presented in the succeeding sections. Another, important observation that has to be noted was that the composition of mixed liquor sterols in terms of the individual sterols was similar in both bioreactors i.e. 10-11% Campe, 4-6% Stigma, 48-53% B-Sito, and 31-34% B-Sitosta (Figure 5-9 and Figure 5-10). 116 5 Removal of Plant Sterols in Lab-Scale Biological Reactors 5.3 Results and Discussions However, the relative proportion of Campe seemed to have decreased and that of fi-Sito appeared to have increased as compared with those of the influent i.e. 23% Campe, 5% Stigma, 43% fi-Sito, and 31% fi-Sitosta (Figure 5-3). This suggests that Campe was probably biodegraded at a relatively higher rate and reverse was true for fi-Sito compared with other sterols present. 5.3.4 S t e r o l M a s s F l o w s En te r ing a n d L e a v i n g the L a b - S c a l e B i o r e a c t o r s Sterol mass flows for Reactor 1 and Reactor 2 are shown in Figure 5-11 and Figure 5-12 respectively. The daily mass flows of sterols entering the bioreactors are shown as Load_1 and Load_2, the daily mass flows leaving the bioreactor systems with treated effluents as FE_1 and FE_2, and with waste secondary sludge as WAS_1 and WAS_2, where the suffixes _1 and _2 denote Reactor 1 and Reactor 2, respectively. These figures also show removal efficiency and estimated biodegradation/transformation of sterols based on daily mass flows. In Figure 5-11 and Figure 5-12, % Removed was estimated by subtracting the effluent sterols mass flow from the influent sterols mass flow, and % Biodegraded was estimated by subtracting the effluent sterols mass flow and the WAS-sterols mass flow from the influent sterols mass flow calculated as percentage of the daily influent sterols mass flow. It was assumed that the difference between the influent mass flow and the effluent plus WAS mass flows was either biodegraded or transformed. Reactor 1 appeared to successfully accommodate the increasing sterol loads of up to about 25 mg/d by day 180 of operation (the 1 s l and 2 n d phase of study), and sustained about 90-95% removal of sterols, out of which 80-90% appeared to have been biodegraded or transformed. During the 3 r d phase, beyond day 180, WAS-sterol mass flows increased rapidly with increasing sterols load (Figure 5-11). This was followed by an increase in effluent sterols mass flows that reduced the calculated removal efficiency of the system to less than 80% by day 220. Under these conditions, a relatively greater loss was observed in sterols biodegradation estimates that approached 50% or less. The sterol removal efficiencies and biodegradation or transformation estimates improved by the 240 th day of operation, when the 117 S Removal of Plant Sterols in Lab-Scale Biological Reactors fi 3 Results and Discussions incoming sterols load was reduced due to an increase in the retention times of the operating system (Figure 5-11 and Figure 5-7). 60 90 120 150 180 210 240 Day —o—Load_1 —a— WAS_1 EffluenM - A - % Removed_1 - * - % Biodegraded_1 Figure 5-11. Daily mass flows of total sterols entering and leaving Reactor 1 Sys tem Reactor 2 system appeared to successfully handle the increasing influent sterol loads of up to about 20 mg/d by day 135 of operation (in the 2 n d phase of study), and sustained about 90% removal of sterols, most of which (~90%) appeared to have been biodegraded or transformed. However, past the 150th day of operation (during the 2 n d phase), treated effluent sterol mass flows increased rapidly with increasing influent sterols load (Figure 5-12). This reduced the removal efficiency of the system to about 60% or less by the 160th day of operation. This was followed by an increase in WAS-sterol mass flows that brought down the estimated biodegradation/transformation of the system to about 50% or less by the 180th day of operation (beginning of the 3 r d phase of study). At this stage, relatively greater difference was observed between the removal efficiency and the estimated biodegradation of sterols 118 5 Removal of Plant Sterols in Lab-Scale Biological Reactors 5.3 Results and Discussions (day 180 to about 210). The loss of reactor performance was recovered latter by the 220 t h day of operation (towards the end of the 3 r d phase) when the influent sterol load was reduced through an increase in the retention times of the operating system (Figure 5-12 and Figure 5-8). Load_2 Effluent_2 -a—WAS_2 -&-% Removed_2 -*— % Biodegraded_2 Figure 5-12. Daily mass flows of total sterols entering and leaving Reactor 2 Sys tem These considerations suggest that the end of the 2 n d phase of study (day 180) presented a critical stage of the operation of both bioreactors. Before approaching this critical operational stage, both of the reactors displayed effective removal as well as biodegradation or transformation of incoming sterols. However, the performance of the reactors started to deteriorate after this critical stage, especially in terms of the estimated biodegradation of sterols, that was reduced more than 50% in both cases. The importance of system retention times (SRT and HRT), influent substrate concentrations and especially the fundamental role of the system SRT, has been discussed 119 5 Removal of Plant Sterols in Lab-Scale Biological Reactors 5.3 Results and Discussions briefly in Section 5.3.3. It is also shown by these results that at a certain critical operational stage, a relatively small change in these variables can considerably influence the performance of biological reactors through the changes in the amount and concentration of biomass present for substrate assimilation. However, the interpretation of these results is complicated because of two main factors. First, a portion (~30-40%) of the influent sterols entered the biological reactors in soluble state, while the rest of the sterols (~ 60-70%) were entering the lab-scale treatment systems in a colloidal and/or particulate state. An important characteristic of the particulate organic substrate is that it is too large to be transported across the microbial cell walls (Grady et al 1999). Thus, it must be acted on by extra-cellular enzymes to be hydrolyzed to release soluble constituents that can be taken up and used as a substrate by the biomass. While the soluble portion of the incoming sterols will be immediately available for biological attack. The hydrolysis solubilization reactions are quite complex and have received little research attention (Grady etal. 1999). Second, it has been seen that the particular chemical nature of plant sterols favors their adsorption to the biomass (McKague and Reeve 2003; Mahmood-Khan and Hall 2003). Bioadsorption will remove the plant sterols from the liquid effluent even if they are not immediately biodegraded in the treatment system. The results further indicate that a relatively small reduction of about 20-30%, in the overall sterols removal, can be related to a considerable loss of about 50% or more in the estimated biodegradation or transformation of sterols during the secondary treatment of PPMES (Figure 5-11 and Figure 5-12). During the last phase of the study, the performance of both reactors improved, indicating that the sterols removal and biodegradation/transformation capacity can be re-established by biological reactors through some operational changes as discussed in Section 5.3.3. It is also important to note that during the last phase of study, for Reactor 1 system, the WAS-sterol mass flows started increasing by about the 160th day of operation, before the effluent sterol mass flows increased (Figure 5-11), whereas for Reactor 2 system, the effluent sterol mass flows started increasing by about the 130th day of operation i.e. before the WAS-sterol mass flows increased (Figure 5-12). This indicated that Reactor 1 effluent sterols were 120 5 Removal of Plant Sterols in Lab-Scale Biological Reactors 5.3 Results and Discussions relatively more attached or adsorbed to the biomass, and at the onset of the reactor performance reduction, it was the WAS-sterols that first started to increase. The Reactor 2 effluent sterols appeared to be in a relatively more dissolved form, that passed through the secondary clarification. It can be recalled that Reactor 2 system was operated at slightly alkaline conditions compared to Reactor 1 system, with a process pH of about 7.6 ± 0.2 during the 2 n d and 3 r d phases of the study (Figure 5-6). The small differences of process pH within the typical range used for secondary treatment of PPMEs may influence specific organic contaminant solubility (Werker and Hall 1999). Thereby, affecting the role of physico-chemical factors during biological treatment of such contaminants as discussed in Section 5.3.2. No previous studies have focused on identifying or quantifying the impacts of treatment system operational changes on plant sterols removal and biodegradation from PPMEs. However, the results highlight the importance of sterols biodegradation/transformation and its shifting role during the secondary treatment of PPMEs in response to the changes in the operating conditions. This illustrates how some relatively small changes in the treatment plant operational conditions (influent loads, SRT, HRT, and pH) can impact the removal as well as biodegradation -.of organic pollutants like plant sterols, and how the effectiveness of secondary treatment systems can be considerably improved or compromised. 5.3.5 C u m u l a t i v e M a s s F l o w s a n d C o n t r i b u t i o n o f B i o d e g r a d a t i o n The average contribution of sterols biodegradation/transformation, in the overall removal of plant sterols, was assessed from the cumulative mass flows entering and leaving the lab-scale biological reactor systems. Daily mass flows were added together to get the cumulative mass flows shown in Figure 5-13 and Figure 5-14. In addition to the cumulative sterol mass flows entering the lab-scale systems with influent, and the mass flows leaving the systems with treated effluent and WAS, Figure 5-13 and Figure 5-14 also show the overall percentage of influent sterols removed (influent minus effluent), retained (influent minus effluent and WAS), and biodegraded/transformed (retained minus accumulated). The legend suffix _1 and _2 indicate the corresponding bioreactor system. Sterols accumulation (mass present in each system) was directly obtained through the analysis of mixed liquor sterol 121 5 Removal of Plant Sterols in Lab-Scale Biological Reactors 5.3 Results and Discussions contents that are also shown in these figures. The % Retained estimates represent the portion of the incoming sterols that was theoretically available for biodegradation or transformation. The cumulative mass flows should present an average picture of the overall sterols removal and biodegradation/transformation of PPME-sterols entering the lab-scale Reactor 1 and Reactor 2 systems. The cumulative mass flows should also compensate for the variations in the accumulated or stored amount of sterols in the biomass, due to the changes in the attained adsorption equilibrium between the sterols and biomass as a result of the operational changes. 6 000 • : : : : : , 100 5 000 + Jp 4 000 - ^ CD CO i E - InfluenM -% Removed 1 •a—Accumulated_1 -t—% Retained 1 -WAS_1 -% Biodegraded_1 -EffluenM -ML Sterols 1 Figure 5-13. Reactor 1 System: Cumulative mass flows, average removal, retention, biodegradation, and ML (mixed liquor) total sterols The cumulative mass flows for Reactor 1 system, confirmed a relatively high removal of about 90%. Almost 70 to 85% of the influent sterols were retained by the system and 65 to 85% of the sterols appeared to have biodegradation or transformed (Figure 5-13). Reactor 2 122 S Removal of Plant Sterols in Lab-Scale Biological Reactors 5.3 Results and Discussions system showed that 50 to 80% of the influent sterols were removed, 45 to 70% were retained by the system and 40 to 70% were biodegraded or transformed (Figure 5-14). This indicated that most of the retained sterols were undergoing biodegradation or transformation. The overall cumulative sterols removal remained around 90% for Reactor 1, and the cumulative removal decreased to about 75% for Reactor 2, in the 3 r d phase of study. The cumulative biodegradation decreased to about 65% for Reactor 1 and to about and 55% for Reactor 2. 2 CO 75 6 000 5 000 + 4 000 3 000 4-2 000 + 1 000 T 100 '-- 90 80 T '-- 70 (mg/l '-- 60 [erols :- 50 ML SI :- 40 i : 30 Remc '-- 20 '-• 10 - 0 -lnfluent_2 -o— Accumulated_2 Effluent_2 - * - W A S _ 2 - % Removed_2 — i — % Retai ned _2 % Biodegraded_2 - A — ML Sterols_2 Figure 5-14. Reactor 2 System: Cumulative mass flows, average removal, retention biodegradation, and ML (mixed liquor) total sterols In general, the cumulative mass flow results demonstrated that biodegradation or transformation contributed to about 90% of the observed removal of sterols achieved during the lab-scale secondary treatment of PPMEs. Although, sterols adsorption to biomass (bio-adsorption) has been anticipated as the major mechanism of sterols removal during full-scale secondary treatment (McKague and Reeve 2003; Mahmood-Khan and Hall 2003) the 123 S Removal of Plant Sterols in Lab-Scale Biological Reactors 5.3 Results and Discussions contribution of biodegradation can be underestimated due to the non-availability of required data. The operation of both of the lab-scale biological reactors showed that biodegradation was actually responsible for a major part (80-90%) of the observed removal of sterols. Hence, the contribution of biodegradation was considerably greater than anticipated initially. The cumulative biodegradation capacity of biological reactors was sensitive to the reactor operational conditions, and it could be lost and gained as discussed earlier. The effect of lost biodegradation on the associated removal efficiency was relatively moderate and did not appear to reflect a similar reduction in the overall removal of sterols, because of the increased contribution of adsorption of sterols to biomass and other secondary solids. A negative correlation between the mixed liquor sterols and biodegradation efficiency suggested that an increase in mixed liquor sterols was a better indicator of the loss of sterols biodegradation instead of the effluent sterol concentration. This was particularly true for Reactor 1, operating at near neutral conditions. The mixed liquor sterols increased 2-4 or more orders of magnitude owing to the decreased biodegradation (Figure 5-13 and Figure 5-14). Therefore, an increase in mixed liquor concentrations of a particular organic pollutant like sterols would probably indicate a loss in biodegradation of the specific pollutant, even if the effluent concentrations do not suggest so. Under these conditions, a continued sub-optimal operation of the treatment system will also increase the probability of releasing a pollutant spike in the treated effluents or a permit violation of pollutant discharge. Relatively lower efficiencies were exhibited by Reactor 2 as compared to those of Reactor 1, in terms of removal as well as biodegradation of sterols. Recalling from Section 5.3.2 that process pH was the major operational difference between the two reactors. The biological Reactor 1 operating at near neutral pH conditions, showed relatively better performance. However, Reactor 2 operated at slightly alkaline pH conditions, showed relatively greater contribution of biodegradation to the observed sterols removal, but less biodegradation overall. Hence, a small difference of pH (from 6.7±0.2 to 7.5±0.2) within the range typically used in full-scale operations, can significantly impact the removal and biodegradation performance of a biological system treating organic pollutants like sterols. Now that biological treatment has shown promising results for an effective removal and degradation/transformation of plant sterols from PPMEs, the operation of secondary systems treating PPMEs can be optimized to achieve efficient treatment of plants sterols. The suggested operating conditions (regarding the process SRT, HRT and pH) for high removal 124 S Removal of Plant Sterols in Lab-Scale Biological Reactors 5.3 Results and Discussions and biodegradation of plant sterols lie within or close to the range normally used at full scale. Therefore, plant sterols treatment and control can be potentially optimized through relatively smaller manipulations of the operational parameters that can allow and maintain the growth of sterols-degrading biomass in the biological reactors. The altered process conditions of the secondary systems are not expected to compromise the treatment and removal of other important pollutants that contribute to the effluent COD, BOD and toxicity. On the other hand, biodegradation kinetic studies can be performed to estimate the kinetic coefficients that can be utilized either for designing the secondary treatment facilities for effective treatment of moderately non-polar organic pollutants like plant sterols, or for modeling the fate of specific organic pollutants like sterols during secondary wastewater treatment. For wastewater treatment plants that are normally working at SRTs significantly lower than the recommended SRT of about 11-12 days for effective biodegradation of plant sterols, an increase in SRT may require additional oxygen, and hence the cost of operation can be increased. However, the secondary treatment plant is also expected to generate decreased amounts of excess secondary sludge and accomplish a greater degree of BOD, COD and probably toxicity removal. These benefits can partially offset the increased cost of a longer SRT. Alternatively, a secondary system can be designed that offers optimized removal of plant sterols or other similar organic pollutant of interest, by maximizing the role of bio-adsorption. In this case, the pollutant will be mainly removed through phase transfer of the pollutant from the liquid to the solid phase or the secondary sludge. Therefore, such a treatment process will require further treatment or proper disposal of the pollutant-rich secondary excess sludge. The use of a treatment process utilizing bio-adsorption of sterols will require further study of the adsorptive behavior of sterols and secondary biomass. This is the focus of the next chapter that describes the study of the adsorptive equilibrium and capacities of inactivated secondary biomass in relation with plant sterols in PPMEs. No published studies have focused on the effects of process variables on the secondary treatment and removal of plant sterols that have been identified as potential 125 S Removal of Plant Sterols in Lab-Scale Biological Reactors 5.4 Conclusions EDCs/HAAs of significant environmental importance. The results obtained in this study have provided useful information that can be used for improving the operation and design of secondary treatment facilities treating PPMEs. This study can also provide some guidance and offer helpful information for further research focused on optimizing the treatment and biodegradation of specific organic pollutants of significant environmental importance. 5.4 Conclusions The operation of two lab-scale bioreactor systems treating plant sterols in PPMEs, suggests that secondary treatment systems can achieve and maintain plant sterol removal efficiencies of up to 90% or more. Biodegradation and bio-adsorption were two major mechanisms of sterols removal from PPMEs. Biodegradation can contribute up to about 80-90% of the total-sterol removal achieved during the biological treatment of PPMEs using suspended growth continuous systems. Sterols biodegradation was found to be sensitive to the relatively small changes within the normal operating range of process parameters: pH, SRT and HRT. Reactor 1 system, operating at a process pH of about 6.7 ± 0.2 and a temperature of about 38-39°C, appeared to be relatively more robust and stable in removing plant sterols from PPMEs. Reactor 1 system could treat a sterols-spiked PPME up to 4500 pg/L in about 11 h HRT and 11 days SRT. Reactor 2 system, operating at the same process temperature and a pH of about 7.6 ± 0.2, showed relatively poor performance and sterols removal capacities. However, Reactor 2 AST system could also treat influent sterols concentrations as high as 4500 pg/L in about 12 h HRT and 11 days SRT. In both cases, further reduction in HRT and SRT deteriorated sterol removal efficiencies especially the biodegradation of plant sterols. An increase in the mixed liquor sterol concentrations indicated a loss of biological treatment of sterols as well as a reduction in the overall removal efficiency of the system. The total sterol removal efficiencies did not fully reflect the loss of sterols biodegradation, because of the increased relative contribution of sterols adsorption to the system biomass. A relatively small reduction of about 20-30% in the overall sterols removal efficiency could be related to a major decline of about 60-70% in sterols biodegradation or transformation. Under 126 5 Removal of Plant Sterols in Lab-Scale Biological Reactors 5.4 Conclusions such conditions, sterols bio-adsorption would become the governing mechanism of sterols removal from PPMEs and the amount of sterols leaving the treatment system with WAS may increase 2-4 or more orders of magnitude of the amount that would normally leave the system during optimal conditions. Such circumstances may also increase the probability of a pollutant spike release in the treated effluents or pollutant discharge permit violation. The design and operation of secondary treatment systems can be optimized for efficient removal and control of sterols or other similar pollutant, utilizing optimized biodegradation process or optimized bio-adsorption process. The latter may require further treatment of pollutant-rich excess sludge. 