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Removal of colour from secondary treated whole mill kraft effluent using dead aspergillus niger as a… Grainger, Sarah 2006

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R E M O V A L OF C O L O U R F R O M S E C O N D A R Y T R E A T E D W H O L E M I L L K R A F T E F F L U E N T U S I N G D E A D ASPERGILLUS  NIGER  A BIOSORBENT  by S A R A H E. G R A I N G E R B.A.Sc, University of Regina, 2001  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERS OF APPLIED SCIENCE  in  THE F A C U L T Y OF G R A D U A T E STUDIES  (Civil Engineering)  THE UNIVERSITY OF BRITISH COLUMBIA AUGUST 2006  © Sarah E. Grainger, 2006  AS  ABSTRACT The pulp and paper industry is widespread in Canada and many other countries. The effluent from this industry is generally highly coloured and typically measures are not taken to prevent or remove this colour due to prohibitory cost and, presently, lack of universal regulation. However, this does not preclude coloured effluents from impacting the public and the environment.  Treatment of pulp mill effluent colour has been approached using a number of different treatment technologies, including lime, membrane filtration, oxidants and adsorption. Unfortunately, biological treatment systems commonly employed to treat pulp and paper effluents are not effective in treatment of colour. However, biological treatment using live fungal biomass, generally white rot fungi, has proven to be effective at pulp mill effluent colour removal. Additionally, biosorption treatment using any biological matter in live or dead form, has been successful in the removal of metals, textile dyes and humic acids from water. Nonetheless, no research has been reported on the use of dead fungal biomass on pulp mill effluent colour. As such, the present study researches the use of dead Aspergillus niger biomass for the treatment of pulp mill effluent colour.  Using a batch test system approach, the present research addressed effluent characterization, pretreatment, effluent pH, biomass washing, mixing, biological inhibition, kinetic rate, isotherm, temperature, molecular weight fraction removed and practical application studies. From these studies it has been determined that autoclave-only pretreatment and initial effluent pH of 4 and 8 provided optimum colour removal. Under biologically inhibited conditions, maximum removal occurred in the first hour of biomass contact with the effluent and the kinetic models by Lagergren (1898) and Ho et al. (1996) roughly described the kinetic rate. The maximum colour removal was over 900 TCU, with a biomass doses in the range of 13-20 g/L. The equilibrium isotherms of the study fit the BET model well, which indicated, with the support of other results of the current study, that this biosorption was predominately due to physical mechanisms. In addition, the application of the biomass in a batch activated sludge process did remove colour.  T A B L E OF CONTENTS  Abstract  ii  Table of Contents  iii  List of Tables  vi  List of Figures  xi  List of Illustrations  xiii  Acknowledgements  xiv  1. Introduction 1.1  Colour  1 1  1.1.1 What is colour?  1  1.1.2 Origins of chromophores in water  2  1.2 Pulp Mill Effluents and Colour  3  1.2.1 Why remove pulp mill effluent colour?  3  1.2.2 Regulation of pulp mill effluent colour  5  2. Background  7  2.1 Colour  7  2.1.1 Color measurement of liquid  7  2.1.2 Parameters affecting colour measurement  8  2.2 Pulp Mills and Their Effluents  8  2.2.1 Pulping  8  2.2.2 Bleaching  9  2.2.3 Effluent management  10  2.2.4 Origins of colour in effluents  11  2.2.5 Lignin  12  2.3 Current Colour Removal Technologies  12  2.3.1 Chemical treatment using aluminum and ferric salts  12  2.3.2 Chemical treatment using lime  13  2.3.3 Other chemicals used to enhance settling  14  2.3.4 Chemical oxidation  14  2.3.5 Advanced oxidation processes  15  2.3.6 Biological treatment  15 iii  2.3.7 Membrane treatment  15  2.3.8 Resin separation and ion exchange processes  16  2.3.9 Activated carbon adsorption  17  2.4 Mechanisms of Adsorption  18  2.4.1 Basics of adsorption  18  2.4.2 Adsorption rates  19  2.4.3 Adsorption isotherms  20  2.4.4 Adsorption of heterogeneous mixtures  22  2.5 Fungal Biosorption  22  2.5.1 Use of biosorption  22  2.5.2 Fungal biosorption  23  2.5.3 Factors affecting dead fungal biomass biosorption  25  2.5.4 Aspergillus niger  26  2.5 Background Conclusions  27  3. Objectives  28  4. Methodologies  29  4.1 General  29  4.1.1 Pulp mill effluents  29  4.1.2 Methods and materials  30  4.1.3 QA/QC  30  4.2 Effluent Characterization and Biomass Production  32  4.2.1 Effluent characterization parameters  32  4.2.2 Molecular weight distribution  32  4.2.3 Biomass production  32  4.3 Batch Biosorption Study  34  4.3.1 Pre-treatment mini-study  35  4.3.2 Effluent pH mini-study  36  4.3.3 Biomass washing mini-study  37  4.3.4 Mixing mini-study  37  4.3.5 Kinetic mini-study  39  4.3.6. Biological inhibition mini-study  40  4.3.7 Isotherm mini-study  41  4.3.8 Removed fraction mini-study  41 iv  4.3.9 Temperature mini-study 4.4 Practical Application Study 5. Results and Discussion 5.1 Effluent Characterization and Biomass Production  41 42 44 44  5.1.1 Effluent characterization  44  5.1.2 Molecular weight distribution  45  5.1.3 Biomass production  48  5.2 Batch Biosorption Study  51  5.2.1 Optimum pretreatment mini-study  51  5.2.2 Optimum pH mini-study  55  5.2.3 Biomass washing mini-study  56  5.2.4 Mixing mini-study.  59  5.2.5 Kinetic mini-study part 1  63  5.2.6 Inhibition mini-study  64  5.2.7 Kinetic mini-study part 2  65  5.2.8 Removed fraction mini-study  77  5.2.9 Temperature mini-study  80  5.3 Practical Application Study  81  6. Conclusions and Further Research Recommendations  83  7. References  85  Appendix 1. Calculations  92  Appendix 2. Effluent Characterization Data  95  Appendix 3. Molecular Weight Distribution Data  96  Appendix 4. Pretreatment Mini-Study Data  98  Appendix 5. pH Mini-Study Data  102  Appendix 6. Biomass Washing Mini-Study  104  Appendix 7. Mixing Mini-Study  Ill  Appendix 8. Kinetic Mini-Study Part 1  123  Appendix 9. Inhibition Mini-Study and Kinetic Mini-Study Part 2  135  Appendix 10. Isotherm Mini-Study  153  Appendix 11. Removed Fraction Mini-Study  158  Appendix 12. Temperature Mini-Study  162  Appendix 13. Practical Application Study  165 v  LIST OF TABLES Table 1. Standard methods for colour measurement  7  Table 2. Pulping processes  9  Table 3. Common bleaching stages  10  Table 4. Membrane filters characteristics  16  Table 5. Definitions of adsorption terms  18  Table 6. BDDT isotherm classifications  21  Table 7. Comparison of adsorption characteristics of life states  25  Table 8. Factors affecting biosorption  26  Table 9. Methods and materials  31  Table 10. Parameters analyzed and rationale  32  Table 11. Possible factors impacting mixing efficiency  38  Table 12. Possible experimental runs  38  Table 13. Biological inhibited test sets  40  Table 14. Results of whole mill treated effluent characterization  44  Table 15. Results of non-linear estimation for colour removal for Lagergren K and Ho et al. k, biosorption rate models, using STATISTICA®  68  Table 16. Isotherm mini-study results compared to Langmuir, Freundlich and BET models  74  Table 17. Practical application of study results  82  Table A . l Worked example values  92  Table A.2 Worked example values  93  Table A.3 Worked example values  93  Table A. 5 Electrical conductivity measurement on raw Western Pulp effluent  95  Table A.6 Chloride measurement on raw Western Pulp effluent  95  Table A.8 Chloride measurement on raw Howe Sound effluent  95  Table A. 9 Colour measurement for the molecular weight distribution on Western Pulp effluent96 Table A. 10 TOC measurement for the molecular weight distribution on Western Pulp effluent. 96 Table A . l 1 Colour measurement for the molecular weight distribution on Howe Sound effluent 97 Table A. 12 TOC measurement for the molecular weight distribution on Howe Sound effluent.97 Table A. 13 Colour measurement at 465 nm for Pretreatment mini-study on Western Pulp effluent  98 vi  Table A. 14 Colour measurement at 400 nm for Pretreatment mini-study on Western Pulp effluent  99  :  Table A. 15 COD measurement at 600 nm for Pretreatment mini-study on Western Pulp effluent 100 Table A. 16 pH measurement for Pretreatment mini-study results on Western Pulp effluent  101  Table A. 17 Colour measurement for pH mini-study on Western Pulp effluent  102  Table A. 18 COD measurement for pH mini-study results on Western Pulp effluent  103  Table A. 19 Colour measurement for original biomass wash method on de-ionized water  104  Table A. 20 COD measurement for original biomass wash method on de-ionized water  104  Table A.21 Test run 1 colour measurement for "after autoclave" biomass wash method on deionized water  105  Table A.22 Test run 1 COD measurement for "after autoclave" wash method on de-ionized water  106  Table A.23 Test run 2 colour measurement for "after autoclave" biomass wash method on deionized water  107  Table A.24 Test run 2 COD measurement for "after autoclave" wash method on de-ionized water  108  Table A.25 Test run 1 colour measurement for "double wash" biomass wash method on deionized water  108  Table A.26 Test run 1 COD measurement for "double wash" biomass wash method on deionized water  109  Table A.27 Test run 2 colour measurement for "double wash" biomass wash method on deionized water  109  Table A.28 Test run 2 COD measurement for "double wash" biomass wash method on deionized water  110  Table A.29 Test run 1 colour measurement mixing study 300 mL flask at 125 rpm  Ill  Table A.30 Test run 1 COD measurement mixing study 300 mL flask at 125 rpm  112  Table A.31 Test run 1 DOC measurement mixing study 300 mL flask at 125 rpm  113  Table A.32 Test run 2 colour measurement mixing study 300 mL flask at 125 rpm  114  Table A.33 Test run 2 COD measurement mixing study 300 mL flask at 125 rpm  115  Table A.34 Test run 2 DOC measurement mixing study 300 mL flask at 125 rpm  116  Table A. 3 5 Test run 1 colour measurement mixing study 300 mL flask at 200 rpm  117  Table A.36 Test run 1 COD measurement mixing study 300 mL flask at 200 rpm  118 vii  Table A.37 Test run 1 DOC measurement mixing study 300 mL flask at 200 rpm  119  Table A.38 Test run 2 colour measurement mixing study 300 mL flask at 200 rpm  120  Table A. 3 9 Test run 2 COD measurement mixing study 300 mL flask at 200 rpm  121  Table A.40 Test run 2 DOC measurement mixing study 300 mL flask at 200 rpm  122  Table A.41 Test run 1 colour measurement kinetic mini-study on Western Pulp effluent  123  Table A.42 Test run 1 COD measurement kinetic mini-study on Western Pulp effluent  124  Table A.43 Test run 1 DOC measurement kinetic mini-study on Western Pulp effluent  125  Table A.44 Test run 1 pH measurement kinetic mini-study on Western Pulp effluent  126  Table A.45 Test run 2 colour measurement kinetic mini-study on Western Pulp effluent  127  Table A.46 Test run 2 COD measurement kinetic mini-study on Western Pulp effluent  128  Table A.47 Test run 2 DOC measurement kinetic mini-study on Western Pulp effluent  129  Table A.48 Test run 2 pH measurement kinetic mini-study on Western Pulp effluent  130  Table A.49 Colour measurement kinetic mini-study on Howe Sound effluent  131  Table A.50 COD measurement kinetic mini-study on Howe Sound effluent  132  Table A.51 DOC measurement kinetic mini-study on Howe Sound effluent  133  Table A.52 pH measurement kinetic mini-study on Howe Sound effluent  134  Table A.53 Test run 1 colour measurement kinetic rate at 4°C on Western Pulp effluent  135  Table A.54 Test run 1 COD measurement kinetic rate at 4°C on Western Pulp effluent  136  Table A.55 Test run 1 DOC measurement kinetic rate at 4°C on Western Pulp effluent  137  Table A.56 Test run 1 pH measurement kinetic rate at 4°C on Western Pulp effluent  137  Table A.57 Test run 2 colour measurement kinetic rate at 4°C on Western Pulp effluent  138  Table A.58 Test run 2 COD measurement kinetic rate at 4°C on Western Pulp effluent  139  Table A.59 Test run 2 DOC measurement kinetic rate at 4°C on Western Pulp effluent  140  Table A.60 Test run 2 pH measurement kinetic rate at 4°C on Western Pulp effluent  140  Table A.61 Colour measurement kinetic rate at 4°C on Howe Sound effluent  141  Table A.62 COD measurement kinetic rate at 4°C on Howe Sound effluent  142  Table A.63 DOC measurement kinetic rate at 4°C on Howe Sound effluent  143  Table A.64 pH measurement kinetic rate at 4°C on Howe Sound effluent  143  Table A.65 Colour measurement kinetic rate with NaN3 addition at room temperature on Western Pulp effluent  144  Table A. 66 COD measurement kinetic rate with NaN3 addition at room temperature on Western Pulp effluent  145 viii  Table A.67 DOC measurement kinetic rate with NaN addition at room temperature on Western 3  Pulp effluent  146  Table A.68 pH measurement kinetic rate with NaN3 addition at room temperature on Western Pulp effluent  146  Table A. 69 Colour measurement kinetic rate with NaN addition at room temperature on Howe 3  Sound effluent  147  Table A. 70 COD measurement kinetic rate with NaN addition at room temperature on Howe 3  Sound effluent  148  Table A.71 DOC measurement kinetic rate with NaN addition at room temperature on Howe 3  Sound effluent  149  Table A. 72 pH measurement kinetic rate with NaN addition at room temperature on Howe 3  Sound effluent  149  Table A.73 Colour measurement kinetic rate with NaF addition at room temperature on Western Pulp effluent  150  Table A.74 COD measurement kinetic rate with NaF addition at room temperature on Western Pulp effluent  151  Table A.75 DOC measurement kinetic rate with NaF addition at room temperature on Western Pulp effluent  152  Table A.76 pH measurement kinetic rate with NaN addition at room temperature on Howe 3  Sound effluent  152  Table A.77 Colour measurement equilibrium isotherms with NaN addition at room temperature 3  on Western Pulp effluent  153  Table A.78 COD measurement equilibrium isotherms with NaN addition at room temperature 3  on Western Pulp effluent  154  Table A.79 DOC measurement equilibrium isotherms with NaN addition at room temperature 3  on Western Pulp effluent  154  Table A.80 pH measurement equilibrium isotherms with NaN addition at room temperature on 3  Western Pulp effluent  155  Table A.81 Colour measurement equilibrium isotherms with NaN addition at room temperature 3  on Howe Sound effluent  155  Table A.82 COD measurement equilibrium isotherms with NaN addition at room temperature 3  on Howe Sound effluent  156  ix  Table A.83 DOC measurement equilibrium isotherms with NaN addition at room temperature 3  on Howe Sound effluent  156  Table A.84 pH measurement equilibrium isotherms with NaN addition at room temperature on 3  Howe Sound effluent  157  Table A. 85 Colour measurement of lg dose of biomass for removed fraction mini-study on Western Pulp effluent  158  Table A. 86 TOC measurement of lg dose of biomass for removed fraction mini-study on Western Pulp effluent  158  Table A. 87 Colour measurement of blank for removed fraction mini-study on Western Pulp effluent  159  Table A. 88 COD measurement of blank for removed fraction mini-study on Western Pulp effluent  159  Table A. 89 Colour measurement of lg dose of biomass for removed fraction mini-study on Howe Sound effluent  160  Table A.90 TOC measurement of lg dose of biomass for removed fraction mini-study on Howe Sound effluent  160  Table A.91 Colour measurement of blank for removed fraction mini-study on Western Pulp effluent  161  Table A.92 COD measurement of blank for removed fraction mini-study on Western Pulp effluent  161  Table A.93 Colour measurement equilibrium isotherms with NaN addition at 35°C on Western 3  Pulp effluent  162  Table A.94 COD measurement equilibrium isotherms with NaN addition at 35°C on Western 3  Pulp effluent  163  Table A.95 DOC measurement equilibrium isotherms with NaN addition at 35°C on Western 3  Pulp effluent  164  Table A.96 pH measurement equilibrium isotherms with NaN addition at 35°C on Western Pulp 3  effluent  164  Table A.97 Colour measurement of batch activated sludge testing on Howe Sound effluent.... 165 Table A.98 pH measurement of batch activated sludge testing on Howe Sound effluent  165  Table A.99 TOC measurement of batch activated sludge testing on Howe Sound effluent  166  Table A. 100 TSS measurement of batch activated sludge testing for Howe Sound effluent  167  Table A. 101 BOD measurement of batch activated sludge testing for Howe Sound effluent.... 167  LIST OF FIGURES Figure 1. Molecular weight distribution by colour  46  Figure 2. Molecular weight distribution by total organic carbon  46  Figure 3. Colour removal efficiencies from WP effluent at 465 nm for various pretreatments after 48 h of contact  52  Figure 4. Colour removal efficiencies from WP effluent at 400 nm for various pretreatments after 48 h of contact  52  Figure 5. Effect of initial effluent pH on biosorption  55  Figure 6. Biomass wash study results where biomass was in contact with distilled-deionized water  57  Figure 7. Mixing mini-study comparing the colour removal efficiency of the biomass with 48 h contact time using 125 mL and 300 mL flask sizes  60  Figure 8. Mixing mini-study comparing the colour removal efficiency of the biomass with 48 h contact time using 125 rpm and 200 rpm shaker speed  61  Figure 9. Total COD results of the sample from the mixing study using biomass doses of 0.2 g, 0.5 g and 0.8 g at 125 rpm and 200 rpm  62  Figure 10. Kinetic study of Western Pulp and Howe Sound effluents at a dose of 0.2 g and time interval up to 52 h  63  Figure 11. Biologically inhibited kinetic study of Western Pulp effluents at a dose of 0.2 g and time interval up to 52 h  64  Figure 12. Biosorption of colour from Western Pulp effluent at various specified time intervals at 4°C and with addition of NaN  66  3  Figure 13. Biosorption of colour from Howe Sound effluent at various specified time intervals at 4°C and with addition of NaN  66  3  Figure 14. Kinetic Study: Lagergren, Ho et al. and measured data - Western Pulp effluent at 4°C ;  69  Figure 15. Kinetic Study: Lagergren, Ho et al. and measured data - Western Pulp effluent with NaN  70  3  Figure 16. Kinetic Study: Lagergren, Ho et al. and measured data - Howe Sound 4°C Figure 17. Kinetic Study: Lagergren, Ho et al. and measured data - Howe Sound NaN  70 3  71  Figure 18. Isotherm Study: Colour removal of both effluents with NaN addition at 32 h at room 3  temperature  72 xi  Figure 19. Isotherm Study: DOC removal of both effluents with NaN addition at 32 h at room 3  temperature  72  Figure 20. Kinetic Study: BET and measured data - Western Pulp effluent with NaN at room 3  temperature  76  Figure 21. Kinetic Study: BET and measured data - Howe Sound effluent with NaN at room 3  temperature  76  Figure 22. Molecular weight distribution of colour for both effluents after treatment  78  Figure 23. Molecular weight distribution of TOC for both effluents after treatment  79  Figure 24. Colour removal at different temperatures  80  Figure 25. Colour removal in practical application study samples using three biomass dose rates 82  xii  LIST OF ILLUSTRATIONS  Illustration 1. Electromagnetic spectrum  2  Illustration 2. Photograph of A. niger agar plate  48  Illustration 3. Microscope view of conidia at the end of broken conidiphores (lOOx)  49  Illustration 4. Biomass in liquid medium just prior to harvesting  50  Illustration 5. Inactivated biomass smear plate  51  xiii  ACKNOWLEDGEMENTS This thesis has been accomplished with the assistance of numerous contributors. Dr. George Fu has provided this simulating topic for my research as well as his experience and knowledge in this field. As well, Dr. Eric Hall's extensive experience in research, wide-based knowledge and patience has made him an indispensable co-supervisor. Throughout my laboratory work the assistance and guidance of Susan Harper and Paula Parkinson has been vital and is greatly appreciated. Further, Howe Sound Pulp and Paper Ltd. Partnership has been very generous in allowing me to use their effluent and laboratory facilities as well as the knowledge and expertise of their staff, specifically, Siew Sim, whose assistance was essential. Appreciation is also extended to Western Pulp Partnership Ltd., for use of their effluent, and their employee Jeanne Taylor, for providing me with information and her time. Lastly, this research was funded by the NSERC grant program, this funding was greatly valued.  xiv  1. INTRODUCTION  1.1 Colour 1.1.1 What is colour? Colour is a part of our daily lives. Most of world's population observes visual colour. Even though it is common to have colour vision deficiencies, it is very rare for a person to see no colour at all (The Canadian Association of Optometrists, 2005). An object's colour is frequently used as a differentiation tool, it can give beauty to an object and colour has been found to affect a person's emotional and physical condition (Nemcsics, 1993). Defining visual colour, however, is complex. The Merriam-Webster's Dictionary defines visual colour as "the aspect of objects and light sources that may be described in terms of hue, lightness, and saturation for objects and hue, brightness, and saturation for light sources" (Merriam-Webster Inc., 2004). The Encyclopedia Britannica gives the following explanations of hue, brightness (lightness), and saturation. "Hue refers to dominant wavelengths. Brightness refers to the intensity or degree of shading. Saturation pertains to purity, or the amount of white light mixed with a hue." (2005 Encyclopedia Britannica Inc., 2005). These definitions and explanations clarify what you see or how to qualitatively describe visual colour, however, they do not explain what colour is.  Matter appears coloured when visible wavelengths are absorbed. For example, an object that looks red actually is absorbing all of the wavelengths in hues other than red, such as green and blue, and is reflecting the wavelengths that are red in hue. As such, the observation of colour requires electromagnetic wavelengths, in the visible region, approximately between 380 -750 nanometers (nm), to reach the observer. In Illustration 1, the electromagnetic spectrum and the visible light spectrum are displayed. As seen in Illustration 1, red hue wavelengths are the longest visible wavelengths (lower energy) and blue hue wavelengths are the shortest visible wavelengths (higher energy). Therefore, in order for water to appear coloured, matter in the water must absorb electromagnetic wavelengths in the visible range (Kuppers, 1973).  1  Wavelength (nanometers) 0.01  1  100  10  4  10  6  10  8  10  G  400  500 600 700 Visible Region  Illustration 1. Electromagnetic spectrum Source: (ACEPT W3 Group, Department of Physics and Astronomy, Arizona State University, 1999).  