127 6 The Bio-Adsorption of Phytosterols on Inactivated Biomass 6.1 Introduction 6. The Bio-Adsorption of Phytosterols on Inactivated Biomass 6.1 Introduction Phytosterols, found in PPMEs, are suspected to be potential EDCs or HAAs for fish and aquatic life. Phytosterols or plant sterols are moderately non-polar compounds that have a natural tendency for adsorbing on to suspended solids and biomass during secondary wastewater treatment of PPMEs. Bio-adsorption, or the adsorption of plant sterols to the secondary solids, is one of the major mechanisms of sterols removal during the secondary treatment of PPMEs (Chapter 5). The use of microbial origin adsorbent materials (bio-adsorbents) such as activated sludge has received increasing attention in recent years as an inexpensive adsorbent that is easily available and can be used for removing organic pollutants from wastewaters through the process of bio-adsorption (Jianlong et al. 2000; Stringfellow and Alvarez-Cohen 1999). Biosorption has been used to remove heavy metal pollutants from wastewaters (Aksu et al. 1999; Raize et al. 2004). Activated sludge, the biomass generated during secondary wastewater treatment, mainly consists of live and dead bacteria, protozoa and other suspended matter. Both bacteria and protozoa can adsorb on, and absorb through, their cell walls and/or membranes. Biological cell walls mainly consist of various organic compounds such as chitin, acidic polysaccharides, lipids, amino acids and other lipoprotein cellular materials (Shaw 1992; Schuler and Kargi 1992; Brock and Madigan 1991). Like micelles, biological cell membranes are organized through hydrophobic bonding with a membrane structure that consists of lipid bimolecular layers with adsorbed and incorporated proteins (Shaw 1992). Microbial cells tend to concentrate and adsorb agglutinating chemicals from their aquatic environments, while lytic agents (or chemicals) tend to penetrate into the lipid bimolecular cellular membranes. The most common site of accumulation of lipophilic substances are the lipid membranes (Flemming 1995). Bacterial cytoplasm is also a potential site for binding and accumulation of pollutants, but for the dissolved portion of the pollutant (Flemming 1995). 128 6 The Bio-Adsorption of Phytosterols on Inactivated Biomass 6.1 Introduction Bio-adsorption of pollutants such as plant sterols bears a significant consequence in aquatic environments as it might serve as the first step in introducing such chemicals into the food chain, thereby enhancing their biodegradation or exposure to other aquatic species like fish. Further, it has been shown that many organic contaminants including plant sterols and other toxic compounds, entering biological secondary wastewater treatment systems with domestic and industrial waste, tend to accumulate in the microbial sludge where they may or may not undergo substantial biodegradation (Mahmood-Khan and Hall 2003; Kostamo et al. 2004; Chapter 5). The organic contaminants adsorbed to microbial sludge may be released back to the environment during the later stages of sludge management and disposal. On the other hand, bio-adsorption can improve the performance of secondary treatment systems such as activated sludge treatment (AST), that are required to treat moderately hydrophobic organic pollutants like plant sterols in PPMEs or other wastewaters. Therefore, it is important to investigate the adsorption phenomenon of plant sterols to secondary sludge or biomass, for obtaining the technical information that may improve the design and operation of treatment plants. In an earlier part of the present study, plant sterols were found to accumulate in secondary sludge, indicating bio-adsorption to be an important mechanism of removal of plant sterols from PPMEs (Mahmood-Khan and Hall 2003). It has been reported that sterols bind to microbial biosolids (McKague and Reeve 2003), and bio-adsorption and biodegradation are two major mechanisms of phytosterols removal from PPMEs during the secondary treatment of PPMEs (Chapter 5). However, the details of sterols adsorption to secondary sludge are not clear and the questions about their adsorption kinetics, adsorptive capacities of secondary solids are important to develop further understanding of the process and improve the treatment and control of plant sterols during the secondary treatment. In this study, the adsorption of three major plant sterols, fi-sitosterol (fi-Sito), li-sitostanol (fi-Sitosta) and campesterol (Campe), was investigated using inactivated biomass as an adsorbent. 6.1.1 Research Questions What are the kinetics of sterols bio-adsorption? 129 6 The Bio-Adsorption of Phytosterols on Inactivated Biomass 6.2 Experimental What is the sorptive capacity of secondary solids? To what extent can bio-adsorption contribute to the overall removal of sterols during the secondary treatment of PPMEs? 6.2 Experimental 6.2.1 Adsorbent (biomass) Secondary sludge was collected from one of the lab-scale biological reactors: described in Chapter 5. At the time of sludge sampling, Reactor 1 was treating un-spiked primary effluent collected from Mill B. The un-spiked primary effluent was used as an influent for more than a month to clear off most of the sterols accumulated in the secondary sludge by the end of the 3 r d phase of the study of sterols biological removal. The reactor had been started previously by inoculation with secondary sludge (return activated sludge or RAS) obtained from the full-scale UNOX-AST systems operating at Mill A and Mill B. The microbial biomass collected from Reactor 1, was centrifuged and washed three times with distilled water, prior to chemical inactivation. 6.2.2 Biomass Inactivation Inactivation of microbial biomass was brought about by the administration of the following three chemical treatments. Treatment 1 (T1). 4% Formaldehyde (from 37% Formalyn) for 10-30 min. Treatment 2 (T2). 1mM Fluoroacetate (Mono-Fluoroacetic acid) for 10-30 min. Treatment 3 (T3). 11% Formaldehyde (from 37% Formalyn) for 10-30 min. 130 6 The Bio-Adsorption of Phytosterols on Inactivated Biomass 6.2 Experimental Chemical inactivation or fixation of the biomass was chosen to preserve cell structures and minimize alterations from the living state. Formaldehyde is an indispensable fixative agent that has been studied extensively (Hayat 1970). An ideal fixative kills the biomass quickly and causes minimum shrinkage or swelling. Since the speed of killing is primarily dependent upon the rate of penetration of a fixative into the living tissue, chemicals of low molecular weight such as formaldehyde are usually most effective (Hayat 1970). Stringfellow and Alvarez-Cohen (1999) used formalyn (2 % formaldehyde) to inactivate concentrated biomass or mixed liquor suspended solids (MLSS) during the study of PAH sorption to biomass. Stringfellow and Alvarez-Cohen (1999) also confirmed that the formalyn treatment did not alter phenanthrene sorption coefficients (Kp) between the treated and untreated cells. Therefore, two levels of formalyn treatment were selected along with an additional treatment using Fluoroacetate, a highly toxic and well known pesticide (WHO and FAO 1975; APA 1997). The inactivation of microbial biomass was monitored respirometrically by head-space analyses for carbon dioxide (C02) over elapsed time, using a Fisher-Hamilton Gas Partitioner equipped with Spectra Physics ™ SP-4290 Integrator and two packed columns: Packed Column 1. 6' long, %" diameter, packed with ethylhexylsebacate on 60-80mesh, DEHS liquid phase Packed Column 2. 6.5' long, 3/16" diameter, packed with 42-60 mesh molecular sieve About 20 mL of active biomass or mixed liquor (for control) and 20 mL of inactivated biomass (for inactivation experiments) were taken in clean glass vials of 50-60 mL capacity equipped with air-tight screw caps. About 200 mg of sodium acetate was added to the biomass and the contents of the glass vials were mixed together and aerated for 10 minutes. The vials were then sealed with air-tight caps having provision for taking head space samples for respirometric measurements. 131 6 The Bio-Adsomtion of Phytosterols on Inactivated Biomass 6.2 Experimental The treatments T1 and 12 did not give satisfactory inactivation results, however, T3 appeared to be effective for inactivating the biomass as demonstrated by the inactivation data in Table D 1 and D 2 and shown in Figure D 2 and D 3 given in Appendix D. Therefore, T3 was used for inactivation of the biomass used in the experiments performed in this study. 6.2.3 S t e r o l s S t o c k S o l u t i o n Primary treated PPMEs (4 L) were filtered through Whatman ™ 934 AH fiberglass filters (47 mm diameter, 1.5 pm pore size) and spiked with plant sterols. The spiking was done as described in Section 5.2.2. The spiked sterols solution was again filtered through Whatman ™ 934-AH fiberglass filter to remove any particulate material greater than 1.5 pm. The filtrate was then used as the sterols containing wastewater, which contained about 5 mg/L of total sterols at about 40°C, for sterols adsorption studies. The 40°C temperature was chosen, as it is the temperature (38-40°C) of the full-scale treatment. 6.2.4 A d s o r p t i o n E x p e r i m e n t s Different amounts of inactivated MLSS, using treatment T3, were introduced into 200 mL of primary effluent containing a known amount of plant sterols in 250 mL Erlenmeyer flasks, in order to give a desired concentration of biomass in each flask (Table 6-1). The contents of each flask were mixed well first by hand shaking and then all the flasks in a batch were placed in a constant-temperature refrigerated incubator-shaker (Innova ™ Model # 4230, New Brunswick Scientific, Edison, NJ, USA) agitating the flasks at about 200 revolutions per minute (rpm) and 39 ± 0.5°C. The incubator temperature and shaking conditions were selected to provide a mixing intensity similar to that of Reactor 1. The flasks were kept in the incubator-shaker for a period of time that was thought to be sufficient for establishing adsorption equilibrium between the adsorbate (phytosterols) and the adsorbent (inactivated secondary sludge). Samples were taken at certain time intervals (Table 6-1) and filtered through Whatman ® 934 AH fiberglass filters (47 mm diameter, 1.5 pm pore size) to separate MLSS and the filtrates were analyzed for phytosterols remaining in solution. 132 6 The Bio-Adsorption of Phytosterols on Inactivated Biomass 6.2 Experimental Table 6-1. M L S S and sampling times for sterols adsorption experiments S e t 1 (Batch #1-7) 1 2 3 4 5 6 7 MLSS (mg/L) 0 20 40 100 200 1000 2000 Sampling Time (h) 0 2 10 24 124 Set 2 (Batch # 8-14) 8 9 10 11 12 13 14 MLSS (mg/L) 0 20 40 100 200 1000 2000 Sampling Time (h) 0 2 10 24 124 169 Set 3 (Batch #15-21) 15 16 17 18 19 20 21 22 MLSS (mg/L) 0 10 20 40 100 200 500 1000 Sampling Time (h) 0 1 2 6 13 24 50 102 170 264 6.2.5 C h e m i c a l A n a l y s i s The modified analytical technique described in Chapter 3: Quantification of Plant Sterols, was used for analyzing the plant sterols. Total suspended solids (TSS), and volatile suspended solids (VSS) solids were analyzed in primary effluent, final effluent and secondary sludge, according to Standard Method 2540 D and 2540 E (Clesceri et al. 1989). Chemical oxygen demand (COD) was measured by the closed reflux colorimetric Standard Method 5220 D (Clesceri et al. 1989), using Hach ™ DR/2000 Spectrophotometer by reading absorbance values at 600 nm. Total organic carbon (TOC) measurements were made by the combustion-infrared Standard Method 5310 B (Clesceri et al. 1989), using Shimadzu ™ TOC-500 (Shimadzu 133 6 The Bio-Adsorption of Phytosterols on Inactivated Biomass 6.2 Experimental Corporation Kyoto, Japan) analyzer. The instrument was calibrated before each batch of samples. 6.2.6 C h a r a c t e r i s t i c s o f S t e ro l S p i k e d P P M E The characteristics of sterol-spiked PPME wastewater are given in Table 6-2. The characteristics of the sterols spiked primary effluent used in the adsorption studies were similar to those used in the lab-scale biological sterols removal studies (Chapter 5), except that the COD and TOC values were somewhat lower, probably because the primary effluents were filtered before and after sterol spiking. Table 6-2. Characteristics of sterol-spiked PPME wastewater used for sterol bio-adsorption studies Sample Characteristic Value Spiked PE COD 736 ± 15 mg/L TOC 560 ± 25 mg/L PH 7.0 ±0.2 Temperature 40-38°C fi-Sito 3600 ± 30 pg/L S-Sitosta 2400 ± 30 pg/L Campe 900 ± 30 pg/L 134 6 The Bio-Adsorption of Phytosterols on Inactivated Biomass 6.3 Results and Discussions 6.3 Results and Discussions Batch adsorption experiments were performed using inactivated secondary sludge as an adsorbent of microbial origin and PPME spiked with plant sterols as adsorbate, to study the bio-adsorption of phytosterols. The secondary sludge, obtained from lab-scale biological reactors treating PPMEs, was inactivated to eliminate the removal of phytosterols through biodegradation. The batch adsorption experiments were carried out for three major phytosterols: fi-sitosterol (fi-Sito), B-sitostanol (li-Sitosta) and campesterol (Campe). These phytosterols were frequently detected in significant amounts during the analyses of PPMEs (Chapter 3 and Chapter 4). The adsorbent, the inactivated secondary solids or MLSS concentration, was varied from 0 (for control) to 2000 mg/L (0, 20, 40, 100, 200, 1000 and 2000 mg/L) and the concentrations of phytosterols remaining in the liquid phase were measured after certain time periods (0, 2, 10, 24, 124, and up to 264 h in other experiments) of continuous mixing at 39 ± 1°C. The results of some batch experiments are shown and discussed in this chapter, while the results obtained from other batch adsorption experiments with similar conditions are shown in Appendix E (E1-E12). 6.3.1 A d s o r p t i o n K i n e t i c s o f P h y t o s t e r o l s The results of kinetic adsorption tests for fi-Sito, li-Sitosta, and Campe are presented graphically in Figure 6-1 to Figure 6-8 that show the liquid phase sterol concentrations (pg/L) with lapsed time (h). Percent removal and dimensionless liquid phase concentrations of fi-Sito are also shown in Figure 6-9 to Figure 6-10 for kinetic considerations. The liquid phase concentrations of B-Sito, B-Sitosta, and Campe in the control batch, with no MLSS, were almost steady and did not show any significant adsorption (Figure 6-1). As the concentration of inactivated biomass (MLSS) was increased from 20 to 2000 mg/L, an increasing reduction in the liquid phase sterol concentrations was achieved (Figure 6-2 to Figure 6-6). This demonstrated a adsorptive tendency between the PPME plant sterols and the inactivated MLSS or biomass. 135 6 The Bio-Adsorption of Phytosterols on Inactivated Biomass 6.3 Results and Discussions 4 000 3 500 3 000 § 2 500 1 | 2 000 o c O 1 500 e $ 1 000 500 0 % — A L-er-- C a m p e — B — f i - S i t o - f i - S i t o s t a i " •' i i " " i 1 1 1 1 1 1 1 1 ' 1 1 1 1 " 1 1 1 1 ' ' i ' 1 1 1 1 i 1 1 1 1 1 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 T i m e (h) Figure 6-1. Phytosterols remaining in solution- 0 mg/L M L S S batch : 4 000 3 500 500 I 1 1 1 1 I -+-0 10 20 30 40 50 60 70 80 90 100 110 120 130 T i m e (h) Figure 6-2. Phytosterols remaining in solution- 20 mg/L M L S S batch 136 6 The Bio-Adsorption of Phytosterols on Inactivated Biomass 6.3 Results and Discussions 4 000 0 f ' 1 ' 1 i ' 1 1 1 i 1 1 1 ' i 1 1 1 1 i 1 1 1 ' i • 1 1 1 i 1 1 1 1 i 1 1 1 ' i 1 1 1 1 i ' 1 1 1 i ' 1 ' ' i ' 1 ' ' i ' ' ' 1 1 0 10 20 30 40 50 60 70 80 90 100 110 120 130 Time (h) Figure 6-3. Phytosterols remaining in solution-100 mg/L MLSS batch 4 000 3 500 —3 000 -Campe —a— B-Sito — A — B-Sitosta 4- 4- + 4- 4- 4- I 1 1 1 1 I 0 10 20 30 40 50 60 70 80 90 100 110 120 130 Time (h) Figure 6-4. Phytosterols remaining in solution- 200 mg/L MLSS batch 137 6 The Bio-Adsorption of Phytosterols on Inactivated Biomass 6.3 Results and Discussions Figure 6-5. Phytosterols remaining in solution-1000 mg/L M L S S batch 4 000 3 500 _ 3 000 _i ]}> "f 2 500 o I 2 000 o c o ^ 1 500 S a> *-» to 1 ooo 4f 500 0 — • — C a m p e —o— f i -S i to — A — f i - S i t o s t a j | f j ! { • i v IT*—— * — • i — • — i - . . . . i . . . . i . . . . i . . . . I ' . ' ' I . . . . I . . . . I . . . . I . . . . I • • "I • • • . j » . • • 0 10 20 30 40 50 60 70 80 90 100 110 120 130 Time (h) Figure 6-6. Phytosterols remaining in solution- 2000 mg/L M L S S batch 138 6 The Bio-Adsorption of Phytosterols on Inactivated Biomass 6.3 Results and Discussions Most of the decrease in the liquid phase sterol concentrations took place within first 24 h of contact. A further decrease in sterol concentrations was also observed between the 24 t h and 122th h, but this decrease was relatively small (Figure 6-2 to Figure 6-6). Therefore, the results indicated that the batch reactor contents were approaching an equilibrium between the adsorbed and non-adsorbed phytosterols after about 122 h of continuous mixing. This was confirmed by subsequent replicate set of batch adsorption experiments (Figure 6-7 and Figure 6-8). The results from Set 2 confirmed that there was no appreciable change in the liquid phase sterol concentrations after 122 h, even when further contact was allowed up to 169 h (Figure 6-7 and Figure 6-8). Hence, most of the adsorption of sterols on to the inactivated biomass was shown to occur within the first 24 h of contact, after which the overall rate of sterols adsorption seemed to have slowed down considerably such that adsorption equilibrium appeared to have been reached after 122 h. Similar liquid phase concentration curves were obtained for all three phytosterols under consideration. However, the shape of the liquid phase concentration curve for B-Sito was slightly different to that for Campe. Campe seemed to reach adsorption equilibrium relatively quickly as compared to both B-Sito and B-Sitosta (Figure 6-2 to Figure 6-8). 4 000 3 500 5 0 0 0 - • — C a m p e _ « _ ( j - S i t o - A - R - S i t o s t a a a • — _ — « — . i i i i 1 • • 1 » • • • • '•I i t i i 0 10 2 0 30 4 0 50 6 0 70 80 90 100 110 120 130 140 1 5 0 1 6 0 170 180 T i m e (h) Figure 6-7. Phytosterols remaining in solution- 40 mg/L M L S S batch (Set 2) 139 6 The Bio-Adsorption of Phytosterols on Inactivated Biomass 6.3 Results and Discussions The kinetic studies showed that the initial rapid decrease in the liquid phase sterol concentrations was followed by a period of more slowly decreasing concentrations. Ultimately the liquid phase concentration tended to have approached a fixed value for a given amount of added inactivated MLSS. When there was no appreciable change in adsorbate concentrations with time elapsed, an adsorption equilibrium was assumed to have been established. 4 000 -r ; ; ; ; 1 : ; ; ; ; ; : : : : , : , II I I I I I I I i I I ! I : • 1 j I 3 500 - j i [ \ \- I \ | \ I j | i | \ ! I 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 Time (h) Figure 6-8. Phytosterols remaining in solution- 2000 mg/L M L S S batch (Set 2) For a given period of contact, the percent removal of sterols increased with increasing amount of inactivated MLSS added (Figure 6-9). This suggested that an increased overall removal of sterols from PPMEs can be achieved by increasing the concentration of MLSS. Moreover, the rate of removal appeared to decrease with elapsed time, but the linear trend of C/Ci versus MLSS concentration for each sampling time or the constant slope of the C/Ci curves, indicated a fixed rate of change in C/Ci (normalized or dimensionless adsorbate concentration) decrease with increasing concentration of MLSS (Figure 6-10). Hence, under the conditions of this experiment, a linear relationship existed between the normalized concentration of B-Sito remaining in solution and the concentration of MLSS between 100 140 6 The Bio-Adsorption of Phytosterols on Inactivated Biomass 6.3 Results and Discussions and 2000 mg/L (Figure 6-10). This means that additional removal of fi-Sito could be achieved in the same amount of time, bv increasina the concentration of MLSS. 100 S-o o X a o 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 Time (h) Figure 6-9. Percentage removal of B-Sito with time and MLSS. C is the concentration remaining in solution at any time t and Ci is the initial concentration. 1.20 o C 2 o o 0.20 4-— • - -Oh - 2 h — A - -10h - X - -24 h — * - -124 h 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 MLSS (mg/L) Figure 6-10. Dimensionless liquid phase concentration of B-Sito and MLSS. C is the concentration in solution at time t and Ci is initial concentration. 141 6 The Bio-Adsorption of Phytosterols on Inactivated Biomass 6.3 Results and Discussions Approximately 57% of fi-Sito was removed after 124 h with an inactivated MLSS of 1000 mg/L. The same extent of removal appears to be achieved in just 2 h at an inactivated MLSS content of about 1700 mg/L (Figure 6-10). Therefore, by increasing the concentration (dose) of the inactivated MLSS from 1000 to 2000 mg/L, similar results were achieved in just 2 h instead of 124 h. The rapid initial decrease in sterols concentration that soon transformed into a slower decrease during the kinetic studies suggested that the extent of sterols adsorption depended upon the available adsorption sites or capacity of the inactivated biomass, which exhausted eventually. If a specific amount of adsorbent contains a specific number of adsorption sites, the adsorbent is expected to hold only a specific amount of the adsorbate. Hence, the amount of adsorbed sterols increased with an increase in the amount of inactivated MLSS. The phytosterols fi-Sito, fi-Sitosta and Campe generally appeared to demonstrate comparable adsorption behavior during all the adsorption experiments (e.g. Figure 6-1 to Figure 6-8). The initial and final liquid phase concentrations of fi-Sito and fi-Sitosta were higher than those of Campe. However, the differences in final concentrations of individual sterols decreased with increasing amounts of the inactivated MLSS, the adsorbent, added (Figure 6-3 and Figure 6-6 or Figure 6-7 and Figure 6-8). Thereby, indicating a positive adsorptive behavior of the tested sterols and a general adsorptive tendency of the inactivated secondary solids for plant sterols. This can be expected because the inactivated secondary solids provide a heterogeneous adsorbent surface composed of mainly lipoprotein cellular materials that are known to have adsorptive tendency for weakly polar and lyophobic (liquid hating) adsorbates that are poorly soluble in aqueous solutions (Shaw 1992; McKay 1995), such as plant sterols. As shown by the kinetic studies, the time required to reach an equilibrium (t-eq) for sterols was about 122 h. This indicates relatively slow adsorption kinetics for plant sterols when compared with the reported t-eq of about 1-72 h for phenol, PCP and some other organic compounds' adsorption to biomass (Aksu 2004). Relatively slow kinetics can be expected for organic pollutants like plant sterols and polychlorinated biphenyls (PCBs) that have log Kow values greater than 6, because the rate of organic pollutant desorption decreases with increasing hydrophobicity of the pollutant (Koelmans et al. 1995). 142 6 The Bio-Adsorption of Phytosterols on Inactivated Biomass 6.3 Results and Discussions Although kinetic adsorption data of plant sterols have not been reported previously in the literature, a general partitioning of sterols to secondary solids has been observed (Kostamo and Kukkonen 2003; McKague and Reeve 2003; Mahmood-Khan and Hall 2003). Hence, the results obtained in this study, not only confirmed a general adsorptive tendency of secondary solids for plant sterols, but also provided more details about the bio-adsorption phenomenon. All three plant sterols readily adsorbed onto the inactivated secondary solids with similar adsorption behavior. Therefore, it is likely that other phytosterols that may be present in PPMEs, will probably follow similar bio-adsorption trends. 6.3.2 A d s o r p t i o n E q u i l i b r i a o f P lan t S t e r o l s The liquid phase phytosterol concentrations in equilibrium with the inactivated secondary solids for three sets of batch adsorption studies are shown in Figure 6-11, Figure 6-12 and Figure 6-13 respectively. A time period of 122 h was considered sufficient for reaching equilibrium, however the time allowed to reach equilibrium for different sets varied from 122 h for Set 1, 169 h for Set 2, to 264 h for Set 3 of the batch experiments. The figures show the estimated (x) amount of phytosterol adsorbed to a unit mass (m) of the inactivated MLSS i.e. the of adsorption capacity (x/m or q) of the inactivated secondary solids at different equilibrium liquid phase concentrations (Ce) of phytosterols for the conditions of this study. Two adsorption regions were observed for each sterol depending upon its Ce, Table 6-3 describes the range of Ce values in each adsorption region. The adsorption capacity, x/m, of the inactivated secondary solids increased with increasing equilibrium concentrations Ce for all three sterols tested. The individual adsorptive capacities were different for each sterol tested. The inactivated secondary solids showed maximum adsorption capacity for Campe, intermediate for fi-Sitosta and minimum for fi-Sito (Figure 6-11 to Figure 6-13). The differences in the observed adsorptive capacities, however, diminished at low Ce values that resulted in low, but similar, adsorption capacities for each phytosterol tested. Moreover, a significant increase in adsorptive capacity was seen for each phytosterol when the Ce was increased (Figure 6-11 to Figure 6-13). 143 6 The Bio-Adsorption of Phytosterols on Inactivated Biomass 6.3 Results and Discussions The observed adsorption phenomenon can probably be explained by the concept of physisorption rather than chemisorption. Chemisorption involves ionic and covalent bond formations, requires high heat of adsorption, and is highly specific as well as irreversible. Physisorption is mainly a surface phenomenon that results from Van der Waals and physical bonds that are produced through a donor-acceptor complexation mechanism where atoms of the surface functional groups donate electrons to the adsorbate (sometimes loosely called sorbate) molecules (Al Duri 1995). In non-specific adsorption, processes of physisorption electron transfer may not be involved, but polarization is likely that creates weak electrostatic bonding, cationic bridging or attraction to provide the energy required. Physisorption requires low heat of adsorption and is usually reversible. In chemisorption, a monolayer is expected due to the irreversibility of the process, whereas in physisorption a multilayer phenomenon is encountered (Al Duri 1995). So, the increasing adsorptive capacities of the inactivated biomass at higher equilibrium concentrations, Ce, of plant sterols may have resulted from a multilayer adsorption process. 0.00 0.50 1.00 1.50 2.00 Ce Sterols mg/L 2.50 3.00 3.50 Figure 6-11. Sterol adsorption capacities of inactivated M L S S (Set 1) The shape of adsorption equilibrium curves depends significantly upon the characteristics of the adsorbate and the adsorbent and subsequent interactions among the 144 6 The Bio-Adsorption of Phytosterols on Inactivated Biomass 6.3 Results and Discussions adsorbates. Wastewaters in general and PPMEs in particular, contain a multitude of organic compounds. Depending upon the composition and concentration of different components, the degree of competition among the adsorbates varies, and thus the adsorption can exhibit different behaviors. The heterogeneity of organics in a multi-component system and the dependence of the isotherm parameters on the initial organic concentration often pose difficulty in describing the exact adsorption pattern of a wastewater system. 45.0 40.0 35.0 -ST 30.0 o> </> — |> 20.0 E 15.0 0.0 — A — Campe -*— fi-Sitosta -«•— B-Sito : _acE~_I—,—,—i—i—,—,—,— — i — i — i — i — i — i — i — i — i — j — i — i — i — i — i i — , — , — , — , — 0.00 0.50 1.00 1.50 2.00 Ce Sterols mg/L 2.50 3.00 Figure 6-12. Sterol adsorption capacities of inactivated M L S S (Set 2) At higher equilibrium concentrations, considerably different adsorptive capacities were observed for each phytosterol. A maximum value for B-Sito was observed for the tested conditions, the corresponding values for Campe and B-Sitosta were not available (Figure 6-11 to Figure 6-13). The initial concentrations of the tested plant sterols were different: about 850 pg/L for Campe, 2300 pg/L for B-Sitosta, and 3500 pg/L for B-Sito. Other experimental conditions were the same. Different initial adsorbate concentrations may influence the equilibrium capacity of a heterogeneous adsorbent. The equilibrium adsorption capacity of activated sludge has been reported to increase with increasing initial concentration of PCP (pentachlorophenol) up to 0.5 mg/L PCP (Jianlong et al. 2000) and the equilibrium adsorption 145 6 The Bio-Adsorption of Phytosterols on Inactivated Biomass 6.3 Results and Discussions capacity of DMP (dimethylphenol) (Gulyas et al. 1999). But similar data for sterols are not available. However, it is possible that the equilibrium adsorption capacity of inactivated secondary solids increases up to a certain concentration and then decreases. This needs to be further investigated. Lee and Kuo (1999) reported an initial increase in the equilibrium sorption coefficients with increasing concentrations of dissolved organic matter, that was followed by a decrease after reaching a maximum. Campe, fi-Sitosta, and fi-Sito have similar steroid nucleus, a characteristic of all sterols, but the hydrocarbon side chain for Campe is different from both li-Sitosta and fi-Sito. l i-Sitosta and fi-Sito have a similar hydrocarbon side chain, but they differ in the degree of saturation due to the double bond present in B-Sito molecule (Figure 2-3). Moreover, the molecular weights of Campe, S-Sitosta, and li-Sito differ slightly with values of 400.7, 416.7, and 414.7 respectively (Table 3-1), and it has been found that lower molecular weight organic compounds are relatively more adsorbable in multi-component wastewater systems (Yuasa et al. 1997). 100.0 T ; ; : ; ; : 1 90.0 [ \ ! ] I | | 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 Ce Sterols mg/L -&— Campe —»— B-Sitosta —©— S-Sito Figure 6-13. Sterol adsorption capacities of inactivated M L S S (Set 3) 146 6 The Bio-Adsorption of Phytosterols on Inactivated Biomass 6.3 Results and Discussions The other important observation was the convergence of the equilibrium adsorptive capacities of the inactivated biomass, to similar values for individual phytosterols at low Ce values. Hence, the observed bio-adsorption of phytosterols revealed a bi-phasic behavior for each sterol tested in this study (Figure 6-11, Figure 6-12 and Figure 6-13). Based upon the results, two adsorption regions A and B could be considered to describe high and low bio-adsorption capacities of the inactivated biomass for phytosterols. The Ce values corresponding to each A and B (high and low capacity) adsorption region are given in Table 6-3. Table 6-3. L o w and high adsorption capacity regions for different sterols (Set 1-3) Region A Region B Sterol High Adsorption Ce (mg/L) Low Adsorption Ce (mg/L) Campe > 0.5 < 0 .4 fi-Sitosta > 1 4 < 1.2 fi-Sito > 2 .4 < 2.3 It can be seen that the observed regions of low and high adsorption were associated with different Ce values for each sterol (Table 6-3). The transition values of Ce, the intermediate values of Ce between the high and the low adsorption regions, were between 0.4-0.5 mg/L for Campe, 1.2-1.4 mg/L for fi-Sitosta, and 2.3-2.4 mg/L for fi-Sito, thereby, suggesting that the Campe was the first sterol and fi-Sito was the last sterol that entered the region of high adsorption capacity when Ce was increased from a low value. This means that with increasing Ce to high values in Region A, the order and the extent of PPME sterols removal through the mechanism of adsorption will be Campe > fi-Sitosta > fi-Sito, and a high removal of sterols through the adsorption mechanism can be expected. However, with decreasing Ce to low values in Region B, a significant decrease in the removal of PPME sterols through bio-adsorption would be expected. 147 6 The Bio-Adsorption of Phytosterols on Inactivated Biomass 6.3 Results and Discussions The matrix effects on sterols solubility or micelle formations due to the presence of a wide range of other organic contaminants in PPMEs may be responsible for such a bi-phasic response, by creating a threshold type of effect on sterols adsorption to the inactivated biomass. When the Ce values are below this threshold the sterols may be relatively more soluble in PPME due to the so called third phase effect of the dissolved organic matter as noted by Li and Kuo (1999) during the sorption of hydrophobic organic pollutants (HOPs). When the Ce values are higher than such a threshold, the dispersive forces may be overcome through the electric-double layer interactions so that the attractive forces predominate, resulting in solvent motivated adsorption and colloidal attachments that are energetically preferable as compared to the aqueous state (Shaw 1992; Weber 2001). This phenomenon may result in an increase in apparent solubility of phytosterols at equilibrium concentrations below the threshold value, so the adsorption capacities of the inactivated biomass decrease. On the other hand, a decrease in apparent solubility of phytosterols, at equilibrium concentrations above the threshold value, increases the adsorption capacities of the inactivated biomass or the adsorbent. The increasing bio-adsorption capacities at higher adsorbate concentrations (Region A) reduce the chances of releasing high concentrations of sterols in treated effluents. However, the reduced bio-adsorption capacities at lower sterol concentrations (Region B) can considerably reduce the sterols removal efficiency mainly because of inefficient adsorption. Hence, a secondary treatment system with simultaneous biodegradation of sterols should be preferred. Biodegradation can provide additional adsorbent surface through regeneration as well as new biomass growth. 6.3.3 Isotherm Modeling Phytosterol equilibrium-adsorption data were used to produce adsorption isotherms presented in Figure 6-14 to Figure 6-16. The estimated values of adsorption constants are given in Table 6-4 to Table 6-6. Additional data related to the adsorption of phytosterols to the inactivated secondary biomass are given in Appendix E. The adsorption isotherms represent adsorbed mass per unit mass of adsorbent (x/m) determined as a function of the equilibrium concentration (Ce) of the adsorbate at a constant 148 6 The Bio-Adsorption of Phytosterols on Inactivated Biomass 6.3 Results and Discussions temperature. The Freundlich isotherm model was used to generate the phytosterols adsorption isotherms because it has been successfully used to describe the adsorption characteristics of heterogeneous adsorbents like secondary sludge and organics in secondary effluents (Chaudhary et al. 2003; Jianlong et al. 2000; Gulyas et al. 1999; Wang and Grady 1994). The model is empirically derived and is defined as x/m = KCe 1 / n (6.1) where x = mass of phytosterol adsorbed (the adsorbate) m = mass of inactivated MLSS (the adsorbent) Ce = Concentration of phytosterol remaining in solution (pg/L) K = Freundlich constant describing the adsorption capacity n = Freundlich constant describing the adsorption affinity or intensity K and n are Freundlich adsorption constants, the values of which depend upon the temperature, the adsorbent and the adsorbate as stated above. A logarithmic plot linearizes Equation (6.1). This enables determination of the exponent n (or 1/n) and K. Equation (6.1) can be re-written as: In (x/m) = In K + (1/n) In Ce (6.2) The fitted Freundlich model produced a correlation coefficient of about 0.9 or better. When tested, the Langmuir adsorption model did not give satisfactory correlation for the observed equilibrium of sterols adsorption. The fitted Freundlich isotherms demonstrated two different equilibrium adsorption regimes (A and B) for each sterol depending upon the equilibrium sterol concentrations Ce, as discussed in the preceding section. The low adsorption regime (Region B) appeared to correspond to the equilibrium concentrations of less than 0.3 mg/L for Campe, 1.0 mg/L for fi-Sitosta and 2.1 mg/L for B-Sito, respectively. The high adsorption regime (Region A) appeared to correspond to the equilibrium concentrations of more than 0.4 mg/L for Campe, 1.2 mg/L for B-Sitosta and 2.2 mg/L for B-Sito, respectively (Figure 6-14 to Figure 6-16). These estimates are slightly less than those presented in the last section (Table 6-3), probably due to the effect of linearization of the isotherm curves for model fitting. However, 149 6 The Bio-Adsorption of Phytosterols on Inactivated Biomass 6.3 Results and Discussions the high adsorption capacity Region A covered most of the observed range used for isotherm generation. In this adsorption region, the estimated capacity constant K values varied in the order of Campe > fi-Sitosta > fi-Sito (Table 6-4 to Table 6-6). 10.0 Ce (mg Sterols/L) Figure 6-14. Set 1: Freundlich isotherms for Campe, B-Sitosta and B-Sito. Legend suffix _a denotes high adsorption Region A, and _b denotes low adsorption Region B Table 6-4. Isotherm model coefficients for different sterols (Set 1) Sterol Region K 1/n N R Campe fi-Sitosta B-Sito A A A 58.70 5.02 0.57 3.31 3.45 3.55 0.30 0.29 0.28 0.92 0.84 0.91 Campe fi-Sitosta fi-Sito B B B 1.05 2.20 2.71 0.16 0.22 0.12 6.33 4.62 8.12 0.99 0.99 0.99 150 ft The Bio-Adc^rptinn of Phv+"«*»™is o n Inactivated Biomass fi 3 Results pnrl Discussions •o (/> O) </> 2 <x> CO E Ce (mg Sterols/L) Figure 6-15. Set 2: Freundlich isotherms for Campe, B-Sitosta and B-Sito. L e g e n d suffix _a denotes high adsorption Region A and _b denotes low adsorption Region B Table 6-5. Isotherm model coefficients for different sterols (Set 2) Region K 1/n Sterol Campe B-Sitosta B-Sito Campe B-Sitosta B-Sito n A A A B B B 181.08 4.10 0.18 3.77 6.12 5.59 4.81 4.21 5.09 0.51 0.57 0.47 0.21 0.24 0.20 1.97 1.75 2.14 R^  0.92 0.94 0.93 0.96 0.98 0.96 151 6 The Bio-Adsorption of Phytosterols on Inactivated Biomass 6 3 Results and Discussions • S-Sito_a o B-Sito_b n B-Sitosta_a • S-Sitosta_b • Campe_a A Campe_b y = 197.11x4 2 7 6 1 R 2 = 0.8846 1000.0 400/6— : y = 7.524x4 3 4 6 9 , f l , 7 R D2 „ M „ y = 0.2811x4 8 3 7 6 R^  = 0.8906, , / R 2 = 0.8957 A / ..v .^ i n n /* / A . * ^ * ^ ^ ^ i A n 1 y = 7.4583X1 2 3 9 4 y = 4.9147x 0 8 6 7 2 1 R2=1 R 2 = 1 I _ w-0 If y = 3 .7537X 0 5 8 1 9 R 2 =1 Ce (mg Sterols/L) Figure 6-16. Set 3: Freundl ich isotherms for Campe, B-Sitosta and B-Sito. Legend suffix _ a denotes high adsorption Region A and _b denotes low adsorption Region B Table 6-6. Isotherm model coefficients for different sterols (Set 3) Sterol Region K 1/n n R2 Campe A 197.11 4.28 0.23 0.88 B-Sitosta A 7.52 4.35 0.23 0.89 B-Sito A 0.28 4.84 0.21 0.90 Campe B 7.46 1.24 0.81 1.00 B-Sitosta B 4.91 0.87 1.15 1.00 B-Sito B 3.75 0.58 1.72 1.00 152 6 The Bio-Adsorption of Phytosterols on Inactivated Biomass 6.3 Results and Discussions In the low adsorption Region B, the estimated K values of the fitted Freundlich Model were similar for the tested sterols. However, in each adsorption region (A or B), the slopes of all three isotherms were approximately identical (Figure 6-14 to Figure 6-16). Therefore, the values of adsorption coefficient n were also anticipated to be similar as confirmed by the estimated n values given in Table 6-4 to Table 6-6. The usefulness of Freundlich isotherm and association adsorption theory to describe organics adsorption behavior in wastewater systems, was demonstrated by Chaudhary et al. (2003) through the successful description and prediction of the adsorption pattern of wastewater organics. However in this study, two distinct models were required to describe the bio-adsorption pattern of PPME phytosterols. Such details of plant sterols bio-adsorption behavior are not available in the literature. However, a biphasic adsorption phenomenon depending upon the sterols solubility in the experimental matrix, may be responsible for such results as discussed in Sec. 6.3.2. When PPME phytosterol concentrations increase, Campe is expected to enter the high adsorption Region A and be removed before the other two sterols. The removal of Campe will be followed by B-Sitosta and B-Sito. B-Sito will be the last sterol to enter the high adsorption region. Therefore, ensuring a high removal of B-Sito may be an important operation and design consideration^ ensure high removal efficiencies of the other two phytosterols. Wang and Grady (1994) examined the sorption of di-n-butyl phthalate (DBP) on live and dead biomass. Upon comparison of the generated sorption isotherms, the DBP sorptive capacity of the dead biomass was apparently higher than that of the live biomass on a unit solids basis. However, when the loss of biomass due to cell lysis and decay, resulting from autoclaving for biomass inactivation, was considered the sorptive capacities of live and dead biomass were the same. It should also be noted that not all of the biomass cells in an aeration basin are active. The active fraction of cells can be typically vary between 60-80% depending upon process conditions and the mean cell residence time (Grady 1999). Therefore, the adsorption behavior of plant sterols observed for inactivated biomass may be assumed to be approximately similar to that of active biomass. Typically, an adsorbent may be expected to exhibit a reduction in its adsorptive capacity when subjected to a mixture of organic adsorbates (Eckenfelder 2000), as almost always is the case for industrial as well as municipal wastewaters. Such multi-component wastewater adsorptive systems are difficult and complex to model, due to the competitive adsorption of 153 6 The Bio-Adsorption of Phytosterols on Inactivated Biomass 6,3 Results and Discussions individual adsorbates and their interactions based upon the composition and concentrations of different components involved. Although the equilibrium adsorption capacity for each individual adsorbate in a mixture is expected to be less than that for single adsorbate present alone, the combined adsorption is usually greater than the sum expected from individual adsorbate data (Eckenfelder 2000). Moreover, the isotherm data are developed by achieving the equilibrium conditions, whereas the field adsorption systems operate in a dynamic environment that is not necessarily in equilibrium. For similar reasons, differences in adsorptive capacities may exist and the isotherm data may typically overestimate the capacity of an operating adsorption system. However, in the present study, the adsorption of sterols to the inactivated secondary sludge was studied in a multi-component system. Therefore, the adsorption capacity estimates obtained in this study are expected to be realistic. Organic matter plays an important role in both preference and capacity of sorption. Dissolved organic matter (DOM) can significantly reduce the adsorption of other organic contaminants by reducing the adsorption capacity of GAC or granular activated carbon (Carter et al. 1992; Carter and Weber 1994). In the presence of simultaneous biodegradation during secondary treatment of PPMEs, a decrease in DOM is expected. The decrease in DOM can subsequently improve the adsorption of organic pollutants from water and wastewater (Weber and LeBoeuf 1999). However, the sorbent biomass is also a dynamic component of the organic matter that contains the living organisms responding to the environmental changes and stress by forming extra-cellular polymeric substances (EPS) or other metabolites that are usually hydrated (Flemming 1995). Depending upon growth conditions, a considerable part of the EPS may consist of extra-cellular enzymes comprised of polysaccharides, proteins and lipids. Hence, the treatment system biomass represents a dynamic system that can continuously modify, synthesize and break down chemicals, and a part of the sorbed chemicals may also be remobilized (Flemming 1995). The complexities involved in different aspects of the bio-adsorption mechanism indicate a considerable need for further research. Therefore, more investigation is necessary to quantify the effect of decreasing levels of DOM present in PPMEs, and changes in biomass due to process operating conditions within the typical range. In summary, bio-adsorption of PPME phytosterols is a variable phenomenon that has been observed to behave differently at low and high equilibrium concentrations of sterols. 154 6 The Bio-Adsorption of Phytosterols on Inactivated Biomass 6.3 Results and Discussions The chances of having a high sterol concentration release in treated effluent, further reduce as the adsorption capacity of the biomass may increase with increasing sterols concentrations. The presence of other adsorbates and DOM may, however, considerably reduce the biomass adsorptive capacities at low concentrations of sterols. This may eventually exhaust the available adsorption capacity of most of the MLSS present, and low concentrations corresponding to Region B for each sterol may be released in the treated effluents. Relatively high concentrations for fi-Sito and relatively low concentrations for Campe observed in treated PPMEs (Mahmood-Khan and Hall 2003) may have resulted from the differences observed in the adsorptive capacities of the secondary biomass present in the full-scale secondary treatment systems (Chapter 4). The observed differences in the biomass adsorptive capacities for individual sterols (Table 6-4 to Table 6-6) may also be responsible for the relatively high removal efficiencies for Campe and comparatively lower removal of fi-Sito (Table 4-9). The differences in individual sterols biodegradation may have also contributed. Phytosterol concentrations typically found in PPMEs, may fall in the low adsorption capacity (Region B) and a reduced sterols adsorption and removal may be experienced across a secondary treatment system. Therefore, the results obtained in this study also suggest that the secondary treatment systems should be designed and operated to enhance biodegradation of sterols by allowing the growth of sterols-degrading biomass for an effective removal of plant sterols or similar organic pollutants. 