Health Canada describes colour in drinking water to be the result of a number of circumstances, " . . . absorption of certain wavelengths of normal "white" light by dissolved or colloidally dispersed substances, by fluorescence in the visible wavelength region from substances that absorb "white" or ultraviolet light, by the presence of coloured suspended solids, and by the preferential scattering of short wavelengths of light by the smallest suspended particles." (Health Canada, 1995). This definition focuses heavily on the particles in the water. Particles can either themselves be chromophores or be attached to chromophores. Chromophore, a word originating from the Greek for "colour bringer", is a chemical group or arrangement that imparts colour (Nassau etal., 1998).  1.1.2 Origins of chromophores in water There are multiple possible origins of colour in water bodies. A l l are a result of chromophores absorbing certain visible wavelengths. Generally, chromophoric molecules either contain metals or are organic substances (Health Canada, 1995).  There are many sources of naturally-occurring chromophores in water. For example, colourimparting metals such as iron, manganese, and copper can be introduced into water from contact with geological formations, such as limestone, and colour-imparting organics can be derived from the degradation of organic matter in the environment, such as natural vegetation in soil runoff (Health Canada, 1995; Health and Welfare Canada, 1992). In addition, sources that do not occur naturally, such as industrial discharges, can also introduce colour into water bodies. The 2  most common coloured water-producing industries are the pulp and paper and textile industries (Health Canada, 1995). The colour imparting substances in pulp and paper mill effluents are the focus of this study.  1.2 Pulp Mill Effluents and Colour  In Canada, the pulp and paper industry extends throughout the country. In 2004, pulp and paper sales revenues were over $21 billion and production of paper and pulp products reached 30,345 thousand tonnes (Pulp and Paper Products Council, 2006). Highly coloured effluents are produced from some pulping and bleaching processes, and it is important to understand the basics of these processes.  Although nearly all pulp mills employ treatment systems, the more commonly used biological treatment systems do not remove colour effectively since effluent colour is predominately recalcitrant to biodegradation (Kemeny and Banerjee, 1997). Therefore, the treated effluent from most mills is highly coloured. There are a number of proven colour removal technologies, however, it is uncommon to see these implemented unless required by regulation due to their prohibitory cost (Springer, 1986).  1.2.1 Why remove pulp mill effluent colour? One of the most dominant issues regarding colour in pulp mill effluents are the aesthetic impacts on the receiving water bodies. Discharges that are made to receiving water bodies, particularly at low dilution ratios, result in the receiving water appearing noticeably coloured. This detracts from visual appeal and recreational value. In addition perception set, i.e. "seeing what is expected to be seen", can impact public opinion about mill effluents (Smith et al., 1995). For example, the general feeling towards pulp mills is that they are large sources of pollution, therefore, the highly coloured water can trigger feelings that discharges are more toxic than in reality. In addition, even though regulations are in place regarding biochemical oxygen demand (BOD), suspended solids and other compounds relating to toxicity, the predominant treatment technique, activated sludge-type technology, does not remove colour effectively. This may give the impression to the unknowing eye that the effluent has not been treated well enough.  3  In addition, in situations where there are downstream water treatment systems, particularly for drinking water, colour intensifies the treatment required and thus displaces responsibility of treatment to the public water utility owner, typically a municipal government, instead of the producer of the effluent (Springer, 1986). In these scenarios, coloured water can pose aesthetic as well as other contamination problems.  Pulp mill effluent colour can affect aquatic systems. A very important issue is light reduction, which can affect the aquatic environment in a variety of ways. The most apparent impact of light reduction is on primary species production. There is a defined relationship between light intensity and algae growth. This not only impacts the primary species but also ripples through the entire food chain to large aquatic life forms such as fish (Rush and Shannon, 1976). Strickland noted that light is a grazing stimulus for zooplankton and is used by fish and other aquatic animals to find food (Strickland, 1958). It has also been suggested that trout avoid habitat with high suspended solids and coloured wastewater. As well, photodegradation is a naturally-occurring process that can break down undesirable compounds, such as pesticides. This process is beneficial to the environment when the by-products are not more toxic than the parent compound (Rush and Shannon, 1976). As such, light reduction is disadvantageous to this process.  Generally, studies have shown that the colour-imparting substances in effluents have no toxic effects. However, there have been a few studies that have isolated toxic substances in pulp mill effluents that are chromophores (Betts et al., 1971; Das et al., 1969). That being said, these findings have not resulted in changes to the overall consensus that colour is not toxic, since it is not consistent for general effluent types and removal of the toxic compounds can be accomplished without colour being removed (Rush and Shannon, 1976).  Nonetheless, colour-imparting substances can be toxic indirectly. Typically colour-imparting substances in the pulp mill effluent are organic compounds that have the capability to form complexes with metals (Rush and Shannon, 1976). These complexes can have impacts in two ways: 1. they can remove metals from the water body that organisms use for normal metabolism, and alternatively,  4  2. they can have direct inhibitory effects on some lower level organisms in the food chain (Springer, 1986).  In addition, lignin-derived colour can be an indicator for the presence of potentially inhibiting compounds and may even directly inhibit lower food chain organisms. Lastly, it has been stated that long term BOD, in the range of 20-100 days, can be exerted by colour bodies (Springer, 1986).  1.2.2 Regulation ofpulp mill effluent colour In Canada, both provincial and federal governments are involved with regulating pulp mill effluent discharges. The Canadian government has set discharge quality and monitoring requirements with the Pulp and Paper Effluent Regulations. These regulations under the Fisheries Act address BOD, total suspended solids (TSS), and toxicity (Government of Canada, Ministry of Fisheries and Oceans, 1992). Furthermore, the provinces give permission for mills to discharge under their various environmental protection acts. In some cases, the provinces have specific regulations regarding pulp mill effluent discharges, such as in British Columbia, Pulp Mill and Pulp and Paper Mill Liquid Effluent Control Regulations, and Ontario, Effluent Monitoring and Effluent Limits - Pulp and Paper Sector (Government of British Columbia, 1990; Government of Ontario, 1993). Provincial effluent quality requirements are generally equal to or more stringent than the federal Pulp and Paper Effluent Regulations.  Although colour limitations are not prescribed by the Pulp and Paper Effluent Regulations, numerous mills, particularly those that discharge to rivers or other water bodies with low dilution ratios, have colour requirements. Permits issued by the Province of British Columbia that do not require specified colour limits state: "Should colour, attributable to the effluent, become objectionable in the receiving environment, the permittee shall implement measures to remove colour forming constituents from the effluent." (Government of British Columbia, Ministry of Water, Land and Air Protection, 2005).  Alberta Environment has taken a more forward approach with color limits and is now imposing colour limits. In the report, Technology Based Standards for Pulp and Paper Wastewater Releases, 2005, the following was stated. 5  "Based on a review of the current performance values for Alberta mills, other Canadian mills, and top performing mills in the U.S. and to allow sufficient flexibility for plant operations during shutdown, start-up and upset conditions, Alberta's standards for colour are a monthly average of 50 kg/ADt (air dried tonne) and a daily maximum of 100 kg/ADt for both new and existing mills. The values may be applied at approval renewal for existing pulp and paper mills, and applied directly to any new mills." (Alberta Environment, 2005).  The United States has a similar regulatory framework to that of Canada whereby the federal Environmental Protection Agency sets nation-wide standards. Individual states, however, have the ability to impose stricter effluent quality requirements (United States Environmental Protection Agency, 2002). Also, like Canada, the United States Environmental Protection Agency (US EPA) does not have general colour requirements. France does have a set limit for colour at 100 mg Pt-Co/L (milligram Platinum Cobalt per litre) for any effluent released into watercourses. Italian legislation for industrial effluents requires colour to be non-visible when dilution is 1:20, and similarly, Spain requires non-visibility at set dilution ratios (TAPPI, 1998).  6  2. BACKGROUND  2.1 Colour  2.1.1 Color measurement of liquid Measurement o f colour in water is typically accomplished in one of two ways, by visual comparison or through values obtained by spectrophotometric methods. Table 1 details the colour measurement methods. Table 1. Standard methods for colour measurement Test  Method  Standards  Nessler Tubes Standard Method 2120B  Visual comparison between tubes of the sample and incremental doses of platinum cobalt Visual comparison with vials o f sample and tinted glass covered distilled water Spectrophotometric measurement of transmittance at 10-30 specified wavelengths, filtered sample Special tristimulus filters and transmittance measured Extension o f 2120D, calculation different, use standards  Platinum-cobalt 1 mg/L equals 1 C U , distilled water blank  Colour Wheel Standard Method 2120B Spectrophotometric Method - Standard Method 2120C  Tristimulus Filter Method - Standard Method 2120D A D M I Tristimulus Filter Method Standard Method 2120 E  Tinted glass corresponding to doses o f platinum-cobalt Distilled water blank, percent transmittance measured Distilled water blank, percent transmittance measured Platinum cobalt standards  Use  Expression o f Results  Naturally coloured water, water in a hue of platinumcobalt  Colour units (CU), true (TCU)or apparent (ACU)  Water o f any colour, particularly industrial effluents  Dominant wavelength, hue, luminance, and purity  (American Public Health Association et. al., 1998)  For the visual comparison method, there are two types of colour measurement, true and apparent colour. Apparent colour is measured with the liquid "as-is", whereas true colour is measured  7  after filtration or centrifugation. The rationale for this distinction is detailed in Section 2.1.2 regarding interference of turbidity in colour measurement.  2.1.2 Parameters affecting colour measurement In the determination of colour, two parameters can alter measurement; turbidity and pH.  Turbidity is the only parameter that interferes with colour measurement. The presence of turbidity will interfere with colour readings since turbidity scatters light, thereby altering absorbance. Turbidity interference is an issue for both visual and spectrophotometric measurement. It is due to this interference that there are two versions of colour measurement, true colour and apparent colour (American Public Health Association et. al., 1998).  The other important parameter in colour measurement is pH. Although pH does not interfere with measurement, it greatly changes the intensity of colour. As the pH of a solution changes, the chemical make up of the solution changes. Chemical structures are altered by the abundance or lack of hydrogen ions in the solution. This affects the formation of molecules or compounds that were not previously in the solution. These changes can, in turn, alter the colour of the solution. Therefore, when measuring colour, pH should be altered to a standard value. In Standard Methods a pH value of 7.6 is standard (American Public Health Association, et. al., 1998).  2.2 Pulp Mills and Their Effluents  2.2.1 Pulping In the creation of paper products, pulp is the fundamental raw material. Wood pulping processes de-fiber and de-knot wood chips in order to obtain the fibrous cellulose material. Pulping can be accomplished by mechanical and/or chemical means. Table 2 describes mechanical, semichemical, and chemical pulping.  8  Table 2 . Pulping processes Type  Principle  Mechanical  Abrasion and friction, fibers fragmented.  Semichemical  Chips are "cooked" then mechanically separated. Chemicals "cook" chips, meaning cellulose fibers are released or separated from other wood components particularly lignin.  Chemical  Predominate Process ThermoMechanical Pulping, chips are steamed and then ground by refiners. Neutral Sulfite Semi-chemical (NSSC) process. Kraft process, "cooks" chips in a solution of sodium hydroxide and sodium sulfate to dissolve lignin.  Notes Softwoods, high pulp yields, poor colour impermanence and fiber strength.  Mainly hardwoods, high pulp yields. L o w raw wood quality requirements, short cooking times, well researched liquor recovery including byproduct recovery, and excellent pulp strength. However, odour, low yield and highly coloured pulp are problematic.  (Fengel and Wegner, 1984; Mechanical Pulping Technical Committee, Pulp and Paper Technical Association of Canada; United States Environmental Protection Agency, 2002) 2.2.2 Bleaching After the pulping process, regardless of method, the pulp has colour. In a large number of cases, white paper products are desired. Therefore, bleaching is a critical step in the pulp and paper process and most problematic in terms of introducing colour to effluents. Bleaching can be performed using lignin-removing or lignin-preserving bleaching procedures and these are selected based on the pulping technique utilized and requirements for the end use of the product (Sjostrom, 1981).  Kraft pulping is almost always followed by lignin-removing bleaching. Lignin-removing bleaching processes involve oxidization and alkaline extraction of lignin compounds. Oxidization breaks down lignin into water- and alkaline- soluble degradation products, and in some cases brightens the pulp (Sjostrom, 1981). The purpose of alkaline extraction is the "removal of lignin degradation products in combination with a neutralization of acidic components formed during oxidation" (Fengel and Wegner, 1984). Table 3 lists commonly used bleaching stages and the corresponding symbols widely used to indicate the processes.  9  Table 3. C o m m o n bleaching stages Stage  Chlorination  Symbol  Use  C  Typically first stage (pre-bleaching), breaks lignin bonds using Ch Dissolve reaction products using NaOH  Alkaline E Extraction Chlorine Dioxide D Delignifies and brightens pulp using CIO2 Ozone Z Delignifies and brightens pulp using O 3 Oxygen O Delignifies using O2, but not selective to lignin Peroxide P Mainly latter stages, brightens using H2O2 (Fengel and Wegner, 1984; TAPPI, 1997)  Bleaching sequences are listed using these symbols in the sequence order. Additionally, subscribed letters indicate a stage that is enhanced with those processes. For example, the bleaching sequence CDEopCD indicates the following stages chlorine, chlorine dioxide, alkaline extraction enhanced with oxygen and hydrogen peroxide, chlorine, and chlorine dioxide. In lignin-removing bleaching processes, at least three of these stages and in some cases up to seven stages are applied. 2.2.3 Effluent management Mills have two potential effluent management approaches: 1. internal process modification to reduce contaminants and effluent quantities, or 2. external treatment processes that are additional to the plant production processes (Springer, 1986). Typically, a combination of these methods is used.  Process modification can be aimed at the reduction of effluent contaminants, such as the use of elemental chlorine-free bleaching, or at reduced water consumption, such as the optimization of production water use (Springer, 1986; TAPPI, 1998). Some mills types have even been able to achieve zero effluent discharge (Forrest, 1992). Process modification improvements can also decrease the amount of colour in effluents. Reduction in colour can be achieved through bleaching sequence modifications and reuse and recycling of high colour streams (Rush and Shannon, 1976).  There are a variety of external treatments for effluent. Physical and physical/chemical treatments are the principal methods for removal of solids. Pulp mills employ physical and 10  physical/chemical effluent treatment methods such as coagulation/flocculation followed by settling or flotation, sandfiltrationor ultrafiltration (TAPPI, 1998). Biological treatments, primarily used for treatment of dissolved or colloidal matter, include aerated stabilization basins, activated sludge treatment systems and anaerobic treatment systems and are widely used in the pulp industry (Springer, 1986).  2.2.4 Origins of colour in effluents The effluent streams in a mill that are the most coloured come from pulp cooking and bleaching processes (Springer, 1986). The effluent from the pulp cooking, suitably referred to as "black liquor", contains "degraded thiolignins, degraded carbohydrates, and small amounts of fatty and resin acids and other extraneous material." (Dugal et al., 1974). The bleach plant effluent also contains lignins, as a result of the further delignification and oxidation of the pulp, as well as small amounts of other compounds. It has been noted that the bleach plant effluent can account for as much as two-thirds of the colour load but only one-third of the effluent volume (Springer, 1986).  The material responsible for imparting colour to these effluents has not been clearly defined. Different theories have been developed regarding its source, such as colour compounds being carbohydrate-derived (Ziobro, 1990). However, the general consensus amongst experts credits lignin and lignin-derived compounds for imparting colour due to lignin's high degree of conjugation (Springer, 1986). For example, a study performed by Suckling and Pasco in 2001 found that carbohydrate-derived chromophores could only contribute slightly to the absorbance of kraft spent liquor. Also, lignin-removing bleaching procedures contribute the most colour to effluents in comparison to pulping and other bleaching processes (Springer, 1986).  In addition to these pulping and bleaching colour sources, a recent study by Milestone et al. (2004) revealed that certain conventional treatment systems can increase colour by an average of 20-40% and up to 50%. The study showed this colour increase occurred in aerated stabilization basins (ASB), but not activated sludge systems, and only in regions of negative or low redox conditions.  11  2.2.5 Lignin Generally speaking, the main macromolecular cell wall components in wood are cellulose, polyoses (hemi-cellulose) and lignin (Fengel and Wegner, 1984). Lignin is a polymeric organic substance that gives strength to wood. It is the last macromolecular substance formed in the plant cell wall and, therefore, is infused within the other cell wall components, including the desired cellulose fibers. The removal of lignin is important in pulping not only because lignin acts like "glue" holding the cellulose fibers together but it also imparts colour to the pulp. Therefore, during the pulping process as much lignin is removed as possible without compromising pulp quality. Softwoods contain more lignin than hardwoods. As well, lignin and most ligninderivatives are high molecular weight molecules (Sjostrom, 1981).  The lignin-based colour theory is supported by characteristics of the pulp mill effluents. Lignin shows predominate absorbance of the short wavelengths in ultraviolet (UV) spectroscopy, which corresponds to the brown-red hue pulp mill effluents (Springer, 1986). In addition, the colour in pulp mill effluents is largely recalcitrant which reflects lignin's high resistance to microbiological degradation (Dugal et al., 1974).  2.3 Current Colour Removal Technologies  Although a description of every technology is not possible, the most feasible and well studied treatment techniques are described in this section.  2.3.1 Chemical treatment using aluminum andferric salts A large number of studies have shown aluminum and ferric salts to be highly effective at removal of colour, with efficiencies ranging from 85-95% (Rush and Shannon, 1976). Aluminum and ferric salts are effective because they act as both a coagulant and a precipitant (Tchobanoglous et al., 2003). In chemical coagulation, colloidal matter is destabilized in order for flocculation to occur, Flocculation is the increase of particle size as a result of particle collisions. Destabilization arises when the stronger repelling electrical charge forces of a colloid no longer prevent attraction to other particles. Alternatively, chemical precipitants alter the state of dissolved and suspended matter in order to alter matter formerly unable to settle, to a form that is able to settle. Although aluminum and ferric salts are very effective for colour removal, there are a number of drawbacks to these removal techniques, in which the main issues are: "1. 12  need for precise pH and zeta potential monitoring and control (this makes process automation difficult), and 2. problems encountered in sludge handling." (Rush and Shannon, 1976).  2.3.2 Chemical treatment using lime The National Council of the Pulp and Paper Industry for Air and Stream Improvement pioneered effluent colour removal technologies using lime (Dugal et al., 1976). Use of lime was an attractive option since it was already being used at most mills in the recovery process, and therefore lime colour treatment could be easily integrated into the mill processes (Rush and Shannon, 1976).  Lime treatment is a precipitation process in which removal is largely  dependant on the formation of insoluble calcium-organic salts.  Massive lime treatment, modified lime treatment and minimum lime treatments have been put into practice in operating mills and studied extensively (Oswalt and Land, 1973; Rush and Shannon, 1976; Spruill, 1973) Massive lime treatment uses lime doses of about 20,000 mg/L and is capable of overall mill effluent colour removal of about 72%. The textbook "Industrial Environmental Control: Pulp and Paper Industry" explains massive lime treatment as follows: (note that green liquor is a liquid formed during the sulfate recovery process and white liquor is the refortified green liquor used for pulping) (Lavigne, 1993) "In the massive lime process, the mill's total lime supply is slaked and reacted with a small-volume, highly colored stream, usually the bleach plant caustic effluent, at a lime concentration of 20,000 mg/L. The lime is settled, dewatered, and used for causticizing green liquor. The color bodies dissolve in the white liquor and eventually find their way into the recovery furnace." (Springer, 1986).  Colour removal using lime has drawbacks. Three of the main issues are: 1. inability to treat the entire effluent stream, 2. dilution of the cooked liquor requires increased treatment and recovery capacities, and 3. foaming in the clarifier and of the cooked liquor (Rush and Shannon, 1976, Springer, 1986).  Modifications to lime treatment were developed, aimed to minimize the problems of the massive lime process. The modified lime treatment replaced a large portion of the colour effluent slaked lime with lime mud from the recarbonation clarifier. This reduced foaming issues (Rush and 13  Shannon, 1976). Minimum lime treatment used lime doses closer to the stoichiometric quantities, which solved problems that developed in the massive lime process but in turn resulted in other issues such as colour reversion (Springer, 1986).  2.3.3 Other chemicals used to enhance settling Beyond the conventional coagulants, there have been experimental trials using synthetic coagulants such as hexamethylene diamine epichlorohydrin polycondensate (HE) and polyethyleneimine (PE) and natural coagulants such as chitosan, that have been successful in the removal of colour (Ganjidoust et al., 1997). Also, conventional chemical settling aids such as lime and ferric chloride have been used in combination to enhance removal already seen with these chemicals independently (Dugal et al., 1976). There are many possible coagulants and chemical variations that have been found to remove colour, all achieving relatively similar results. It can, therefore, be concluded that coagulant and precipitant technologies can be effective for the removal of colour, however cost and practicality of use are limiting factors.  2.3.4 Chemical oxidation Chemical oxidization is a treatment process wherein colour molecules are destroyed rather than removed. Ozone, hydrogen peroxide, chlorine dioxide, chlorine and hypochlorite, peracetic acid and Caro's acid have all been studied for the treatment of coloured pulp effluents (Springer, 1986). However, cost is a major restrictive issue with the use of oxidants. In addition, less costly oxidants such as chlorine and hypochlorite have other unwanted effects. Chlorination with chlorine or hypochlorite forms unwanted halogenated organic by-products. (Hao et al., 2000) Oxidants have also been employed as a pretreatment to biological treatment, as they can break down recalcitrant organics.  Ozonation is effective both as a treatment and pretreatment method. Bauman and Lutz found that the application of 30-40 part per million (ppm) of ozone on secondary effluent reduced colour by 60-70 percent (%), however, it did result in an increase of BOD (Bauman and Lutz, 1974). Ozone as a pretreatment has also proven to be effective in the breakdown of recalcitrant colour compounds (Bijan and Mohseni, 2004). The main issue with ozone is its acquisition, in that onsite generation is generally required which affects cost.  14  2.3.5 A dvanced oxidation processes Advanced oxidation processes are based on the formation of extremely reactive radical species, namely hydroxyl radicals (Hao et al., 2000). Utilization of ozone, ozone and hydrogen peroxide, Fenton's reagent  (H2O2 and Fe II), UV/peroxide/ozone, and UV and titanium dioxide are all  examples of known combinations that will form hydroxyl radicals (Hao et al., 2000; Sevimli, 2005). In a comparison of homogeneous and heterogeneous ozone applications by Mansilla et al., 1997, ozone used in conjunction with titanium oxide or zinc oxide achieved nearly 40% removal of colour in the first minute in the bleach process effluent compared to just ozone, which required 15 minutes to achieve 30% removal. Sevimli (2005) performed Fenton's oxidation studies in which 95% of colour was removed from biological treated pulp and paper effluents.  2.3.6 Biological treatment Biological treatment is an important category in the treatment of pulp mill effluent colour. As mentioned in Section 1.2.3, conventional biological treatment technologies, including activated sludge and aerated stabilization basins, are the most widely used effluent treatment processes in the pulp and paper industry, yet they are not effective in the removal of colour. Process enhancements, such as oxidation pretreatment, have the ability to increase the biological removal of colour through breakdown of the recalcitrant colour bodies into readily degradable matter.  Non-conventional biological treatments using white rot fungus have proven to be successful. A full discussion of these treatments can be found in Section 2.4.  2.3.7 Membrane treatment Membrane treatment, primarily ultrafiltration, has also been successful in the removal of colour. Membranefiltrationis the separation of particulate, colloidal and dissolved constituents down to the fractions of a micron size range from a liquid (Tchobanoglous et al., 2003). In membrane treatment, the semi-permeable membrane produces two liquid streams; the permeate, or the treated stream, and the concentrate, or the liquid that does not pass through the membrane. The driving forces used for treatment, either hydrostatic pressure difference as in reverse osmosis, or dialysis type membranes that use concentration gradients and electrical potential, can distinguish membranes types. Membrane systems that use hydrostatic pressure will be the focus of this  15  discussion. Table 4 lists these membranes and the typical operating ranges and mechanisms for removal.  Table 4. Membrane filters characteristics Membrane Type  General Separation Mechanism  Minimum particle size removed, urn  Microfiltration Sieve Ultrafiltration Nanofiltration Sieve + solution/diffusion + exclusion Reverse Osmosis (Tchobanoglous et al., 2003)  0.08-2.0 0.005-0.2 0.001-0.01 0.0001 -0.001  A range of membrane processes from ultrafiltration through reverse osmosis has been studied for treatment of pulp mill colour. Nevertheless, ultrafiltration has been the membrane process of choice. In 1973, Freemont et al. (1973) did a study for the US EPA using ultrafiltration and found colour removal efficiencies on kraft pulp bleachery effluents to be in the range of 90-97%. Ultrafiltration and nanofiltration membranes achieved 92% and 100% colour removal, respectively, on first stage alkaline extraction effluents (Rosa & Depinho, 1995).  The problems associated with membranefiltrationof pulp effluents are concentrate disposal, membrane fouling, and cost. Additionally, membrane treatment in pulp mills is generally seen as a polishing step or is relegated for use on specific effluent streams (Springer, 1986).  2.3.8 Resin separation and ion exchange processes Ion exchange is a term that has been loosely used for processes that include true ion exchange, a combination of ion exchange and adsorption and in some cases only adsorption. What appears to be common with this group of technologies is the use of resins that require regeneration. The fundamentals of adsorption will be explained in Section 2.4.  Ion exchange processes displace ions from a resin, an insoluble exchange material, with ions in a solution. There are many categories of resin types; strong-acid cation, weak-acid cation, strongbase anion, weak-base anion, and heavy-metal selective chelating resin (Tchobanoglous et al., 2003). Generally weak-base anionic resins are most effective on pulp mill effluents. It is important to note that the higher capacity the resin, the shorter lifespan it will have. Generally, the lifespan of resins is short, up to five years (Springer, 1986). 16  Under the title of ion exchange, two notable processes were developed that have been found to be successful for the treatment of coloured effluents; the Rohm-Haas process and the UddeholmKamyr process. The Rohn-Hass process is an adsorption-based process, since the resin used, Amberlite XAD-8, a highly cross-linked hydrophilic, porous polymer, contains no ion exchange groups (Rock et al., 1974). Nonetheless, this process was able to remove 70-95% of colour from a combined alkaline extraction and chlorine effluent. The Uddeholm-Kamyr process works on the principles of both ion exchange and adsorption using a weak-base anionic resin (Rush and Shannon, 1976). This approach has been shown to remove 80-95% of colour from caustic extraction effluent.  2.3.9 Activated carbon adsorption Activated carbon is charred organic material that is heated to just below 700°C under low oxygen conditions to prevent combustion, and then activated by exposure to oxidizing gases, such as steam or CO2, again at high temperatures (Tchobanoglous et al., 2003). This creates the all-important porous structure of activated carbon. Activated carbon is used in two forms, powdered or granular, and is referred to as powdered activated carbon (PAC) or granular activated carbon (GAC). G A C is used in a filter column and, therefore, generally requires its own apparatus. On the other hand, PAC can be added virtually anywhere. PAC has been added to the head end of the aeration tanks in activated sludge systems (United States Naval Facilities Engineering Service Center, 2002). Activated carbon has a relatively short lifespan but can be regenerated.  Activated carbon alone has been found to remove over 50% of colour in pulp effluents (Jacksonmoss et al., 1992). Interestingly, low molecular weight particles are more readily adsorbed by activated carbon and this makes addition of carbon an excellent polishing step (Rush and Shannon, 1976). Most colour removal technologies, such as lime, remove the larger molecular weight portions. The patented ISEP system "allows continuous operation of all processing steps in any staged sorption process without resorting to slurried transport of the sorbent material or use of batch sequencing techniques." (Springer, 1986). ISEP uses three steps; acidification, settling and activated carbon adsorption and can achieve 90-95% colour removal from final effluent (Springer, 1986).  17  2.4 Mechanisms of Adsorption  2.4.1 Basics of adsorption Table 5 provides definitions of terms important for understanding adsorption.  Table 5. Definitions of adsorption terms Definition A process where contaminants move, or concentrate, from one phase to another. Adsorption Process whereby ions or molecules in one phase have a tendency to accumulate on the surface of another phase. Absorption Partitioning of specific constituent from one phase to another. Adsorbate A substance that is being removed from the liquid phase at the interface. Adsorbent The solid, liquid or gas phase on which the adsorbate accumulates. Bulk liquid The liquid phase where the constituent concentration is uniform due to advection and dispersion. Stagnant liquid A film of liquid next to the adsorbent where the constituent concentration is decreasing from the bulk liquid. film (Tchobanoglous et al., 2003; Sawyer et al., 2003) Term Sorption  Adsorption, like absorption, falls under the general category of sorption, however, as described in Table 5, these processes are quite different. Adsorption occurs in four general steps: 1. bulk solution transport, 2. film diffusion transport, 3. pore transport, in the case of a porous absorbent, and 4. adsorption (Tchobanoglous et al., 2003).  Bulk solution transport describes the movement of the adsorbate through the bulk liquid to the stagnant liquid film (Tchobanoglous et al., 2003). Next, the adsorbate is transferred through the stagnant film layer by diffusion to the entrance of the pores of the adsorbent. This is called film diffusion transport. Pore transport is the movement of the adsorbate through the pores by molecular diffusion via the pore liquid and/or diffusion along the surface of the adsorbent. Lastly adsorption occurs where the adsorbate attaches to the adsorbent at an available adsorption site.  Adsorption is a result of many potential mechanisms. Wastewater Engineering - Treatment and Reuse by Tchobanoglous et al. (2003), lists the following adsorption forces: coulombic-unlike 18  charges, point charge and a dipole, dipole-dipole interactions, point charge neutral species, van der Waals forces, covalent bonding with reaction and hydrogen bonding. Generally, these forces are classified into two categories; physical and chemical adsorption (Sawyer et al., 2003). Physical adsorption is attributed to the existence of weak attractive forces, or van der Waals' forces, between molecules. Physically adsorbed particles are not fixed to a particular site on the solid surface but can move around on the surface, allowing more than one layer to form. This type of adsorption is typically quite reversible. Chemical adsorption, often entitled chemisorption, involves a strong adsorption force that generally only allows a single layer of adsorbed molecules to form. Also, molecules are not free to move along the surface and the adsorption is rarely reversible.  2.4.2 A dsorption rates An important characteristic of an adsorbent is the rate at which adsorption equilibrium occurs between the adsorbent and the adsorbate at a specific initial concentration. Two equations have been commonly used to describe adsorption rates, the Lagergren equation and the Ho et al. equation. (Ho et al., 1996; Lagergren, 1898)  The Lagergren equation is a first order rate equation, although it is commonly referred to as a pseudo-first order rate equation "to distinguish kinetics equation based on sorption capacity of solid from concentration of solution, ..." (Ho, 2004). Equation 1 is the Lagergren equation, \n(q -q,) = \n(q )-Kt e  e  (1)  and will be used in the present study in the re-arranged form, Equation 2, as follows, .q,=q (l-e- ') K  e  (2)  where q is the amount of colour removed, per mass of adsorbent (mg/g), at equilibrium, q is e  t  the amount of colour adsorbed, per mass of biomass (mg/g), at time t (h) and K (1/h) is the rate constant per unit time (Lagergren, 1898).  Ho et al. discovered that the Lagergren equation, although it applied very well to some situations, did not describe all situations. Therefore, they developed the following, Equation 3, of the pseudo-second order to describe a multi-component system, t  \  t  — = 2+— I, Qe Qe  (3)  2k  19  which was re-arranged for use in the present study as Equation 4,  '  l + 2kq t  (4)  e  where k (g/mg-h) is the rate of sorption (Ho, 2004).  2.4.3 Adsorption isotherms Isotherms are used to describe equilibrium between the quantity of adsorbate in solution and the quantity of adsorbate on the adsorbent at a given temperature. This equilibrium is based on properties and quantities of both the adsorbate and adsorbent. Solubility, molecular structure, molecular weight, polarity, and hydrocarbon saturation are important characteristics of the adsorbate (Tchobanoglous et al., 2003). The widely used Langmuir, Freundlich and Brunauer, Emmet and Teller (BET) isotherms are described below. The Langmuir isotherm describes a monolayer surface adsorption on an ideal surface. The Langmuir theory is based on the kinetic concept that there is continual bombardment of molecules onto the surface and a simultaneous desorption of molecules from the surface to maintain a zero rate of accumulation at equilibrium (Do, 1998). The assumptions of this model are: 1. the surface is homogeneous, i.e. adsorption energy is consistent at all sites, 2. molecules are adsorbed at a definite, localized site, and 3. each site can only accommodate one molecule.  The Langmuir model is described by Equation 5, Q°bC  < ^cy  q  where q  e  =  ... ( 5 )  (mg/g) is the amount of constituent adsorbed per unit weight of adsorbent at  concentration C, Q° (mg/g) is the amount of constituent adsorbed per unit weight of adsorbent in forming a complete mono-layer, b is a constant related to energy of adsorption and C (mg/L) is the measured concentration of the constituent, in solution, at equilibrium (Sawyer et al., 2003).  The Freundlich equation is a long-used empirical equation, commonly applied to describe activated carbon adsorption. In spite of its empirical origins, this equation can be derived from the Langmuir equation by assuming that the adsorbent surface has a heterogeneity of adsorption 20  energies and that sites having the same adsorption energies, are grouped together (Do, 1998). Equation 6 is the Freundlich equation. q.'K C "  (6)  u  F  where K  F  and Mn are the Freundlich constants indicating adsorption capacity of adsorbent and  changes in affinity for the adsorbate with adsorption density, respectively (Sawyer et al., 2003). The Freundlich isotherm becomes linear when n = 1, meaning that all adsorption sites have equal affinity for the adsorbates.  Multi-layer adsorption can be described by the BET theory.  BET theory makes the same  assumptions as the Langmuir theory, however, more than one layer of adsorbate can form. Consequently, the BET theory applies the kinetic principles of adsorption and desorption of the Langmuir theory to every layer. Equation 7 is the BET equation BCQ" °  q  (C -C)[\ s  +  {B-\){CIC j\ s  where C (mg/L) is the saturation concentration of constituent in solution, and B is the constant s  regarding energy at the surface (Sawyer, et al., 2003).  In addition, the Brunaner, Deming, Deming, Teller (BDDT) classification system typifies real world experimental results into five categories of isotherms. This classification is listed Table 6.  Table 6. BDDT isotherm classifications Type I II III  IV V  Standard example Adsorption of oxygen on charcoal at 183°C Adsorption of nitrogen on iron catalysts at-195°C Adsorption of bromine on silica gel at 79°C, water on glass Adsorption of benzene on ferric oxide gel at 50°C Adsorption of water on charcoal at 100°C  Comments Langmuir isotherm type (monolayer coverage) Demonstrates the BET adsorption mechanism Type where adsorption is not favourable at low pressure, hydrophobic adsorbent Similar to type II except has a finite limit Similar to type III except has a finite limit  (Do, 1998)  21  Types I through III can be described by the BET theory (Do, 1998). Types IV and V do not follow the BET theory as it assumes there is a infinite number of layers of molecules and types IV and V have a finite limit.  The International Union of Pure and Applied Chemistry (IUPAC) has accepted the five types described by Brunaner, Deming, Deming, Teller described above, as well as a sixth type that details a macroporous structure where stepwise multilayer adsorption occurs (Sing et al., 1985).  2.4.4 Adsorption of heterogeneous mixtures Compared to a pure solution, a mixture generally shows a decrease in total adsorbance for a specific compound, however, the total adsorbance capacity for the mixture may be greater than it is for that specific compound (Tchobanoglous et al., 2003). Competition for adsorption sites is dependant on the size of the molecules being adsorbed, adsorption affinities and relative concentrations. That being said, the isotherms described in Section 2.4.3 can be applied to heterogeneous mixtures.  2.5 Fungal Biosorption  2.5.1 Use of biosorption Biosorption is a loose term used to describe the removal of contaminants from a solution by sorption on biological material (Zhou and Banks, 1991). Biosorption consists of an assortment of removal mechanisms including adsorption and precipitation, as well as, in the case of living biological matter, metabolic activity (Gadd, 1992). Any biological matter can be a biosorbent, yet, research in this field has focused on microorganisms of all classes, including algae, bacteria, fungi and yeasts. Further, the definition of biosorption includes organisms in any life state, i.e. dead or alive, any component of the organism, and any matter the organism may have produced.  The majority of work classified under the category of biosorption has been in the treatment of metals, dyes and pulp mill effluents. This work has been quite successful and thus research has been extensive, investigating a wide range of biosorbents. Several researchers have utilized fungus, treating a variety of metals, dye types and pulp mill effluent streams (Banat et al., 1996; Fu and Viraraghavan, 2001a; Garg and Modi, 1999; Ho and McKay, 2003; Kapoor and Viraraghavan, 1995; Polman and Breckenridge, 1996). Dyes, and in some cases metals, are 22  chromophoric substances. As well, numerous chromophores exist in the heterogeneous mixtures of pulp mill effluents. There has also been work in the area of naturally occurring chromophores like humic acids. Zhou and Banks found thatfilamentousfungus Rhisopus arrhizus was successful in the removal of colour from naturally coloured waters (Zhou and Banks, 1991).  The major reason for the popularity of research in biosorption is economics. In comparison to conventional treatment technologies biosorption has the potential to be low cost in addition to being relatively easy to implement (Banat et al., 1996).  2.5.2 Fungal biosorption In the realm of biosorption research, there has been particular focus on the use of fungal biomass. Fungal biomass has been applied in both living and dead states, but the removal mechanisms are different, although some commonality exists.  Living fungal cells utilize metabolic activity to treat waters. There are generally two categories of live fungi used; white rot fungi and other fungi.  White rot fungi, also known as wood-rotting fungi, which fall under the category of Basidiomycetes, were first used to treat pulp mill effluents (Demain and Soloman, 1986; Hawker and Linton, 1979). Notably, Fukuzumi et al. (1977), and, Eaton et al. (1980), reported successes with the use of the species Tinctoporia species and Phanerochaete chrysosporium, respectively. As a result, white rot fungi have been researched extensively for the breakdown of recalcitrant compounds in pulp and paper effluents and a variety of dyes. A large number of researchers have used Phanerochaete chrysosporium, whereas others have selected different species of white rot fungi, for example, Trametes versicolor, Coriolus versicolor, and Ceriporiopsis subverispora (Bergbauer et al., 1991; Bilgic et al., 1997; Glenn and Gold, 1983; Mittar et al., 1992; Nagarathnamma et al, 1999; Pastigrigsby et al., 1992; Royer et al., 1991; Sundman et al., 1981). Phanerochaete chrysosporium is used in the registered process MYCOR® for the treatment of pulp mill effluents (Springer, 1986). White rot fungi are unique as they produce ligninolytic enzymes: laccase, manganese peroxides (MnP) and lignin peroxidase (LiP), to biodegrade recalcitrant organics such as lignin and lignin-derived compounds found in pulp mill effluents. In addition to biodegradation, white rot fungi cells are capable of facilitating adsorption to the cell wall (Fu and Viraraghavan, 2001a). 