6.3.4 Sterols Bio-adsorption and Removal during Secondary Treatment Results from the present study indicate that a sterols removal (C/Ci) that was achieved in 124 h with an adsorbent dose of about 500 mg/L, can be achieved in just 2 h by increasing the inactivated MLSS dose to about 1500-2000 mg/L (Figure 6-10). The activated sludge secondary wastewater treatment systems studied in this research project typically maintained a concentration of about 4000 mg/L MLSS. At such a concentration of MLSS, it is reasonable to expect that most of the phytosterols entering the treatment system are probably adsorbed onto the secondary solids within the first 2-4 h of mixing in the aeration basin. The total 155 6 The Bio-Adsorption of Phytosterols on Inactivated Biomass 6.3 Results and Discussions amount of sterols removed through adsorption will depend upon the level of sterols saturation of the MLSS present in the aeration basin and the growth of new biomass. Depending upon the operating conditions of the treatment system, a portion of the adsorbed sterols can be biodegraded. If the operating conditions are suitable for sterols biodegradation, about 80% of the dissolved and adsorbed sterols are expected to be biodegraded or transformed (Chapter 5). The growth of new biomass, coupled with sterols biodegradation, can act as an adsorbent-regeneration process and increase the available adsorbent capacity. In this case, the effectiveness of the combined bio-adsorption process will be mainly influenced by bio-regerieration of the adsorbent surface through adsorbate biodegradation and growth of new biomass (Klimenko et al. 2002). Bio-regeneration takes place as a result of interactions among the microorganisms and biodegradation or bio-transformation of the adsorbed molecules. The narrowest micro-pores of the adsorbent (or the dead microorganisms, non-degraders and other suspended matter) become unavailable once occupied by the adsorbate molecules. Therefore it can be accepted that further removal or decrease in sterols content occurs due to the biodegradation and bio-regeneration of the occupied part of the biomass as well as growth of new biomass. The intensity of bio-regeneration is an important factor influencing the effectiveness of secondary treatment systems or the processes that mainly depend upon bio-adsorption for an effective removal of a specific pollutant. Therefore, treatment systems with combined bio-adsorption and biodegradation would generally have a good capability to handle the normal loads of phytosterols and some shock loads that may hit the treatment plant during abnormal operation of the mills. As shown in Chapter 5, the removal of phytosterols in secondary wastewater systems is a dynamic process involving both biodegradation and bio-adsorption. A particular set of operating conditions may favor both of these mechanisms in which case biodegradation may be the primary mechanism of removal of phytosterols entering the treatment system. However, the high-rate process conditions of a secondary treatment system can substantially reduce (about 50% or more) biodegradation of sterols. Under these circumstances bio-adsorption becomes the governing mechanism of sterols removal (Chapter 5). Under such conditions, most of the available adsorptive capacity of the system MLSS can be used up or exhausted, especially at lower sterols concentrations (Sec. 6.3.4), depending upon the dynamic equilibrium between the sterols and the biomass. Therefore, the overall sterols removal efficiency can decline quickly, resulting in elevated concentrations of sterols 156 6 The Bio-Adsorption of Phytosterols on Inactivated Biomass 6.4 Conclusions discharged in the final treated effluent. This may partially explain the variable sterol removal efficiency observed during the survey of full-scale treatment systems presented in Chapter 4. This argument is further supported by the mass balance estimates that revealed little biodegradation of phytosterols during the full-scale treatment survey. A loss of sterols biodegradation would increase accumulation of sterols in secondary solids through bio-adsorption. Subsequently, the sterols leaving the system with WAS would increase, indicating a shift in the principal mechanism responsible for observed sterols removal from biodegradation to bio-adsorption. Therefore, the specific pollutant treatment efficiency will be related to the dynamic adsorption capacity of the systems MLSS, and the relative contributions of bio-adsorption and biodegradation mechanisms will depend upon the process operating conditions. 6.4 Conclusions Sterols adsorption was investigated using inactivated secondary solids as an adsorbent for three majonplant sterols: fi-Sito, fi-Sitosta and Campe. All the three plant sterols readily adsorbed on the inactivated secondary solids following similar adsorption trends. Other phytosterols, which can be present in PPMEs, are also expected to follow similar trends of adsorption to secondary solids and sludge. Almost 80% or more of the adsorption equilibrium was achieved during the first 10-20 hours for the three phytosterols and the achievement of complete equilibrium appeared to take more than a few days. For an adsorbent dose of 2000 mg/L of the inactivated MLSS more than 80% of the phytosterols were removed within the first 2 to 4 h. The Langmuir adsorption model could not give satisfactory correlation for the observed equilibrium of sterols adsorption. The Freundlich adsorption model was fitted to the phytosterols equilibrium adsorption data with a correlation coefficient of about 0.9. The fitted Freundlich isotherms revealed two different equilibrium adsorption regimes, a high adsorption Region A and a low adsorption Region B, for each sterol, depending upon the equilibrium concentration of sterols. The high adsorption capacity Region A, appeared to correspond to equilibrium concentrations of more than 0.3-0.4 mg/L for Campe, 0.7-1.0 mg/L for fi-Sitosta and 1.5-2.0 mg/L for fi-Sito, respectively. The low adsorption capacity Region B, appeared to 157 6 The Bio-Adsorption of Phytosterols on Inactivated Biomass 6.4 Conclusions correspond to equilibrium concentrations of less than 0.3 mg/L for Campe, 0.7 mg/L for fi-Sitosta and 1.5 mg/L for B-Sito, respectively. In the high adsorption Region A, at a specific value of Ce, the inactivated MLSS exhibited the maximum adsorptive capacity for Campe, an intermediate capacity for B-Sitosta, and the lowest adsorptive capacity for B-Sito. In the low adsorption Region B, the inactivated biomass appeared to have considerably less, but comparable adsorptive capacities for each phytosterol tested. In the high adsorption Region A, Freundlich adsorption capacity coefficient K varied in the order of Campe > B-Sitosta > B-Sito. While the adsorption intensity coefficient n had same or similar values for all three plant sterols. In the low adsorption Region B, both K and n values were similar for each phytosterol tested. For PPME sterol concentrations falling in the low adsorption capacity Region B, a secondary treatment system with simultaneous biodegradation seems to be preferable. 158 7 Overall Conclusions & Recommendations 7.1 Overall Conclusions 7. Overall Conclusions & Recommendations At this stage it is important to summarize the overall results and conclusions obtained from the research project, and this is the objective of this chapter. Therefore, the important questions of interest are: • What are the overall findings and conc lus ions? • What is the engineering significance of the work done? • How can this information be used for future research projects? An effort has been made, within the scope of this thesis, to present the answers of the above mentioned three questions in the succeeding sections. 7.1 Overall Conclusions 7.1.1 C a n p l an t s t e r o l s b e s u c c e s s f u l l y quan t i f i ed i n P P M E s ? A gas-liquid chromatographic technique was modified for effective isolation, extraction and analysis of plant sterols from PPMEs. The modified procedure involved liquid-liquid extractions with MTBE (methyl-t-butyl ether) and optimum silylation derivatization with BSTFA (methyl-t-butyl ether) followed by GC/MS analysis. The modified technique was successfully used for sterols extraction and analysis from primary- and secondary-treated PPMEs and secondary sludges. Optimum silylation derivatization is required for effective chromatographic determination of the sterols extracted from PPMEs. Optimum silylation was achieved by incubating the extracted sterols at 70°C for 4 h. Silylation duration was reduced from 12 to 4 h, that reduced the time required for sterols analysis and improved the method sensitivity. 159 7 Overall Conclusions & Recommendations 7.1 Overall Conclusions 7.1.2 Which sterols are present in (local) P P M E s ? Are they removed dur ing secondary treatment? The modified analytical technique was applied in different surveys of two Canadian pulp and paper mills that revealed li-sitosterol (fi-Sito), fi-sitostanol (li-Sitosta) and campesterol (Campe) as the key sterols present in PPMEs. Stigmasterol (Stigma), ergosterol (Ergo) and cholesterol (Chole) are also present but in relatively minor quantities. fi-Sito, li-Sitosta and Campe accounted for about 70% of the total sterols present in PPMEs and secondary sludges. Total sterol concentrations were 250-2500 pg/L in primary effluents, 100-700 pg/L in biologically treated final effluents, 20,000-30,000 pg/L in return activated sludge (RAS), and 20,000-40,000 pg/L in waste activated sludge (WAS). High sterols concentrations in RAS and WAS indicated significant accumulation of plant sterols in secondary solids. Plant sterols were generally removed from PPMEs through the secondary biological wastewater treatment provided by the UNOX-AST systems at both mills. The average total sterols removal efficiencies varied from 58 to 93%, and the observed removal did not suggest significant biodegradation of sterols. A significant portion of the total sterols entering the UNOX-AST systems at both mills was discharged without biodegradation. As much as 34% of the incoming sterols may leave with secondary-treated final effluents to the receiving waters and another 30% or more may leave with WAS. A single pulp mill may discharge about 20 kg/day sterols in its treated effluents only. Sorption to secondary solids and limited biodegradation were the suggested mechanisms of sterols removal. 7.1.3 To what extent can biodegradation contribute to the overall removal of plant sterols during the biological secondary treatment of P P M E s ? Two lab-scale biological reactors equipped with secondary clarifiers were operated continuously to treat primary-treated PPMEs spiked with phytosterols. The results suggest that suspended growth AST systems can achieve a high level of sterols removal, and the removal efficiencies can be maintained at about 90% and above. Bio-adsorption and biodegradation or transformation are the two major mechanisms of sterols removal. 160 7 Overall Conclusions & Recommendations 7.1 Overall Conclusions Biodegradation can contribute up to about 80% or more of the total sterol removal achieved through the biological treatment of PPMEs. Hence, biodegradation can play a major role in sterols removal. Sterols biodegradation was, however, sensitive to the normal operating range of the process variables of pH, HRT, and SRT. An increase in the system MLSS or secondary sludge sterols correlated with a loss in sterols biodegradation and a reduction in the overall removal efficiency of sterols. A reduction of about 20-30% in the overall sterols removal was found to be associated with a major decline of about 50% or more in the sterols biodegradation. Under the conditions bio-adsorption may be the principal mechanism of sterols removal and this may increase the amount of sterols leaving the treatment system with WAS by 2-4 or more orders of magnitude. This also reduces the available adsorptive capacity of the system and potentially increases the chance of a sterols shock load passing through the treatment system. Lab-scale biological Reactor 1, operating at a pH of 6.7 ± 0.2 and a temperature of 38 ± 1°C, appeared to be relatively more robust and effective in removing sterols from primary-treated PPMEs than the lab-scale biological Reactor 2, operating at similar process conditions but a pH of 7.6 ± 0.2. Both lab-scale AST systems (Reactor 1 and Reactor 2) could treat an influent concentration of total sterols up to 4500 pg/L with an HRT of about 11-12 h and an SRT of about 11-12 d, but Reactor 2 showed lower stability and sterols removal capacity. Further reductions in system SRT and HRT increased the relative amounts of sterols associated with the biomass and reduced the sterol removal efficiencies for both reactors as sterols biodegradation/transformation was particularly affected. 7.1.4 W h a t are the k i n e t i c s o f s t e r o l s b i o - a d s o r p t i o n a n d w h a t i s the a d s o r p t i v e b e h a v i o r o f p lant s t e r o l s w i t h s e c o n d a r y s o l i d s ? Adsorption of plant sterols to secondary solids was investigated using inactivated secondary solids as an adsorbent for three major plant sterols: fi-Sito, fi-Sitosta and Campe. All the three plant sterols readily adsorbed on to the inactivated secondary solids and appeared to exhibit comparable adsorption kinetics. About 80% of the adsorption occurred within the first 10-20 h for all three phytosterols and an equilibrium appeared to have been established by 122 h of contact. The overall rate 161 7 Overall Conclusions & Recommendations 7.1 Overall Conclusions of adsorption appeared to increase with increasing amount of inactivated MLSS, the adsorbent. With an adsorbent dose of about 2000 mg/L of the inactivated MLSS, more than 80% of the phytosterols were removed within 2-4 h of contact. The inactivated secondary solids exhibited similar equilibrium adsorption capacities for the sterols tested at lower equilibrium concentrations (Ce < 500 pg/L), but the equilibrium adsorption capacities considerably increased at higher equilibrium concentrations (Ce > 300 pg/L for Campe, > 700 pg/L for fi-Sitosta, and > 1700 pg/L for fi-Sitosta). The order of adsorption capacities on the inactivated secondary solids was Campe > fi-Sito > fi-Sitosta i.e. maximum for Campe and minimum for fi-Sito. The Langmuir adsorption model could not give satisfactory correlation for sterols adsorption equilibrium isotherms. However, the Freundlich model was successfully fitted by dividing the phytosterols equilibrium adsorption data in the two sets that had a correlation coefficient of about 0.9 or better. Hence, two different equilibrium adsorption regimes, a high adsorption Region A and a low adsorption Region B, for each sterol were considered depending upon the respective Ce values for each sterol tested. In the high adsorption Region A (high Ce), the Freundlich adsorption capacity coefficient K varied between 58-197 for Campe, 4-7.5 for fi-Sitosta and 0.2-0.5 for fi-Sito respectively. While the adsorption intensity coefficient n was found to be relatively less affected and had similar values varied between 0.2-0.3 for each sterol tested. In the low adsorption Region B (low Ce), both of the coefficients K and n, exhibited similar values for each phytosterol tested. The values for K ranged from 1 to 7 and for n from 1 to 8. these results suggest that a relatively low removal of sterols through bio-adsorption mechanism should be expected when the incoming sterol concentrations fall in the low adsorption Region B. Therefore, the secondary treatment systems with simultaneous biodegradation of sterols would be generally more effective for the treatment and control of plant sterols in PPMEs. 162 7 Overall Conclusions & Recommendations 7.2 New Knowledge & Engineering Significance 7.2 New Knowledge & Engineering Significance 7.2.1 C o n t a m i n a n t Quan t i f i c a t i on A standard technique for analyzing plant sterols in PPMEs has not been published. However, the need for having a reliable method for the analysis of a particular contaminant in environmental samples cannot be over-emphasized. An analytical technique was modified for the detection and quantification of (plant) sterols, suspected EDCs/HAAs, from a complex mixture of PPMEs. The modified technique was used to measure phytosterol concentrations in specific pulp and paper wastewaters, and the expected concentration ranges before and after secondary wastewater treatment were estimated. These analyses provided estimates of pollutant removal efficiencies across the treatment systems, mass balances as well as pollutant discharges to the receiving water environments. This is the first study of its kind. Such information is important for evaluating the effectiveness of a wastewater treatment system and assessing the impact of any operational as well as design changes for improving the level of pollutant treatment if required. The pollutant discharge information is required for subsequent risk assessment, contaminant discharge limit considerations, the decisions about treatment requirements, treatment method selection, and the management and disposal considerations and programs for the excess secondary sludge that may be contaminant rich. 7.2.2 P o l l u t a n t R e m o v a l t h r o u g h S e c o n d a r y B i o l o g i c a l T r e a t m e n t The level of treatment of phytosterol provided by the full-scale state-of-the-art UNOX-AST wastewater treatment systems at two mills was assessed and a range of the specific pollutant removal efficiencies was obtained through different mill surveys. This provided more detailed information about the individual and total sterols removal, as well as the involved mechanisms of sterols removal. Such information is not readily available in the published literature, especially for Canadian mills. The results are helpful for planning and designing 163 7 Overall Conclusions & Recommendations 1.2 New Knowledge & Engineering Significance further research and experimental work for the improvement and optimization of the treatment of plant sterols and similar other moderately hydrophobic organic contaminants that may be present in the forest products industry wastewaters. Lab-scale biological treatment studies offered more flexibility for research work and provided promising results that suggested a high potential for treating such pollutants through biological processes. The operation of the lab-scale reactors quantified the role of sterols biodegradation in the overall removal of sterols during secondary biological treatment. This also estimated the impact of changes in process control variables: SRT, HRT and pH, on the sterols biodegradation and overall secondary treatment, and provided recommendations about the selection of these important process variable. This provides some guidance and a starting point for further research that may lead to improve the design and operation practices for the treatment of pollutants that may otherwise pass through the secondary treatment currently provided. These studies are also the first of their kind, when there is not much published research work available regarding the impact of process variables on the secondary treatment of plant sterols. 7.2.3 P o l l u t a n t B i o - a d s o r p t i o n a n d W a t e r / W a s t e w a t e r r e u s e Adsorption of sterols to secondary biomass, bio-adsorption, was identified as one of the major mechanisms of sterols removal during secondary treatment, in addition to biodegradation. Sterols bio-adsorption kinetic and batch equilibrium studies were conducted using inactivated secondary solids to generate adsorption isotherms for three key plant sterols found in PPMEs, and the adsorptive capacities of the inactivated biomass adsorbent were estimated. The knowledge of pollutant adsorption kinetics and isotherm curves, is of fundamental importance in the design as well as operation of treatment systems utilizing the adsorption process. Such information for plant sterols is not available in the published literature. During the high rate operating conditions that do not allow effective biodegradation, the effectiveness of a secondary treatment process in removing sterols or similar pollutants mainly depends upon bio-adsorption. The increased role of the bio-adsorption mechanism during such operation of secondary systems treating phytosterol-containing PPMEs, may influence the bio-reactor size or minimum residence time required for achieving a certain level 164 7 Overall Conclusions & Recommendations 7.2 New Knowledge & Engineering Significance of pollutant removal or expected effluent concentrations of phytosterols. The pollutant of interest can thus be accumulated/concentrated in secondary solids, and removed or collected with excess sludge from the treatment system for further treatment and disposal. The handling of pollutant-rich waste secondary sludges, may be relatively easier than dealing with dilute but comparatively much larger volumes of industrial wastewaters. Expanding anthropogenic activities that interfere with Earth's normal hydrologic cycle, steadily deteriorate water qualities and blur the boundaries between natural waters, water supplies and wastewaters. This entails us to a virtual continuum of developing the means of transforming water of any quality into a water of required quality for a particular use (Weber and LeBouef 1999). Increased reuse of water seems to be the obvious demand for a sustainable civilization. However, increased reuse of water significantly impacts source water quality and product water value. Water reuse can be increased through the primary forms of either Cascading: the sequential use of water of deteriorating quality for purposes having decreasingly stringent quality requirements or Recycling: the repeated use of water for the same purpose. A combination scheme of Closing the Loop on the Cascading Reuse of water seems to be the ultimate configuration of the recycling scheme that presents the greatest technology challenge and calls for advanced treatment of water/wastewater to meet the increasing use-related standards. Adsorption is one of the processes that can be used for such an advanced treatment of water/wastewater. Granular/powdered activated carbon (GAC/PAC) is a broad-spectrum sorbent of choice for removing an assortment of organics and heavy metals (Weber 1972; Weber and Smith 1986). The broad-spectrum properties of GAC/PAC that are advantageous in general water recovery and reuse, may not be desirable for specific pollutant and targeted solute recoveries and reuses. The initial cost as well as chemical and thermal regeneration costs of GAC/PAC become exceedingly expensive when low effluent concentrations must be achieved at a large scale. So there is a need to explore alternative technologies and bio-adsorption offers such an alternative. Cost effectiveness and good removal performance are the main attractions of bio-adsorption that uses cheap and easily available adsorbents of biological origin like activated sludge process wastes and sea weeds (Aksu 2004). Relatively high adsorptive capacities of inactivated biomass for plant sterols have indicated the general selectivity of microbial-origin adsorbents for organic pollutants present in industrial wastewaters. 