23  Successful removal of recalcitrant organics is also possible with other fungal types. However, since they do not have the lignin-modifying enzymes, they rely on other mechanisms for removal. Zhou and Banks observed biodegradation of humic acids with Rhizopus arrhizus by use of the cell's metabolic energy (Zhou and Banks, 1991). This process is thought to involve the uptake of compounds, or their degradation products by the cell, but this process occurred as a second phase of removal. Thefirstphase of removal involved adsorption of constituents to the cell wall (Zhou and Banks, 1991). In addition to Rhizopus arrhizus, other fungi have been used to remove recalcitrant organic material, such as Rhizopus oryzae and Aspergillus niger (Kapoor, 1998; Nagarathnamma and Bajpai, 1999).  Some research has shown success using dead fungal biomass for removal of metals, humic acids, phenol and dyes. In these studies, removal of dead biomass was a result of adsorption, almost exclusively physical (Fu and Viraraghavan, 2000; Gallagher et al., 1997; Kapoor et al., 1999; Rao, 2001, Zhou and Banks, 1991). Many studies have attributed the majority of removal to chitin/chitosan in the cell wall of the fungus (Banks and Parkinson, 1992; Zhou and Banks, 1991). Chitin is a complex amino-polysaccharide that is found in the cell wall of many moulds, as well as other organic matter such as crab shell, and chitosan is the deacetylatized form of chitin (Hawker and Linton, 1979).  A benefit to the use of dead biomass is the employment of pretreatments. Alteration of biomass cell structure or surface charge can significantly change the biosorption ability of fungal biomass as it is usually negatively charged. A wide variety of chemical and physical modifications have been tested with varying results, depending on the characteristics of the targeted constituents (Fu and Viraraghavan, 2001b; Gallagher et al., 1997; Kapoor, 1998). For example, Gallagher et al. (1997), found alkali pretreatment using NaOH increased adsorption of humic acids as it increased anionic sites on the cell wall by exposing the chitin/chitosan complex. In addition, acid pretreatment, using acids such as HC1 and H2SO4, have been used in attempt to neutralize or change the surface charge of the biomass, and salt pretreatment, using such salts as NaHC03 or CaCU, can also affect the surface charges (Fu and Viraraghavan, 2001b). Autoclaving increases the surface area and monolayer volume of a cell (Gallagher et al., 1997). As well, recent studies have isolated certain cell components for use as absorbents. For example, chitosan beads have been reported to be successful biosorbents (Chiou et al., 2003).  24  As both live and dead fungi have proven to be potential biosorbents, it is important to compare the two life states. Table 7 is a comparison of the adsorption characteristics of living and dead cells.  Table 7. Comparison of adsorption characteristics of life states Issue Biosorption capability  Operation  Dyes  Live Excellent  Dead Excellent  Humic acids  Excellent  Excellent  Metals  Excellent  Excellent  Pulp mill effluents Additional chemicals  Good/Excellent  No work found.  Must ensure readily available food source and nutrients. Either suspended or fixed film, such as a rotating biological contactor. Short biomass life span. Need to ensure solution to be treated in not toxic to biomass.  Chemical elution for regeneration.  Configuration  Other  1  Powder addition or immobilized biomass in filter column. Used either in powdered or column forms.  Citation Fu and Viraraghavan (2001a) Zhou and Banks (1991) Kapoor and Viraraghavan (1995) Bilgic et al. (1997)  Fu and Viraraghavan (2001a)  Notable, in Table 7, is the absence of research in the area of pulp mill effluents using dead biomass.  2.5.3 Factors affecting deadfungal biomass biosorption In dead biomass studies, researchers have noted that certain constituents or physical properties of the solution affect biosorption. Table 8 lists these constituents and physical properties.  25  Table 8. Factors affecting biosorption Property pH  Metal ions  Affect on biosorption Both increase or decrease depending on target contaminant and pH. Increased humic acid uptake.  Ionic strength  Increased humic acid uptake.  Temperature  Physical adsorption generally decreases with increased temperature. Chemical interactions increase with temperatures.  Mechanism Charge neutralization or change.  Citation Fu and Viraraghavan (2001b)  Bridging of cell wall surface charge. Surface chemistry forming an electric double layer. Physical adsorption surface loading capacity increases with decreasing temperature.  Zhou and Banks (1991) Zhou and Banks (1993) International Union of Pure and Applied Chemistry (1971)  Additionally, Zhou and Banks studied the effects of biomass culture age and growth medium on adsorption performance (Zhou and Banks, 1993). They found that biomass age did affect biosorption performance, with four days being the optimum age for Rhizopus arrhizus. In terms of growth medium, they also found this to have an effect, with Rhizopus arrhizus grown in potato dextrose liquid medium being the most effective compared to other Rhizopus arrhizus biomasses. Both of these improvements were related to an increase in chitin/chitosan content of the cells (Zhou and Banks, 1993).  2.5.4 Aspergillus niger A variety of dead fungal biomasses have been used as biosorbents such as Myrothecium verrucaria, Rhisophus arrhizus, and Rhizopus oryzae, including Aspergillus niger (A. niger), which has been used in the removal of metals, dyes and phenol (Fu and Viraraghavan, 2000; Gallagher et al., 1997; Mou et al., 1991; Rao, 2001; Zhou and Banks 1991).  A. niger is classed as Fungi Imperfecti, essentially a fungi that reproduces asexually (Hawker and Linton, 1979). It also falls in the sub-class Hyphomycete, commonly called moulds, which is a diverse group of fungi that produces conidia (a spore type). In the case of A. niger, conidia are grown on the end of a conidiphore, a stem-like structure that grows from the fungal mass or mycelium. Another popular fungal strain in this category is Pencillium. A. niger is characterized by its "mould-like" appearance with a white-creamy coloured mass and black conidia.  26  A. niger exhibits many advantageous characteristics for use as a biosorbent. First and very importantly, is its wide spread use in industry. The US EPA's Aspergillus niger Final Risk Assessment stated that A. niger is most commonly used for the production of enzymes such as aamylase, amyloglucosidase, cellulases, lactase, invertase, pectinases, and acid proteases, and the fermentation production of citric acid (United States Environmental Protection Agency, 1997). This is very important as there is the potential to use waste A. niger as an economical biosorbent supply source. In addition, A. niger is a classified under Biosafety Level 1 by the US Center for Disease Control. "Biosafety Level 1 represents a basic level of containment that relies on standard microbiological practices with no special primary or secondary barriers recommended, other than a sink for handwashing." (U.S. Department of Health and Human Services, 1999). Lastly, due to the effective reproductive techniques of A. niger, biomass growth is effortless (Hawker and Linton, 1979).  2.5 Background Conclusions  Coloured pulp mill effluent discharges can affect the public via aesthetic related issues, the aquatic environment and downstream water treatment systems. Although there has been a great deal of research regarding the prevention and treatment of coloured effluents, economical treatment solutions have not been developed. As a result, pulp mills have generally not adopted colour-removing techniques, unless required by law.  In addition, research using live fungi for removal of colour in pulp mill effluents and dead fungal biomass for removal of dyes, metals and other contaminates has been widespread. Nonetheless, based on the information collected, it appears that no research has occurred using dead fungal biomass for the removal of pulp mill effluent colour. Therefore, dead fungal biomass biosorption has potential to be an economical effluent colour removal technology for the pulp and paper industry.  27  3. OBJECTIVES  The overall objective of this study was to determine if inactivated fungal biomass oiA.niger effectively removes colour from pulp mill effluent. This overall objective was achieved through three minor objectives. 1. To determine if dead fungal biomass A. niger would remove colour from treated whole mill effluent in a laboratory setting. 2. To optimize and understand the mechanisms for removal of colour from treated whole mill effluent in a laboratory setting using dead fungal biomass A. niger. 3.  To determine if dead fungal biomass A. niger would remove colour from primary clarified effluent in an activated sludge process.  28  4. METHODOLOGIES  The current research consisted of three main phases: 1. effluent characterization and biomass production, 2. batch biosorption study, and 3. practical application study.  4.1 General  4.1.1 Pulp mill effluents Two effluents were studied in this research. Thefirsteffluent was collected on April 20, 2005, from the former Western Pulp Partnership Ltd. pulp mill near Squamish, British Columbia. The sample was whole mill effluent after secondary treatment. This mill produced a northern bleached softwood kraft (NBSK) pulp for fine paper manufacturing using cedar, hemlock and fir and used a CDEopCD bleaching process. The bleach sequence symbols are explained in Section 2.2.2. The effluent treatment was an activated sludge UNOX process. The UNOX process uses oxygen instead of air for aeration to achieve a high rate oxygen activated sludge system. This effluent was used throughout the batch study for all mini-studies, and will be referred to as Western Pulp effluent.  The second effluent was collected on January 5, 2006, from the Howe Sound Pulp and Paper Ltd. Mill in Port Mellon, British Columbia and was also treated whole mill effluent. This mill also produces NBSK pulp, using hemlock and cedar, as well as newsprint, which is pulped via thermo-mechanical process using a variety of spruce, pine, fir, hemlock and balsam. The bleaching process used is ODEopDnD; where n stands for nitrogen compounds. The remaining symbols are detailed in Section 2.2.2. This mill also uses the activated sludge UNOX process for effluent treatment. This effluent was only used for the adsorption rate kinetics, biological inhibition and isotherm mini-studies and will be referred to as the Howe Sound effluent. In addition, the practical application study took place at this mill.  29  Both of these effluents were grab samples collected on one occasion and stored in the Environmental Engineering Laboratory cold room at 4°C at the University of British Columbia for the duration of the study. No further preservation was used.  4.1.2 Methods and materials Numerous test methods where used within each phase. Table 9 details these test methods and the materials used. All glassware and containers were washed with detergent, then rinsed with tap water and, finally, rinsed three times with distilled water. All glassware, such as Petri dishes and flasks used for growth of biomass, was also washed and rinsed similarly to the other glassware but with the addition of autoclaving for 30 minutes (min) at 122 °C and 124 kilo Pascal (kPa). Prior to use, all effluent samples werefilteredwith a 1.5 micrometer (uzn)fiberglassfilterpaper, except the effluent used in the Molecular Weight Distribution and Practical Analysis Studies.  4.1.3 QA/QC In order to ensure quality in measurement, every test performed was duplicated to avoid gross error. In addition, unless warranted by consistent results, every batch test was also duplicated. Blanks and/or reagent blanks were also analyzed for each parameter. In addition, all measurements of quantities removed were compared against experimental blanks that had been processed the same way as the samples.  30  Table 9. Methods and materials Parameter Colour  pH COD  Electrical conductivity  Method HACH Method 8025 - Platinum Cobalt Standard Method with pH adjustment following Standard Method 2120 C Spectrophotometric. Samples are centrifuged and filtered with 0.22 urn filter. Standard Method 4500-H B. Electrometric Method Standard Method 5220 D - Closed Reflux Method, Hach digester Method 8000 (EPA Approved) and DR/2000 Standard Method 2520 B. - Electrical Conductivity Method +  Materials Filter apparatus and paper, H S0 at cone, so that amount does not exceed 3% of sample, DR2000 spectrophotometer 2  4  pH meter, buffer solution materials Hach COD digester, 0.25 N of K Cr 0 , Ag S0 reagent, 2  2  7  2  4  Electrode, 0.01 M of KC1 for standards  Molecular size distribution  Different membranes and TOC analyzer. Amicon stir cell 8200. Micropore Y M 500, 1000, 3000, 10000, and 100000 Dalton membranes.  Amicon Stirred Cell and different membrane sizes.  Total/Dissolved Organic Carbon Biochemical Oxygen Demand  Standard Method 5310B - High Temperature Combustion Method Standard Method 5210B - 5-Day BOD Test  Shimadzu TOC analyzer  Total Suspended Solids  Standard Method 2540D - Total Suspended Solids Dried at 103-105°C  Chloride  Standard Method 4500G - Mercuric Thiocyanate Flow Injection Analysis  60 mL incubation bottles, nutrients, dilution water, dissolved oxygen meter, nitrification inhibitor Drying oven, scale, filter apparatus, Millipore type AP40 filter Lachat flow injector  Standards Platinum-cobalt Fisher Chemical 500 standard. Standards at 50, 100, 300 and 500 for 465 nm. pH 4, 7 and 10, Fisher Chemical 800, 500,300, 100, 50 and 0 CODasmg/L. Zero and one standard @ 1412 umhos/cm  50 and 500 mg/L ofC. Dilution water blanks  Distilled water filter blank 0, 50, 75, 100, 200,400 mg/L  Notes Measured at pH of 7.6 and at 465 nm in accordance with Pulp and Paper Technical Association of Canada standards.  Make calibration curve @ 600 nm.  Filter effluent through each membrane sequentially beginning with the largest andfilteringthe effluent passing with the next largest membrane. Measure organic content of each fraction with TOC measurement. Acidify sample to pH < 3firstfor DOC  4.2 Effluent Characterization and Biomass Production  4.2.1 Effluent characterization parameters Table 10 lists the parameters that were analyzed for characterization of effluent.  Table 10. Parameters analyzed and rationale Parameter  Rationale  Colour Determination of process efficiency. Effluent pH can alter biosorption efficiency. pH COD Indicates organic content. Electrical conductivity Potential factor in biosorption efficiency. Organic carbon Quantifies molecular size fractions. Molecular size distribution Identification of colour imparting substances. The methods and materials used were presented in Table 9.  4.2.2 Molecular weight distribution In order to better understand the composition of the effluent, as well as to determine which fraction of the colour and COD is removed through dead A. niger biosorption, molecular size distribution was determined. Molecular weight distribution was performed as described in Table 9.  Lignin and its derivates are generally high molecular weight compounds, therefore, the  molecular weight distributions of the effluent should show this tendency (Sjostrom, 1981). In addition, certain removal mechanisms have tendencies to remove specific molecular weight fractions. For example, in a study done for the US EPA by Dugal et al. (1974), effluent characterization before and after lime treatment was performed and it was found that the lime process removed the higher molecular weight molecules.  Therefore, the molecular size  distribution can give insight into the potential removal characteristics of the dead A. niger biomass.  4.2.3 Biomass production Fungus Aspergillus niger was produced and harvested in preparation for the batch and practical application study. A freeze-dried culture of A. niger was ordered from the American Type Culture Collection (ATCC), A T C C #11414. Biomass production included the following steps: 1. cultivation of biomass on agar plates, 2. cultivation of biomass in a liquid medium, and 32  3. harvesting biomass.  All culturing and harvesting steps were performed using aseptic techniques.  All media and  glassware was autoclaved for 30 min at 122°C and 124 kPa before use.  The freeze-dried culture required activation, which was performed by immersing the culture in distilled-deionized autoclaved water for 45 min. The solution was then transferred to potato dextrose agar plates, pouring approximately 10 milliliters (mL) of solution per plate. The plates were incubated for 7 to 10 days at 22°C upside down. Cultures from the incubated plates were transferred by streaking to fresh plates. This occurred once before the culture was used for biomass cultivation in the liquid medium. The cultures were maintained on agar plates for the duration of both Batch and Practical Application Studies. The plate cultures were refrigerated at 4°C where they were maintained for up to 2 months, at which time the culture was transferred to fresh plates.  The biomass to be harvested was cultivated in a liquid medium for four days.  The liquid  medium contained the following constituents: dextrose - 20 g/L (grams/litre); peptone - 10 g/L; yeast extract - 3 g/L. Two-litre Erlenmeyer flasks, containing 1 litre (L) of liquid medium, were inoculated with the fungus from the agar plates. The flasks were covered with a plug to protect from contamination while allowing aerobic conditions and placed on a rotary shaker at 125 rotations per minute (rpm) at room temperature (approximately 20°C). Each biomass culture was matured for four days.  After the culture had aged for four days the biomass harvesting commenced. Initial separation of the biomass from the growth medium was achieved by filtering the biomass and liquid medium mixture through a 150 \im sieve. The initial biomass washing regime consisted of a distilled water wash, to remove any remaining medium, followed by autoclaving the cells for 45 minutes. The distilled water wash consisted of washing the biomass over a 150 urn sieve with distilled water until the pH of the rinse water equaled that of distilled water. However, the observance of excessive COD initiated a mini-study of into the washing methods. This mini-study and results will be described in more detail in Section 4.3.3. As a result of the mini-study there was a switch to a double wash protocol that consisted of the original wash regime being followed by a second wash after the cells were autoclaved for 45 minutes. 33  Following washing, the biomass was dried for 36 h at approximately 60°C. After the biomass was dried, it was ground to a powder with an electronic coffee grinder. Particles that passed through a 250 pm sieve were used.  In order to ensure the pre-treated biomass was no longer viable, a portion of the finished pretreated biomass was returned to an agar plate and incubated, as explained in the preceding section.  4.3 B a t c h B i o s o r p t i o n S t u d y  The batch biosorption study consisted of eight key mini-studies on the following: 1. optimum pretreatment method, 2. effluent pH, 3. biomass washing method, 4. mixing requirements, 5. adsorption rate kinetics, 6. biological inhibition, 7. equilibrium isotherms, 8. removed fraction, and 9. temperature effect.  For each batch study the biomass was separated from the effluent by centrifuging the sample at approximately 3,500 rpm for 15 min, followed by filtering the sample with a 0.22 urn membrane filter. Originally, a 0.45 um membrane filter was used, however, the sample turbidity interfered with colour measurement and therefore finer filtration was required. The Howe Sound effluent almost always required more than one filter paper per sample. For example, for the same dose and same biosorption time, the Western Pulp 45 mL centrifuged sample only required one filter paper for this volume, whereas the Howe Sound effluent normally required at least two. As a result, there was potential for more variability in the Howe Sound results since more processing was required. As a note, in order to be properly compared, the experimental blanks were also filtered using two papers for Howe Sound effluent experiments.  34  4.3.1 Pre-treatment mini-study The purpose of the pre-treatment mini-study was to determine which pre-treatment yielded the highest colour removal efficiency. Subsequent to harvesting biomass, the pre-treatments were applied. In total, eight different methods of pre-treatment were utilized. The eight biomass pretreatment methods included the following:  1. Type A - autoclaving for 30 minutes at 122°C and 124kPa and then biomass was dried at 60 to 70°C for 36 h in a drying oven, 2. Type B - contacting 100 g wet weight pellicles with IL of 0.1 Molar (M) NaOH solution for 1 h at room temperature, 3. Type C - contacting 100 g wet weight pellicles with 1L of 0.1 M HC1 solution for 1 h at room temperature, 4. Type D - contacting 100 g wet weight pellicles with IL of 0.1 M H2SO4 solution for 1 h at room temperature, 5. Type E - contacting 100 g wet weight pellicles with IL of 0.1 M CaCh solution for 1 h at room temperature, 6. Type F - contacting 100 g wet weight pellicles with IL of 0.1 M NaHC03 solution for 1 h at room temperature, 7. Type G - contacting 100 g wet weight pellicles with IL of 0.1 M Na2C03 solution for 1 h at room temperature, and 8. Type H - contacting 100 g wet weight pellicles with IL of 0.1 M NaCl solution for 1 h at room temperature. Type B thru H pre-treatments were also autoclaved and dried as listed in Type A.  After pre-treatment and prior to drying, the biomass was washed with distilled water until the wash water reached the original pH of the distilled-deionized water. The biomass was then dried and ground.  The pre-treatment batch mini-study was performed on the Western Pulp effluent and consisted of eight separate batch tests, each using 75 mL of effluent at original pH and 0.2 g of pre-treated biomass. Each pre-treatment method was tested twice, although in some instances the second trial was scaled down due to the amount of biomass that was recovered after drying. Experiment blanks were analyzed concurrently to determine the effects of this process on colour and COD. 35  The effluent and biomass was put into a 125 mL Erlenmeyer flask and placed on the shaker/incubator for 48 hours (h) at 125 rpm. After 48 h of contact, the mixture was centrifuged and thenfilteredwith a 0.22 um membrane filter under vacuum pressure.  The adsorption performance of Type A thru H pre-treated biomass was determined by analysis of residual colour in thefiltrateat both 400 and 465 nm and COD. Colour was measured at two wavelengths since the highest measurement of absorbance in the visible spectrum on the raw effluent was at 400 nm. Therefore the results obtained at 400 nm showed colour that was not measured at 465 nm. Based on the results for colour removal, the optimum pre-treatment method, autoclave only, was selected and used in the remainder of the study.  4.3.2 Effluent pH mini-study The pH mini-study illustrated the effect of pH on biosorption and gave an indication of optimal effluent pH ranges.  This mini-study was performed on Western Pulp effluent. Prior to adding the biomass to the effluent, the pH of the effluent was altered. The adjustment of pH was achieved using sulfuric acid (H2SO4) and sodium hydroxide (NaOH) depending on the pH target. The initial effluent pH was adjusted to the following pH levels 2, 4, 6, 8, 10, and 12. Batch tests were performed, each using 75 mL of one of the six pH-adjusted effluents and 0.2 g of the biomass in a 125 mL flask. Replicate batch tests were performed on each pH-adjusted effluent, resulting in 12 tests plus an experimental blank at original pH. These flasks were then placed on the shaker/incubator for 48 h at 125 rpm. After the 48 h of contact, the mixture was filtered with a 0.22 um membrane filter under vacuum. Further, based on the results of this test, experimental blanks at pH 2, 4, 6, and 8, were performed in order to determine the quantity of removal that occurred as a result of the pH adjustment and how much removal was due to the biomass biosorption.  The adsorption performance at each of the initial effluent pH levels was determined by analysis of colour and COD. Although pH did have an effect on biosorption, it was determined that a more practical approach would be to perform the remaining studies using effluent at the original pH.  36  4.3.3 Biomass washing mini-study An important part of the biomass harvesting process was the biomass washing step. The original biomass washing protocol was a single wash of the cells prior to autoclaving. However, through results obtained using the biomass, it became evident that the addition of the fungus to liquid resulted in an increase in dissolved matter in the liquid. Therefore, attention was turned to the fungal wash methods.  In order to determine the levels of colour and COD introduced to a liquid by the fungus, the prepared fungal biomass was added to de-ionized water in the same manner as during normal testing (ie, 75 mL of water, 0.2 g of biomass, on shaker at 125 rpm). These batch tests were sampled after different time intervals within a 2-day period, at 15 min, 24 and 48 h. An experimental blank of distilled-ionized water was used. Measurement of the colour and COD was used to determine the appropriate biomass washing method.  As the original biomass wash method was not sufficient, two other biomass wash methods were tested.  One method involved autoclaving the biomass while still in the growth medium,  followed by washing and draining of the biomass. This wash method is referred to as the "wash after autoclaving" method. The other biomass wash method consisted of washing the biomass both before and after autoclaving. This method is referred to as the "double wash" method. For both of these methods, washing was continued until the wash water pH was that of the distilled water.  Based on the values of colour and COD, it was found that the "double wash" method was the most effective and, therefore, it was used for preparation of the biomass for the reminder of this study.  4.3.4 Mixing mini-study Due to observations of incomplete mixing in the original set of batch adsorption tests, it was determined that analysis of mixing efficiency was required. In the original mixing method, a flask size of 125 mL containing 75 mL of effluent at a rotational speed of 125 rpm was used.  37  It was determined that two main factors may affect mixing on a rotary shaker: 1. the relationship of flask size to amount of effluent it contains; and 2. the rotational speed of the shaker. In addition, it was anticipated that the dose of fungus added to the effluent might also impact the mixing efficiency. Table 11 lists these variables and the ranges in which they may impact mixing. Note that the effluent amount in each test was 75 mL.  Table'll. Possible factors impacting mixing efficiency  Setting 1 - Low Setting 2 - Medium Setting 3 -High  Variable 1 Shaker Speed (rpm) 125 160 200  Variable 2 Flask Size (mL) 125  Variable 3 Biomass Dose (g) 0.2 0.5 0.8  300  Table 12 lists the experimental runs performed based on the variables listed in Table 11.  Table 12. Possible experimental runs Run Number Shaker Speed (rpm) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18  125 125 125 160 160 160 200 200 200 125 125 125 160 160 160 200 200 200  Flask Size (mL) 125 125 125 125 125 125 125 125 125 300 300 300 300 300 300 300 300 300  Biomass Dose (g) 0.2 0.5 0.8 0.2 0.5 0.8 0.2 0.5 0.8 0.2 0.5 0.8 0.2. 0.5 0.8 0.2 0.5 0.8  38  For each experimental run, batch adsoprtion tests were carried out with adsorption times of 0.25, 1, 4 and 24 h. Effluents that had been shaking without the addition of fungus for 24 h were used as experimental blanks. Again, Western Pulp effluent was used for this mini-study.  There were 18 possible experimental runs using these combinations of factors. From prior testing, Run 1 was analyzed at times 0.25, 1, 4, and 24 h, as well as additional times, and, therefore, repeating this experiment was not required. Also, Runs 2 and 3 had been partially executed but with samples taken only at 48 h. Although analysis was not complete due to missed sampling times, these previous test results give some indication of the expected results. Based on the data previously obtained the following experimental plan was proposed. 1. Runs 10, 11, and 12 were undertaken to confirm if flask size was a factor. All of these tests were run simultaneously for 48 h only. 2. Runs 16, 17, and 18 were completed in order to determine if there was an impact on mixing efficiency between rotational speeds of 125 rpm and 200 rpm. All of these tests were run simultaneously. 3. Further testing would be completed if necessary, such as Runs 13, 14, and 15.  Based on the results obtained, runs 10, 11, 12, 16, 17, and 18 were performed. It was determined that using a flask size of 300 mL at 125 rpm was sufficient for complete mixing for all doses tested and, therefore, this flask size and rotational speed was used in all further testing. Each of the adsorption tests was performed twice and colour, COD, DOC and pH were measured.  4.3.5 Kinetic mini-study The purpose of the kinetic mini-study was two-fold, to determine the rate at which the biosorption occurred, and to determine the equilibrium time for adsorption for the following isotherm mini-study.  This mini-study used 75 mL of effluent and 0.2 g of pre-treated biomass per flask. Experimental blanks using only effluent with shaking for 48 h were used, to determine the effect of this process on colour. Samples were collected at times 0, 0.08, 0.17, 0.25, 0.5, 0.75, 1, 4, 8, 12, 19, 24, 32, 40, 44, 48 and 52 h. This mini-study was performed on both Western Pulp and Howe Sound effluents, however, only the Western Pulp experiments were duplicated. The adsorption performance at each time was determined by analysis of colour, COD, DOC and pH. 39  The results of this mini-study were used to determine the equilibrium time beyond which no additional significant colour removal occurred. Further, the kinetics of the adsorption were studied by comparing the data with the Lagergren (1898) and Ho et al. (1996) equations.  4.3.6. Biological inhibition mini-study Initial results from kinetic testing showed an additional peak of colour removal occurring at roughly 30 plus h, after the initial adsorption period slowed. The mechanisms that caused this delayed colour removal were not clear. It is possible that these mechanisms were chemical, biological or bid-chemical. In order to help identify what mechanisms were responsible, biological inhibitors were utilized to determine if the delayed colour removal effects were related to biological activity. As there was only one factor in this study, inhibition, only three tests sets were required. They are listed in Table 13.  Table 13. Biological inhibited test sets Test Set # 1 .2 3  Inhibitor Environmental temp < 4°C Addition of Sodium Azide Addition of Sodium Fluoride  The test sets were sampled at set intervals of 0, 1, 8, 19, 24, 32, 40, 48, and 52 hour for test set 1 and the same intervals, less the 19 and 52 hour intervals, were used for test sets 2 and 3. Colour, COD, DOC and pH were analyzed for each sample. In test set 1, the incubator and the effluent was at 4°C before commencing the test set. For test sets 2 and 3, chemical addition of sodium azide (NaN ) and sodium fluoride (NaF) at 1 milli-molar (mM) and 50 micro-molar (uM), 3  respectively, was used based on information found regarding doses of these chemicals for biological inhibition (Carlson and Suttie, 1967; Desseaux et al., 2002; Hongslo et al., 1974; Rachenko et al., 2004; William Ramey, University of British Columbia, personal communication, January 15, 2006; Vyas and Molitoris 1995). These chemicals were added to the solution and mixed for 15 minutes before the addition of the biomass.  All three test sets were performed on Western Pulp effluent. Based on the results of these two methods, set 1 and 2 were applied to the Howe Sound effluent in order to obtain kinetic data results for both effluents that were not influenced by biological activity. Test set 1 for both effluents was duplicated, however, test set 2 was not duplicated as it was felt that the results were 40  consistent with the other test and duplication was not required.  4.3.7 Isotherm mini-study The isotherm mini-study provided a description of the adsorption characteristics and information about biomass doses required. The experimental time for this mini-study was determined by the results of the kinetic study. Therefore, each isotherm adsorption period was 32 h.  Unlike previous mini-studies, this mini-study used biomass amounts of 0.1, 0.5, 1.0, 2.0 and 3.0 grams to 75 mL of effluent, which gave doses of 1.33, 6.67, 13.33, 26.67 and 40.00 g/L. These tests were run with effluent inhibited by NaN at 1 mM. Both Western Pulp and Howe Sound 3  effluents were tested once. In addition, an experiment blank using effluent was run for each set of batch tests. The samples were tested for colour, COD, DOC and pH. The data were compared to three isotherms, the Langmuir, Freundlich and BET.  4.3.8 Removedfraction mini-study In order to better understand the biomass removal, molecular weight distributions were determined again, however, this time on the effluent samples of the isotherm mini-study. Both the effluent blank and 1.0 g biomass batch test samples were fractionated using the Amicon stir cell and membrane. In this mini-study only 3,000, 10,000 and 100,000 Dalton (Da) membranes were used. This mini-study detailed the fractions being removed or added by the addition of 1.0 g of biomass to 75 mL of effluent.  4.3.9 Temperature mini-study In the inhibition mini-study, a biologically hindering ambient temperature was used as an inhibitor. It was observed that batch tests run at 4°C showed less removal than tests at room temperature, i.e. 20°C.  In order to better understand this occurrence, batch studies were  performed at 35°C, similar to the kinetic rate mini-study, at time intervals 1, 8, 20, 24, 32, 40, 48 h. The effluent was inhibited with NaN at a 1 mM concentration. These results were compared 3  with the results of the kinetic study.  41  4.4 Practical Application Study  After completing the Batch Study and determining that the fungus was successful under controlled and optimum conditions, a simulated real world application was tested. The practical application testing was conducted on-site at the Howe Sound Pulp and Paper Partnership Ltd. pulp mill on February 22, 2006.  In order to mimic the activated sludge system used at the plant, samples were collected from the primary clarified effluent line as well as from the return activated sludge (RAS) line. Due to extremely high dosing of nutrients to the activated sludge system in the days prior sampling, addition of further nutrients beyond what already existed in the RAS sample was not required.  The experiment, although intended to be a scaled down replication of the existing continuous activated sludge system at the mill, was tested using a batch process. Five batch tests ran simultaneously in 500 mL Erlenmeyer flasks. Each flask was sealed with a bung that had an air input and output. Air was supplied by aquarium bubblers and dispersed by small diffusers that hung, by tubing, down from the bung submerged in the RAS/effluent mixture. Each batch test contained 300 mL of a mixture of effluent and RAS at the same ratio as used by the mill, 100:55, i.e. 194 mL of effluent with 106 mL of RAS (Siew Sim, personal communication, February 22, 2006). Each flask was also mixed with a magnetic stir bar. This was to ensure complete mixing occurred, which may not have been achieved by aeration alone.  The average residence time of each step in the mill's activated sludge process was used for the batch test. The mill's activated sludge system had a four to six hour residence time in the aeration chamber followed by eleven to twelve hours of settling (Howe Sound Pulp and Paper Ltd. Partnership, 2006; Siew Sim, personal communication, February 22, 2006). After the biomass was added, aeration was started immediately and continued for 4 h. After the aeration was terminated, the activated sludge was left to settle for 12 h. After settling, the samples were decanted and put on ice until transported to the University of British Columbia, Environmental Engineering Laboratory for analysis.  42  Of the five batch tests, two were experimental blanks or controls that contained only the effluent and RAS. The other three were dosed with fungal biomass in one of three quantities, 0.8, 4.0 and 8.0 g, which were equivalent to dosages of 2.67, 13.33 and 26.67 g/L, respectively. To determine the effect of the biomass on the treatment system, COD, BOD, TOC, TSS and colour was measured. The removal efficiencies of the controls were compared to the results obtained when adding the fungus. Although this testing was very crude, it would indicate potential for use in the activated sludge process.  43  5. RESULTS AND DISCUSSION  5.1 Effluent Characterization and Biomass Production  5.1.1 Effluent characterization In order to gain a better understanding of the characteristics of the effluents, both were analyzed for colour, COD, DOC, pH, electrical conductivity, and chlorides. The results of this analysis are listed in Table 14.  Table 14. Results of whole mill treated effluent characterization Western Pulp Colour (TCU) 1750 COD (mg/L) 370 DOC (mg/L) 190 BOD (mg/L)* 12.1 7.6 PH EC (uS) 2100 Chlorides (mg/L) 156 * Yearly average data obtained from the plants  Howe Sound 1050 310 140 10.8 7.4 3000 146  Colour was obviously a very important factor as it was the focus constituent of the study. Comparing the colour of the Western Pulp and the Howe Sound effluent shows that the Western Pulp whole mill effluent was significantly higher in colour. This could have been a result of the pulping and bleaching processes used, process efficiency and/or wood type. As northern softwood was used at both mills, using generally the same species of wood, it was unlikely that wood type would have a significant impact on colour. More likely, this difference was a result of the pulping and bleaching processes and their efficiencies.  Howe Sound employs two pulping processes, kraft and thermo-mechanical, as opposed to Western Pulp, which used kraft pulping only. Thermo-mechanical pulp is used to make newsprint and, therefore, was not bleached. The kraft process, which produces a physically stronger pulp, does not remove colour very effectively and, therefore, a bleaching step is required (Fengel and Wegner, 1984). As the bleaching process is the major contributor of coloured effluents, it was likely the thermo-mechanical effluent had a small impact on the amount of colour in the effluent stream through dilution of colour from the kraft stream. 44  In addition, the bleaching sequence can also affect the colour of the effluents. Based on real plant data, bleach sequences using oxidative species instead of chlorine produce significantly less effluent colour (Springer, 1986). The Howe Sound mill uses an ODEopDnD non-elemental chlorine sequence as opposed to the Western Pulp mill that used a CDEopCD chlorine based sequence. As noted previously, the major contributor of coloured effluents in a pulp mill is the bleaching system. As such, it was conceivable that the difference in bleaching sequence had a significant impact on the difference in colour.  Lastly, the Howe Sound mill's pulping and bleaching processes are more modern than Western Pulp's and, therefore, runs "tighter" mill operations. This could have impacted the reduction of colour in the mill's effluents.  The other constituents measured, similarly to colour, also showed the Western Pulp effluents having higher levels than Howe Sound, with the exception of electrical conductivity. This supports the theory that the bleaching processes and the mill's ability to run a "tight" operation are differentiating factors in the condition of the effluent.  The COD and DOC values can indicate the organic loading of the effluent and what fraction is recalcitrant. From data supplied by the mills, the yearly BOD averages for Western Pulp in 2004 and Howe Sound in 2005 were 12.1 mg/L and 10.8 mg/L, respectively (Howe Sound Pulp and Paper Ltd. Partnership, 2006; Jeanne Taylor, electronic mail, May 4, 2005). Therefore, it was assumed that most of the organic matter in both of the effluents was recalcitrant. Electrical conductivity, pH and chlorides are all parameters that have the potential to affect biosorption. Therefore these factors may impact the biosorption process occurring in a specific effluent.  5.1.2 Molecular weight distribution Using the Amicon stir cell and various sized membranes, a molecular weight size distribution was determined. It should be noted that, unlike the effluent characterization, the samples were not pre-filtered with a 1.5 pm fiberglass filter paper prior to the analysis. Figures 1 and 2 summarize the colour and TOC of each fraction for the Western Pulp and Howe Sound effluents.  45  HS - Colour  0  300  • WP - Colour  600  900  1200  1500  1800  2100  Colour (TCU)  Figure 1. Molecular weight distribution by colour  I WP - T O C  HS-TOC  :  0  25  50  75  100  125  150  175  T O C (mg/L)  Figure 2. Molecular weight distribution by total organic carbon  200  The results of the molecular weight study shed light on the distribution of chromophores in the effluents. As opposed to the Howe Sound effluent, which contained predominantly molecules that were larger than 10,000 Da, the Western Pulp effluent contained two predominant ranges of high and low molecular weight colour components. These results were consistent with the generally accepted theory that the colour was primarily composed of lignin-related compounds, since they are typically higher molecular weight compounds (Sjostrom, 1981). In addition, both high and low molecular weight recalcitrant organic compounds have been found in pulp mills (Konduru, etal., 2001).  The difference in colour distributions was possibly related to the variation in bleaching sequence and the effluent minimization strategies used by the plants. However, the colour distribution data also indicated that dilution by the fhermo-mechanical pulping effluent stream is not a significant factor. Both of these assumptions were based upon the substantial difference in colour distributions. If dilution by the thermo-mechanical stream was a major factor, the Howe Sound colour distribution would be more similar to the Western Pulp, albeit at smaller concentrations. On the other hand, the molecular weight distributions of the organic matter, TOC, were quite similar for the two effluents. This did not allow for clear conclusions regarding the source of the effluent colour difference and made the previous assumptions based on the colour distribution questionable.  This raised issues regarding colour measurement itself. Colour measurement of a liquid is a function of light absorbance, not a specific measurement of the amount of chromophoric materials. Therefore, unlike measurement of a specific compound such as carbon where it is a direct function of the quantity of the material, the quantity of coloured materials is not measured but is instead the amount of light those materials absorb. So, in comparing two different single molecules, one may be a "stronger" chromophore than the other and, therefore, have a higher absorbance which results in a higher colour reading. Nevertheless, the quantity of the material could be the same for each coloured molecule. This means that it is difficult to make conclusions regarding sources of colour since the strength of the chromophores is also a factor. The Western Pulp mill's pulping and bleaching sequence may have created stronger chromophores in the low molecular weight region that were not formed by the Howe Sound pulping and bleaching processes or those absent chromophores were recycled.  47  5.1.3 Biomass production The A. niger biomass grew easily both on the plates and in the liquid medium. Three streak plates were maintained at any one time to ensure the continuous supply of biomass for inoculation of the liquid medium for biomass production. On the plates, the biomass exhibited a whitish-creamy colour mass or mycelium, with black spores-head on topside only. These characteristics were consistent with descriptions of A. niger given by Olds (1975). Illustration 2 is a photograph of a plate.  Illustration 2 . Photograph of A. niger agar plate  In order to further clarify the identity of the fungal species, the biomass was observed under a microscope at an optical magnification of 100 times (lOOx). Illustration 3 is a microscope image of broken conidiphore and conidia of the biomass. The conidiphore was broken as a result of poor slide preparation techniques.  48  Illustration 3. Microscope view of conidia at the end of broken conidiphores (lOOx)  This microscope image verifies the biomass as A. niger.  