165 7 Overall Conclusions & Recommendations 7.3 Recommendations 7.3 Recommendations 7.3.1 B i o l o g i c a l T rea tmen t o f S e m i - H y d r o p h o b i c O r g a n i c P o l l u t a n t s The studies in this project focused mainly on the suspended growth AST system. However, the removal of plant sterols and other similar organic pollutants across other types of treatment systems like ASBs and membrane bioreactors need to be investigated. The biodegradation of such pollutants was seen to be a dynamic phenomenon that was readily influenced by the process operating conditions, especially when the system was operating at HRT shorter than 10 h and SRTs shorter than 10 days. Further optimization of residence times needs to be investigated, especially for HRT, if the SRT cannot be reduced further. An investigation into sterol biodegradation kinetics of sterol-degrading active biomass is recommended in this regard. Relatively better performance and operation of the suspended growth bioreactor operating at near-neutral pH conditions (Reactor 1), suggested that slightly alkaline conditions may not be preferred for the treatment of semi-hydrophobic organic pollutants. The effect of process pH need to be further explored to understand the solubility-related variations and their impact on biodegradation of such contaminants and the overall performance of the secondary treatment system. Other important factors include the study of the impact of process temperature, if the secondary system needs to be operated at temperatures other than 38-40°C. Optimized biodegradation of sterols or similar pollutants may enhance the performance of the secondary process through increased utilization of other organic substrate that is relatively easily biodegradable as compared to sterols. 7.3.2 O r g a n i c Po l lu t an t B i o - a d s o r p t i o n S t u d i e s The tested plant sterols were adsorbed onto the inactivated biomass, however, the adsorptive capacity of the inactivated biomass appeared to be significantly different for 166 7 Overall Conclusions & Recommendations 7.4 Thesis Writing individual sterols. This needs to be compared with sterols bio-adsorption data obtained through using active biomass, to check for any the difference of behavior between the active and the inactivated biomass. Other important factors that need further exploration include the effect of agitation rate, process pH, dissolved organic matter and temperature depending upon the needs of particular treatment systems that may use bio-adsorption as the principal mechanism of sterols removal especially during the high-rate operation fashion when long residence times are not feasible for the overall treatment process. Proper handling, stabilization and disposal of excess secondary sludge is also important, because of the expected high levels of sterols or other adsorbed pollutant in secondary sludge. The relatively low adsorptive capacity of the inactivated biomass for fi-Sito as compared to that for the other sterols tested: fi-Sitosta and Campe, suggests that fi-Sito can be used as a key plant sterol for making the treatment system design and operation decisions. If fi-Sito was effectively removed, other plant sterols are expected to be removed as well. A considerable reduction in the adsorptive capacities of the inactivated biomass for the plant sterols at lower equilibrium concentrations also indicated that secondary treatment systems that have simultaneous biodegradation may be preferable for ensuring a high level of treatment, even at low sterol concentrations, as plant sterols have been suspected to induced EDC/HAA effects even at trace concentrations. 7.4 Thesis Writing Some action strategies for thesis writing are given in Appendix F for the benefit of enthusiastic Ph.D. candidates of future, who may read this thesis for various reasons. The objective is to ease the task of writing and getting to the finish line by incorporating some simple strategies in daily routine work. 167 References 8. References Abramovitch, R. A., Micetich, R. G., and Smith, S. J. 1963. Extractives from Populus tremuloides (Aspen Poplar) heartwood. Tappi, 46 (1): 37-40. Ahokas, J. T., Holdway, D. A., Brennan, S. E., Goudey, R. W., and Bibrowska, H. 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Environmental Science and Technology, 29: 2140-2146. 180 9. Appendices 9.1 Appendix A. Chemical Analysis of Plant Sterols 9. Appendices 9.1 Appendix A. Chemical Analysis of Plant Sterols A 1. Target plant sterol standards, surrogate and internal s tandard Name Code Chemical Name (Company, purity) Formula MW MW (d) RT (min.) Chole Cholesterol (Sigma, 98%) C27H46O 386.70 458.70 17.83 Ergo Ergosterol (Sigma, 90%) C28H44O 396.33 468.33 19.03 Campe Campesterol (Sigma, 98%) C28H48O 400.70 472.70 19.37 Stigma Stigmasterol (Fluka, 98%) C29H48O 412.67 484.67 19.73 B-Sito fi-Sitosterol (Sigma, 98%) C29H50O 414.70 486.70 20.68 fi-Sitosta fi-Sitostanol (Sigma, 98%) C29H52O 416.71 488.71 20.89 IS-3 Dotriacontane (Aldridge, 97%) C32H66 450.90 - 18.65 {MW=Molecular Weight; MW (d)=Silylated Molecular Weight; IS=lnternal Standard; RT= Approximate Retention Time} 181 9. Appendices 9.1 Appendix A. Chemical Analysis of Plant Sterols A 2. Peak area and silylation time for B-Sito, B-Sitosta and Chole at 65 and 70°C Cone. Silylation at 65°C (ug/L) B-Sitosterol B-Sitostanol Cholesterol 1 h 3 h 1 h 3 h 1 h 3 h 10.0 73.6 617.6 32.5 146.7 453.8 755.5 50.0 3830.0 5597.8 2553.8 3185.8 4033.5 5356.8 100.0 11331.1 14363.9 7908.5 9821.9 11884.1 14166.6 250.0 31411.1 37702.1 23188.5 26286.8 31547.9 38052.7 Cone. Silylation at 70°C (pg/L) B-Sitosterol B-Sitostanol Cholesterol 1 h 3 h 1 h 3 h 1 h 3 h 10.0 476.7 2385.0 598.8 2225.8 1090.9 2689.5 50.0 5567.8 17431.6 5058.3 14786.5 6169.7 17257.4 100.0 13147.0 34540.0 13573.2 26923.1 14236.0 32555.0 250.0 73441.3 83781.4 61117.3 70107.0 75125.4 80199.5 40 000 35 000 30 000 -\ P 25 000 co co | 20 000 < jj> 15 000 10 000 5 000 -\ 0 000 01 h n 3 h 10 50 100 250 B-Sitostanol (pg/L) 90 000 80 000 70 000 60 000 o O 5 50 000 -I co s> < 40 000 co a> Q. 30 000 20 000 10 000 0 000 s 1 hr 13 hr 10 50 100 250 B-Sitostanol (ug/L) 182 a Appendices. A3. 9.1 Appendix A. Chemical Analysis of Plant Sterols Unsilylated normalized peak area of fi-Sito & Camp Time 7 5 u g / L 1 0 0 u g / L 150 u g / L 2 0 0 u g / L 2 5 0 p g / L 75 u g / L 100 u g / L 150 u g / L 2 0 0 u g / L 2 5 0 u g / L 1.00 0.00 0.00 0.43 0.56 0.90 0.00 0.00 0.36 0.47 0.83 2.00 0.00 0.00 0 22 0.63 7.73 0.00 0.00 0.18 0.49 0.67 3.00 0.00 0.00 0.00 0.70 1.60 0.00 0.00 0.00 0.51 1.23 4.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 5.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 183 a Appendices A 4. 9.1 Appendix A. Chemical Analysis of Plant Sterols Normalized peak area of fi-Sito & Camp (Silylation at 70°C) Time 75 pg/L 100 ug/L 150 ug/L 200 ug/L 250 ug/L 75 ug/L 100 ug/L 150 ug/L 200 ug/L 250 ug/L 1.00 1.07 1.89 1.30 2.64 3.45 1.21 2.12 1.57 2.78 3.88 2.00 2.20 3.23 1.52 3.23 3.44 2.22 2.99 1.72 3.28 3.84 3.00 2.69 5.03 7.65 9.57 11.95 2.44 4.46 6.69 9.20 12.12 4.00 3.20 8.82 13.78 17.63 18.99 2.70 7.96 11.66 15.50 17.50 5.00 3.18 8.76 12.56 16.14 18.20 2.65 8.23 12.37 15.76 16.28 184 a Appendices A 5. 9.1 Appendix A. Chemical Analysis of Plant Sterols Peak area of plant sterols at 50 pg/L (Silylation at 70°C) Time (h) B-Sito B-Sitosta Chole Campe Stigma 1.0 684961 459331 433078 502786 2.0 890817 - 615682 - 453699 3.0 4338698 3628081 3448036 3892559 3420198 4.0 7419780 5875982 6384544 6891880 5974460 5.0 5625623 4558740 4837752 5159380 4466222 185 9. Appendices 9 1 Appendix A. Chemical Analysis of Plant Sterols A 6. Peak area of plant sterols at 100 pg/L (Silylation at 70°C) Time (h) B-Sito B-Sitosta Chole Campe Stigma 1.0 1898698 1187220 1456646 1446158 2716558 2.0 4914397 2694514 2953369 3032163 1216734 3.0 23563916 18107807 19479299 20778871 18459261 4.0 30793194 23505760 26450700 27742693 24250579 5.0 27911743 21330350 24353873 25331990 22075729 Plant Sterols (100 pg/L) j2 2.5E+07 •[ ' c 3 2 2.0E+07 1 1.5E+07 | 1.0E+07 5.0E+06 0.0E+00 186 a Appendices 9.1 Appendix A. Chemical Analysis of Plant Sterols Peak area of plant sterols at 200 pg/L (Silylation at 70°C) Time (h) B-Sito B-Sitosta Chole Campe Stigma 1.0 12970352 5358715 7170380 6864795 5190285 2.0 8292593 4987905 6074580 5748373 5778887 3.0 49831111 31886792 39276582 38468417 33534369 4.0 44540431 33203545 39227359 39905481 34355906 5.0 48378860 36228528 41550638 42065290 36903042 Plant Sterols (200 pg/L) 6.0E+07 5.0E+07 c 4.0E+07 3 CD £ 3.0E+07 co S> cc | 2.0E+07 Q. 1.0E+07 0.0E+00 2 3 Time (h) 187 9. Appendices 9.1 Appendix A. Chemical Analysis of Plant Sterols A 8. Peak area of plant sterols at 500 pg/L (Silylation at 70°C) Time (h) B-Sito B-Sitosta Chole Campe Stigma 1.0 43629597 11424371 23881944 24078509 36039424 2.0 63589421 33162442 39180019 40446502 21249735 3.0 1.31E+08 72244486 93301467 91447172 81509638 4.0 1.55E+08 113477518 136874411 137530556 120418758 5.0 1.41E+08 103198575 124220110 123821271 108930795 Plant Sterols (500 pg/L) CO CD Q . 1.8E+08 1.6E+08 1.4E+08 f 1.2E+08 1.0E+08 8.0E+07 -f 6.0E+07 4.0E+07 2.0E+07 0.0E+00 -B-Sito —a— B-Sitosta —X—Chole A Campe Stigma 2 3 Time (h) 188 9. Appendices 9.1 Appendix A. Chemical Analysis of Plant Sterols A 9. Variation in normalized peak area of plant sterols with silylation incubation time (200pg/L; Silylation at 70°C) Silylation Time (h) Chole (18.08 min) Chole-I Chole-ll Chole-Ave B-Sito (20.96 min) B-Sito-I B-Sito-ll B-Sito-Ave IS3 (18.88 min) R1 R2 0.05 2.250 2.137 2.193 3.506 3.329 3.417 410.0 503.5 0.25 2.875 2.782 2.829 3.964 3.703 3.833 436.8 494.0 0.50 3.849 3.774 3.812 4.681 4.577 4.629 427.5 442.8 0.50 4.206 4.635 4.420 5.063 5.224 5.143 555.4 426.8 0.75 4.140 4.003 4.072 4.988 4.609 4.799 511.1 474.3 1.00 5.314 4.859 5.086 6.076 5.635 5.856 347.6 475.8 1.00 5.157 5.197 5.177 5.989 6.106 6.047 507.1 512.1 1.50 5.338 4.799 5.068 6.126 5.545 5.835 424.6 588.0 2.00 5.097 4.649 4.873 5.991 5.342 5.666 546.2 752.3 4.50 5.280 5.299 5.290 6.221 5.978 6.100 569.6 585.3 7.50 3.794 3.643 3.719 4.467 4.238 4.353 522.8 577.1 A 10. Average normalized peak area of five plant sterols and derivatization incubation time (250pg/L; Silylation at 70°C) Silylation 18.14 min 19.67 min 20.05 min 21.03 min 21.25 min Time (hr) Chole Campe Stigma B-Sito B-Sitosta 0.5 1.882 1.890 1.642 2.124 1.530 1.0 2.173 2.153 1.872 2.386 1.755 1.5 1.849 1.833 1.592 2.030 1.495 2.0 2.102 2.086 1.814 2.343 1.721 2.5 2266 2.231 1.945 2.492 1.825 3.0 2.224 2.171 1.884 2.394 1.760 4.0 2.277 2.289 1.986 2.557 2.198 5.0 2.131 2.113 1.840 2.383 1.746 7.0 2.186 2.173 1.893 2.433 1.788 12.0 2.262 2.250 1.956 2.516 1.850 24.0 2.281 2.245 1.965 2.548 1.861 189 a Appendices A11. 9.1 Appendix A. Chemical Analysis of Plant Sterols Standard curve for plant sterols (Silylation 4 h at 70°C) Cone. 17.83 18.61 19.01 19.38 19.71 20.66 20.87 (mg/L) Chole IS-3 Ergo Campe Stigma B-Sito B-Sitosta 0.0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.5 0.08 196.28 0.02 0.03 0.12 0.10 0.09 0.8 0.14 184.28 0.00 0.10 0.20 0.17 0.14 1.0 0.22 207.66 0.02 0.16 0.29 0.25 0.23 2.5 0.21 841.72 0.09 0.17 0.25 0.23 0.20 5.0 2.14 247.24 1.19 1.28 2.26 2.21 1.74 7.5 3.19 233.38 1.86 2.24 3.39 3.33 2.81 10.0 4.22 229.92 2.45 2.97 4.42 4.36 3.69 12.5 4.79 259.12 2.85 3.40 4.96 4.90 4.13 15.0 6.07 260.58 3.62 4.30 6.31 6.14 5.16 17.5 7.65 227.42 4.47 5.30 7.84 7.57 6.38 20.0 7.82 259.06 4.73 5.56 8.07 7.86 6.63 25.0 9.45 244.54 5.75 6.67 9.85 9.61 8.10 27.0 11.12 273.12 6.79 7.86 11.59 11.32 9.59 30.0 12.12 234.12 7.36 8.62 12.62 12.26 10.35 35.0 13.47 221.90 8.20 9.54 14.06 13.61 11.53 40.0 15.31 206.86 9.19 10.67 15.77 15.27 12.93 50.0 18.21 213.40 11.05 12.86 19.08 18.45 15.66 190 9. Appendices 9.1 Appendix A. Chemical Analysis of Plant Sterols 21 Sterol Concentration (x 10-20 ug/L or mg/L; according to extract/sample volume ratio) 191 9. Appendices 9.1 Appendix A. Chemical Analysis of Plant Sterols A 12. Chromatogram of different plant sterols: STD-25 1 15< -15 3 o. S3 i as . ipsccoi /I f- j J I "3 • JW 8 I 8; "8 i j ; ii 2<s.oc 20m 21.00 192 9. Appendices 9.1 Appendix A. Chemical Analysis of Plant Sterols A 13. Plant sterols detected in P P M E s before biological treatment 2850000? 2 £*soooe\ i i 1CC03C» 0 M A j li i U \ it I t /s ^ A* I \ .1 V 193 9. Appendices 9.1 Appendix A. Chemical Analysis of Plant Sterols A 14. Plant sterols detected in P P M E s after biological treatment S • .-*»• M m 2S0CS3< W U > , A J t r i I I I v. v : rev ' •y x V;y y y y c y ^ y y ? •yvy>^-r^yy yy t y ;••>»•< "f-Tr>-yy^ 194 9. Appendices 9 1 Appendix A Chemical Analysis of Plant Sterols A 15. Plant sterols or phytosterols found in PPMEs C2H5 Cholesterol Ergosterol 195 9. Appendices 9.1 Appendix A. Chemical Analysis of Plant Sterols A 16. Plant Sterols MDL and QA/QC Cone. Cholesterol ( C ) RT= 18.06 min Peak Area SD MDL (mg/L) r1 r2 r3 r4 r5 Average 3.747*SD 0.5 0.048 0.051 0.056 0.062 0.054 0.0060 0.0226 1.0 0.088 0.087 0.094 0.094 0.099 0.092 0.0049 0.0182 2.0 0.253 0.267 0.281 0.275 0.295 0.274 0.0157 0.0588 4.0 0.661 0.665 0.653 0.640 0.640 0.652 0.0117 0.0438 6.0 1.487 1.366 1.413 1.348 1.277 1.378 0.0781 0.2926 8.0 1.785 1.786 1.648 1.627 1.600 1.689 0.0896 0.3358 10.0 2.191 2.132 2.060 2.024 1.982 2.078 0.0839 0.3142 15.0 3.713 3.674 3.412 3.299 3.280 3.475 0.2057 0.7708 20.0 4.614 4.438 4.300 4.171 4.164 4.337 0.1909 0.7153 Cone. Ergosterol (T1a) RT=19.27 min Peak Area SD MDL (mg/L) r1 r2 r3 r4 r5 Average 3.747*SD 0.5 #DIV/0! #DIV/0! 1.0 #DIV/0! #DIV/0! 2.0 0.055 0.055 #DIV/0! #DIV/0! 4.0 0.159 0.139 0.157 0.152 0.159 0.153 0.0085 0.0318 6.0 0.403 0.382 0.381 0.376 0.376 0.384 0.0113 0.0422 8.0 0.513 0.500 0.480 0.476 0.473 0.488 0.0171 0.0641 10.0 0.652 0.640 0.630 0.606 0.610 0.627 0.0195 0.0731 15.0 1.212 1.173 1.122 1.095 1.079 1.136 0.0553 0.2073 20.0 1.522 1.493 1.433 1.391 1.385 1.445 0.0608 0.2278 196 9. Appendices 9.1 Appendix A. Chemical Analysis of Plant sterols Cone. Stigmasterol (T2) RT=19.96 min Peak Area SD MDL (mg/L) r1 r2 r3 r4 r5 Average 3.747*SD 0.5 0.085 0.084 0.092 0.090 0.096 0.089 0.0048 0.0182 1.0 0.152 0.153 0.152 0.151 0.153 0.152 0.0008 0.0031 2.0 0.426 0.423 0.431 0.417 0.424 0.424 0.0051 0.0190 4.0 0.994 0.970 0.925 0.876 0.878 0.929 0.0532 0.1992 6.0 2.088 1.977 1.936 1.822 1.681 1.901 0.1552 0.5817 8.0 2.488 2.508 2.312 2.178 2.087 2.314 0.1856 0.6954 10.0 3.029 2.939 2.794 2.648 2.594 2.801 0.1851 0.6935 15.0 5.231 5.100 4.609 4.477 4.328 4.749 0.3957 1.4826 20.0 6.191 6.067 5.624 5.295 5.253 5.686 0.4314 1.6164 Cone. B-Sitosterol (T3) RT=20.95 min Peak Area SD MDL (mg/L) r1 r2 r3 r4 r5 Average 3.747*SD 0.5 0.075 0.073 0.072 0.079 0.083 0.077 0.0044 0.0163 1.0 0.130 0.134 0.134 0.134 0.136 0.134 0.0023 0.0087 2.0 0.379 0.367 0.395 0.386 0.385 0.382 0.0100 0.0374 4.0 0.924 0.892 0.862 0.821 0.823 0.864 0.0444 0.1662 6.0 1.955 1.876 1.838 1.738 1.620 1.805 0.1298 0.4864 8.0 2.354 2.379 2.224 2.097 2.015 2.214 0.1584 0.5934 10.0 2.894 2.804 2.702 2.561 2.540 2.700 0.1525 0.5716 15.0 4.992 5.010 4.401 4.287 4.117 4.561 0.4137 1.5500 20.0 5.790 5.750 5.332 5.108 5.058 5.408 0.3468 1.2994 197 9. Appendices 9.1 Appendix A. Chemical Analysis of Plant Sterols Cone. S-Sitostanol (T4) RT=21.15 min Peak Area SD MDL (mg/L) r1 r2 r3 r4 r5 Average 3.747*SD 0.5 #VALUE! 0.073 0.075 0.078 0.083 #VALUE! #VALUE! #VALUE! 1.0 0.123 0.131 0.137 0.131 0.132 0.131 0.0051 0.0190 2.0 0.374 0.355 0.224 0.357 0.352 0.332 0.0611 0.2289 4.0 0.869 0.823 0.781 0.738 0.733 0.789 0.0577 0.2163 6.0 1.820 1.718 1.636 1.556 1.429 1.632 0.1495 0.5602 8.0 2.204 2.162 2.011 1.881 1.803 2.012 0.1736 0.6503 10.0 2.735 2.585 2.489 2.324 2.314 2.489 0.1786 0.6690 15.0 4.494 4.509 3.871 3.748 3.587 4.042 0.4315 1.6169 20.0 5.153 5.061 4.685 4.451 4.379 4.746 0.3499 1.3109 Cone. Dotriacontane (IS3) 10 mg/L RT=18.86 min Peak Area (mg/L) r1 r2 r3 r4 r5 Average 0.5 438.00 648.40 662.20 919.90 1184.80 770.66 1.0 480.30 620.20 659.00 801.40 914.10 695.00 2.0 411.40 508.90 667.70 786.90 984.80 671.94 4.0 657.80 626.10 801.20 873.80 947.80 781.34 6.0 704.90 747.00 766.50 851.70 1117.30 837.48 8.0 779.90 645.80 850.20 944.30 1076.00 859.24 10.0 815.60 821.30 987.20 946.70 1094.90 933.14 15.0 985.80 964.30 1247.10 1284.20 1304.40 1157.16 20.0 1070.00 1080.20 1337.60 1396.30 1336.40 1244.10 198 9 Appendices 9 1 Appendix A Chemical Analysis of Plant Sterols 7.0 6.0 « 5 . 0 + • < 4.0 1 3.0 Q. 2.0 B-Sitosterol (B-Sito) r1 .. .•- - r2 - - - A- - r3 • -A- -• r4 • - X- - r5 9 — — Average I ' ' 1 1 I 1 1 1 1 6 8 10 12 14 Concentration (mg/L; x10 pg/L) 16 18 20 Cholesterol (Chole) 6 8 10 12 14 Concentration (mg/L; x10 ug/L) B-Sitostanol (B-Sitosta) 20 6 8 10 12 14 Concentration (mg/L; x10 ug/L) 9 Appendices 9 1 Annendix A Chemical Analysis of Plant Sterols 200 9. Appendices 9.1 Appendix A. Chemical Analysis of Plant Sterols A 17. Regression for line of fit and residual plots for B-Sito 50 45 40 35 + _j 30 "B> I 20 15 10 5 4 0 fi-Sito Line Fit Plot A A A A*g A A Aft A A A A £ A AA BH& A A A A A A observed a Predicted -+- •+-5000 10000 15000 fi-Sito 1 20000 25000 fi-Sito Residual Plot CO M -2 + </) * -4 -6 -8 A A 50tOA A 100A0 A A 15000 20000 A A A 25000 -10 fi-Sito 201 9. Appendices 9.1 Appendix A. Chemical Analysis of Plant Sterols 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Concentration (mg/L; x10 ug/L) 202 9. Appendices 9.2 Appendix B. Plant Sterols in PPMEs 9.2 Appendix B. Plant Sterols in PPMEs B 1. Plant sterols (Mg/L) in primary treated PPMEs at Mill B Primary Effluent Ergo Campe Stigma fi-Sito fi-Sitosta Total Sterols Weekl 18 243.6 77.4 328.2 176.7 843.9 Week 2 46.4 302.2 149.8 347.2 263.5 1109.1 Week 3 33.3 339.5 147 384.7 308.1 1212.6 Week 4 15.1 374.4 167.1 396.1 234.2 1186.9 Week 5 89.3 321.3 122.4 384.2 263.6 1180.8 Week 6 19 230.3 77.9 344.8 185.7 857.7 Week 7 61.7 321 155.1 387.7 297.4 1222.9 Week 8 22.3 238.4 86.9 313 176.5 837.1 Average 38.1 296.3 123.0 360.7 238.2 1056.4 SD 26.2 53.1 37.2 31.3 53.5 177.3 RSD (%) 68.8 17.9 30.3 8.7 22.5 16.8 SE 9.9 20.1 14.1 11.8 20.2 67.0 t*SE= ± 23.5 47.4 33.3 28.0 47.8 158.5 95% Cl-Low 14.7 248.9 89.7 332.7 190.4 897.9 95% Cl-Hi 61.6 343.8 156.2 388.8 286.0 1214.9 203 9. Appendices 9 2 Armendix B Plant Sterols in PPMEs B 2. Summary of 8-week monitoring results* for primary effluent at Mill B 1400 1200 1000 o » c 8 o O 800 600 e 400 CD c/> 200 - Total SI erols --- fi-Sito _ Car tosta 0 -€>• Ergo Stigma Plant Sterols {* For each vertical box shown, the lower edge represents 25 percentile and the upper edge indicates 75 t h percentile; a continuous line shows 50 t h percentile or median and a dotted line at the arithmetic mean (in the inside or the outside of the boxes). The bars extending above and below the boxes show the upper and the lower 95% confidence interval limits} 204 9. Appendices 9.2 Annendix B. Plant Sterols in PPMEs B 3. Plant sterols (ug/L) in secondary treated P P M E s at Mill B Final Effluent Ergo Campe Stigma B-Sito B-Sitosta Total Sterols Weekl 13.6 49.2 19.1 69.2 45.4 196.5 Week 2 19.5 43.5 16.7 60.9 38.7 179.3 Week 3 27.9 47.6 20.2 63.4 40.5 199.6 Week 4 24.1 44.6 25.5 60.9 41.1 196.2 Week 5 29 45 28.7 50.8 37 190.5 Week 6 26 38.3 55.3 98.8 61.7 280.1 Week 7 11.7 17.7 14.7 32.4 18 94.5 Week 8 16 19.3 19.4 29.2 18 101.9 Average 21.0 38.2 25.0 58.2 37.6 179.8 SD 6.7 12.5 13.1 21.9 14.3 59.1 RSD (%) 32.0 32.9 52.4 37.7 38.1 32.9 SE 2.5 4.7 4.9 8.3 5.4 22.3 t*SE= ± 6.0 11.2 11.7 19.6 12.8 52.8 95% Cl-Low 15.0 26.9 13.3 38.6 24.8 127.0 95% Cl-Hi 27.0 49.4 36.6 77.8 50.3 232.7 205 9. Appendices 9.2 Appendix B. Plant Sterols in PPMEs B 4. Summary of 8-week monitoring results* for final effluent at Mill B 300 250 200 1 150 C <D O o 100 2 CO 55 50 o 4 Campe Ergo fi-Sito Total Sterols " X " li-Sifosta Stigma • F ™ 1 X Z . . . . X -j i i i j i i i i j i i i i j i i i i j i i i i j_i i i i j ' i i i_ Plant Sterols {* For each vertical box shown, the lower edge represents 25 t h percentile and the upper edge indicates 75 t h percentile; a continuous line shows 50 t h percentile or median and a dotted line at the arithmetic mean (in the inside or the outside of the boxes). The bars extending above and below the boxes show the upper and the lower 95% confidence interval limits} 206 a Appendices 9.2 Appendix B. Plant Sterols in PPMEs B 5. Removal of plant sterols from P P M E s through secondary treatment at Mill B Sample Primary Effluent Final Effluent Removal % Week 1 843.9 196.4 76.7 Week 2 1109.1 179.3 83.8 Week 3 1212.6 199.6 83.5 Week 4 1186.9 196.2 83.5 Week 5 1180.8 190.4 83.9 Week 6 857.7 280.1 67.3 Week 7 1222.9 94.4 92.3 Week 8 837.1 101.9 87.8 Average 1056.4 179.8 82.4 SD 177.3 59.1 7.5 RSD (%) 16.8 32.9 9.1 SE 67.0 22.3 2.8 t*SE= ± 158.5 52.9 6.7 95% Cl-Low 897.9 126.9 75.7 95% Cl-Hi 1214.9 232.6 89.1 t(n-1) = 2.365 207 9. Appendices 9.2 Appendix B. Plant Sterols in PPMEs B 6. P P M E s and sludge sampling locations around the U N O X - A S T secondary treatment system at Mill A and Mill B - On-site Sampl ing Program I Final ! Effluent ! I Primary