The biomass used for biosorption testing was produced in liquid medium and formed whitecreamy coloured globules approximately two centimeters in diameter. Illustration 4 is a photograph of the biomass prior to harvesting.  49  Illustration 4. Biomass in liquid medium just prior to harvesting  The biomass was grown in the liquid medium for four days and was then harvested. The biomass was then dried and inactivated by autoclaving for 45 min. In order to ensure that the biomass was in fact not viable after autoclaving for 45 min, biomass that was pretreated and dried was smeared onto agar plates of potato dextrose agar and incubated for 7 days. Of the two such smear plates that were prepared neither showed regrowth of biomass. Illustration 5 is a picture of one of the smear plates.  50  Illustration 5. Inactivated biomass smear plate.  5.2 Batch Biosorption Study  Due to the large amount of data obtained within the batch biosorption study, only data vital to the discussion will be presented within this Section. Other data obtained are provided in the Appendices.  5.2.1 Optimum pretreatment mini-study The first mini-study was used to determine the optimal biomass pretreatment method. Colour was measured at both 400 and 465 nm, since at 400 nm, the total absorbance of the original effluent was higher than at 465 nm. Figure 3 and 4 displays the colour removal efficiencies at 465 nm and 400 nm, respectively, for the various pre-treatment methods on Western Pulp effluent.  51  35%  Figure 3. Colour removal efficiencies from WP effluent at 465 nm for various pretreatments after 48 h of contact  > o  o o  •4-*  a  <D U I*  <u  04  #  &  <P -cP J>  Pretreatment type  Figure 4. Colour removal efficiencies from WP effluent at 400 nm for various pretreatments after 48 h of contact 52  The above data directed attention to the difference in absorbance from the same samples measurement at different wavelengths. It is important to note that for each batch test, two colour samples were taken and these same duplicates were both measured at 400 and 465 nm. Again, issues of colour measurement were raised. Figures 3 and 4 illustrate the percentage colour removal efficiencies, but, they do not indicate the difference between the experiment blank readings at 400 and 465 nm, which produced absorbances of 0.258 and 0.152, respectively. These experiment blank results were used as reference points from which to measure percent removal in order to analyze the pretreatment efficiencies. Again, it must be noted that colour measurement is not a measure of quantity of chromophores but a measure of absorbed light. It was concluded that light at 400 nm was absorbed more readily by the chromophores in the pulp mill effluent than that at 465 nm.  When analyzing which pretreatment method was most appropriate for use in this research, the values obtained at 400 nm had more significance than those at 465 nm, since the 400 nm values represented the removal of more colour. Although the removal efficiencies after pretreatments with NaOH, HC1, C a C h , and N a H C 0 3 were notable, ultimately the autoclaving-only pretreatment was selected for further use. This was primarily due to the simplicity of this pretreatment process and the choice was reinforced by the lack of obvious superiority of any of the other pretreatments. The autoclaving-only pretreatment did not involve the use of chemicals or additional soaking time, but only involved autoclaving of the cells, which was also required with every pretreatment process. This is more practical for real world application.  Other studies using inactive biosorbents have also selected autoclaving only for pretreatment. Zhou and Banks (1991), for use on humic acids, and Gallagher et al (1997), for use on dyes and metals, found that autoclaving the biomass increased biosorption. For removal of Basic Blue 9 dye, Fu and Viraraghavan (2000), selected autoclaving only pretreatment for further use in that study. Fu and Viraraghavan, also found that NaOH, CaC^, and Na2C03 provided similar removal efficiencies to the autoclave-only pretreatment. In fact, generally there were many similarities in the effects of pretreatment on biosorption between Fu and Viraraghavan's study on Basic Blue 9 and the present study results at 400 nm, with a few distinct differences. NaOH pretreatment has also been used by Banks and Parkinson (1992), and Zhou and Banks (1993), for humic acid removal, Gallagher et al. (1997), for use on dyes and metals, and, Rao (2001), for 53  removal of phenol, and was found to provide increased removal efficiency. In another study by Fu and Viraraghavan (2002), on Congo Red dye, pretreatments with HCI, H2SO4, CaCl3 and NaHCC>3 were the most effective, with NaHCC>3 pretreatment being selected for use. Like the Basic Blue 9 dye pretreatment results, there were many parallels between the pretreatment results of the Congo Red and the current study.  The data obtained in this mini-study provided a great deal of information regarding the chromophoric material and biosorption using A. niger. Based on the results of colour absorbance at 400 nm, it was possible to assume that the effects of electrostatic adsorption were not significant for removal of the chromophores, since the pretreatments generally seemed to slightly reduce biosorption rather than increase it when compared to autoclaving-only pretreatment. HCI and NaHCC>3 pretreatments showed similar removals to autoclave-only pretreatment for measurement at 400 nm; these pretreatments were used with the intent of altering the fungal surface charge of the fungal biomass which is typically negative (Fu and Viraraghavan, 2001b).  NaOH is used to expose chitin in the fungal cell walls and, since this pretreatment enhanced the removal of colour that absorbs at 400 and 465 nm, it can be assumed that the colour was biosorbed on chitin sites.  Colour removal efficiencies at 465 nm provided similar conclusions for most effective pretreatment, although it was apparent that the chromophores absorbing light at this wavelength were removed by slightly different mechanisms, since some pretreatments were more successful at removing colour bodies reflected at 400 nm. Again, autoclaving and NaOH pretreatments showed substantial removal, which indicated a physical type adsorption not dependant on electrostatic surface charges.  Evident from the results of the molecular weight distribution measurements, the effluent colour was composed of a wide variety of molecules. Therefore, defining the mechanisms responsible for the removal of the chromophores was difficult since many may have been working at once. The different chromophores in the effluent could have been removed by different biosorption mechanisms.  54  J. 2.2 Optimum pH mini-study The alteration of the effluent pH did have an effect on the biosorption capability. Figure 5 displays the results of initial pH on colour removalfromWestern Pulp effluent. "Total" refers to removal of colour through the entire process of pH adjustment and biosorption, and "fungus only" describes the removal that occurred only due to biosorption, i.e. subtracting the removal associated with pH adjustment from the overall removal efficiency. In every situation pH was adjusted to experimental pH, the experiment was performed, the sample was filtered and finally the pH was adjusted to pH 7.6 prior to colour measurement.  i i Total 100% |  • Just Fungus  •  :  —  90% 80% |  — - ---  -  70%  pH7.6  pH2  pH4  pH 6  pH 8  pH 10  pH 12  Effluent pH  Figure 5. Effect of initial effluent pH on biosorption  The initial effluent pH not only altered the biosorption efficiency but also the colour quantity in the effluent after filtration. For example, by adjusting the effluent pH to 2, 70% of the colour was removed without biosorption. This indicated that a reduction of pH to very low values precipitated some of the chromophoric material, which then was filtered from the samples. This 55  also occurred at pH 4. The reverse happened at pH 8, whereby the colour bodies became more soluble and, therefore, colour that would have previously beenfilteredby the removal of the biomass was not removed. Essentially, the biomass was exposed to more colour and thus appeared to be more successful.  There is no clear trend regarding the effect of the initial effluent pH on the biosorption efficiency. Typically, biosorption occurs more readily under either basic or acidic conditions but generally not both. However other variations have been reported (Fu and Viraraghavan, 2001a; Rao, 2001; Zhou and Banks, 1993). Again, the reason that a clear trend was not seen for the effluent colour could be a result of the "blend" of chromophores in the effluent. It appeared that pH 4 and 8 were the most effective initial pHs. This could have been related to an electrostatic mechanism of biosorption. Neutralization of the negative surface charges could have enhanced the removal at pH 4.  5.2.3 Biomass washing mini-study Due to the observance of excessive COD levels in the filter effluent in the previous batch tests, the biomass washing mini-study was performed. This study involved addition of the biomass to distilled-deionized water. The biomass and distilled-deionized water mixture was shaken on the rotary plate for the specified time periods. COD was measured from the biomass and water mixtures afterfilteringwith the 0.22 urn filters.  Figure 6 displays the results of this study using Western Pulp effluent. Please note, as stated in Section 4.3.3, "wash before autoclaving" means wash before autoclaving only, "wash after autoclaving" means wash after autoclaving only and "double wash" means washing before and after autoclaving.  56  • Wash before autoclaving Wash after autoclaving I Double wash  0.25  1  24  48  Time (hrs)  Figure 6. Biomass wash study results where biomass was in contact with distilled-deionized water  One of the duplicate test results was removed for time 1 h for the "wash after autoclaving" test, since it was more than 100% higher than the other sample taken at this time and the rest of the wash after results. A sample at time 1 hour was not taken for the "wash before autoclaving" test.  From Figure 6 it is quite evident that the "double wash" method was the most effective as the COD results showed the double wash methodfilteredsamples to have significantly less COD after contact with biomass in all the specific time periods. Further the "wash after autoclaving" method also proved to be more effective than the originally utilized "wash before autoclaving" method since the COD concentrations remaining in thefilteredsamples were higher than with the "wash before autoclaving" method.  With the "wash before autoclaving" method, the rinsing was only done prior to autoclaving. It was hypothesized that autoclaving disturbed the cell wall, thereby releasing organic matter that was within the cells. With no washing after autoclaving, the released matter remained with the biomass and in turn was released to the liquid samples. In addition, since the cells were live 57  when the washing occurred, it was their nature to try to equilibrate the external liquid concentration with the liquid in the cell (Oxford University Press, 2004). This could explain why it was difficult to achieve a wash water pH equivalent to that of distilled water. If the cells were rupturing due to this concentration difference, salts would have been released during biomass washing.  To determine why the "wash after autoclaving" method was less effective than the "double wash" method was challenging. In theory, the after autoclave wash should have remedied the issue of organic matter being released from the cells as a result of the autoclaving. However, other explanations are possible. For example, since the biomass was autoclaved in the medium, this could have allowed additional medium constituents to enter the cells as a result of the cell disruption due to autoclaving and, therefore, become difficult to remove during washing.  Overall the "double wash" method produced the "cleanest" biomass of the three wash methods tested and this approach was used from this point on.  Although the results of this Biomass Washing Study had potential to negate the data from the previous Optimum Pre-Treatment and Optimum pH Mini-Studies, the results from this Biomass Washing Study for colour showed no significant difference in the colour values after the addition of biomass into distilled-deionized water. For example for the "double wash" method the average colour value of the water was approximately 20 T C U whereas the "wash before autoclaving" method used in the previous studies had an average of approximately 40 TCU. Therefore the difference in colour was not significant in this study where colour removal values were in the hundreds of T C U range. Optimum pretreatment and pH was selected based on colour removal, not COD removal.  Interference with biosorption could have occured from the high COD levels found in the "wash before autoclaving" biomass washing method. However, although the Optimum Pre-Treatment and Optimum pH Mini-Studies were not repeated with "double wash" prepared biomass, it was felt that the previous test results would be indicative enough regardless of some interference from the COD. Additionally, the selection of the use of autoclave only and original pH made from these mini-studies were also based on practicality of process and not explicitly on the highest colour removal. 58  5.2.4 Mixing mini-study In addition to the observance of excessive COD levels, there was also concern that mixing was not complete. To ensure that the batch testing was performed effectively, a study was carried out to analyze three variables: flask sizes, shaker, speed and dose, using Western Pulp effluent. From this test the filtered samples were analyzed for colour removal. The combination of mixing variables that provided the most colour removal indicated the most effective mixing.  Figure 7 shows results from runs 1 and 10 (125 rpm; 125 mL and 300 mL, respectively; 0.2 g) and runs 2 and 3 (125 rpm; 125 mL; 0.5 g and 0.8 g, respectively), and runs 11 and 12 (125 rpm; 300 mL; 0.5 g and 0.8 g, respectively), at 48 h, which compares the different flask sizes and biomass doses and the amount of colour that was removed. Additionally, Figure 7 also shows test results at 0.2 g (runs 2 and 3) with the biomass in contact with the effluent for 24 h, labeled "0.2 *ks test". These results are included since the results obtained from the mixing test at 0.2 g for 48 h contact were not consistent with other data obtained from the kinetic testing at 0.2 g. Since the data obtained in the kinetic study were more consistent with the trends of other results at dose rates 0.5 and 0.8 g, it was suspected that the data at 0.2 g from the mixing test was erroneous. As the kinetic study data for 0.2 g appears more appropriate, it was used for analysis.  59  1200  u  1000  H > o  800  a  400  I  200  "o U  600  0.2 *from mixing test  0.2 *from ks test  0.5  0.8  Biomass Dose (g) •  125 ml flask  f l 300 ml flask  Figure 7. M i x i n g mini-study comparing the colour removal efficiency of the biomass with 48 h contact time using 125 m L and 300 m L flask sizes  Figure 7 shows that the test results indicated that the 300 m L flask size allowed for more mixing to occur than the 125 m L flask size, since colour removal was greater at 300 m L .  Figure 8, compares the results at different shaker speeds 125 rpm and 200 rpm, runs 10-12 (125 rpm; 300 m L ; 0.2 g, 0.5 g and 0.8 g, respectively) and 16-18 (200 rpm; 300 m L ; 0.2 g, 0.5 g and 0.8 g, respectively) on Western Pulp effluent with results o f the total colour removed.  60  ;iqo&£  • Dtosie0.8g-125 rpm  B D o s e 0 . 8 g - 200 rpm  SDosefj.5g- 125 rpm  B Dose 0.5g»200 rpm  B Dose 0,2g - 125 rpm  • Dose 0.2g~ 200 rpm  Figure 8. Mixing mini-study comparing the colour removal efficiency of the biomass with 48 h contact time using 125 rpm and 200 rpm shaker speed  The results for times 0.25, 1 and 4 h indicate that there was no improvement from the increased shaking speed since the amount of colour removed by the biomass did not show any consistent increase colour removal. However, the results at time 24 h showed a consistently greater colour removal, which indicated a benefit from increased shaker speed. It was likely that the increased speed did result in increased colour removal since the long-term removal at 24 h showed a consistent increase. Nonetheless, it was not completely clear that 200 rpm improved mixing, particularly due to the high level of error for colour measurement.  Observance of mixing at different flask sizes and rotation speeds showed that at 125 rpm, the 125 mL flask did not allow full suspension of all the biomass, particularly at the higher doses, 0.5 and 0.8 g. However, with the larger flask size, 300 mL, at 125 rpm all of the biomass was suspended. Further, both flask sizes at 200 rpm showed suspension at all doses. Therefore, at the time of research, it was assumed that increased shaking speed did not result in increased mixing. However, in retrospect, based on further analysis, this may not have been the correct assumption. The colour results shown in Figure 8 did not unquestionably indicate that the 61  increased speed from 125 rpm to 200 rpm was beneficial, however, the COD results for the same test run provides another perspective.  Figure 9 shows the total COD results for the mixing study for runs 10-12 (125 rpm; 300 mL; 0.2 g, 0.5 g and 0.8 g, respectively) and 16-18 (200 rpm; 300 mL; 0.2 g, 0.5 g and 0.8 g, respectively). 1200  0.25  • Dose 0 . 8 g « 125 rpm BLDoseOJg* 200 rpm.  1  24  Sample at time t (h) B Dose 0.8g - 200 rpm • Dose 0.5g -125 rpm t l Dose 0.2g -125- rpm • Dose 0,2g - 200 rpra  Figure 9. Total C O D results of the sample from the mixing study using biomass doses of 0.2 g, 0.5 g and 0.8 g at 125 rpm and 200 rpm  Note the results shown for 0 h indicated the initial total COD of the effluent prior to biomass addition. The results show a definite improvement in total COD levels in the effluent with an increased shaker speed. Most of the runs showed higher levels of total COD, compared to the runs with the same dose, at 125 rpm than for 200 rpm. Since, from visual observance while testing, the fungus appeared suspended using a 300 mL flask size at both 125 rpm and 200 rpm, perhaps the increased shaker speed improved bulk solution transport, therefore enhancing the absorption. This could mean that the bulk solution transport was a limiting factor.  Finally, it appears that further testing of runs 14 and 15 and perhaps 13 (160 rpm; 300 mL; 0.5 g, 0.8 g and 0.2 g, respectively) should have been performed to clarify the results of this study. It is 62  likely that the increased shaking speed was increasing colour absorption at doses of 0.5 and 0.8 g. However, the results obtained for this study were not very clear. As well, mixing at 200 rpm was quite violent and since biomass production occurred at 125 rpm, maintaining the shaker speed at 125 rpm allowed biomass production and testing to occur simultaneously on the same shaker. Therefore, a shaker speed of 125 rpm was warranted.  5.2.5 Kinetic mini-study part 1. The kinetic mini-study was performed to determine both the kinetic rate and the approximate equilibrium point of absorption. This was determined from comparison of colour removal efficiencies at different contact periods. Both effluents were utilized in this mini-study.  Figure 10 displays the amount of colour removed from Western Pulp and Howe Sound effluent through time with a biomass dose of 0.2 g.  • Western Pulp  10  Howe Sound * one data set  20  30  40  50  60  Time (h)  Figure 10. Kinetic study of Western Pulp and Howe Sound effluents at a dose of 0.2 g and time interval up to 52 h  With the initial observation, it appeared that there were two phases of removal, one that plateaued around time 8 to 15 h, and another that began around 19 to 32 h. This raised the question; what caused the removal for this second phase? This may have been a result of 63  chemical, biological or biochemical mechanisms. The appearance of a second phase of removal at such a time leads one to suspect that biological mechanisms are the cause. In response, testing was performed on the biomass under biologically-inhibited conditions.  5.2.6 Inhibition mini-study  The inhibition mini-study was used to determine i f biological activity was causing this second phase of removal or i f it was a result of other mechanisms. This was determined by comparing the colour removal efficiencies using three inhibition methods; a reduced temperature of 4°C, addition of N a N and addition of NaF. 3  Figure 11 shows the results of various biologically inhibiting conditions or biological chemical inhibitors on Western Pulp effluent on the colour removal efficiency of the biomass.  Time (h)  Figure 11. Biologically inhibited kinetic study of Western Pulp effluents at a dose of 0.2 g and time interval up to 52 h  Note that the results for N a N and NaF testing were obtained from single runs, whereas the test 3  runs at an ambient temperature of 4°C was run twice. Both the N a N and temperature-inhibited 3  treatments showed only a very small increase in colour removal at time 19 to 32 h. Therefore, it 64  was reasonable to assume that biological activity contributed to the delayed removal observed more predominately in the Kinetic Study Part 1 where no biological inhibitor was utilized. Therefore, in an effort to "clean up" the biosorption results to allow for a less complicated description of the biosorption mechanisms, N a N and temperature inhibition were used in the 3  kinetic rate mini-study.  A colour removal increase did seem to occur after time 24 h for the effluent that was inhibited with NaF. It could be hypothesized that NaF was unable to inhibit the biological activity that was occurring in the batch tests, however, it could also be hypothesized that the dose in which it was applied that was incorrect. The dose selected for this study may have been too low to be effective.  In addition, the results at 4°C compared to the N a N at room temperature revealed an increased 3  biosorption under the N a N test conditions, which may have been a result of the N a N being run 3  3  at a room temperature (approximately 20°C). These results prompted the temperature ministudy. Further discussion regarding these differences will be presented below.  5.2.7 Kinetic mini-study part 2 The kinetic rate study was continued using two of the previously tested biological inhibitors; an experimental temperature of 4°C and N a N addition. It was intended with the inhibition of 3  biological activity, the biosorption kinetic rate analysis of would have reduced interference providing a more precise comparison to the Lagergren and Ho et al. equations.  Figures 12 and 13 illustrate the results of the inhibited kinetic studies of Western Pulp and Howe Sound effluents, respectively, of colour removed from the effluent at the specified time intervals under the two biologically inhibited conditions.  65  Time (h)  Figure 12. Biosorption of colour from Western Pulp effluent at various specified time intervals at 4°C and with addition of NaN  3  Time (h)  Figure 13. Biosorption of colour from Howe Sound effluent at various specified time intervals at 4°C and with addition of NaN3 66  The results of colour removal due to biosorption using Western Pulp effluent, shown in Figure 12, indicated that removal of colour occurred significantly within the first 8 h of biosorption, with emphasis on the first hour, then continued slowly until measurement ceased at 48 h. The colour removal data from biosorption of Howe Sound effluent, shown in Figure 18, on the other hand, indicated more immediate removal, with most of the colour removal occurring in the first hour. After that point, biosorption reached a plateau, with only a slight further increase through time. The results of biosorption of colour using Howe Sound effluent appeared to have more variability and this could have been a result of the difficulty experienced whilefilteringthe effluent. The filtering of the Howe Sound effluent with or without addition of the biomass was always more difficult than for the Western Pulp effluent. It was hypothesized that this is a result of the higher electrical conductivity levels in the Howe Sound effluent as seen in the Effluent Characterization Study results.  The results obtained from this mini-study were similar to other studies using dead biomass for biosorption in which removal took anywhere from 1 to 48 h to reach equilibrium, depending on initial effluent pH for dead A. niger used for the removal of metals, dyes and phenol (Fu and Viraraghavan, 2000; Fu and Viraraghavan, 2001b; Fu and Viraraghavan, 2002; Kapoor et al., 1999; Rao, 2001). However, in some studies, equilibrium took 3 days or 4 weeks to achieve as in the case of studies done by Zhou and Banks (1993), and Gallagher et al. (1992). As well, maximum removal periods vary; although, some have been reported to be as rapid as the first 10 min of contact (Banat et al., 1996).  To determine if the rate of biosorption fit either the Lagergren or Ho etal. equations, the data were analyzed using STATISTICA® version 6.1 statistical analysis software. Using a GaussianNewton estimation method and a 95% confidence level, the following results were obtained. Please note that 1 TCU = 1 mg of Pt-Co/L and this unit was used to describe the concentration of colour removal.  Table 15 presents the kinetic constants estimated for the Lagergren and Ho et al. equations by non-linear regression analysis of the measured data and, also, the statistical comparison of the resulting equation with the measured data.  67  Table 15. Results of non-linear estimation for colour removal for Lagergren K and Ho et al. k, biosorption rate models, using STATISTICA® Effluent  Western Pulp 4°C  Western Pulp NaN 3  Howe Sound 4°C  Howe Sound NaN * 3  N=9 Std. error t(7) p-level .N=7 Std. error t(5) p-level N=9 Std. error t(7) p-level N=7 Std. error t(5) p-level  de (mg/g) 158.68 10.14 n/a n/a 195.02 17.97 n/a n/a 137.11 7.13 n/a n/a 155.63 11.87 n/a n/a  Lagergren K (1/h) 45.77 n/a n/a n/a 53.08 n/a n/a n/a 50.96 n/a. n/a n/a 51.51 n/a n/a n/a  R 0.89  0.87  0.92  0.91  (mg/g) 158.68 12 13.71 0.000003* 195.02 21 9.40 0.000230* 137.11 8 17.08 0.000001* 155.63 14 11.34 0.000093*  Ho et al. k (g/mg-h) 345.14 7686233 0.00004 1.0 435.92 19157976 0.000023 1.0 294.59 3896835 0.00008 1.0 351.60 8237917 0.00004 1.0  R 0.89  0.87  0.92  0.91  * note: extremely high results for t = 0 were thrown out and replaced by qt = 0 at t = 0 this indicates that the model parameters estimated were statistically significant (t-test) at a 95% confidence level  #  For the Lagergren model, the q and K estimated by non-linear regression using STATISTICA  0  e  indicated that the values were degenerated and therefore may not be correct. Therefore, the statistical values for t-test, p-level and standard error for K are not applicable. The estimated values of k obtained for the Ho et al. model were statistically significant at a confidence level of 95% for q but not for k. This indicates that 95% of the measured data fit the Ho et al. equation e  with the q values generated by STATISTICA®, however, the estimated k did not fit with 95% of 0  the measured values. The p-level indicates the probability of the relationship of the variable as being a "fluke", i.e. indicating the statistical significance of the relationship (StatSoft Inc., 2006). Therefore the lower the p-level the less likely the measured data's fit with the model is a "fluke".  Nonetheless, based on the R values, the correlation co-efficient, it can be said that correlation of the measured data to the Lagergren and Ho et al. model, in general, was good. The range of possible R values is between 0 and 1, therefore values between 0.87 and 0.92 show good  68  correlation. The model could be used to predict the kinetic rates of colour biosorption of fungal biomass on the treated whole mill pulp effluents used in this study.  Figures 14 to 17 show the comparison of the measured kinetic rate data obtained from the Kinetic Mini-Study Part 2 with the Lagergren and Ho et al. equations with constants estimated by the non-linear estimation of the S T A T I S T I C A program, using two different biological inhibitors on both the Western Pulp and Howe Sound effluents. The constants obtained from non-linear estimation based on the Lagergren and Ho et al. models provided nearly identical curves for the intervals over which the data were measured and therefore overlap in the Figures 14 to 17.  •  Measured  • Lagergren and Ho et al.  250  200  £  150  6, & 100 Lagergren : q, = 159(1- e^)  , H o et a l . : q, = T T  50  10  20  30  40  2x345x(159) xf ^ J ^ . (2x345x(159rx0 2  50  60  Time (h)  Figure 14. Kinetic Study: Lagergren, Ho et al. and measured data - Western Pulp effluent at 4°C  69  •  Measured  • Lagergren and Ho et al.  300  2x436x(195) xf Ho et al.: q = 1+(2X436X(195) X0 2  Lagergren : q, = 195(1 - e^ ) x,)  10  t  30  20  2  40  50  60  Time (h)  Figure 15. Kinetic Study: Lagergren, Ho et al. and measured data - Western Pulp effluent with NaN3 • Lagergren and Ho et al.  Measured  180 160 140  1  120 100  a  r  2x295x(l37) x7 Ho et al.: q, = l + (2x295x(137) xt) 2  80  Lagergren : q, = 137(1 - V" " ) 51  0  2  60 40 20  10  20  30  40  50  60  Time (h)  Figure 16. Kinetic Study: Lagergren, Ho et al. and measured data - Howe Sound 4°C  70  • Lagergren and Ho et al.  Measured 250  200  150  100  2x352x(156) xr 2  Lagergren: 4 = 156(1- e {  )  52x,)  Ho etal.: 4 =  1 + (2X352X(156) X0 2  50  10  20  30  40  50  60  Time (h)  Figure 17. Kinetic Study: Lagergren, Ho et al. and measured data - Howe Sound NaN3  5.2.8 Isotherm mini-study The results of the isotherm mini-study were very informative as they gave an indication of the biosorption mechanisms of the biomass for removal of colour. The isotherm mini-studies were conducted on both effluents with addition of NaN3 as an inhibitor. Colour removal was measured and used to determine equilibrium dose and the best fit isotherm model, either the Langmuir, Freundlich or BET.  Figures 18 and 19 show the colour removed by biosorption obtained in the isotherm testing using different doses of biomass.  71  Dose (g/L)  Figure 18. Isotherm Study: Colour removal of both effluents with NaN addition at 32 h at 3  room temperature  • 400 -,  :  Western Pulp  •  —  Howe Sound •  —  Dose (g/L)  Figure 19. Isotherm Study: DOC removal of both effluents with NaN3 addition at 32 h at room temperature  72  The test results for colour removal showed the maximum removal values for each effluent at approximately 900 TCU, which is a significant amount of colour removed. This reflects a removal of approximately 55% of the Western Pulp and 75% of the Howe Sound effluent colour. This is roughly a moderate-to-good removal efficiency compared to other removal colour removal technologies available.  The test results for DOC removal indicated that when applying the biomass to the Howe Sound effluent, DOC removal was possible, up to approximately 200 mg/L. However, above a certain dose of biomass, the amount of DOC introduced by the addition of the biomass was greater than that removed. The DOC removal of the biomass on the Western Pulp effluent did not provide enough removal at any point that was able to counter the amount of DOC introduced by the biomass.  It was difficult to explain the difference of addition of organics between the two effluents after treatment since colour removal for Western Pulp effluent was very similar to Howe Sound effluent. It would be more logical to see a consistent removal of constituents from each effluent. One explanation of the difference in organics loading may be a result of differing chemical strengths of the effluents.  In order to better understand the type of adsorption that was occurring, non-linear estimation of the Langmuir, Freundlich and BET constants was performed and then correlation of the resulting equations were applied to the measured data. Table 16 indicates the correlation of the measured data to these models. Again, STATISTICA® version 6.1 statistical analysis software was used, employing the Gaussian-Newton estimation method and a 95% confidence level.  73  Table 16. Isotherm mini-study results compared to Langmuir, Freundlich and BET models Effluent  Western Pulp  Howe Sound  Langmuir  N=6 Std. error t(4) p-level N=6 Std. error t(4) p-level  0° (mg/g) 84.99 126.92 0.0000 1.00 101.20 141 0.718 0.5124  B 1350 2xl0 0.6696 0.5398 487.83 120377653 0.0000 1.0  Freundlich R  K  0.47  n/a n/a n/a n/a n/a n/a n/a n/a  9  0.40  F  N  n/a n/a n/a n/a n/a n/a n/a n/a  * this indicates that the model parameters estimated were statistically significant (t-test)  BET R  0° (mg/g) 36.75 0 4.74 7.75 0.0015* 0 126.64 19.34 6.55 0.0028* at a 95% confidence level  B  R  3.59 3.98 0.90 0.418 0.99 0.40 2.51 0.0663  0.97  0.99  Of the estimated data listed in Table 16, only values for Q° obtained from the BET model were statistically significant at a 95% confidence level; the estimated constant was verified to be correct with a 95% of the measured data. Estimation of the constants of the Freundlich equation was not possible, since, the measured data could not fit the Freundlich equation.  Based on the R values of the Langmuir and BET equations, the correlation of the measured data with these models, in general, is sufficient for prediction of biomass dose rates. The BET model at R values of 0.97 and 0.99 for Western Pulp and Howe Sound effluent, respectively, shows a superior correlation than the Langmuir equation at 0.47 and 0.40 for Western Pulp and Howe Sound effluent, respectively. The BET model applies the Langmuir model to each monolayer and, therefore, it was logical that the Langmuir applied poorly yet the BET model applied so well. The excellent correlation with the BET model confirmed the assumption that biosorption with dead A. niger biomass was a physical process. This also led to the conclusion that multilayer adsorption was occurring.  Figures 20 and 21 show the measured isotherm data, inhibited by NaN , and the BET equation 3  using the constants estimated with non-liner regression by STATISTICA .  75  •  Measured  BET  250 !  1600 Concentration (mg/L) Figure 20. Kinetic Study: B E T and measured data - Western Pulp effluent with NaN at 3  room temperature •  Measured  BET  300  Ce (mg/L) Figure 21. Kinetic Study: B E T and measured data - Howe Sound effluent with NaN3 at room temperature 76  5.2.8 Removedfraction mini-study  Isotherm Mini-Study samples at a dose of 1.0 gram and the experimental blank, for both effluents, were analyzed by molecular weight fractionation in order to identify the fractions removed by biosorption.  Figures 22 and 23 illustrate the total colour and TOC, respectively, in each fraction of the samples of dose 0 g and 1.0 g for the Western Pulp and Howe Sound effluents.  Generally, colour removal occurred in all the molecular weight fractions of both effluents except for those fractions below 500 Da. The data in Figure 22 also indicated that colour in the <500 Da fraction increased slightly after contact with the biomass. It was difficult to determine if this was due to test method error or to a true increase. Notably, most of the colour removal from the Western Pulp effluent occurred in the 10,000 to 100,000 Dalton range, whereas with the Howe Sound effluent, colour was removed in the 500 to 3,000 Da and the 10,000 to 100,000 Da ranges. This lower molecular weight colour of 500 to 3,000 Da was not present in the experiment blank of the Western Pulp effluent, meaning there was no colour fraction in the effluent to be removed. Therefore, it was concluded that dead A. niger biomass removed the medium-to-high molecular weight fractions of colour but was ineffective for very low, less than 500 Da, and very high, greater than 100,000 Da, fractions. The medium-to-high molecular weight fractions have also been reported to have been removed in colour treatment processes such as lime treatment (Dugal et al., 1974).  The TOC data in Figure 23 also revealed that organics were being removed by the biomass, however, an increase of organics in a different fraction was resulting. Again removal seemed to be occurring in the mid-range of molecular fractions as seen with colour. However, the increase in very high molecular weight fractions was significant. This occurred more dramatically for the Western Pulp effluent, which confirmed previous results indicating post biosorption COD levels were higher than those before biosorption, ie. after the addition of the biomass to the effluent. This also occurred for the Howe Sound effluent to a smaller extent, which was not as noticeable since it was masked by organic removal of other fractions. These results support the results of the Biomass Washing Mini-Study where organics were being introduced to the sample fluids, the treated plant effluent or distilled-deionized water, from the addition of the biomass. Nonetheless, the biomass was still effective at colour removal. Biosorption of Howe Sound effluent was still 77  • WP blank  WPl.Og  IS HS blank  HS 1.0  • WP blank  0  WP 1.0 g  25  50  75  E HS blank  100  125  150  HS 1.0 g  175  200  225  TOC (mg/L)  Figure 23. Molecular weight distribution of TOC for both effluents after treatment  able to show an overall removal of organics when biomass doses were not excessive, ie. the doses where additional biomass provided no further colour removal. A s there was organic removal occurring by the fungus it would be interesting to research i f other problematic pulp mill  79  effluent constituents, such as those measured as absorbable chlorinated organics, AOX, is removed with these organics.  5.2.9 Temperature mini-study From the previous kinetic data it was observed that temperature affected the rate of color removal. Therefore, a test run was performed at 35°C with the addition of the NaN3 inhibitor to identify the effects of temperature.  Figure 24 shows the total colour removed with an experimental temperature at 35°C with NaN3 addition and the previous results of Western Pulp effluent at 4°C without NaN3 and 20°C with NaN . 3  Figure 24. Colour removal at different temperatures  Unfortunately, it appears that the inhibitor dose of 0.1 mM was not enough to prevent biological activity at the higher temperature of 35°C. However, it was evident throughout time and prior to 20 h the removal ability at 35°C was greater than removal at lower temperatures. This was unexpected since it was assumed that biosorption with dead A. niger was a physical phenomenon. Physical adsorption should decrease as temperature increases (International Union 80  of Pure and Applied Chemistry, 1971). Even though this was contrary to the principles of physical adsorption, this does not imply that physical adsorption was not the principal biosorption mechanism. Instead, this could indicate that chemical mechanisms were limiting factors in the biosorption of pulp mill effluent colour to dead A. niger biomass. In the mixing study it was observed that although the biomass was suspended, more rapid mixing might have increased biosorption. In combination with the results of this mini-study, in which temperature increased biosorption, the results of the mixing mini-study may have indicated that the physical process of adsorption was readily occurring but the transport of the colour to the adsorption sites was limiting. As noted in Section 2.4.1 Basics of Adsorption, there is a series of steps that occurs prior to the absorbate reaching the adsorption site; bulk liquid transport, the absorbate being moved to the vicinity of the stagnant liquid film, and diffusion of the absorbate through the stagnant film layer. Mixing impacts the bulk liquid transport and diffusion is based in chemistry, which is influenced by temperature. Therefore the temperature effect could be a result of the increased diffusion of the adsorbate. 5.3 Practical Application Study  The application of biomass in a simulated real world treatment system was assessed to determine the feasibility of the practical use of dead A. niger biomass. This study was performed at the Howe Sound pulp and paper mill on February 22, 2006. Batch testing was performed that mimicked the treatment process the plant used, using effluent and return activated sludge samples taken from the transfer pipes to the aeration tank. Table 17 details the colour, pH, TSS, TOC and BOD measurements of the five batch test run, Blanks 1 and 2 and a dose of 0.8 g, 4.0 g and 8.0 g in 300 mL of mixed liquor. In Table 17, the Blank sample indicates the average of experimental Blanks 1 and 2 where no biomass was added. Figure 25 shows the total colour removed at each dose.  81  Table 17. Practical application of study results Dose (g) Blank 0.8 4.0 8.0  Dose (g/L) 0 2.67 13.33 26.67  Colour (TCU) 995 595 440 340  pH  TSS (mg/L)  8.45 8.14 7.82 7.75  23 20 47 220  TOC (mg/L) 113 123 185 >189  BOD (mg/L) 43 118 730 >1000  700  2.67  13.33  26.67  Dose (g/L)  Figure 25. Colour removal in practical application study samples using three biomass dose rates  The application of the biomass in a batch activated sludge system did result in colour removal that increased with an increase in dose. However, the purpose of this study was only to determine if colour could be removed in a real world application and, therefore, no optimization of this process was studied. Therefore, it was concluded that with further research there is potential for use of dead A. niger biomass, in powdered form, in an activated sludge process.  82  6. CONCLUSIONS AND FURTHER RESEARCH RECOMMENDATIONS Based on the research performed, the following conclusions have been made.  1. Based on colour removal efficiencies, autoclave only pretreatment was the most effective for measurement of colour at 400 nm. Autoclave only pretreatment was also selected based on its ease in preparation.  2. Initial effluent p H did have an effect on the biosorption rate and also the solubility of chromophores. In terms of colour removed by biosorption only, p H 4 followed by p H 8 showed the greatest removal.  3. Biomass washing and the mixing of the biomass and effluent did affect colour and organic removal efficiencies.  4. At approximately 24 h of biosorption, a second phase of removal occurred. This second phase did not occur when the effluent was inhibited by temperature or chemicals and therefore was contributed to biological activity.  5. The kinetic rate study revealed that colour removal by biosorption occurred most readily in the first 8 h with Western Pulp effluent, and within the first hour for Howe Sound effluent. Lagergren and Ho et al. models can be used to describe the kinetic removal rates, however, the correlation was reasonable but not exceptional.  6. For both effluents the maximum colour removal was over 900 T C U , with a biomass dose of about 20 g/L and 13 g/L for Western Pulp and Howe Sounds effluent, respectively. The isotherm study data fit the B E T isotherm model very well and the Langmuir isotherm model moderately. This indicated that adsorption was occurring in a multi-layer fashion.  7. It was found that the biomass removed medium to high molecular weight molecules between 500-100,000 Daltons. 83  8. Temperature increase also increased biosorption of colour in the Western Pulp effluent between 4 and 3 5 ° C  9. Based on data from the pretreatment, pH, kinetic rate, isotherm and removed fraction mini-studies, it was concluded that physical adsorption was the main mechanism used in biosorption of effluent colour on autoclaved A. niger biomass. In addition, the limiting factors in this study appeared to be bulk solution transport, and stagnant film diffusion.  10. There is potential for use of dead fungal biomass in activated sludge systems. Application of biomass in an activated sludge system did show colour removal from the effluent but did impact the TSS, TOC, and BOD removal efficiency. No further testing was done to improve this process.  As well, the follow areas could be researched further:  1. Limiting factors that affect biosorption. With the data obtained in the mixing and temperature mini-studies it was observed that bulk solution transport and stagnant layer diffusion was limiting.  2. The current study found that organic matter was also removed by the biomass. Research to identify this organic matter could reveal further benefits of A. niger biosorption. For example, more toxic organic constituents such as those measured as AOX, may be biosorbed.  3. The current study showed organics being introduced by the biomass into the effluent. It would be important to determine if other pretreatment forms could counter this organic addition yet still provide good colour removal.  4. As it was found that biosorption with A. niger could remove colour within an activated sludge system, further studies could be performed to determine if biosorption could occur without upsetting the activated sludge system.  84  7. REFERENCES 2005 Encyclopedia Britannica Inc. 2005, Encyclopedia Britannica On-line, "color". Available: http://www.britannica.com/ [2005, July 29]. ACEPT W3 Group, Department of Physics and Astronomy, Arizona State University 1999, Patterns in Nature, Light and Optics. Available: http://acept.asu.edu/PiN/rdg/color/color.shtml [2006, April 5]. Alberta Environment 2005, Technology Based Standards for Pulp and Paper Mill Wastewater Releases. 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Ziobro, G.C. 1990, "Origin and nature of kraft color .1. Role of aromatics", Journal of Wood Chemistry and Technology, vol. 10, no. 2, pp. 133-149.  