j Effluent j Primary Sludge UNOX Bio-Reactor • Outlet Box Secondary Clarifier • \ \ \ \ I f i y W n o * ~® r ...Waste Sludge Secondary Recycle Sludge = Sampling Point 208 9. Appendices 9.2 Appendix B. Plant Sterols in PPMEs B 7. Sampl ing stations around UNOX-AST secondary treatment sys tem at Mill B - On-site Sampling Program II P8 209 a Appendices 9.2 Appendix B. Plant Sterols in PPMEs B 8. Plant sterols variation during secondary treatment Sample Cycle Chole Ergo Campe Stigma B-Sito B-Sitosta Total Sterols UNOX AST Influent P1 1 0.00 0.00 240.96 86.46 328.53 177.91 833.85 2 0.00 39.84 294.39 158.89 345.06 259.90 1098.07 3 0.00 25.91 326.91 157.66 378.69 300.14 1189.32 4 0.00 11.81 493.65 234.14 548.74 321.69 1610.02 5 0.00 33.62 446.40 505.78 564.07 420.31 1970.18 6 0.00 11.21 675.91 583.59 848.40 493.71 2612.82 7 0.00 51.70 308.76 165.83 380.64 289.64 1196.56 8 0.00 0.00 611.30 262.08 816.78 466.17 2156.33 Ave 0.00 21.76 424.79 269.30 526.36 341.18 1583.39 UNOX Bioreactor Cell A P2 1 0.00 54.41 628.49 609.59 1055.69 721.76 3069.95 2 0.00 0.00 1141.84 578.10 2176.24 1126.41 5022.60 3 0.00 0.00 475.65 175.83 1016.04 495.49 2163.01 4 0.00 0.00 471.62 101.66 1053.48 478.31 2105.06 5 0.00 0.00 627.82 773.87 1786.33 1206.28 4394.31 6 0.00 0.00 549.72 437.76 1419.91 842.30 3249.68 7 0.00 0.00 247.48 31.13 504.83 350.90 1134.35 8 0.00 9.07 598.82 378.36 1265.44 729.86 2981.54 Ave 0.00 7.93 592.68 385.79 1284.74 743.91 3015.06 UNOX Bioreactor Cell B P3 1 0.00 70.53 1369.57 900.40 2923.23 1389.13 6652.85 2 0.00 15.51 1223.63 811.19 1905.50 1993.37 5949.21 3 0.00 0.00 662.60 277.53 1483.72 777.60 3201.45 4 0.00 0.00 603.23 217.72 1314.13 668.01 2803.08 5 0.00 54.41 515.25 581.32 1210.94 1070.56 3432.48 6 0.00 27.20 559.24 399.52 1262.53 869.29 3117.78 7 0.00 0.00 316.16 144.69 855.10 285.10 1601.05 8 0.00 13.60 437.70 272.11 1058.82 577.19 2359.41 Ave 0.00 22.66 710.92 450.56 1501.75 953.78 3639.66 210 9. Appendices 9.2 Appendix B. Plant Sterols in PPMEs UNOX Bioreactor Cell C P4 1 0.00 25.99 811.95 646.33 2177.00 976.29 4637.57 2 0.00 0.00 544.50 541.47 893.13 494.53 2473.63 3 0.00 0.00 380.33 109.23 859.18 447.27 1796.01 4 0.00 0.00 456.70 119.60 1155.49 471.64 2203.42 5 0.00 0.00 540.69 266.07 1145.06 473.00 2424.82 6 0.00 0.00 675.86 332.59 1431.32 591.25 3031.03 7 0.00 0.00 272.93 102.46 572.49 1492.24 2440.13 8 0.00 0.00 459.24 164.97 1053.24 463.97 2141.42 Ave 0.00 3.25 517.78 285.34 1160.86 676.27 2643.50 UNOX-AST Effluent P5 1 0.00 8.47 772.49 448.87 1871.28 813.70 3914.81 2 0.00 168.14 554.74 609.71 922.20 621.27 2876.06 3 0.00 0.00 460.67 251.61 997.78 570.20 2280.27 4 0.00 0.00 400.35 177.40 869.40 439.13 1886.29 5 0.00 0.00 652.63 617.33 1364.91 880.75 3515.62 6 0.00 52.45 769.67 204.91 1301.34 627.17 2955.54 7 0.00 0.00 223.00 32.77 456.57 1863.98 2576.32 8 0.00 62.41 689.53 241.51 1139.76 596.25 2729.46 Ave 0.00 36.43 565.39 323.01 1115.40 801.56 2841.80 Final Secondary Effluent P6 1 0.00 6.24 29.61 15.16 42.53 28.18 121.72 2 0.00 9.63 26.33 13.49 37.69 24.32 111.48 3 0.00 14.37 28.48 15.18 38.89 25.17 122.10 4 0.00 42.65 94.66 38.86 132.31 90.18 398.66 5 0.00 0.00 137.66 111.18 276.16 141.50 666.50 6 0.00 0.00 74.28 55.67 150.33 72.88 353.15 7 0.00 0.00 59.11 32.67 163.34 56.77 311.89 8 0.00 0.00 45.30 30.79 115.41 46.26 237.76 Ave 0.00 9.11 61.93 39.13 119.58 60.66 290.41 211 9. Appendices 9.2 Appendix B. Plant Sterols in PPMEs Sample Cycle Chole Ergo Campe Stigma B-Sito B-Sitosta Total Sterols Recycle Secondary Sludge P7 1 0.00 0.00 3604.79 752.53 8346.36 2672.05 15375.72 2 0.00 296.04 5108.96 1360.03 11344.72 3876.40 21986.14 3 0.00 213.79 3848.99 1020.88 10405.45 3034.81 18523.93 4 0.00 416.85 6051.43 1580.51 18585.29 5043.67 31677.76 5 0.00 296.32 5517.36 1384.42 14726.76 4407.91 26332.78 6 0.00 0.00 6985.74 3481.71 17616.78 6679.05 34763.28 7 0.00 87.31 6551.59 3669.54 16695.69 6644.98 33649.11 8 0.00 0.00 4162.03 2092.53 10750.81 4061.61 21066.98 Ave 0.00 163.79 5228.86 1917.77 13558.98 4552.56 25421.96 Waste Secondary Sludge P8 1 0.00 82.46 4379.96 2651.04 9072.09 4944.72 21130.27 2 0.00 492.98 11663.43 6629.50 22779.58 9442.48 51007.97 3 0.00 466.83 5964.80 4101.01 10896.94 5499.81 26929.38 4 0.00 76.71 7108.36 1717.15 18137.98 5680.08 32720.28 5 0.00 119.98 5075.54 1374.55 15316.13 4454.61 26340.81 6 0.00 594.67 10646.84 3053.50 34743.18 9555.93 58594.11 7 0.00 339.78 8553.62 5113.66 15697.45 9273.88 38978.39 8 0.00 318.21 6483.82 4003.07 11047.22 9220.26 31072.57 Ave 0.00 311.45 7484.55 3580.43 17211.32 7258.97 35846.72 212 9. Appendices 9.2 Appendix B. Plant Sterols in PPMEs B 9. Variation of PPME plant sterols around UNOX-AST system UNOX-AST Influent 3,000 2,500 _ 2'000 I u> 1,500 2 S OT 1,000 500 ^ — ^ 1 2 3 4 5 6 7 8 A v e Sampling Cycle ——Ergo A Campe - X — Stigma —•—B-Sito —a— B-Sitosta —e—Total Sterols UNOX Bioreactor Cell A 6,000 1 2 3 4 5 6 7 8 A v e Sampling Cycle —•—Ergo —tx—Campe X Stigma —B—B-Sito • B-Sitosta —«—Total Sterols 213 9. Appendices 9.2 Appendix B. Plant Sterols in PPMEs UNOX Bioreactor Cell B 8,000 4 5 6 Sampling Cycle UNOX Bioreactor Cell C 6,000 5,000 4 —«— Ergo X Stigma — B — fi-Sitosta -A—Campe -a—B-Sito -e—Total Sterols 4 5 6 Sampling Cycle 214 9. Appendices 9.2 Appendix B. Plant Sterols in PPMEs UNOX-AST Effluent 4,500 n Sampling Cycle —»— Ergo —A— Campe —K— Stigma - a — B-Sito — B — fi-Sitosta —©—Total Sterols Final Secondary Effluent 800 -, 700 4 1 2 3 4 5 6 7 8 A v e Sampling Cycle —*—Ergo —A— Campe x Stigma - « — fi-Sito —•— fi-Sitosta —©—Total Sterols 215 9. Appendices 9.2 Appendix B. Plant Sterols in PPMEs Recycle Secondary Sludge 40,000 i 1 2 3 4 5 6 7 8 Ave Sampling Cycle — • — E r g o — A — C a m p e — X — S t i g m a — « — 1 3 - S ito • B - S i t o s t a — o — To ta l S t e r o l s 70,000 60,000 Waste Secondary Sludge 4 5 6 Sampling Cycle • E r g o - B - S i t o - A — C a m p e -H — B - S i t o s t a 8 Ave S t i g m a « — T o t a l S t e r o l s 216 9. Appendices 9.2 Appendix B. Plant Sterols in PPMEs B 10. Average sterols at the U N O X - A S T system at Mill B . Cycle PE Cell-A Cell-B Cell-C SE FE RAS WAS 1 833.85 3069.95 6652.85 4637.57 3914.81 121.72 15375.72 21130.27 2 1098.07 5022.60 5949.21 2473.63 2876.06 111.48 21986.14 51007.97 3 1189.32 2163.01 3201.45 1796.01 2280.27 122.10 18523.93 26929.38 4 1610.02 2105.06 2803.08 2203.42 1886.29 398.66 31677.76 32720.28 5 1970.18 4394.31 3432.48 2424.82 3515.62 666.50 26332.78 26340.81 6 2612.82 3249.68 3117.78 3031.03 2955.54 353.15 34763.28 58594.11 7 1196.56 1134.35 1601.05 2440.13 2576.32 311.89 33649.11 38978.39 8 2156.33 2981.54 2359.41 2141.42 2729.46 237.76 21066.98 31072.57 Ave 1583.39 3015.06 3639.66 2643.50 2841.80 290.41 25421.96 35846.72 Sample 1 2 3 4 5 6 7 8 Average PE 833.85 1098.07 1189.32 1610.02 1970.18 2612.82 1196.56 2156.33 1583.39 Cell-A 3069.95 5022.60 2163.01 2105.06 4394.31 3249.68 1134.35 2981.54 3015.06 Cell-B 6652.85 5949.21 3201.45 2803.08 3432.48 3117.78 1601.05 2359.41 3639.66 Cell-C 4637.57 2473.63 1796.01 2203.42 2424.82 3031.03 2440.13 2141.42 2643.50 SE 3914.81 2876.06 2280.27 1886.29 3515.62 2955.54 2576.32 2729.46 2841.80 FE 121.72 111.48 122.10 398.66 666.50 353.15 311.89 237.76 290.41 RAS 15375.72 21986.14 18523.93 31677.76 26332.78 34763.28 33649.11 21066.98 25421.96 WAS 21130.27 51007.97 26929.38 32720.28 26340.81 58594.11 38978.39 31072.57 35846.72 217 9 Appendices 9.2 Appendix B. Plant Sterols in PPMEs B 11. Total sterols variation at different sampling stations around the U N O X - A S T system at Mill B . Total Sterols 0 i i i i i i i i i 1 2 3 4 5 6 7 8 Ave Sampling Cycle 218 9. Appendices 9.2 Appendix B. Plant Sterols in PPMEs B 12. Total sterols variation across the UNOX-AST system. 219 E Appendices 9.2 Appendix B. Plant Sterols in P P M E s 1.15 h 220 9.2 Appendix B. Plant Sterols in PPMEs 9. Appendices B 13. Total sterols variation in recycle and waste secondary s ludge from the U N O X -A S T bioreactor. 221 9. Appendices 9.3 Appendix C. Biological Removal of Plant Sterols 9.3 Appendix C. Biological Removal of Plant Sterols C 1. Schemat ic of small-scale suspended growth A S T system: C pH control , D diffuser aerator, E final effluent, F influent feed, H heated water, M stirrer motor, N Nutrients, P air supply pump, R recycle, T temperature control, S mechanical stirrer, V control valve Heated Water Heated Water 222 9.3 Appendix C. Biological Removal of Plant Sterols 9. Appendices C 2. Lab-scale Reactor 1 & 2 influent sterol concentrat ions (ug/L) Day Campe Stigma B-Sito B-Sitosta Influent 40 213.4 42.3 517.1 261.0 1033.8 45 275.8 88.4 649.4 282.7 1296.3 50 257.6 99.8 653.8 371.5 1382.8 55 244.1 94.6 619.4 351.9 1310.0 60 213.4 82.0 647.4 411.2 1354.0 65 236.1 72.5 661.3 388.8 1358.7 70 318.8 30.6 653.1 264.3 1266.8 75 344.3 135.8 971.2 637.9 2089.3 80 487.8 115.8 910.6 461.4 1975.6 85 344.3 135.8 971.2 637.9 2089.3 90 362.5 145.3 985.4 669.5 2162.7 95 491.1 51.5 966.9 594.7 2104.3 100 685.3 57.3 1111.2 764.8 2618.6 105 699.9 143.2 1171.8 640.1 2655.0 110 654.9 158.8 1094.8 729.7 2638.2 115 616.5 204.2 1072.5 783.7 2676.9 120 482.0 145.6 1370.2 746.8 2744.6 123 599.9 143.2 1171.8 640.1 2555.0 126 382.3 141.6 1470.1 746.8 2740.9 129 372.6 145.8 1369.6 880.6 2768.6 132 824.9 98.5 1165.6 1172.6 3261.6 135 854.0 105.0 1435.6 1186.6 3581.2 138 742.1 403.7 1234.9 1025.1 3405.8 141 757.2 411.9 1260.1 1046.0 3475.3 144 742.1 450.0 1334.3 980.5 3506.9 147 1224.2 108.3 833.4 1401.8 3567.7 150 1124.0 254.0 1070.0 992.4 3440.3 153 1103.0 403.7 1234.9 970.2 3711.8 156 1079.9 203.0 1434.0 925.0 3641.9 159 676.3 222.3 1539.2 1207.9 3645.7 162 791.2 219.3 1425.2 1088.2 3523.9 165 776.1 269.3 1597.1 920.4 3563.0 223 9 Appendices 9 3 Appendix C Biological Removal of Plant Sterols Day Campe Stigma B-Sito B-Sitosta Total Sterols 168 725.3 324.8 1339.6 1207.9 3597.6 171 691.9 235.3 1345.1 1087.1 3359.3 174 791.2 286.3 1597.1 828.4 3503.0 177 731.2 242.4 1513.1 968.9 3455.6 180 769.0 269.3 1517.3 1022.7 3578.3 183 845.0 211.1 1604.1 902.5 3562.7 186 733.3 255.8 1517.3 1022.7 3529.1 189 845.3 189.0 1455.3 1126.6 3616.2 192 1081.9 207.1 1688.5 1274.3 4251.8 195 1224.2 171.9 1507.0 1486.4 4389.5 197 1154.2 125.3 1730.3 1371.3 4381.1 199 1006.3 163.2 1764.8 1532.6 4466.9 201 988.9 158.3 1990.5 1512.8 4650.5 203 1056.3 283.5 1681.2 1437.3 4458.3 205 1215.3 288.6 1654.8 1358.9 4517.6 207 969.9 198.3 1974.8 1421.3 4564.3 209 868.8 256.0 2051.2 1470.8 4646.8 211 1085.2 266.5 1685.0 1526.5 4563.3 213 776.7 250.9 1983.5 1528.6 4539.7 215 914.7 170.8 1952.0 1513.6 4551.0 217 1019.7 214.3 1850.0 1487.7 4571.7 219 859.7 86.6 2170.5 1447.5 4564.3 221 948.6 66.8 2371.9 1283.9 4671.3 223 1119.7 108.4 2053.1 1389.8 4670.9 225 1046.0 128.6 2154.9 1334.7 4664.2 227 1153.2 46.6 2200.6 1222.9 4623.4 229 986.5 55.1 2189.0 1353.6 4584.2 231 895.6 125.0 2168.4 1426.5 4615.5 233 956.4 128.4 2059.6 1433.9 4578.3 235 889.4 148.9 2155.1 1368.9 4562.3 237 824.9 46.0 2294.1 1383.8 4548.8 239 813.4 52.5 2174.3 1362.0 4402.2 241 955.0 118.6 2023.2 1427.3 4524.1 243 938.6 59.6 2088.3 1495.8 4582.3 245 1012.4 187.4 2037.9 1354.6 4592.3 224 9. Appendices 9.3 Appendix C. Biological Removal of Plant Sterols Day Campe Stigma 6-Sito B-Sitosta Total Sterols 247 1081.7 38.9 2014.6 1490.2 4625.4 249 1017.2 58.6 2048.4 1431.8 4556.0 251 983.7 77.6 2017.0 1539.9 4618.2 253 933.1 96.3 2167.5 1457.9 4654.8 255 937.6 87.6 2085.7 1533.2 4644.2 257 916.9 85.9 2074.7 1561.7 4639.1 Overall Average 791.0 163.8 1541.8 1090.0 3586.6 Overall % 19.2 3.7 46.1 31.1 100.0 C 3. Reactor 1 effluent sterols (pg/L), removal efficiency (%), flow rate (l/d), HRT (h) and PH day Campe Stigma B-Sito B-Sitosta EffluentJ RemovaM HRT_1 Flow_1 PH_1 40 11.6 18.5 50.7 37.7 118.5 88.5 29.1 3.3 6.5 45 15.7 28.8 60.2 39.2 143.9 88.9 28.7 3.4 6.5 50 11.7 17.8 49.4 29.2 108.1 92.2 29.1 3.3 6.5 55 19.6 29.7 73.3 33.1 155.8 88.1 29.5 3.3 6.5 60 17.1 15.5 49.7 31.9 114.2 91.6 24.0 4.0 6.5 65 9.3 4.7 28.9 11.6 54.6 96.0 24.0 4.0 6.5 70 10.8 4.8 32.5 12.1 60.2 95.2 23.1 4.2 6.5 75 11.7 0.0 30.8 14.8 57.3 97.3 24.0 4.0 6.5 80 11.2 0.0 28.7 51.3 91.2 95.4 24.0 4.0 6.5 85 42.2 30.7 139.5 94.8 307.2 85.3 23.4 4.1 6.6 90 12.5 4.1 44.6 26.6 87.8 95.9 20.0 4.8 6.6 95 11.0 0.0 37.7 21.3 70.1 96.7 20.0 4.8 6.6 100 11.1 0.0 90.5 135.3 236.9 91.0 20.4 4.7 6.6 105 10.6 0.0 103.3 150.1 264.0 90.1 20.2 4.8 6.6 110 84.6 9.7 94.7 107.8 296.7 88.8 20.4 4.7 6.6 115 61.8 43.6 70.1 56.3 231.8 91.3 17.5 5.5 6.6 120 95.6 8.0 46.9 36.6 187.0 93.2 16.3 5.9 6.6 123 81.6 4.9 39.2 31.0 156.7 93.9 16.0 6.0 6.6 126 19.6 15.0 59.5 42.7 136.9 95.0 15.7 6.1 6.6 225 9. Appendices 9.3 Appendix C. Biological Removal of Plant Sterols day Campe Stigma B-Sito B-Sitosta EffluentJ RemovaM HRT_1 F lowJ PH_1 129 28.1 27.8 46.6 33.7 136.2 95.1 16.0 6.0 6.6 132 20.7 7.9 44.7 26.6 99.9 96.9 16.0 6.0 6.7 135 14.2 5.9 39.7 29.7 89.5 97.5 16.0 6.0 6.7 138 15.6 7.1 49.5 36.1 108.3 96.8 16.0 6.0 6.7 141 25.2 14.0 50.8 35.5 125.5 96.4 16.0 6.0 6.7 144 20.0 7.1 52.1 38.8 118.0 96.6 16.0 6.0 6.7 147 29.6 6.3 107.0 61.7 204.7 94.3 16.0 6.0 6.7 150 50.2 17.4 120.8 68.6 256.9 92.5 16.0 6.0 6.7 153 51.6 42.9 115.1 77.6 287.2 92.3 16.0 6.0 6.7 156 40.9 20.5 83.4 55.0 199.8 94.5 13.7 7.0 6.7 159 39.5 4.0 139.3 81.5 264.3 92.7 13.7 7.0 6.7 162 44.8 18.3 111.6 68.4 243.1 93.1 13.7 7.0 6.7 165 45.3 18.6 108.2 69.0 241.0 93.2 13.7 7.0 6.7 168 34.6 17.5 84.3 60.8 197.2 94.5 13.7 7.0 6.7 171 61.5 69.1 116.0 98.0 344.6 89.7 13.5 7.1 6.6 174 28.9 23.3 64.1 42.5 158.8 95.5 13.7 7.0 6.6 177 54.7 35.3 123.5 81.9 295.5 91.4 13.7 7.0 6.6 180 27.0 12.3 84.7 58.9 182.9 94.9 13.7 7.0 6.6 183 39.6 52.8 107.7 74.8 275.0 92.3 12.0 8.0 6.6 186 13.2 10.8 63.2 33.7 120.9 96.6 12.0 8.0 6.6 189 13.2 10.8 63.2 33.7 120.9 96.7 12.0 8.0 6.6 192 13.5 8.4 63.1 36.0 121.0 97.2 12.0 8.0 6.6 195 23.6 23.9 138.7 56.0 242.2 94.5 12.0 8.0 6.6 197 35.8 34.3 117.4 55.2 242.7 94.5 12.0 8.0 6.6 199 65.5 30.0 102.0 65.5 263.0 94.1 12.0 8.0 6.5 201 81.9 20.7 445.2 235.7 783.5 83.2 12.0 8.0 6.6 203 54.9 14.0 309.9 141.4 520.2 88.3 12.0 8.0 6.6 205 61.7 17.7 333.7 163.8 576.9 87.2 12.0 8.0 6.6 207 36.6 20.9 202.7 101.5 361.7 92.1 12.0 8.0 6.6 209 80.9 25.6 449.5 228.5 784.6 83.1 12.0 8.0 6.6 211 63.8 25.6 362.6 186.7 638.7 86.0 12.0 8.0 6.6 213 75.1 42.8 345.4 187.6 650.9 85.7 12.0 8.0 6.6 215 122.0 76.0 569.1 324.3 1091.4 76.0 10.7 9.0 6.6 217 74.2 48.5 315.5 316.8 755.0 83.5 10.7 9.0 6.6 219 86.8 51.7 379.0 378.2 895.7 80.4 9.6 10.0 6.5 226 9. Appendices 9,3 Annenrtix C. Biological Removal of Plant Sterols day Campe Stigma B-Sito B-Sitosta EffluentJ RemovaM HRT_1 Flow_1 PH_1 221 62.7 27.3 295.4 353.8 739.1 84.2 9.6 10.0 6.6 223 101.3 54.1 447.5 313.4 916.4 80.4 9.6 10.0 6.6 225 120.8 62.8 502.3 426.1 1112.0 76.2 9.1 10.5 6.6 227 101.4 60.8 481.0 415.4 1058.6 77.1 9.1 10.5 6.8 229 104.5 58.3 470.7 400.2 1033.7 77.5 9.6 10.0 6.8 231 62.6 40.8 328.9 348.2 780.6 83.1 9.6 10.0 6.8 233 64.3 40.3 344.5 306.0 755.0 83.5 9.6 10.0 6.8 235 64.7 30.6 317.6 266.6 679.5 85.1 9.6 10.0 6.8 237 80.8 23.8 420.5 232.7 757.9 83.3 9.6 10.0 6.8 239 132.2 16.2 333.3 303.8 785.5 82.2 9.6 10.0 6.8 241 59.5 22.9 289.1 167.3 538.9 88.1 10.7 9.0 6.8 243 31.1 11.8 149.4 91.4 283.7 93.8 10.7 9.0 6.8 245 36.3 14.8 190.2 111.5 352.9 92.3 10.7 9.0 6.8 247 31.5 6.2 205.2 55.5 298.3 93.5 10.7 9.0 6.8 249 18.7 10.5 109.6 44.2 183.0 96.0 10.7 9.0 6.8 251 28.5 17.7 61.5 165.3 272.9 94.1 10.7 9.0 6.8 253 8.9 6.7 32.9 94.5 143.1 96.9 10.7 9.0 6.7 255 30.4 19.9 129.2 129.8 309.2 93.3 10.7 9.0 6.8 227 9. Appendices 9.3 Appendix C. Biological Removal of Plant Sterols C 4. Reactor 2 effluent sterols (ug/L), removal efficiency (%), flow rate (l/d), HRT (h) and PH day Campe Stigma B-Sito B-Sitosta Effluent_ 2 Removal_2 HRT_2 Flow_2 PH_2 40 161.1 29.9 292.1 176.0 659.1 36.2 24.3 3.95 7.0 45 87.4 40.0 284.4 104.5 516.3 60.2 23.4 4.1 7.0 50 119.1 27.9 250.1 134.0 531.1 61.6 23.4 4.1 6.9 55 117.4 70.0 314.4 112.5 614.3 53.1 24.0 4.0 6.9 60 94.3 78.9 207.3 125.4 505.9 62.6 24.0 4.0 6.9 65 125.4 35.8 229.4 139.6 530.2 61.0 24.0 4.0 7.1 70 125.4 17.9 114.7 69.8 327.8 74.1 23.1 4.15 7.1 75 0.0 0.0 30.2 37.3 67.5 96.8 24.0 4.0 7.1 80 0.0 0.0 0.0 36.6 36.6 98.1 24.0 4.0 7.1 85 71.84 0 210.24 104.16 386.2 81.5 23.4 4.1 7.1 90 67.04 0 198.08 93.04 358.2 83.4 20.0 4.8 7.3 95 0.0 0.0 65.4 82.5 147.9 93.0 20.0 4.8 7.4 100 130.3 20.1 109.2 106.8 366.4 86.0 20.4 4.7 7.4 105 0.0 0.0 55.7 72.0 127.7 95.2 20.2 4.75 7.4 110 35.8 0.0 138.2 94.5 268.5 89.8 20.4 4.7 7.4 115 34.1 0.0 148.1 97.4 279.6 89.6 16.6 5.8 7.7 120 0.0 0.0 24.5 63.9 88.4 96.8 16.3 5.9 7.6 123 0.0 0.0 31.5 58.6 90.1 96.5 16.0 6.0 7.7 126 0.0 0.0 59.0 51.0 110.0 96.0 16.0 6.0 7.6 129 0.0 0.0 54.3 43.6 97.9 96.5 16.0 6.0 7.7 132 79.2 19.1 182.9 103.8 385.0 88.2 15.7 6.1 7.8 135 158.4 38.2 365.7 207.6 769.9 78.5 16.0 6.0 7.7 138 133.2 35.8 191.9 143.3 504.2 85.2 13.7 7.0 7.6 141 114.5 64.1 205.7 103.0 487.3 86.0 13.7 7.0 7.8 144 270.4 56.2 305.8 299.1 931.5 73.4 13.7 7.0 7.3 147 193.5 41.1 277.4 291.8 803.9 77.5 13.7 7.0 7.5 150 240.7 14.3 394.9 251.7 901.6 73.8 13.7 7.0 7.6 153 261.2 39.0 334.0 214.5 848.7 77.1 12.2 7.9 7.7 156 194.1 57.4 445.1 218.3 914.9 74.9 12.0 8.0 7.7 159 328.7 82.3 696.5 356.9 1464.4 59.8 12.0 8.0 7.7 162 134.5 147.6 757.5 274.5 1314.2 62.7 12.0 8.0 7.7 228 9 Appendices 9 3 Appendix C Biological Removal of Plant Sterols day Campe Stigma B-Sito B-Sitosta Effluent_2 Removal_2 HRT_2 Flow_2 PH_2 165 137.3 150.6 773.0 280.2 1341.0 62.4 12.2 7.9 7.7 168 137.3 150.6 773.0 280.2 1341.0 62.7 11.9 8.1 7.7 171 368.3 107.8 533.2 315.7 1325.0 60.6 12.0 8.0 7.6 174 281.8 168.6 397.1 222.3 1069.9 69.5 10.7 9.0 7.6 177 305.8 118.9 567.3 313.0 1305.0 62.2 10.7 9.0 7.6 180 264.1 126.7 489.8 260.8 1141.4 68.1 10.7 9.0 7.6 183 482.9 100.3 546.1 534.1 1663.4 53.3 10.7 9.0 7.5 186 345.6 73.4 495.4 521.1 1435.5 59.3 10.7 9.0 7.6 189 170.7 175.8 815.3 345.7 1507.5 58.3 10.7 9.0 7.6 192 267.4 162.8 774.6 390.5 1595.2 62.5 9.1 10.5 7.6 195 208.0 105.2 686.1 322.6 1321.9 69.9 9.1 10.5 7.6 197 307.6 94.7 570.3 361.6 1334.3 69.5 9.1 10.6 7.6 199 248.2 0.0 578.9 331.9 1159.0 74.1 9.0 10.7 7.6 201 123.7 115.5 408.6 340.8 988.6 78.7 9.1 10.6 7.6 203 164.9 58.9 528.5 152.0 904.4 79.7 9.1 10.5 7.6 205 157.8 105.4 527.5 392.1 1182.8 73.8 9.1 10.5 7.6 207 134.4 81.4 430.8 546.6 1193.2 73.9 10.5 9.1 7.6 209 147.4 82.0 508.2 490.5 1228.1 73.6 10.5 9.1 7.6 211 94.4 52.5 325.2 350.6 822.6 82.0 10.4 9.2 7.6 213 118.0 65.6 508.5 411.1 1103.2 75.7 10.4 9.3 7.6 215 107.1 42.0 354.4 284.5 788.0 82.7 10.7 9.0 7.6 217 61.7 0.0 184.1 165.2 411.0 91.0 10.9 8.8 7.6 219 94.4 52.5 385.1 313.9 845.9 81.5 10.9 8.8 7.6 221 40.0 0.0 127.5 123.4 290.9 93.8 10.7 9.0 7.6 223 96.4 0.0 287.7 101.9 485.9 89.6 10.7 9.0 7.5 225 39.6 0.0 126.9 36.5 203.0 95.6 10.7 9.0 7.5 227 154.3 92.4 326.9 272.6 846.2 81.7 10.9 8.8 7.5 229 39.6 0.0 126.9 36.5 203.0 95.6 10.7 9.0 7.5 231 50.6 0.0 210.3 79.4 340.3 92.6 10.7 9.0 7.7 233 39.6 0.0 126.9 36.5 203.0 95.6 10.7 9.0 7.7 235 62.8 61.3 205.5 113.9 443.5 90.3 10.7 9.0 7.7 237 67.1 42.0 189.7 109.2 408.0 91.0 10.7 9.0 7.7 239 62.8 61.3 205.5 113.9 443.5 89.9 10.7 9.0 7.7 241 67.1 42.0 189.7 109.2 408.0 91.0 10.7 9.0 7.7 229 9. Appendices 9 3 Annendix C Biological Removal of Plant Sterols C 5. Reactor 1 mixed liquor sterols (ug/L), SRT (d), and WAS flow (mUd) day Campe Stigma B-Sito B-Sitosta ML Sterols_1 SRT_1 WAS_1 80 286.4 81.7 1337.5 967.4 2673.0 26.7 150.0 85 409.4 137.5 1466.9 1102.6 3116.4 26.7 150.0 90 324.1 159.3 1584.2 1135.4 3203.0 26.7 150.0 95 271.8 141.0 2181.5 1326.6 3920.9 26.7 150.0 100 269.7 119.7 1624.6 881.2 2895.2 22.9 175.0 105 230.1 90.0 1618.9 993.7 2932.6 22.9 175.0 110 365.2 276.5 4937.1 2054.5 7633.3 22.9 175.0 115 145.1 100.1 4140.4 1656.7 6042.4 22.9 175.0 120 385.6 138.6 1924.2 906.2 3354.5 20.0 200.0 123 227.6 0.0 2386.2 1217.8 3831.6 20.0 200.0 126 204.5 0.0 1922.8 841.9 2969.2 20.0 200.0 129 170.4 0.0 2428.4 788.2 3387.0 20.0 200.0 132 136.8 0.0 2135.3 718.7 2990.8 20.0 200.0 135 758.4 272.7 2855.1 1098.4 4984.7 16.0 250.0 138 697.3 216.1 2946.3 1052.3 4912.1 16.0 250.0 141 901.8 322.9 3461.3 1068.7 5754.6 16.0 250.0 144 841.0 290.0 3311.3 1014.1 5456.3 16.0 250.0 147 876.8 347.2 3248.3 1031.7 5504.0 13.3 300.0 150 1298.5 308.6 4492.8 1430.9 7530.8 13.3 300.0 153 494.2 88.5 2143.5 738.1 3464.3 13.3 300.0 156 697.6 102.2 2765.5 1003.6 4568.8 13.3 300.0 159 558.7 68.4 2667.2 946.1 4240.4 13.3 300.0 162 591.6 101.2 2694.7 956.4 4343.9 11.4 350.0 165 1537.8 241.7 7320.2 3149.0 12248.7 11.4 350.0 168 1294.4 295.7 6389.0 2813.0 10792.1 11.4 350.0 171 1659.4 258.2 8131.7 3561.9 13611.1 11.4 350.0 174 1498.9 283.7 7531.1 3281.0 12594.7 11.4 350.0 177 707.4 695.4 4113.0 2570.1 8086.0 10.0 400.0 180 684.1 555.2 3569.8 2295.7 7104.8 10.0 400.0 183 706.0 596.2 3572.6 2317.1 7191.9 10.0 400.0 186 726.3 605.7 3788.0 2393.4 7513.4 10.0 400.0 230 9 Appendices 9 3 Appendix C Biological Removal of Plant Sterols day Campe Stigma B-Sito B-Sitosta ML SterolsJ SRT_1 WAS_1 189 1020.4 840.6 5165.1 3286.7 10312.8 8.9 450.0 192 1139.2 889.9 5934.4 3686.5 11649.9 8.9 450.0 195 1687.2 1290.5 8641.2 5655.1 17274.0 8.9 450.0 197 1857.2 1464.8 9331.4 6162.0 18815.4 8.9 450.0 199 2604.7 1780.0 11229.1 8686.7 24300.4 8.0 500.0 201 3214.5 2161.0 11496.2 10077.1 26948.8 8.0 500.0 2574.4 1226.5 14496.4 7711.5 26008.9 8.0 500.0 207 2549.6 1075.3 14287.6 7511.1 25423.6 8.0 500.0 209 2854.1 1667.8 13414.0 9122.9 27058.9 8.0 500.0 211 3332.2 1967.9 16123.3 10703.5 32126.8 7.3 550.0 213 3498.1 1935.6 14163.9 10951.0 30548.6 7.3 550.0 215 3529.1 1954.8 14823.1 11064.7 31371.6 7.3 550.0 217 3843.9 2195.6 13046.2 11658.0 30743.6 6.7 600.0 219 3921.7 2238.5 13110.3 11912.8 31183.4 6.7 600.0 221 4265.4 2465.1 11553.5 12695.1 30979.1 7.3 550.0 223 4586.8 2619.3 11322.7 13372.1 31900.9 7.3 550.0 225 4012.9 2258.7 12381.4 12165.5 30818.5 8.0 500.0 227 3910.8 2240.3 12696.9 11985.3 30833.3 8.0 500.0 229 3199.1 1829.2 14653.8 10413.7 30095.8 8.0 500.0 231 3658.2 2023.6 13195.8 11151.3 30028.9 8.0 500.0 233 2757.3 2295.6 13337.7 11466.6 29857.1 8.0 500.0 235 2576.2 2191.5 12753.6 10654.7 28176.1 8.0 500.0 237 1864.4 1402.8 11383.6 7501.0 22151.8 8.0 500.0 239 1673.8 1270.4 9873.8 6564.8 19382.7 10.0 400.0 241 912.1 1087.9 6531.8 4720.7 13252.6 10.0 400.0 243 731.7 1080.5 5654.2 4224.8 11691.3 10.0 400.0 245 995.4 1070.5 6608.7 5160.4 13835.1 10.0 400.0 247 822.9 798.4 5171.4 3878.8 10671.5 10.0 400.0 | 730.2 857.8 4635.3 3545.7 9769.1 10.0 400.0 l 745.1 875.3 4729.9 3618.1 9968.4 10.0 400.0 231 9 . Appendices 9 . 3 Appendix C. Biological Removal of Plant Sterols C 6. Reactor 2 mixed liquor sterols (ug/L.), SRT (d), and WAS flow (mL/d) day Campe Stigma B-Sito B-Sitosta ML Sterols_2 SRT_2 WAS_2 80 254.9 104.1 1219.0 644.9 2222.9 22.2 180.0 181.4 130.7 1241.0 801.1 2354.2 22.2 180.0 : J m * * 238.0 250.9 3192.9 1571.3 5253.0 22.2 180.0 367.8 253.2 2431.2 1181.5 4233.7 22.2 180.0 523.6 174.5 1821.4 672.6 3192.1 18.2 220.0 "*123 384.7 72.1 1453.0 705.7 2615.5 18.2 220.0 126 315.3 40.0 1872.1 625.9 2853.3 18.2 220.0 129 304.6 122.3 1533.1 470.0 2430.0 18.2 220.0 132 452.3 157.0 1496.2 725.4 2830.8 18.2 220.0 135 567.2 148.0 2494.0 898.7 4108.0 14.8 270.0 138 976.3 145.9 3345.0 1349.7 5817.0 14.5 275.0 141 828.3 153.3 3154.6 1267.2 5403.5 14.5 275.0 144 839.1 343.2 2874.3 1116.0 5172.6 14.5 275.0 147 906.4 309.2 3387.5 1307.0 5910.0 14.5 275.0 150 463.3 0.0 2333.6 725.5 3522.3 14.5 275.0 153 461.2 0.0 2428.4 766.1 3655.7 14.5 275.0 156 415.9 0.0 2119.0 617.4 3152.3 12.1 330.0 159 526.9 0.0 2431.2 733.0 3691.1 12.1 330.0 162 512.5 0.0 2611.6 805.6 3929.6 12.1 330.0 165 846.4 0.0 3936.2 1284.1 6066.6 12.1 330.0 168 429.0 50.0 2510.8 810.8 3800.5 12.1 330.0 171 466.1 109.7 2803.3 853.7 4232.8 11.1 360.0 174 1781.9 59.9 4435.0 3611.4 9888.3 11.1 360.0 177 1308.7 156.9 7395.5 3305.7 12166.8 11.1 360.0 180 2573.0 86.5 6403.9 5214.7 14278.1 11.1 360.0 183 1454.1 174.3 8217.2 3673.0 13518.6 11.1 360.0 186 1646.2 196.8 9242.9 4375.6 15461.5 10.0 400.0 189 1767.0 772.4 8555.8 5034.2 16129.4 10.0 400.0 192 1693.1 421.3 9595.0 5698.9 17408.3 10.0 400.0 195 2354.9 1136.0 11326.8 7335.9 22153.7 8.0 500.0 197 2458.3 1195.4 11627.4 7633.5 22914.6 8.0 500.0 232 9 Appendices 9.3 Appendix C. Biological Removal of Plant Sterols day Campe Stigma B-Sito B-Sitosta ML Sterols_2 SRT_2 WAS_2 199 2354.9 1136.0 11326.8 7335.9 22153.7 8.0 500.0 201 2458.3 1195.4 11627.4 7633.5 22914.6 8.0 500.0 20$ 2777.9 1335.3 12414.1 8454.9 24982.2 8.0 500.0 207 3068.9 1470.7 13027.9 9224.1 26791.5 8.9 450.0 209 2893.6 1391.0 12931.4 8807.2 26023.1 9.1 440.0 211 3068.9 1470.7 13027.9 9224.1 26791.5 10.0 400.0 213 2777.9 1335.3 12414.1 8454.9 24982.2 10.0 400.0 215 2946.2 1411.9 12506.7 8855.1 25719.9 11.4 350.0 217 2762.0 1323.6 11725.1 8301.7 24112.4 11.4 350.0 219 2257.4 986.8 10930.3 6431.3 20605.9 11.4 350.0 221 1653.4 411.4 9370.1 5565.3 17000.3 11.4 350.0 223 1513.7 376.7 8578.6 5095.2 15564.2 11.4 350.0 227 1767.0 772.4 8555.8 5034.2 16129.4 11.4 350.0 231 1404.3 349.5 7958.5 4726.9 14439.1 11.4 350.0 2S7 1339.3 333.3 7589.8 4507.9 13770.2 11.4 350.0 C 7. Reactor 1 daily mass flow of total sterols (mg/d), removed (%), biodegraded (%), and non-degraded (%) Day Load_1 EffluentJ WAS_1 Removed_1 Biodegraded_1 Non-degraded_1 40 3.4 0.4 0.2 88.5 82.3 17.7 45 4.3 0.5 6.3 88.9 82.7 17.3 50 4.6 0.4 OS 92.2 85.7 14.3 55 4.3 0.5 0.3 88.1 81.9 18.1 60 5.4 0.5 0.3 91.6 85.2 14.8 65 5.4 0.2 0.3 96.0 91.2 8.8 70 5.3 0.2 0.3 95.2 90.5 9.5 75 8.4 0.2 0,4 97.3 92.4 7.6 80 7.9 0.4 0.4 95.4 90.3 9.7 85 8.6 1.3 0.5 85.3 79.8 20.2 233 9. Appendices 9.3 Appendix C. Biological Removal of Plant Sterols 90 10.4 0.4 0.5 95.9 91.3 8.7 95 10.1 0.3 0.6 96.7 90.8 9.2 100 12.3 1.1 0.5 91.0 86.8 13.2 105 12.6 1.3 0.5 90.1 86.0 14.0 110 12.4 1.4 1.3 88.8 78.0 22.0 115 14.7 1.3 1.1 91.3 84.2 15.8 120 16.2 1.1 0.7 93.2 89.0 11.0 123 15.3 0.9 0.8 93.9 88.9 11.1 126 16.7 0.8 0.6 95.0 91.5 8.5 129 16.6 0.8 0.7 95.1 91.0 9.0 132 19.6 0.6 0.6 96.9 93.9 6.1 135 21.5 0.5 1.2 97.5 91.7 8.3 138 20.4 0.6 1.2 96.8 90.8 9.2 141 20.9 0.8 1.4 96.4 89.5 10.5 144 21.0 0.7 1.4 96.6 90.2 9.8 147 21.4 1.2 1.7 94.3 86.5 13.5 150 20.6 1.5 2.3 92.5 81.6 18.4 153 22.3 1.7 1.0 92.3 87.6 12.4 156 25.5 1.4 1.4 94.5 89.1 10.9 159 25.5 1.9 1.3 92.7 87.8 12.2 162 24.7 1.7 1.5 93.1 86.9 13.1 165 24.9 1.7 4.3 93.2 76.0 24.0 168 25.2 1.4 3.8 94.5 79.5 20.5 171 23.9 2.4 4.8 89.7 69.8 30.2 174 24.5 1.1 4.4 95.5 77.5 22.5 177 24.2 2.1 3.2 91.4 78.1 21.9 180 25.0 1.3 2.8 94.9 83.5 16.5 183 28.5 2.2 2.9 92.3 82.2 17.8 186 28.2 1.0 3.0 96.6 85.9 14.1 189 28.9 1.0 4.6 96.7 80.6 19.4 192 34.0 1.0 5.2 97.2 81.7 18.3 195 35.1 1.9 7.8 94.5 72.3 27.7 197 35.0 1.9 8.5 94.5 70.3 29.7 199 35.7 2.1 12.2 94.1 60.1 39.9 201 37.2 6.3 13.5 83.2 46.9 53.1 203 35.7 4.2 13.3 88.3 51.2 48.8 234 9. Appendices 9.3 Appendix C. Biological Removal of Plant Sterols 205 36.1 4.6 13.0 87.2 51.2 48.8 207 36.5 2.9 12.7 92.1 57.3 42.7 209 37.2 6.3 13.5 83.1 46.7 53.3 211 36.5 5.1 17.7 86.0 37.6 62.4 213 36.3 5.2 16.8 85.7 39.4 60.6 215 41.0 9.8 17.3 76.0 33.9 66.1 217 41.1 6.8 18.4 83.5 38.7 61.3 219 45.6 9.0 18.7 80.4 39.4 60.6 221 46.7 7.4 17.0 84.2 47.7 52.3 223 46.7 9.2 17.5 80.4 42.8 57.2 225 49.0 11.7 15.4 76.2 44.7 55.3 227 48.5 11.1 15.4 77.1 45.3 54.7 229 45.8 10.3 15.0 77.5 44.6 55.4 231 46.2 7.8 15.0 83.1 50.6 49.4 233 45.8 7.6 14.9 83.5 50.9 49.1 235 45.6 6.8 14.1 85.1 54.2 45.8 237 45.5 7.6 11.1 83.3 59.0 41.0 239 44.0 7.9 7.8 82.2 64.5 35.5 241 40.7 4.8 5.3 88.1 75.1 24.9 243 41.2 2.6 4.7 93.8 82.5 17.5 245 41.3 3.2 5.5 92.3 78.9 21.1 247 41.6 2.7 4.3 93.5 83.3 16.7 249 41.0 1.6 4.0 96.0 86.3 13.7 251 41.6 2.5 3.9 94.1 84.7 15.3 253 41.9 1.3 3.9 96.9 87.6 12.4 255 41.8 2.8 4.0 93.3 83.8 16.2 235 9 Appendices 9 3 Annendix C, Biological Removal of Plant Sterols C 8. Reactor 2 daily mass flow of total sterols (mg/d), removed (%), biodegraded (%), and non-degraded (%) Day Load_2 Effluent_2 WAS_2 Removed_2 Biodegraded_2 Non-degraded_2 40 4.1 2.6 0*1 36.2 32.6 67.4 45 5.3 2.1 0.3 60.2 54.2 45.8 50 5.7 2.2 0 3 61.6 55.4 44.6 55 5.2 2.5 0.3 53.1 47.8 52.2 60 5.4 2.0 0.3 62.6 56.4 43.6 65 5.4 2.1 0.3 61.0 54.9 45.1 70 5.3 1.4 0.3 74.1 68.9 31.1 75 8.4 0.3 04 96.8 91.9 8.1 80 7.9 0.1 0.4 98.1 93.1 6.9 85 8.6 1.6 0.4 81.5 76.8 23.2 90 10.4 1.7 0.4 83.4 79.4 20.6 95 10.1 0.7 0.4 93.0 88.7 11.3 100 12.3 1.7 0.9 86.0 78.3 21.7 105 12.6 0.6 0.9 95.2 88.1 11.9 110 12.4 1.3 0.8 89.8 83.7 16.3 115 15.5 1.6 0.7 89.6 84.9 15.1 120 16.2 0.5 0.7 96.8 92.4 7.6 123 15.3 0.5 0.6 96.5 92.7 7.3 126 16.4 0.7 0.6 96.0 92.2 7.8 129 16.6 0.6 0.5 96.5 93.2 6.8 132 19.9 2.3 0.6 88.2 85.1 14.9 135 21.5 4.6 1.1 78.5 73.3 26.7 138 23.8 3.5 1.6 85.2 78.5 21.5 141 24.3 3.4 1.5 86.0 79.9 20.1 144 24.5 6.5 1.4 73.4 67.6 32.4 147 25.0 5.6 1.6 77.5 71.0 29.0 150 24.1 6.3 1.0 73.8 69.8 30.2 153 29.3 6.7 1.0 77.1 73.7 26.3 156 29.1 7.3 1.0 74.9 71.3 28.7 159 29.2 11.7 1.2 59.8 55.7 44.3 236 Appendices 9 3 Appendix C Biolnnir-.al Removal nf Plant Sterols 162 28.2 10.5 1.3 62.7 58.1 41.9 165 28.1 10.6 2.0 62.4 55.3 44.7 168 29.1 10.9 1.3 62.7 58.4 41.6 171 26.9 10.6 1.5 60.6 54.9 45.1 174 31.5 9.6 3.6 69.5 58.2 41.8 177 31.1 11.7 4.4 62.2 48.2 51.8 180 32.2 10.3 5.1 68.1 52.1 47.9 183 32.1 15.0 4.9 53.3 38.1 61.9 186 31.8 12.9 6.2 59.3 39.9 60.1 189 32.5 13.6 6.5 58.3 38.5 61.5 192 44.6 16.7 7.0 62.5 46.9 53.1 195 46.1 13.9 11.1 69.9 45.9 54.1 197 46.4 14.1 11.5 69.5 44.9 55.1 199 47.8 12.4 11.1 74.1 50.9 49.1 201 49.3 10.5 11.5 78.7 55.5 44.5 203 46.8 9.5 11.5 79.7 55.1 44.9 205 47.4 12.4 12.5 73.8 47.5 52.5 207 41.5 10.9 12.1 73.9 44.8 55.2 209 42.3 11.2 11.5 73.6 46.5 53.5 211 42.0 7.6 10.7 82.0 56.4 43.6 213 42.0 10.2 10.0 75.7 51.9 48.1 215 41.0 7.1 9.0 82.7 60.7 39.3 217 40.2 3.6 8.4 91.0 70.0 30.0 219 40.2 7.4 7.2 81.5 63.5 36.5 221 42.0 2.6 6.0 93.8 79.6 20.4 223 42.0 4.4 5.4 89.6 76.6 23.4 225 42.0 1.8 5.5 95.6 82.6 17.4 227 40.7 7.4 5.6 81.7 67.8 32.2 229 41.3 1.8 0.0 95.6 95.6 4.4 231 41.5 3.1 5.1 92.6 80.5 19.5 233 41.2 1.8 5.0 95.6 83.3 16.7 235 41.1 4.0 5.0 90.3 78.0 22.0 237 40.9 3.7 4.8 91.0 79.3 20.7 239 39.6 4.0 3,6 89.9 80.9 19.1 241 40.7 3.7 37 91.0 81.9 18.1 237 9. Appendices 9 3 Appendix C Biological Removal nf Plant Sterols C 9. Reactor 1 cumulative mass flows of total sterols influent (mg), effluent (mg), W A S (mg), removed (%), retained (%), accumulated (mg), ML sterols (mg/L), biodegraded (%) Day Influent _1 Effluent _1 WAS_1 Removed Retained Accumulate J _1 d _1 ML Sterols_1 Biodegraded_1 40 136.5 15.6 8.5 88.5 82.3 21.384 2 673 66.7 45 158.2 18.0 9.8 88.6 82.4 21.384 2.673 68.9 50 181.0 19.8 11.3 89.0 82.8 21.384 71.0 55 202.3 22.4 12.6 88.9 82.7 21.384 2.673 72.1 60 229.4 24.6 14.3 89.3 83.0 21.384 2.673 73.7 65 256.5 25.7 15.6 90.0 83.9 21.384 2.673 75.5 70 282.8 27.0 16.9 90.5 84.5 21.384 2.673 76.9 75 324.6 28.1 18.9 91.3 85.5 21.384 2.673 78.9 80 364.1 30.0 20.9 91.8 86.0 21.384 2.673 80.2 85 407.0 36.3 23.3 91.1 85.4 24.931 3.116 79.2 90 458.9 38.4 25.7 91.6 86.0 25.624 3.203 80.5 95 509.4 40.0 28.6 92.1 86.5 31.367 3.921 80.4 100 570.9 45.6 31.1 92.0 86.6 23.161 2.895 82.5 105 633.9 51.9 33.7 91.8 86.5 23.461 2.933 82.8 110 695.9 58.9 40.4 91.5 85.7 61.067 7.633 77.0 115 769.6 65.2 45.7 91.5 85.6 48.339 6.042 79.3 120 850.5 70.7 49.0 91.7 85.9 26.836 3.355 82.8 123 896.5 73.6 51.3 91.8 86.1 30.653 3.832 82.7 126 946.7 76.1 53.1 92.0 86.4 23.754 2.969 83.8 129 996.5 78.5 55.1 92.1 86.6 27.096 3.387 83.9 132 1055.2 80.3 56.9 92.4 87.0 23.926 2.991 84.7 135 1119.7 81.9 60.7 92.7 87.3 39.877 4.985 83.7 138 1181.0 83.9 64.4 92.9 87.4 39.297 4.912 84.1 141 1243.5 86.1 68.7 93.1 87.6 46.037 5.755 83.8 144 1306.7 88.3 72.8 93.2 87.7 43.651 5.456 84.3 147 1370.9 91.9 77.7 93.3 87.6 44.032 5.504 84.4 150 1432.8 96.6 84.5 93.3 87.4 60.246 7.531 83.2 153 1499.6 101.7 87.6 93.2 87.4 27.714 3.464 85.5 156 1576.1 105.9 91.7 93.3 87.5 36.551 4.569 85.1 238 Appendices 9 3 Appendix C Binlnpir.al Remnx/al nf Plant Sterols 159 1652.7 111.5 95.5 93.3 87.5 33.924 4.240 85.4 162 1726.7 116.6 100.1 93.2 87.5 34.751 4.344 85.4 165 1801.5 121.7 113.0 93.2 87.0 97.990 12.249 81.5 168 1877.0 125.8 124.3 93.3 86.7 86.337 10.792 82.1 171 1948.6 133.1 138.6 93.2 86.1 108.889 13.611 80.5 174 2022.2 136.5 151.8 93.3 85.7 100.758 12.595 80.8 177 2094.7 142.7 161.5 93.2 85.5 64.688 8.086 82.4 180 2169.9 146.5 170.0 93.2 85.4 56.838 7.105 82.8 183 2255.4 153.1 178.7 93.2 85.3 57.535 7.192 82.7 186 2340.1 156.0 187.7 93.3 85.3 60.107 7.513 82.7 189 2426.9 158.9 201.6 93.5 85.1 82.502 10.313 81.7 192 2528.9 161.8 217.3 93.6 85.0 93.199 11.650 81.3 195 2634.2 167.6 240.7 93.6 84.5 138.192 17.274 79.3 197 2704.3 171.5 257.6 93.7 84.1 150.523 18.815 78.6 199 2775.8 175.7 281.9 93.7 83.5 194.404 24.300 76.5 201 2850.2 188.3 308.8 93.4 82.6 215.591 26.949 75.0 203 2921.6 196.6 335.3 93.3 81.8 208.071 26.009 74.7 205 2993.8 205.8 361.3 93.1 81.1 203.389 25.424 74.3 207 3066.9 211.6 386.8 93.1 80.5 216.471 27.059 73.4 209 3141.2 224.2 413.8 92.9 79.7 257.015 32.127 71.5 211 3214.2 234.4 449.2 92.7 78.7 244.389 30.549 71.1 213 3286.9 244.8 482.8 92.6 77.9 250.973 31.372 70.2 215 3368.8 264.4 517.3 92.2 76.8 245.949 30.744 69.5 217 3451.1 278.0 554.2 91.9 75.9 249.467 31.183 68.7 219 3542.4 295.9 591.6 91.6 74.9 247.833 30.979 67.9 221 3635.8 310.7 625.7 91.5 74.2 255.207 31.901 67.2 223 3729.2 329.1 660.8 91.2 73.5 246.548 30.818 66.8 225 3827.1 352.4 691.6 90.8 72.7 246.666 30.833 66.3 227 3924.2 374.6 722.4 90.5 72.0 240.767 30.096 65.9 229 4015.9 395.3 752.5 90.2 71.4 240.231 30.029 65.4 231 4108.2 410.9 782.5 90.0 70.9 238.857 29.857 65.1 233 4199.8 426.0 812.4 89.9 70.5 225.409 28.176 65.1 235 4291.0 439.6 840.6 89.8 70.2 177.215 22.152 66.0 237 4382.0 454.8 862.7 89.6 69.9 155.062 19.383 66.4 239 4470.1 470.5 878.2 89.5 69.8 106.020 13.253 67.5 241 4551.5 480.2 888.8 89.5 69.9 93.530 11.691 67.9 239 9 Appendices 9 3 Appendix C Biological Removal nf Plant Sterols 243 4634.0 485.3 898.2 89.5 70.1 110.680 13.835 67.8 245 4716.6 491.6 909.3 89.6 70.3 85.372 10.671 68.5 247 4799.9 497.0 917.8 89.6 70.5 78.152 9.769 68.9 249 4881.9 500.3 925.7 89.8 70.8 79.747 9.968 69.2 251 4965.0 505.2 933.5 89.8 71.0 253 5048.8 507.8 941.4 89.9 71.3 255 5132.4 513.4 949.3 90.0 71.5 C 10. Reactor 2 cumulative mass flows of total sterols influent (mg), effluent (mg), W A S (mg), removed (%), retained (%), accumulated (mg), ML sterols (mg/L), biodegraded (%) Day Influent 2 Effluent 2 WAS_2 Removed 2 Retained Accumulated _2 2 ML Sterols_2 Biodegraded_2 40 163.3 104.1 5.9 36.2 32.6 17.783 2 223 21.7 45 189.9 114.7 7.5 39.6 35.6 17.783 2.223 26.3 50 218.3 125.6 9.3 42.5 38.2 17.783 2.223 30.1 55 244.5 137.9 10.7 43.6 39.2 17.783 2*22$ 32.0 60 271.5 148.0 12.4 45.5 40.9 17.783 34.4 65 298.7 158.6 14.0 46.9 42.2 17.783 2.223 36.3 70 325.0 165.4 15.4 49.1 44.4 17.783 2.223 38.9 75 366.8 166.8 17.4 54.5 49.8 17.783 2.223 44.9 80 406.3 167.5 19.4 58.8 54.0 17.783 2.223 49.6 85 449.1 175.4 21.4 60.9 56.2 17.784 2.223 52.2 90 501.0 184.0 23.5 63.3 58.6 18.833 2.354 54.8 95 551.5 187.6 25.7 66.0 61.3 19.200 2.400 57.9 100 613.1 196.2 30.4 68.0 63.0 42.024 5.253 56.2 105 676.1 199.2 34.9 70.5 65.4 40.000 5.000 59.5 110 738.1 205.5 38.7 72.2 66.9 33.870 4.234 62.3 115 815.8 213.6 42.3 73.8 68.6 32.000 4.000 64.7 120 896.7 216.2 45.8 75.9 70.8 25.537 3.192 67.9 123 942.7 217.9 47.6 76.9 71.8 20.924 2.615 69.6 126 992.1 219.8 49.4 77.8 72.9 22.827 2.853 70.6 129 1041.9 221.6 51.0 78.7 73.8 19.440 2.430 72.0 132 1101.6 228.6 52.9 79.2 74.4 22.646 2.831 72.4 135 1166.0 242.5 56.2 79.2 74.4 32.864 4.108 71.6 240 fl Appendices 9 3 Appendix C Bioloaioal Removal of Plant Sterols 138 1237.6 253.1 61.0 79.5 74.6 46.536 5.817 70.9 141 1310.5 263.3 65.5 79.9 74.9 43.228 5.403 71.6 144 1384.2 282.9 69.8 79.6 74.5 41.381 5.173 71.5 147 1459.1 299.8 74.6 79.5 74.3 47.280 5.910 71.1 150 1531.4 318.7 77.5 79.2 74.1 28.179 3.522 72.3 153 1619.3 338.8 80.6 79.1 74.1 29.245 3.656 72.3 156 1706.7 360.8 83.7 78.9 74.0 25.218 3.152 72.5 159 1794.2 395.9 87.3 77.9 73.1 29.529 3.691 71.4 162 1878.8 427.5 91.2 77.2 72.4 31.437 3.930 70.7 165 1963.2 459.2 97.2 76.6 71.7 48.533 6.067 69.2 168 2050.7 491.8 101.0 76.0 71.1 30.404 3.801 69.6 171 2131.3 523.6 105.6 75.4 70.5 33.862 4.233 68.9 174 2225.9 552.5 116.2 75.2 70.0 79.106 9.888 66.4 177 2319.2 587.7 129.4 74.7 69.1 97.334 12.167 64.9 180 2415.8 618.6 144.8 74.4 68.4 114.225 14.278 63.7 183 2512.0 663.5 159.4 73.6 67.2 108.149 13.519 62.9 186 2607.3 702.2 178.0 73.1 66.2 123.692 15.461 61.5 189 2704.9 742.9 197.3 72.5 65.2 129.035 16.129 60.5 192 2838.8 793.2 218.2 72.1 64.4 139.266 17.408 59.5 195 2977.1 834.8 251.4 72.0 63.5 177.229 22.154 57.6 197 3070.0 863.1 274.3 71.9 62.9 183.317 22.915 57.0 199 3165.6 887.9 296.5 72.0 62.6 177.229 22.154 57.0 201 3264.2 908.9 319.4 72.2 62.4 183.317 22.915 56.8 203 3357.8 927.9 342.4 72.4 62.2 184.000 23.000 56.7 205 3452.7 952.7 367.4 72.4 61.8 199.858 24.982 56.0 207 3535.7 974.4 391.5 72.4 61.4 214.332 26.792 55.3 209 3620.3 996.8 414.4 72.5 61.0 208.185 26.023 55.3 211 3704.3 1011.9 435.8 72.7 60.9 214.332 26.792 55.1 213 3788.2 1032.3 455.8 72.7 60.7 199.858 24.982 55.4 215 3870.2 1046.5 473.8 73.0 60.7 205.759 25.720 55.4 217 3950.6 1053.7 490.7 73.3 60.9 192.899 24.112 56.0 219 4031.0 1068.6 505.1 73.5 61.0 164.847 20.606 56.9 221 4115.0 1073.9 517.0 73.9 61.3 136.002 17.000 58.0 223 4199.1 1082.6 527.9 74.2 61.6 124.514 15.564 58.7 225 4283.1 1086.3 538.8 74.6 62.1 124.800 15.600 59.1 227 4364.4 1101.1 550.1 74.8 62.2 129.035 16.129 59.2 9. Appendices 9.3 Appendix C. Biolnpical Removal of Plant Sterols 229 4447.0 1104.8 550.1 75.2 62.8 129.600 16.200 59.9 231 4530.0 1110.9 560.2 75.5 63.1 115.513 14.439 60.6 233 4612.4 1114.6 570.3 75.8 63.5 115.200 14.400 61.0 235 4694.6 1122.6 580.4 76.1 63.7 115.200 14.400 61.3 237 4776.4 1129.9 590.0 76.3 64.0 110.162 13.770 61.7 239 4855.7 1137.9 597.2 76.6 64.3 110.400 13.800 62.0 241 4937.1 1145.2 604.6 76.8 64.6 110.400 13.800 62.3 242 9 Appendices 9.4 Appendix D. Inactivation of Biomass 9.4 Appendix D. Inactivation of Biomass D 1 . Inactivation of Biomass and Head Space Monitoring for C 0 2 . Treatment 1 (T1)= 4% Formaldehyde (37% Formalyn) for 10-30 min. Treatment 2 (T2)= 1 mM Fluoroacetate (Mono-Fluoroacetic acid) for 10-30 min. Treatment 3 (T3)= 11% Formaldehyde (37% Formalyn) for 10-30 min. Time Inactive-TI (h) C 0 2 o 2 0.00 0.45 214.00 0.58 5.60 210.60 0.75 5.40 206.80 1.17 5.00 211.20 1.50 4.95 209.60 3.08 4.57 211.00 4.33 4.63 208.60 4.83 4.20 211.00 Time lnactive-T2 (h) C 0 2 o 2 0.00 0.47 216.96 0.25 0.79 212.50 1.50 1.30 215.20 2.50 1.80 212.60 14.50 1.60 215.70 15.00 1.79 211.80 16.50 1.75 211.00 20.00 1.90 214.70 Time Active-1 (h) c o 2 o 2 0.00 0.45 214.00 0.34 1.43 163.00 0.78 2.00 210.80 1.33 2.39 211.10 1.75 2.20 214.40 3.83 2.72 214.80 4.58 3.00 209.50 4.83 3.00 Time Active-3 (h) c o 2 o 2 0.00 0.41 216.70 1.50 1.30 205.30 2.00 2.00 211.70 2.25 2.30 205.30 3.00 2.80 204.40 15.83 6.90 196.60 16.00 6.80 195.20 18.50 7.60 197.60 243 9 Appendices 9.4 Appendix D. Inactivation of Biomass Time lnactive-T3 (h) C 0 2 o 2 0.00 0.45 216.00 0.50 0.68 209.70 1.00 0.70 213.10 2.00 0.76 212.60 6.60 0.78 212.80 Time lnactive-T3 (h) c o 2 o 2 0.00 0.50 216.00 1.00 0.40 219.60 2.50 0.41 215.10 3.75 0.51 216.20 5.00 0.52 217.40 12.00 0.56 216.80 14.00 0.55 212.60 15.00 0.50 Time Active-2 (h) c o 2 o 2 0.00 0.45 216.00 0.75 1.70 210.80 2.25 2.70 182.30 6.00 3.40 214.90 6.92 3.90 209.00 Time Fresh Biomass (h) C 0 2 o 2 0.00 0.50 216.00 0.90 1.57 206.30 2.50 2.50 196.60 6.50 3.20 209.00 7.00 3.70 202.90 Time Active (h) c o 2 o 2 0.00 0.44 218.00 2.00 1.30 213.60 3.00 1.70 209.60 3.00 1.70 209.60 6.00 4.70 200.00 13.00 7.70 192.70 15.00 6.80 194.30 Time Active-4 (h) C 0 2 o 2 1.50 1.30 205.30 2.00 2.10 208.00 2.25 1.80 208.80 2.70 1.90 204.80 3.25 1.93 207.10 3.75 2.10 203.10 5.00 2.30 206.00 8.50 2.30 205.00 10.00 3.40 200.50 244 9 Appendices 9.4 Appendix D. Inactivation of Biomass T i m e co2 o2 N 2 (h) Air Air Air 0.00 0.45 214.00 794.80 0.50 0.44 221.80 826.20 1.00 0.41 216.70 807.30 2.00 0.40 216.20 806.50 4.00 0.67 221.30 823.30 8.00 0.40 216.60 806.50 12.00 0.38 215.80 448.40 18.00 0.50 221.00 828.00 24.00 0.40 222.00 827.80 48.00 0.35 218.00 815.00 Average 0.44 218.34 778.38 Scintillations Counting for labeled C Q 2 using C14 labeled glucose Control Inactive 4% Formaldehyde 11 % Formaldehyde Active Biomass No. Low High 10 min 35 min 10 min 35 min Low High 1 800.0 1266.0 2688.0 2468.0 1562.0 1215.0 29539.0 40968.0 2 870.0 1500.0 2688.0 2468.0 1562.0 1215.0 29212.0 42113.0 Average 835.0 1383.0 2688.0 2468.0 1562.0 1215.0 29375.5 41540.5 245 9. Appendices 9.4 Appendix D. Inactivation of Biomass D 2. Head space C 0 2 for active (1,2 & fresh) and inactive b iomass (T1 & T3) 246 9. Appendices 9 4 Appendix D Inactivation of Biomass D 3. Head space C 0 2 for active (3 & 4) and inactive biomass (T2 & T3) 247 9. Appendices 9.5 Appendix E. Adsorption of Plant Sterols 9.5 Appendix E. Adsorption of Plant Sterols E 1. Set 1: Ba tch 1-7 data for sterols adsorption to the inactivated secondary s ludge Batch-1 Control 0 mg/L MLSS Campe B-Sito B-Sitosta Campe B-Sito B-Sitosta Time (h) (MQ/L) (Mg/L) (M9/L) Time (h) (C/Ci) (C/Ci) (C/Ci) 0.0 853.51 3661.19 2358.21 0.0 1.00 1.00 1.00 2.0 796.43 3441.05 2246.55 2.0 0.93 0.94 0.95 10.0 848.22 3497.94 2291.04 10.0 0.99 0.96 0.97 24.0 857.22 3463.96 2258.48 24.0 1.00 0.95 0.96 124.0 830.13 3456.12 2273.52 124.0 0.97 0.94 0.96 169.0 830.34 3444.25 2277.89 169.0 0.97 0,94 0.97 Batch-2 20 mg/L MLSS Campe B-Sito B-Sitosta Campe B-Sito B-Sitosta Time (h) (M9/L) (MQ/L) (M9/L) Time (h) (C/Ci) (C/Ci) (C/Ci) 0.0 853.51 3661.19 2358.21 0.0 1.00 1.00 1.00 2.0 760.35 3359.76 2058.45 2.0 0.89 0.92 0.87 10.0 687.01 3194.31 1916.29 10.0 0.80 0.87 0.81 24.0 666.01 3195.99 1801.35 24.0 0.78 0.87 0.76 122.0 627.97 3035.10 1579.11 122.0 0.74 0.83 0.67 Batch-3 40 mg/L MLSS Campe B-Sito B-Sitosta Campe B-Sito B-Sitosta Time (h) (Mg/L) (Mg/L) (Mg/L) Time (h) (C/Ci) (C/Ci) (C/Ci) 0.0 853.51 3661.19 2358.21 0.0 1.00 1.00 1.00 2.0 712.57 3284.73 2000.11 2.0 0.83 0.90 0.85 10.0 651.90 3106.06 1742.74 10.0 0.76 0.85 0.74 24.0 597.05 2972.73 1619.53 24.0 0.70 0.81 0.69 122.0 505.04 2661.92 1464.90 122.0 0.59 0.73 0.62 248 9 Appendices 9.5 Appendix E. Adsorption of Plant Sterols Batch-4 100 mg/L MLSS Campe B-Sito B-Sitosta Campe B-Sito B-Sitosta Time (h) (ug/L) (ug/L) (ug/L) Time (h) (C/Ci) (C/Ci) (C/Ci) 0.0 853.51 3661.19 2358.21 0.0 1.00 1.00 1.00 2.0 752.28 3335.82 2035.43 2.0 0.88 0.91 0.86 10.0 605.53 3077.52 1682.84 10.0 0.71 0.84 0.71 24.0 547.69 2846.47 1492.30 24.0 0.64 0.78 0.63 122.0 449.79 2463.07 1317.11 122.0 0.53 0.67 0.56 Batch-5 200 mg/L MLSS Campe B-Sito B-Sitosta Campe S-Sito B-Sitosta Time (h) (ug/L) (M9/L) (pg/L) Time (h) (C/Ci) (C/Ci) (C/Ci) 0.0 853.51 3661.19 2358.21 0.0 1.00 1.00 1.00 2.0 686.72 3182.84 1870.35 2.0 0.80 0.87 0.79 10.0 575.34 2988.70 1662.01 10.0 0.67 0.82 0.70 24.0 506.32 2608.07 1395.20 24.0 0.59 0.71 0.59 122.0 435.64 2330.03 1282.04 122.0 0.51 0.64 0.54 Batch-6 1000 mg/L MLSS Campe B-Sito B-Sitosta Campe B-Sito B-Sitosta Time (h) (ug/L) (Mg/L) (Mg/L) Time (h) (C/Ci) (C/Ci) (C/Ci) 0.0 853.51 3661.19 2358.21 0.0 1.00 1.00 1.00 2.0 509.12 2500.47 1464.56 2.0 0.60 0.68 0.62 10.0 432.91 2173.62 1262.29 10.0 0.51 0.59 0.54 24.0 369.84 1850.40 996.19 24.0 0.43 0.51 0.42 122.0 274.26 1511.01 725.40 122.0 0.32 0.41 0.31 Batch-7 2000 mg/L MLSS Campe B-Sito S-Sitosta Campe B-Sito B-Sitosta Time (h) (Mg/L) (Mg/L) (Mg/L) Time (h) (C/Ci) (C/Ci) (C/Ci) 0.0 853.51 3661.19 2358.21 0.0 1.00 1.00 1.00 2.0 192.16 1089.89 632.21 2.0 0.23 0.30 0.27 10.0 140.35 768.02 407.05 10.0 0.16 0.21 0.17 24.0 111.65 562.40 306.42 24.0 0.13 0.15 0.13 122.0 60.34 331.61 176.09 122.0 0.07 0.09 0.07 249 9. Appendices 9.5 Appendix E. Adsorption of Plant Sterols E 2. Set 1: Ba tch 1-7 figures showing sterols adsorption to the inactivated secondary sludge 4,000 3,500 d 3,000 3 § 2,500 co | 2.000 c 3 1,500 o & 1,000 500 0 1 ad 2 ad 0MLSS : 4 » — * —A— • -Can ipe -~B f J-Sitc ) —A--U-Sitostc • — • — 4 — ' ' • • ' 1 1 1 1 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 Time (hr) 1 ad 20 M L S S 130 250 9. Appendices 9.5 Appendix E. Adsorption of Plant Sterols 1 ad 100 MLSS o r . . . . i . . . . j . . i . j . . . i i . . . . i . . . i i i . . . i 1111 i 111. j. i ' ' j ' r , 11111, i 1 1 ! i 0 10 20 30 40 50 60 70 80 90 100 110 120 130 Time (hr) 251 9. A p p e n d i c e s 9.5 Appendix E . Adsorption of Plant Sterols 1_ad 200 MLSS 0 10 20 30 40 50 60 70 80 90 100 110 120 130 Time (hr) 1_ad 1000 MLSS 0 10 20 30 40 50 60 70 80 90 100 110 120 130 Time (hr) 252 9. Appendices 9.5 Appendix E Adsorption of Plant Sterols E 3. Set 1: Liquid phase sterol concentration variation with inactivated M L S S dose 253 9. Appendices 9.5 Appendix E. Adsorption of Plant Sterols R-Sitostanol 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 MLSS (mg/L) Campesterol on c o 1 c s c o o 8 1,000 900 800 700 600 500 400 300 200 100 0 • L •9—^ ^ ' < * _•—Ohr - « - 2 h r - A - 1 0 h r _ H _ 2 4 h r - * - 1 2 4 h r :L*xX~ """^  ': \ | ; | ; | . . . . . . . 1 1 1 ' — - • • • • 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 MLSS (mg/L) 254 9. Appendices 9.5 Appendix E. Adsorption of Plant Sterols E 4. Set 1: Sterol equilibrium concentrations, adsorption capacit ies of the inactivated secondary sludge and adsorption isotherms B-Sito B-Sitosta Campe Ce (mg/L) Qe=x/m Ce Qe=x/m Ce Qe=x/m mg/L mg/g mg/L mg/g mg/L mg/g 0.33 2.36 0.18 1.51 0.06 0.68 1.51 2.85 0.73 2.05 0.27 0.86 2.33 7.36 1.28 5.80 0.44 2.37 2.46 12.68 1.32 10.83 0.45 4.32 2.66 25.68 1.46 22.75 0.51 8.99 3.04 32.00 1.58 39.38 0.63 11.56 45 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 Ce Sterols mg/L 255 9. Appendices 9 5 Appendix E. Adsorption of Plant Sterols 256 9. Appendices 9 5 Appendix E. Adsorption of Plant Sterols E 5. Set 2: Ba tch 8-14 data for sterols adsorption to the inactivated secondary s ludge Batch-8 Control 0 mg/L MLSS Campe B-Sito B-Sitosta Campe B-Sito B-Sitosta Time (h) (M9/L) (Mg/L) (Mg/L) Time (h) (C/Ci) (C/Ci) (C/Ci) 0.0 853.51 3661.19 2358.21 0.0 1.00 1.00 1.00 2.0 796.43 3441.05 2246.55 2.0 0.93 0.94 0.95 10.0 848.22 3497.94 2291.04 10.0 0.99 0.96 0.97 24.0 857.22 3463.96 2258.48 24.0 1.00 0.95 0.96 124.0 830.13 3456.12 2273.52 124.0 0.97 0.94 0.96 169.0 830.34 3444.25 2277.89 169.0 0.97 0.94 0.97 Batch-9 20 mg/L MLSS Campe B-Sito B-Sitosta Campe B-Sito B-Sitosta Time (h) (Mg/L) (pg/L) (Mg/L) Time (h) (C/Ci) (C/Ci) (C/Ci) 0.0 853.51 3661.19 2358.21 0.0 1.00 1.00 1.00 2.0 675.89 3219.88 1921.99 2.0 0.79 0.88 0.82 10.0 622.69 3017.64 1775.23 10.0 0.73 0.82 0.75 24.0 592.34 2881.32 1716.48 24.0 0.69 0.79 0.73 124.0 554.66 2888.78 1652.97 124.0 0.65 0.79 0.70 169.0 574.98 2822.19 1627.27 169.0 0.67 0.77 0.69 BatCh-10 40 mg/L MLSS Time (h) Campe B-Sito S-Sitosta Campe B-Sito B-Sitosta (Mg/L) (Mg/L) (Mg/L) Time (h) (C/Ci) (C/Ci) (C/Ci) 0.0 853.51 3661.19 2358.21 0.0 1.00 1.00 1.00 2.0 708.58 3094.75 1835.51 2.0 0.83 0.85 0.78 10.0 596.12 2927.64 1714.42 10.0 0.70 0.80 0.73 24.0 550.94 2776.37 1582.62 24.0 0.65 0.76 0.67 124.0 522.94 2787.37 1467.78 124.0 0.61 0.76 0.62 169.0 521.98 2711.31 1497.68 169.0 0.61 0.74 0.64 257 9. Appendices 9.5 Appendix E. Adsorption of Plant Sterols Batch-11 100 mg/L MLSS Campe B-Sito B-Sitosta Campe B-Sito B-Sitosta Time (h) (ug/L) (Mg/L) (Mg/L) Time (h) (C/Ci) (C/Ci) (C/Ci) 0.0 853.51 3661.19 2358.21 0.0 1.00 1.00 1.00 2.0 672.96 3186.49 1823.99 2.0 0.79 0.87 0.77 10.0 585.00 2888.51 1624.69 10.0 0.69 0.79 0.69 24.0 535.54 2649.00 1494.17 24.0 0.63 0.72 0.63 124.0 506.75 2482.67 1305.00 124.0 0.59 0.68 0.55 169.0 483.80 2410.24 1331.70 169.0 0.57 0.66 0.56 Batch-12 200 mg/L MLSS Campe B-Sito B-Sitosta Campe B-Sito B-Sitosta Time (h) (Mg/L) (Mg/L) (Mg/L) Time (h) (C/Ci) (C/Ci) (C/Ci) 0.0 853.51 3661.19 2358.21 0.0 1.00 1.00 1.00 2.0 610.58 3097.67 1687.37 2.0 0.72 0.85 0.72 10.0 533.68 2780.41 1530.06 10.0 0.63 0.76 0.65 24.0 479.36 2544.84 1359.66 24.0 0.56 0.70 0.58 124.0 433.96 2208.85 1142.85 124.0 0.51 0.60 0.48 169.0 400.44 2096.66 1091.03 169.0 0.47 0.57 0.46 Batch-13 1000 mg/L MLSS Campe S-Sito B-Sitosta Campe B-Sito B-Sitosta Time (h) (Mg/L) (Mg/L) (Mg/L) Time (h) (C/Ci) (C/Ci) (C/Ci) 0.0 853.51 3661.19 2358.21 0.0 1.00 1.00 1.00 2.0 356.16 1936.96 985.88 2.0 0.42 0.53 0.42 10.0 268.43 1578.28 771.45 10.0 0.31 0.43 0.33 24.0 192.26 1196.23 528.81 24.0 0.23 0.33 0.22 124.0 131.71 687.29 291.28 124.0 0.15 0.19 0.12 169.0 103.84 586.86 256.80 169.0 0.12 0.16 0.11 258 9. Appendices 9.5 Appendix E. Adsorption of Plant Sterols Batch-14 2000 mg/L MLSS Campe B-Sito B-Sitosta Campe B-Sito B-Sitosta Time (h) (ug/L) (MQ/L) (Mg/L) Time (h) (C/Ci) (C/Ci) (C/Ci) 0.0 853.51 3661.19 2358.21 0.0 1.00 1.00 1.00 2.0 155.56 848.77 426.90 2.0 0.18 0.23 0.18 10.0 78.20 467.94 225.89 10.0 0.09 0.13 0.10 24.0 64.11 278.50 152.59 24.0 0.08 0.08 0.06 124.0 51.33 113.41 62.14 124.0 0.06 0.03 0.03 169.0 30.16 148.63 82.19 169.0 0.04 0.04 0.03 E 6. Set 2: Batch 8-14 figures showing sterols adsorption to the inactivated secondary sludge 1 ad 2 ad 0 M L S S 4 , 0 0 0 3 , 5 0 0 i i 3 , 0 0 0 2 , 5 0 0 c 2 , 0 0 0 8 1 ,500 I 1 ,000 5 0 0 0 K ^ — : f . z t • — A — — A £ Campe _B_s -S i to -A—B-Si tosta — • — — • • _i—i i i 1 1 1 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0 Time (hr) 259 9. Appendices 9 5 Annendix E. Adsorption of Plant Sterols 4,000 3,500 4 | ^ 3,000 -F-^o-o 2,500 - f CD | 2,000 c O 1,500 f o $ 1,000 - f 500 2 ad 20 MLSS -Campe -a—fi-Sito — A — U-Sitosta -H + -H 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 Time (hr) 4,000 3,500 2 ad 40 MLSS 2 § 2,500 5 | 2,000 c O 1,500 2 g 1,000 | : : i ; i —•—Campe —a— B-Sito —A— B-Sitosta - M ^ j i i i '• i i i j j i -i j | \ -^ T"*' f-A—i j J : ! ! Hi ' • j j r—"-;A 1 ^ '—*—i j i j i ^  j * -~-• • • j • • • • j • • • • i • • > • i •... j . . . . I • . . . j.... j . . . . j •... j . . . . j... • j • • • • j.. • • |. • • • j • • • • j •... j . . . • 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 Time (hr) 260 9. Appendices 9.5 Appendix E. Adsorption of Plant Sterols 2 ad 100 MLSS Campe —a—B-Sito —A—B-Sitosta ^ t--I ' 1 ' 1 I ' ' 1 ' I ' 1 ' ' I ' ' ' ' I ' ' ' 1 I I 1 1 1 ' I I " " I 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 Time (hr) 261 9. Appendices 9.5 Appendix E. Adsorption of Plant Sterols 2 ad 1000 M L S S 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 Time (hr) 2_ad 2000 MLSS 4 ,000 -, : ; : : : ; : : ; r 3,500 -f Time (hr) 262 9. Appendices 9.5 Appendix E. Adsorption of Plant Sterols E 7. Set 2: Liquid phase sterol concentration variation with inactivated M L S S dose ^-Sitosterol 4,000 3,500 5" 3,000 I § 2,500 2 | 2,000 c 3 1,500 o £ 1,000 2,500 c v 1,500 (A * T T ; 4 - •—0 hr -o— 2 hr vs. - A - 1 0 . 5 h r - -- * - 2 4 hr - * - 1 2 4 hr -•—169 hr - ' 1 1 1 1 ' ' ' • i ' 1 1 1 1 1 1 1 • 1 1 1 i • • • 1 1 1 1 1 1 ' 1 1 1 1 • ' • 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 MLSS (mg/L) ft-Sitostanol i i i : ! V ! ! f - » - 0 hr -o—2 hr i -j \ 1 -A—10.5hr - M - 2 4 hr -- * - 1 2 4 hr - •—169 hr -- 1 1 ' 1 1 1 • ' i ' 1 ' 1 1 ' ' 1 1 i ' ' 1 1 1 ' 1 1 1 i 1 ' ' 1 i ' 1 1 1 ' • ' 1 1 1 1 • I I 1 I • 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 MLSS (mg/L) 263 9. Appendices 9.5 Annenriix E. Adsorption of Plant Sterols Campesterol 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 MLSS (mg/L) E 8. Set 2: Sterol equilibrium concentrations, adsorption capacit ies of the inactivated secondary sludge and adsorption isotherms B-Sito B-Sitosta Campe Ce Qe=x/m Ce Qe=x/m Ce Qe=x/m mg/L mg/g mg/L mg/g mg/L mg/g 0.15 2.46 0.08 1.56 0.03 0.69 0.59 3.77 0.26 2.52 0.10 1.03 2.10 8.52 1.09 6.76 0.40 2.55 2.41 13.21 1.33 10.69 0.48 3.98 2.71 24.45 1.50 21.93 0.52 8.57 2.82 42.65 1.63 36.97 0.57 14.21 264 9. Appendices 9.5 Appendix E. Adsorption of Plant Sterols o> T3 _3 W CO I E 45.0 40.0 35.0 30.0 25.0 20.0 15.0 10.0 5.0 0.0 : — A — Campe _ ' . -•— fi-Sitosta -o— fi-Sito • - • • — 1 — — i — i — i — i — j . — i — i — i — , — 0.00 0.50 1.00 1.50 2.00 Ce Sterols mg/L 2.50 3.00 1000.0 CD O) TJ J3 CO O) J2 s CD CO E X 10.0 Ce (mg Sterols/L) 265 9. Appendices 9 5 Appendix E. Adsorption of Plant Sterols E 9. Set 3: Batch 15-22 data for sterols adsorption to the inactivated secondary s ludge Batch-15 Control 0 mg/L MLSS Campe B-Sito B-Sitosta Campe B-Sito B-Sitosta Time (h) (pg/L) (M9/L) (M9/L) Time (h) (C/Ci) (C/Ci) (C/Ci) 0.0 860.40 3749.53 2517.38 0.0 1.00 1.00 1.00 1.0 669.20 3416.24 2181.73 1.0 0.78 0.91 0.87 2.0 659.58 3371.88 2191.51 2.0 0.77 0.90 0.87 6.0 677.74 3327.75 2059.66 6.0 0.79 0.89 0.82 12.5 653.27 3197.52 1897.96 12.5 0.76 0.85 0.75 24.0 837.92 3652.64 2370.55 24.0 0.97 0.97 0.94 50.0 856.80 3521.03 2414.88 50.0 1.00 0.94 0.96 102.0 823.45 3615.68 2290.77 102.0 0.96 0.96 0.91 170.0 815.69 3618.85 2324.12 170.0 0.95 0.97 0.92 264.0 819.10 3630.83 2330.87 264.0 0.95 0.97 0.93 Batch-16 10 mg/L MLSS Campe B-Sito B-Sitosta Campe B-Sito B-Sitosta Time (h) (pg/L) (Mg/L) (Mg/L) Time (h) (C/Ci) (C/Ci) (C/Ci) 0.0 860.40 3749.53 2517.38 0.0 1.00 1.00 1.00 1.0 591.79 3161.59 1789.70 1.0 0.69 0.84 0.71 2.0 612.66 3259.23 1868.73 2.0 0.71 0.87 0.74 6.0 628.34 3161.52 1684.62 6.0 0.73 0.84 0.67 12.5 548.57 3055.03 1586.10 12.5 0.64 0.81 0.63 24.0 530.81 2958.91 1646.01 24.0 0.62 0.79 0.65 50.0 737.22 3422.68 1984.09 50.0 0.86 0.91 0.79 102.0 594.24 3076.27 1598.98 102.0 0.69 0.82 0.64 170.0 595.00 3076.64 1674.79 170.0 0.69 0.82 0.67 264.0 595.20 3079.33 1665.74 264.0 0.69 0.82 0.66 266 9. Appendices 9.5 Appendix E. Adsorption of Plant Sterols Batch-17 20 mg/L MLSS Campe B-Sito B-Sitosta Campe B-Sito B-Sitosta Time (h) (ug/L) (Mg/L) (Mg/L) Time (h) (C/Ci) (C/Ci) (C/Ci) 0.0 860.40 3749.53 2517.38 0.0 1.00 1.00 1.00 1.0 575.04 3046.94 1629.65 1.0 0.67 0.81 0.65 2.0 603.78 3174.04 1800.79 2.0 0.70 0.85 0.72 6.0 575.63 3023.52 1714.79 6.0 0.67 0.81 0.68 12.5 559.25 2825.56 1533.78 12.5 0.65 0.75 0.61 24.0 509.09 2677.51 1421.19 24.0 0.59 0.71 0.56 50.0 702.89 3279.96 1911.20 50.0 0.82 0.87 0.76 102.0 554.10 2785.84 1525.69 102.0 0.64 0.74 0.61 170.0 532.87 2697.82 1420.55 170.0 0.62 0.72 0.56 264.0 524.76 2774.15 1481.81 264.0 0.61 0.74 0.59 Batch-18 40 mg/L MLSS Campe B-Sito B-Sitosta Campe B-Sito B-Sitosta Time (h) (Mg/L) (Mg/L) (Mg/L) Time (h) (C/Ci) (C/Ci) (C/Ci) 0.0 860.40 3749.53 2517.38 0.0 1.00 1.00 1.00 1.0 631.59 3124.14 1831.86 1.0 0.73 0.83 0.73 2.0 634.65 3119.51 1736.44 2.0 0.74 0.83 0.69 6.0 592.65 3090.04 1680.55 6.0 0.69 0.82 0.67 12.5 521.65 2693.73 1545.25 12.5 0.61 0.72 0.61 24.0 496.84 2588.71 1436.76 24.0 0.58 0.69 0.57 50.0 621.98 3098.23 1519.03 50.0 0.72 0.83 0.60 102.0 510.71 2641.27 1415.04 102.0 0.59 0.70 0.56 170.0 500.90 2600.29 1375.96 170.0 0.58 0.69 0.55 264.0 498.50 2526.39 1323.69 264.0 0.58 0.67 0.53 Batch-19 100 mg/L MLSS Campe S-Sito B-Sitosta Campe B-Sito B-Sitosta Time(h) (pg/L) (pg/L) (pg/L) Time (h) (C/Ci) (C/Ci) (C/Ci) O0 860.40 3749.53 2517.38 O i lJO V00 1.00 1.0 610.53 3022.12 1765.25 1.0 0.71 0.81 0.70 267 9 Appendices 9.5 Appendix E. Adsorption of Plant Sterols 2.0 595.60 2943.95 1693.30 2.0 0.69 0.79 0.67 6.0 575.91 2927.93 1658.54 6.0 0.67 0.78 0.66 12.5 548.87 2544.73 1509.36 12.5 0.64 0.68 0.60 24.0 501.38 2433.01 1412.80 24.0 0.58 0.65 0.56 50.0 534.31 2625.55 1377.55 50.0 0.62 0.70 0.55 102.0 467.20 2393.26 1249.52 102.0 0.54 0.64 0.50 170.0 470.75 2461.37 1286.40 170.0 0.55 0.66 0.51 264.0 467.44 2407.11 1257.08 264.0 0.54 0.64 0.50 Batch-20 200 mg/L MLSS Campe B-Sito B-Sitosta Campe B-Sito B-Sitosta Time (h) (ug/L) (ug/L) (pg/L) Time (h) (C/Ci) (C/Ci) (C/Ci) 0.0 860.40 3749.53 2517.38 0.0 1.00 1.00 1.00 1.0 638.73 3202.01 1851.67 1.0 0.74 0.85 0.74 2.0 623.79 3057.85 1770.15 2.0 0.72 0.82 0.70 6.0 569.42 2831.56 1679.27 6.0 0.66 0.76 0.67 12.5 541.81 2563.51 1347.95 12.5 0.63 0.68 0.54 24.0 519.09 2336.73 1315.29 24.0 0.60 0.62 0.52 50.0 469.67 2275.08 1238.86 50.0 0.55 0.61 0.49 102.0 425.52 2234.25 1166.36 102.0 0.49 0.60 0.46 170.0 395.93 2155.89 1129.59 170.0 0.46 0.57 0.45 264.0 402.84 2250.69 1135.41 264.0 0.47 0.60 0.45 Batch-21 500 mg/L MLSS Campe B-Sito B-Sitosta Campe B-Sito B-Sitosta Time (h) (ug/L) (ug/L) (Mg/L) Time (h) (C/Ci) (C/Ci) (C/Ci) 0.0 860.40 3749.53 2517.38 0.0 1.00 1.00 1.00 1.0 569.75 2964.84 1751.07 1.0 0.66 0.79 0.70 2.0 526.15 2686.20 1548.82 2.0 0.61 0.72 0.62 6.0 500.85 2421.09 1434.17 6.0 0.58 0.65 0.57 12.5 483.97 2285.21 1308.63 12.5 0.56 0.61 0.52 24.0 419.49 2083.43 1176.05 24.0 0.49 0.56 0.47 50.0 383.53 1951.07 1057.66 50.0 0.45 0.52 0.42 102.0 338.74 1877.40 958.41 102.0 0.39 0.50 0.38 268 9. Appendices 9.5 Appendix E. Adsorption of Plant Sterols Batch-22 1000 mg/L MLSS Campe B-Sito B-Sitosta Campe B-Sito B-Sitosta Time (h) (ug/L) (M9/L) (Mg/L) Time (h) (C/Ci) (C/Ci) (C/Ci) 0.0 860.40 3749.53 2517.38 0.0 1.00 1.00 1.00 1.5 466.01 2413.59 1410.01 1.0 0.54 0.64 0.56 2.0 382.21 2115.18 1102.57 2.0 0.44 0.56 0.44 6.0 312.36 1767.82 928.25 6.0 0.36 0.47 0.37 12.5 269.96 1597.85 829.77 12.5 0.31 0.43 0.33 24.0 235.16 1374.77 712.87 24.0 0.27 0.37 0.28 50.0 214.27 1274.45 614.08 50.0 0.25 0.34 0.24 102.0 202.72 1192.59 573.04 102.0 0.24 0.32 0.23 170.0 200.68 1073.63 526.40 170.0 0.23 0.29 0.21 E 10. Set 3: Batch 15-22 figures showing sterols adsorption to the inactivated secondary sludge 3 ad 0 M L S S 5 0 0 +• o [, i , j , , , i , , , j i , , i , , , i , , , i , , , [ , , , i , , , j , i , i , , , i , , , i , , , 11, , 0 2 0 4 0 6 0 80 100 120 140 160 180 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 Time (hr) 269 9. Appendices 9.5 Appendix E. Adsorption of Plant Sterols 3 ad 10 MLSS 4,000 3,500 3,000 2,500 A 2,000 4 <S 1,500 •5 W 1,000 500 0 -Campe —a—fi-Sito — A — fi-Sitostak •I 11 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 Time (hr) 3 ad 20 M L S S B[ | | | | I i i B 1 1 j —•—Campe —n— fc-Sito —A—B-Sitosta ; M I M I —A—j -f-f i ' o I ! ! ! i i | ' -" " " i i i "i i i i •....^..•^•.^.".•r-r-z-i:-:-.- L t.. — +*• 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 Time (hr) 270 9. Appendices 9.5 Appendix E. Adsorption of Plant Sterols 3 ad 40 M L S S 4,000 3,500 4 3,000 4 2,500 A 2,000 S 2 1,500 Si to • [ —•—Campe —a—fi-Sito — A — fi-Sitosts V B • k — - A — -- A r - A A — A <> . — • — - • u m m t • — — ' • — • i i i i i i 11 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 Time (hr) 4,000 3 ad 100 M L S S 3,500 4 p. 3,000 "g 2,500 33 19 b c u 2,000 Si, & CO 1,000 500 0 —•—Campe —a—fi-Sito —A—fi-Sitosta 4 i -a • — ^ — A < > ' "''•'III ' • — • — 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 Time (hr) 271 9. Appendices 9.5 Appendix E. Adsorption of Plant Sterols 3 ad 200 MLSS 4,000 -Campe - a — fi-Sito —A—fi-Sitosta ' I I 1 [ I' 2 0 4 0 . 6 0 80 100 120 140 160 180 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 Time (hr) 3_ad 500 MLSS 4,000 , : ; : 1 , ; ; II i | | ] | | | 3,500 -- [ i j I | | i 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 Time (hr) 272 9. Appendices 9.5 Appendix E. Adsorption of Plant Sterols 4 , 0 0 0 3 ad 1000 MLSS 0 2 0 4 0 6 0 80 100 120 140 160 180 2 0 0 2 2 0 2 4 0 2 6 0 2 8 0 Time (hr) 273 9. Appendices 9.5 Appendix E. Adsorption of Plant Sterols E 11. Set 2: L iquid phase sterol concentration variation with inactivated M L S S dose B-Sitosterol 4,000 -I ; ; : : : ; , : : ; , 500 + o T' . . . i . . . . i . i . . j . . , . i i i i , i , . . . i . i i , i , i i i j i i i , j , , , , i , , , , 0 100 200 300 400 500 600 700 800 900 1000 1100 MLSS (mg/L) B-Sitostanol 3,000 | : : : : : : 0 [",.., i , , , , j . . . . i . . . . i . . . . j . . . , i . . . . j I I I , j , , , , j i , , , j , , , , 0 100 200 300 400 500 600 700 800 900 1000 1100 MLSS (mg/L) 274 9. Appendices 9.5 Appendix E. Adsorption of Plant Sterols Campe sterol 1,000 T i ; : : ; ; o r 1 1 1 1 i 1 1 1 1 i 1 1 1 1 i 1 1 • 1 i 1 1 r 1 i 1 ' ' ' i 1 1 1 • I ' ' ' ' i ' ' ' ' i 1 1 ' ' i 1 ' ' ' 1 0 100 200 300 400 500 600 700 800 900 1000 1100 MLSS (mg/L) E 12. Set 2: Sterol equilibrium concentrations, adsorption capacit ies of the inactivated secondary sludge and adsorption isotherms B-Sito S-Sitosta Campe Ce Qe=x/m Ce Qe=x/m Ce Qe=x/m mg/L mg/g mg/L mg/g mg/L mg/g 1.03 3.82 0.49 2.63 0.19 0.95 1.72 5.15 0.80 4.04 0.30 1.71 2.25 8.59 1.14 7.51 0.40 2.89 2.41 14.52 1.26 13.20 0.47 4.53 2.53 31.68 1.32 30.44 0.50 9.65 2.77 49.87 1.48 52.38 0.52 17.38 3.08 68.12 1.67 85.76 0.60 27.12 275 9. Appendices 9.5 Appendix E. Adsorption of Plant Sterols 100.0 j 90.0 ; 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 Ce Sterols mg/L Campe - a — B-Sitosta —©— B-Sito 9. Appendices 9.6 Appendix F. Thesis Writing 9.6 Appendix F. Thesis Writing Completing a Ph.D. is normally a long process, and the initial high motivation of the candidate can drop to moderate as well as low levels more than once during the whole process. Additionally, after all the hard & demanding research work, thesis writing may particularly demand the most concentrated effort. The huge amount of work required as well as the attached expectations along with the normal activities of everyday life can easily make the task overwhelming. The objective of this section is to ease the task, provide help & support to the candidate for getting to the finish line. F 1. Action Strategies 1. START [The Secret of Successful Scholarly Writing] No matter how stuck you are. Learn how to Begin again & again & again ...Start before feeling fully ready & Stop before being fully exhausted, for another easy but early start. 2. DO [Detach from the Destination & Be Present Fully for the Journey] Do your BEST without the fear of the OUTCOME. 3. BREAK INTO 10-20 [Do-able Smaller Tasks] Reframe the "Over-whelming Task" into 10 to 20 'Manageable' Small Steps. Rank them & Start with No.1. Just do one Small Task at a time. 4. CONSTRUCTIVELY PROCRASTINATE [Make a 2-Column High & Low Priority List] Commit a set amount of time for writing e.g. 8 to 12 pm. Start with high-priority items. Eliminate the Urgent. Get the Critical Tasks (rocks) done, others will fall in place, automatically. Attend to the low-priority items in your break. 5. FORCE A DRAFT [Free-Write for 30 min. Without Stopping] Schedule a Time to Write. Sit down & Take a Deep Breath. Review your notes for 15 min. & Free-Write for 30 min. Do not Stop Writing; No editing; No re-reading; No stopping. For necessities write a 'quick note' for yourself but do not let anything break your flow. 6. ASK A FRIEND [Seek Help From a Friend] Write to a friend. Ask specific questions for feedback & Revise your Draft based upon your friends' margin comments. 277 9. Appendices 9.6 Appendix F Thesis Writing 7. GET AN EXPERT'S ADVICE [Your Supervisor & Committee] The experts' advice can save a lot of wasted effort. Read 'Robert Bioce 1994. How writers Journey to Comfort & Fluency: A Psychological Adventure', Praeger Publishers, Westport, Connecticut, USA, or any other good book. 8. GET THAT ATTITUDE [Take a Strong Position] Cheer up! 'You can do it'. Pick a path and follow it. For now, that is the way it is. Tell your audience "What to think" as if you know at this stage. 9. MASTER THE FORM [Know the Acceptable Technical Structure] Master the style(s) acceptable to your department, University or a journal. 10. SUSTAIN A MODEST DAILY SCHEDULE [Day after Day after Day...] Maintain a moderate amount of daily writing & tie it to a positive contingency e.g. at least 30 to 60 min. of writing before checking the news or opening your e-mail. The need to maintain a brief daily session (BDS) cannot be over emphasized. BDSs can help you achieve more with less difficulty & pain (Dr. Boice 1992). 11. GET THE MAIN BODY FIRST [Write the Main Body 1st] Leave the Introduction & Conclusions till after you are done with the Main Body of your chapter, paper or article. 12. JUST DO ITI [Prioritize Your Work & Update Your To-Do List] Continuously ask yourself "Does this activity help me achieve my top priorities?" "Does this activity help me achieve my top priorities?"... 13. JUST SAY NO! [Refrain from over-booking your time] Say No to sidetracking and socializing activities before or during your writing. Continuously ask yourself "Does this activity help me achieve my top priorities?' 14. GET TO THAT TASK [Banish worry thought] Stop worrying & start doing 278 9. Appendices 9.6 Appendix F. Thesis Writing F 2. Further Motivation and Support Words of Wisdom from New Ph.D.'s. (with thanks to Dr. Ben Dean) "A Ph.D. is just a license to learn." You're not expected to know it all. You are expected to understand the current debates in the field, how to ask intelligent questions and how to go about gathering the information needed to answer those questions. In fact, that's what you will be best qualified ("licensed") to do when the whole process is over." Lance Baugh, Ph.D. Don't expect to get any stage of your dissertation done. The lit review will never be done. The data collection will never be done. Ditto for the data analysis, first draft, and even the final polished version. At a certain point on the calendar, just declare that it's good enough and move on to the next stage. It's much easier to come back to a stage if you need to, than to move on to the next stage in the first place, so move on anyway." Judith Levy, Ph.D. "A PhD is not a measure of intelligence, but one of perseverance and determination. It was only by being single-minded and determined not to let this thing beat me that I stuck at it." Barbara A. Kee, Ph.D. Inspirational Quotes "And that man can have nothing but what he strives for, and (the fruit of) his striving will soon come in sight. Then he will be rewarded for (his effort) to the fullest." Quran (53:39 to 53.41) "Seeking knowledge is a duty of every muslim man and woman." Mohammed, The Messenger of Allah "Striving for excellence motivates you; striving for perfection is demoralizing." Harriet Braiker "Effort only fully releases its reward after a person refuses to quit." Napoleon Hill "Challenges are what make life interesting; overcoming them is what makes life meaningful." Joshua J . Marine A dear friend was giving me advice on completing my dissertation. I believe his words were: "beat that thing with a stick until its dead." Judith Levy "Obstacles don't have to stop you. If you run into a wall, don't turn around and give up. Figure out how to climb it, go through it, or work around it." Michael Jordan "There is no perfect time to write. There's only now." Barbara Kingsolver "The secret of success is constancy of purpose." Benjamin Disraeli 279 9. Appendices 9.6 Appendix F. Thesis Writing "I have learned that success is to be measured not so much by the position that one has reached in life as by the obstacles which he has had to overcome while trying to succeed." Booker T. Washington "I am always doing that which I can not do, in order that I may learn how to do it." Pablo Picasso "Start by doing what's necessary, then what's possible, and suddenly you are doing the impossible." Francis of Assisi "Adversity has the effect of eliciting talents, which in prosperous circumstances would have lain dormant." Horace "The beginning of knowledge is the Discovery of something we do not understand." Frank Herbert "New Knowledge is the most valuable commodity on earth. The more truth we have to work with, the Richer we become." Kurt Vonnegut F 3. References & Resources Boice, R. 1994. How Writers Journey to Comfort and Fluency. A Psychological Advanture. Praeger Publishers, Westport, CT, USA. Dean, B. J . 1997-2004. Sample Issues. Archive of past issues. The All-But-Dissertation Survival Guide™, http://www.abdsurvivalauide.org/secret.html. MentorCoach.com™, http://www.ecoach.com. 4400 East West Highway, Bethesda, MD, USA. Islamic Server of MSA-USC http://www.usc.edu/dept/MSA/reference/reference.html Northey, M., and Proctor, M. 1998. Writer's Choice: A Portable Guide for Canadian Writers. Prentice Hall Canada Inc., Scarborough, Ontario. Phillips, E. M., and Pugh, D. S. 1994. How to Get a Ph.D. : A handbook for Students and their Supervisors. 2 n d Edition. Open University Press, Buckingham, UK. Questions and Answers about Islam, www.islamaa.com The Holy Quran, http://quran.al-islam.com/ Traditions of the Prophet Muhammad, http://hadith.al-islam.com/Bayan/ 280 

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