91  APPENDIX 1. CALCULATIONS Colour Colour values are determined through absorbance measurement of standard values and extrapolated accordingly. The following is a work example of colour value determination. Table A. 1 lists the values used.  Table A . l Worked example values Sample Blank Std. 50 Std. 100 Std. 300 Std. 500 500 Daltons  Absorbance 0 0.024 0.039 0.082 0.143 0.027  Using Microsoft Excel 2004 a linear equation was determined using the standard values. For the values of obtain in Table A. 1 the Equation A. 1 was found.  y = 0.0003x + 0.0063  (A.l)  Where x represents the absorbance value and y is the colour value (TCU). This equation has a R =0.9905 and provides a y value of 78. This value is then multiplied by the dilution ratio, which in this example is 5, and provided a final colour value of 389 TCU for a x value of 0.027.  COD COD values are determined through absorbance measurement of standard values and extrapolated accordingly. The following is a worked example of COD value determination. Table A.2 lists the values used.  92  Table A . 2 Worked example values  Sample Blank Std. 100 Std. 300 Std. 500 Std. 800 HCI HCI HCI HCI  Absorbance 0 0. 036 0.114 0.197 0.317 0.146 0.141 0.137 0.134  0.145 0.141 0.137 0.134  Value 0 0. 036 0.114 0.197 0.317 0.145 0.141 0.137 0.134  0.145  Average  0.139  Using Microsoft Excel 2004 a linear equation was determined using the standard values. For the values of obtain in Table A.2 the Equation A.2 was found. y = 0.0003* + 0.0063  (A2)  Where x represents the absorbance value and y is the COD value (mg/L). This equation has a R =0.9997 and provides a y value of 356 for a x value of 0.139. This value is then multiplied by 2  the dilution ratio, which in this example is 2, and provided a final colour value of 718 mg/L. DOC and TOC Originally, the DOC and TOC values were provided by the instrument, however, due to instrument malfunction midway through the research, measurement of plotted peak height was then used. Peak height measurement of standards was used to determine the DOC and TOC values. The following is a worked example of this TOC value determination. Table A.3 lists the values used.  Table A.3 Worked example values  Sample Blank Std. 50 Std. 500 t=0 •t=0  5 16 158 67 63  Height of peak (mm) 8 5 15 17 138 130 66 67 60 63  5 17 127  Value 6 16 138 67 62  Average  64  Using Microsoft Excel 2004 a linear equation was determined using the standard values. For the values of obtain in Table A.3 the Equation A.3 was found. y = 3.0119x + 6.3303  (A.3) 93  Where x represents the peak height value and y is the D O C or T O C value (mg/L). This equation has a R =0.9905 and provided a y value of 223 mg/L for x of 64. 2  TSS TSS values were determined by the following Equation A 4 . T O (  Where W  ,  [(W«w«pfc  ~  ^filter ) ~~ ^blank  (A4)  i represents the dried weight of the filter containing the solids of the sample and the  samp  e  filter dish, Wf,i, stands for the blank filter andfilterdish prior to filtering and drying, Wbiank er  indicates a filter that passed distilled water at the same volume as the sample and then dried, and V represents the volume of sample.  In the practical application study for Blank 1 W i vj&s 1.1218 g, Wf,i was 1.1215 g, Wbiank samp  e  ler  was -0.0003 g and V is 0.015 L . This provided a TSS value of 40 mg/L.  BODs BOD5 values were determined using Equation A 5 . BOD =  (A5)  -BOD, dilution bottle  5  sample  Where Dc7j jjai stands for initial dissolved oxygen content, DOf i indicates final dissolved n t  ma  oxygen content, BODdu thn represents the B O D of the dilution water, V u  5  volume of sample added to the bottle, and V  botile  sample  e  is the total volume of the B O D bottle.  In the practical application study for the 4.0 g sample mg/L, BODdUuHon was 1.91 mg/L, V  i describes the  samp  DOinitiai  was 9.81 mg/L, DOf i was 3.72  was 0.5 m L and V  ma  bott]e  was 60 mL. This provided a  BOD value of 729 mg/L. 5  94  APPENDIX 2. EFFLUENT CHARACTERIZATION DATA Table A.5 Electrical conductivity measurement on raw Western Pulp effluent Sample 1  micro Siemens 2100  Table A.6 Chloride measurement on raw Western Pulp effluent Sample J  Chloride (mg/L) 156  2  155  Table A.7 Electrical conductivity measurement on raw Howe Sound effluent Sample 1  micro Siemens 3000  Table A.8 Chloride measurement on raw Howe Sound effluent Sample  ~T~ 2  Chloride (mg/L) 145 146  APPENDIX 3. MOLECULAR WEIGHT DISTRIBUTION DATA  Table A.9 Colour measurement for the molecular weight distribution on Western Pulp effluent Sample  Absorbance  Blank  0  Std. 50  0.024  Std. 100  0.039  Std. 300  0.082  Std. 500  0.143  500 Daltons  0.027  1,000 Daltons  0.033  3,000 Daltons  0.035  10,000 Daltons  0.038  100,000 Daltons  0.093  Total  0.114  Note: absorbance measured on U n i c a n U V 300  Table A.10 TOC measurement for the molecular weight distribution on Western Pulp effluent Sample Blank  Instrument V a l u e  Average  0  500 Daltons  95  500 Daltons  95  500 Daltons  97  1000 Daltons  96  1000 Daltons  99  1000 Daltons  97  3000 Daltons  109  3000 Daltons  97  3000 Daltons  98  10000 Daltons  98  10000 Daltons  97  10000 Daltons  94  100000 Daltons  160  100000 Daltons  173  100000 Daltons  165  Total  193  Total  179  96  97  101  96  166 186  Table A . l l Colour measurement for the molecular weight distribution on Howe Sound effluent Sample Blank  Absorbance  Std. 50  0 0.018  0.019  Value 0 0.019  Std. 100 Std. 300  0.042 0.105  0.042 0.105  0.042 0.105  Std. 500 500Daltons 1000 Daltons 1000 Daltons  0.177 0.005 0.006 0.006  0.177 0.004 0.007 0.007  0.177 0.005 0.007 0.007  3000 Daltons 3000 Daltons 10000 Daltons 10000 Daltons 100000 Daltons 100000 Daltons Total  0.008 0.01 0.019 0.018 0.062 0.064  0.008 0.008 0.02 0.016 0.064 0.065  0.11  0.111  0.008 0.009 0.020 0.017 0.063 0.065 0.111  Total  0.129  0.131  0.130  Average  0.005 0.007 0.009 0.018 0.064 0.120  Table A.12 TOC measurement for the molecular weight distribution on Howe Sound effluent Sample 500 Daltons 500 Daltons 1000 Daltons 1000 Daltons  32 39 33 32  Instrument Value (mg/L) 32 40 35 32  Average (mg/L) 32 39 34 34  3000 Daltons 3000 Daltons 10000 Daltons  33 33 32  33 33 32  39 32 32  10000 Daltons 100000 Daltons 100000 Daltons Total  30 50  32 47 49 60  31  49 58  29 51 51 60  Total  55  56  57  58  36 33 34  50  97  APPENDIX 4. PRETREATMENT MINI-STUDY DATA Table A.13 Colour measurement at 465 nm for Pretreatment mini-study on Western Pulp effluent Sample  Absorbance  Blank  Value  0  Std. 50  0.023  0.025  0.024  Std. 100  0.051  0.053  0.052  Std. 300  0.116  0.115  0.116  Std. 500  0.190  0.192  HC1  0.142  0.140  0.141  0.141  HC1  0.141  0.142  0.140  0.141  HC1  0.135  0.137  0.136  HC1  0.137  0.139  0.138  Filter Blank  0.152  0.149  Filter Blank  0.152  Autoclaved  0.196  Autoclaved  0.196  0.197  Autoclaved  0.119  0.120  0.118  Autoclaved  0.125  0.130  0.132  NaOH  0.121  0.118  NaOH  0.114  0.118  NaOH  0.107  0.106  0.107  NaOH  0.106  0.112  0.109  H S0  0.191  0.152  0.151  0.152  0.152  0.198  0.198  0.197 0.197 0.129  0.118  0.128  0.129  0.123  0.124  4  0.130  0.131  0.131  H S0  4  0.128  0.128  0.128  NaCl  0.167  0.163  0.165  NaCl  0.163  0.164  NaCl  0.138  0.143  2  NaCl  0.138  0.138  0.138  0.108  0.109  0.109  NaHC0  3  0.109  0.115  0.109  0.111  NaHC0  3  0.152  0.149  0.152  0.151  3  0.151  0.15  0.148  0.150  0.119  CaCl  2  0.118  CaCl  2  0.121  0.119  0.118  0.119  CaCl  2  0.121  0.124  0.120  0.122.  CaCl  2  0.12  0.123  0.118  0.120  0.130  0.130 0.133  2  3  0.129  Na C0 2  3  0.132  0.133  Na C0 2  3  0.132  0.128  Na C0  3  0.119  0.119  2  0.113  0.128  0.141  3  Na C0  0.124  0.164 0.143  NaHC0  NaHC0  0.197*  0.117  0.124  2  0.152  0.120  0.129  4  0.139  0.119  H2SO4 H S0  2  Average  0  0.152  0.130  0.119  0.129  0.120  0.130 0.119  0.128  Assumed filter breakthrough  98  Table A.14 Colour measurement at 400 nm for Pretreatment mini-study on Western Pulp effluent Sample Blank HC1 HC1 HC1 HC1 Filter Blank Filter Blank Autoclaved Autoclaved Autoclaved Autoclaved NaOH NaOH NaOH  3  3  2  3  0.180  0.217 0.173 0.183 0.203 0.200 0.184 0.183 0.209 0.213 0.185 0.185 0.200 0.203 0.194 0.199 0.184 0.201  0.212*  0.205 0.197 0.186 0.184 0.209 0.214 0.186 0.183 0.200 0.201 0.195 0.202  0.198 0.194  0.226  0.205 0.198 0.199 0.227  0.203 0.230  0.201 0.195 0.201 0.228  0.225 0.220  0.225 0.219  0.226 0.226  0.225 0.222  0.208  0.206  0.209  0.208  2  2  0.178 0.258 0.257 0.207  0.201 0.192 0.202  2  2  0.177 0.258 0.257 0.206 0.215 0.173 0.185 0.200 0.200 0.184  0.176 0.195  3  2  Na C0 Na C0  0.178 0.253 0.258 0.208 0.216 0.175 0.183  0.178 0.204  3  2  3  0.178 0.263 0.256 0.206 0.219 0.17 0.182 0.204 0.204  0.178 0.202  NaCl NaCl NaCl NaCl  2  0.186 0.181  0.193  H2SO4  Na C0  0.185 0.183  3  H2SO4  CaCl Na C0  0.186 0.178  3  H2SO4  CaCl CaCl CaCl  0.187 0.181  0.186 0.209 0.187  H2SO4  NaHC0 NaHC0  0.174  Value 0 0.175  0.183 0.181 0.209 0.211 0.185 0.186 0.200 0.204 0.196 0.199 0.184 0.202  NaOH  NaHC0 NaHC0  0 0.178  Absorbance 0 0.174  0.183 0.210 0.214 0.184 0.184 0.200 0.204 0.190 0.197 0.181 0.193 0.189  0.190 0.177 0.200  Average  0.258  0.178  0.193  0.198  0.199  0.188  0.199  0.221  * Assumed filter breakthrough  99  Table A.15 COD measurement at 600 nm for Pretreatment mini-study on Western Pulp effluent Sample  Absorbance  Blank  Value  0  0  Std. 100  0.036  0. 036  Std. 300  0.114  0.114  Std. 500  0.197  0.197  Std. 800  0.317  0.317  HCI  0.146  0.145  HCI  0.141  0.141  0.141  HCI  0.137  0.137  0.137  HCI  0.134  0.134  0.134  Filter Blank  0.125  0.126  0.126  Filter Blank  0.125  0.125  0.125  Autoclaved  0.175  0.175  0.175  Autoclaved  0.178  0.178  0.178  Autoclaved  0.153  0.152  0.153  0.145  0.145  Autoclaved  0.154  0.154  0.154  NaOH  0.129  0.129  0.129  NaOH  0.125  0.125  0.125  NaOH  0.12  0.119  0.120  NaOH  0.123  0.123  H S0  4  0.121  0.125  H S0  4  0.122  0.121  0.122  H S0  4  0.145  0.145  0.145  4  0.148  0.148  0.148  0.152  0.151  0.152  2  2  2  H S0 NaCl 2  0.123 0.121  0.151  0.15  0.151  NaCl  0.144  0.143  0.144  NaCl  0.137  0.136  0.137  NaHC0  3  0.122  0.122  0.122  NaHC0  3  0.121  0.121  0.121  NaHC0  3  0.143  0.143  0.143  NaHC0  3  0.146  0.142  0.144  CaCl  2  0.124  0.123  0.124  CaCl  2  0.123  0.123  0.123  CaCl  2  0.121  0.124  0.123  CaCl  2  0.123  0.124  0.124  2  3  0.138  0.138  0.138  2  3  0.136  0.139  0.138  3  0.131  0.132  0.132  3  0.127  0.127  0.127  Na C0 2  Na C0 2  0.139 0.125 0.177* 0.153  0.124  0.122  NaCl  Na C0 Na C0  Average  0.134  0.146  0.133  0.123  0.134  * Assumed filter breakthrough  100  Table A.16 pH measurement for Pretreatment mini-study results on Western Pulp effluent Pre-treatment Blanks Autoclaved  pH 8.69 8.05  Autoclaved  8.31  HC1 HC1  8.00 7.90  NaOH NaOH  2  3  8.26 8.62 8.32 8.38 8.06 8.18 8.38 8.14 8.35 8.36 8.14  2  3  8.30  H2SO4 H2SO4  NaCl NaCl NaHC0 NaHC0 CaCl CaCl 2  2  Na C0 Na CQ  3  3  101  APPENDIX 5. PH MINI-STUDY D A T A  Table A.17 Colour measurement for pH mini-study on Western Pulp effluent Sample Blank  Absorbance 0  Value 0  Std. 50 Std. 100 Std. 300 Std. 500 pH2 pH2 pH2 pH2 pH4 pH4 pH4 pH4 pH6 pH6 pH6 pH6 pH8 pH 8 pH8 pH8 pH 10 pH 10  0.015 0.042 0.107 0.184  0.016 0.043 0.107 0.184  0.016 0.043 0.107 0.184  0.019 0.018 0.016 0.014 0.040  0.097 0.095  0.015 0.018 0.017 0.016 0.037 0.040 0.048 0.040 0.089 0.087 0.096 0.094  0.061 0.066 0.074  0.061 0.066 0.072  0.073  pH 10  0.081 0.082 0.094  0.075 0.081 0.080 0.082  0.017 0.018 0.017 0.015 0.039 0.040 0.047 0.040 0.090 0.087 0.097 0.095 0.061 0.066 0.073 0.074  pH pH pH pH pH  10 12 12 12 12  0.040 0.046 0.040 0.090 0.086  0.081 0.080  0.085 0.092  Average  0.017  0.041  0.092  0.069  0.081 0.080 0.082 0.084  0.082  0.093 0.094 0.090  0.095 0.090 0.093  0.093 0.090 0.093  Original pH Original pH Filter blank  0.083 0.085 0.127  0.084  0.084  0.081 0.125  0.083 0.126  0.083  Filter blank  0.129  0.127  0.128  0.127  0.093  0.093  102  Table A.18 COD measurement for pH mini-study results on Western Pulp effluent Sample Blank Std. 100  Absorbance 0 0 0.036 0.036 0.114 0.115 0.197 0.195  Value 0 0.036 0.115 0.196  0.318  0.317  0.318  pH2 pH2 pH2 pH2  0.156 0.156 0.122 0.122  0.156 0.160 0.121 0.122  0.156 0.158 0.122 0.122  pH.4 pH4 pH4 pH4 pH6  0.150 0.152 0.122  0.149 0.149 0.122  0.123 0.110 0.102 0.107  0.123 0.109 0.103 0.103 0.098 0.123 0.123 0.126 0.129 0.137 0.137 0.148  0.150 0.151 0.122 0.123 0.110  Std. 300 Std. 500 Std. 800  pH6 pH6 pH6 pH 8 pH 8 pH 8 pH 8 pH 10 pH 10 pH 10 pH pH pH pH  10 12 12 12  pH 12 Original pH Original pH Filter blank Filter blank  0.097 0.124 0.121 0.125 0.127 0.137 0.136 0.149 0.151 0.284 0.265 0.271 0.277 0.122  0.150 0.283 0.266 0.272  Average  0.139  0.136  0.103 0.105 0.098 0.124 0.122 0.126 0.128 0.137 0.137 0.149 0.151 0.284  0.104  0.125  0.143  0.266 0.272  0.277  0.277  0.274  0.120  0.123 0.120  0.123 0.120  0.121  0.120  0.118  0.119  0.121  0.123  0.122  0.121  103  APPENDIX 6. BIOMASS WASHING MINI-STUDY  Table A.19 Colour measurement for original biomass wash method on de-ionized water Sample Blank Std. 50 Std. 100 Std. 300 Std. 500  Absorbance 0 0.017 0.047 0.110 0.184  t=24 h t=24h t=48h t=48h Filter blank t=48 h  0.018 0.047 0.110 0.185 0.019 0.026 0.015 0.026 0.005 0.002 0.001  Filter blank t=48 h  0.001  0.001  t=0.25 h t=0.25h  Value 0  0.019 0.027 0.014 0.029 0.006 0.002 0.003  0.031 0.006  Average  0.018 0.047 0.110 0.185 0.019 0.027 0.015 0.029 0.006 0.002 0.002  0.023  0.001  0.002  0.022 0.004  Table A.20 COD measurement for original biomass wash method on de-ionized water Sample Blank Std. 100 Std. 300  Average  t=0.25 h t=24 h  0 0.036 0.115 0.196 0.317 0.041 0.045 0.062  0.036 0.115 0.196 0.317 0.041 0.045 0.062  Value 0 0.036 0.115 0.196 0.317 0.041 0.045 0.062  t=24 h t=48h  0.063 0.050  0.062 0.050  0.063 0.050  0.062  t=48h  0.048  0.048  0.049  Filter blank t=48 h  0.003  0.048 0.002  Filter blank t=48 h  0.003  o:ooi  0.002  Std. 500 Std. 800 t=0.25 h  Absorbance  0.043  0.003 0.002  104  Table A.21 Test run 1 colour measurement for "after autoclave" biomass wash method on de-ionized water Sample Blank  Absorbance  Value  Average  Std. 50  0 0.017  0.017  0 0.017  Std. 100 Std. 300  0.041 0.106  0.040 0.106  0.041 0.106  Std. 500 t=0.25 h t=0.25 h t=l h t=l h t=4h t=4h t=24h t=24h t=48 h t=48h Filter blank t=48 h  0.178 0.005 0.002 0.003 0.004 0.001 0.006  0.178 0.004 0.003 0.002 0.003 0.002 0.005 0.002 0.003 0.002 0.004 0.001  0.002  0.001 0.003 0.001 0.005 0.001  0.178 0.003 0.003 0.001 0.001 0.003 0.004 0.002 0.002 0.003 0.003 0.000  Filter blank t=48 h  0.002  0.003  0.003  0.002  0.003  0.004 0.002 0.003  105  Table A.22 Test run 1 COD measurement for "after autoclave" wash method on de-ionized water Sample Blank Std. 100 Std. 300 Std. 500 Std. 800 t=0.25 h t=0.25 h t=l h t=l h t=4h t=4 h t=24h t=24 h t=48h t=48h Filter blank t=48 h Filter blank t=48 h  Absorbance 0 0.035 0.035 0.114 0.114 0.196 0.196 0.316 0.316 0.038 0.038 0.037 0.036 0.064 0.064 0.065 0.065 0.029 0.028 0.030 0.030 0.025 0.025 0.026 0.026 0.032 0.030 0.024 0.023 0.002 0.000 -0.001 -0.001  Value 0 0.035 0.114 0.196 0.316 0.038 0.037 0.064 0.065 0.029 0.030 0.025 0.026 0.031 0.024 0.001 -0.001  Average  0.037 0.065 0.029 0.026 0.027 0  106  Table A . 2 3 Test run 2 colour measurement for "after autoclave" biomass wash method on de-ionized water Sample Blank  0  Std. 50  0.013  0.012  0.013  Std. 100 Std. 300  0.037 0.103  0.036 0.101  0.037 0.102  Std. 500 t=0.25 h  0.178 0.000 0.001 0.004  0.177 0.000 0.000 0.003 0.000  0.178 0.000 0.001 0.004  t=0.25 h t=l h t=l h t=4h 1=4 h t=24h t=24h  Absorbance  0.001 0.001 0.002  t=48 h t=48h Filter blank t=48 h  0.003 0.005 0.002 0.003 0.002  Filter blank t=48 h  0.005  0.003 0.002 0.004 0.005  Value 0  0.001 0.002 0.002  0.003 0.002 0.004  0.004 0.005 0.003 0.003 0.003  0.005  0.005  Average  0.000 0.002 0.002 0.004 0.003 0.004  107  Table A.24 Test run 2 COD measurement for "after autoclave" wash method on de-ionized water Sample Blank Std. 100  Absorbance 0 0.03  0.03  Value 0 0.030  Std. 300 Std. 500  0.112 0.201  0.112 0.201  0.112 0.201  Std. 800 t=0.25 h t=0.25 h t=l h t=l h t=4 h t=4h  0.310 0.023 0.022 0.033 0.021 0.025 0.021  0.310 0.022 0.022 0.030  0.310 0.023 0.022 0.032  0.019 0.024 0.021  0.020 0.025 0.021  t=24h t=24 h  0.033 0.033 0.032  0.033 0.032 0.032 0.032 -0.003  0.032  0.001  -0.001  t=48h t=48h Filter blank t=48 h  0.032 -0.002  0.032 0.030 0.032 0.032 -0.003  Filter blank t=48 h  0.000  0.001  Average  0.022 0.026 0.023 0.032  Table A.25 Test run 1 colour measurement for "double wash" biomass wash method on deionized water Sample Blank Std. 50 Std. 100  Absorbance 0 0.016 0.017 0.042 0.043  Value 0 0.017 0.043  Std. 300 Std. 500  0.108 0.185  0.108 0.185  t=0.25 h  0.001  0.108 0.185 0.001  t=0.25 h t=24h t=24h t=48h  0.003 0.000  0.005 0.001  0.001 0.004 0.001  0.000 0.001  0.000 0.001  0.000 0.001  0.000  t=48h  0.005  0.003 0.002  0.002  -0.001  0.000  Filter blank t=48 h  0.001  0.001 0.002  Filter blank t=48 h  -0.001  -0.001  0.003  Average  0.003  108  Table A.26 Test run 1 COD measurement for "double wash" biomass wash method on deionized water Sample Blank  Absorbance 0 0.035  0.034  0.114  0.114 0.196  Std. 800 t=0.25 h t=0.25 h t=24h  0.196 0.315 0.008 0.008 0.013  t=24h t=48h t=48 h Filter blank t=48 h  0.012 0.005 0.008 -0.002  Filter blank t=48 h  -0.001  Std. 100 Std. 300 Std. 500  Value 0 0.035  Average  0.114 0.196  0.008  0.012 0.004 0.008 -0.002  0.315 0.008 0.008 0.013 0.012 0.005 0.008 -0.002  0.006  0.001  0.000  -0.001  0.315 0.008 0.007 0.013  0.013  Table A.27 Test run 2 colour measurement for "double wash" biomass wash method on deionized water Sample Blank Std. 50 Std. 100 Std. 300 Std. 500 t=0.25 h t=0.25 h  Absorbance 0 0.019 0.040 0.109 0.178 0.004  0.017 0.041 0.105 0.177 0.002  Value 0 0.018 0.041  Average  0.001  0.107 0.178 0.003 0.004  0.002 0.005 0.004  0.003 0.005 0.004 0.005 0.005  0.004  0.006 0.003  0.005  0.004  0.003  t=l h t=lh  0.006 0.004 0.004  t=24 h  0.004  t=24h t=48h  0.004 0.004  t=48h Filter blank t=48 h  0.007 0.004  0.005 0.006 0.004 0.002  Filter blank t=48 h  0.006  0.001  0.003 0.004  109  Table A.28 Test run 2 COD measurement for "double wash" biomass wash method on deionized water Sample Blank Std. 100 Std. 300 Std. 500  Absorbance 0 0.033  Value 0 0.033  Average  0.115 0.204  0.033 0.115 0.205  0.315 0.008 0.009 0.010  0.314 0.008 0.010 0.010  0.315 0.008 0.010 0.010  0.009 0.008 0.008 0.017 0.017 0.001  0.010 0.008 0.008 0.016 0.017 0.002  0.010  t=24 h 1=24 h 1=48 h t=48h Filter blank t=48 h  0.011 0.008 0.008 0.015 0.017 0.002  Filter blank t=48 h  0.005  0.006  0.006  0.004  Std. 800 t=0.25 h t=0.25 h t=l h 1=1 h  0.115 0.205  0.009  0.008 0.017  110  APPENDIX 7. MIXING MINI-STUDY  Table A.29 Test r u n 1 colour measurement mixing study 300 m L flask at 125 r p m Sample Blank  Absorbance 0  Value  Average  0  Std. 50  0.019  0.016  0.018  Std. 100 Std. 300  0.039 0.102  0.041  0.040  0.103  0.103  Std. 500  0.176  0.176  0.176  d=0, t=0  0.124  0.126  0.125  d=0, t=0  0.122  0.124  0.123  d=0.8 g, t=0.25 h  0.093  0.092  0.093  d=0.8 g, t=0.25 h  0.088 0.099  0.090 0.103  0.089 0.101  0.091  d=0.5 g, t=0.25 h d=0.5 g, 1=0.25 h d=0.2 g, 1=0.25 h d=0.2 g, t=0.25 h  0.098 0.104  0.101  0.100  0.103  0.100 0.104  0.102  0.102  0.102  0.103  d=0.8 g, t=l h  0.081  d=0.8 g, t=l h  0.079  0.081 0.078  0.081 0.079  0.080  d=0.5 g, t=l h  0.091  0.090  d=0.5 g, t=l h  0.092  0.088 0.094  d=0.2 g, t=l h  0.098  0.096  0.097 0.092  d=0.8 g, t=4 h  0.091 0.083  0.092 0.083  0.083  d=0.8 g, t=4 h  0.077  0.079  0.078  d=0.5g,t=4h  0.088  0.088  0.088  d=0.5 g, 1=4 h  0.085  0.084  0.085  d=0.2 g, t=4h  0.097  0.096  0.097  d=0.2 g, t=4 h  0.097  0.098  0.098  d=0.8 g, t=24 h  0.077  0.075  0.076  d=0.8 g, t=24 h  0.077  0.076  0.077  d=0.5 g, t=24 h  0.082  0.082  0.082  d=0.5g,t=24h  0.083  0.083  0.083  d=0.2 g, t=24 h  0.089  0.089  0.089  d=0.2g,t=24h  0.086  0.088  0.087  d=0, t=24 h  0.119  0.120  0.120  d=0,1=24 h  0.119  0.116  0.118  d=0.2 g, t=l h  0.093  0.124  0.091 0.094 0.081 0.086 0.097 0.076 0.083 0.088 0.119  111  Table A.30 Test r u n 1 C O D measurement mixing study 300 m L flask at 125 r p m Sample Blank  Absorbance 0  Value  Average  0  Std. 100  0.022  0.023  0.023  Std. 300  0.116  0.117  0.117  Std. 500  0.195  0.196  0.196  Std. 800  0.313  0.313  0.313  d=0, t=0  0.114  0.114  0.114  d=0, t=0 d=0.8 g, t=0.25 h  0.112  0.112  0.112  0.166  0.167  d=0.8 g, t=0.25 h  0.167 0.174  0.175  0.175  0.171  d=0.5 g, t=0.25 h d=0.5 g, t=0.25 h  0.158 0.144  0.158 0.143  0.158 0.144  0.151  d=0.2 g, t=0.25 h  0.123  0.123  0.123  d=0.2 g, 1=0.25 h d=0.8 g, t=l h  0.126 0.177  0.126  0.126  0.177  0.177  d=0.8 g, t=l h d=0.5 g, t=l h  0.175  0.176 0.159  •0.176 0.160  0.176  0.160  d=0.5 g, t=l h  0.159  0.159  0.159  0.159  d=0.2 g, t=l h  0.124  0.124  0.124  d=0.2 g, t=l h  0.126  0.126  0.126  d=0.8 g, t=4 h  0.184  0.186  d=0.8 g, t=4 h  0.226  0.188 0.226  d=0.5 g, t=4 h  0.164 0.164  0.166 0.164  0.165  d=0.5 g, t=4 h d=0.2 g, t=4 h  0.130  0.129  0.130  d=0.2 g, t=4 h  0.129  0.131  0.130  d=0.8 g, t=24 h  0.231  0.231  0.231  d=0.8 g, t=24 h  0.232  0.236  0.234  d=0.5 g, t=24 h  0.191  0.191  0.191  d=0.5 g, t=24 h  0.192  0.191  0.192  d=0.2 g, t=24 h  0.137  0.139  0.138  d=0.2 g, t=24 h  0.139  0.140  0.140  d=0, t=24 h  0.125  0.124  0.125  d=0, t=24 h  0.124  0.124  0.124  0.226 0.164  0.113  0.125  0.125 0.206 0.165 0.130 0.233 0.191 0.139 0.124  112  Table A.31 Test run 1 DOC measurement mixing study 300 mL flask at 125 rpm Sample d=0, t=0 d=0, t=0 d=0.8 g, t=0.25 h d=0.8 g, t=0.25 h d=0.5 g, t=0.25 h d=0.5 g, t=0.25 h d=0.2 g, t=0.25 h d=0.2 g, t=0.25 h d=0.8 g, t=l h d=0.8 g, t=l h d=0.5 g, t=l h d=0.5 g, t=l h d=0.2 g, t=l h d=0.2 g, t=l h d=0.8 g, t=4 h d=0.8 g, t=4 h d=0.5 g, t=4 h d=0.5 g, t=4 h d=0.2 g, t=4 h d=0.2 g, t=4 h d=0.8 g, t=24 h d=0.8 g, t=24 h d=0.5 g, t=24 h d=0.5 g, t=24 h d=0.2 g, t=24 h d=0.2 g, t=24 h d=0, t=24 h d=0, t=24h  DOC (mg/L) 283  Average (mg/L)  239  261  373 393 322  383  311 294 269 379 389 328 334 270 287 403 377 345 339 259 227 414  317 282 384 331 279 390 342 243  413 264  414  311 247 243 214  288  165  190  245  113  Table A.32 Test run 2 colour measurement mixing study 300 m L flask at 125 r p m Sample Blank  Absorbance 0  Value  Std. 50  0.015  0.018  0.017  Std. 100 Std. 300  0.040 0.102  0.041 0.102  0.041 0.102  Std. 500  0.177  0.175  0.176  d=0.8 g, t=0.25 h d=0.8 g, t=0.25 h  0.090  0.091 0.091 0.099 0.101  0.091 0.091 0.100 0.101  0.104 0.104  0.103 0.104  0.081  0.082  d=0.5 g, t=0.25 h  0.091 0.101  d=0.5 g, 1=0.25 h  0.101  d=0.2 g, 1=0.25 h d=0.2 g, 1=0.25 h  0.102  Average  0  0.091 0.101  d=0.8 g, t=l h  0.103 0.082  d=0.8 g, 1=1 h d=0.5g,t=lh  0.081 0.098  0.082 0.097  0.082 0.098  0.082  d=0.5 g, t=l h d=0.2 g, t=l h d=0.2 g, t=l h  0.096 0.108  0.097 0.107  0.097  0.102  d=0.8 g, t=4 h d=0.8 g, t=4 h  0.073 0.076 0.090 0.090  0.105 0.074 0.077  0.097 0.108 0.104 0.074 0.077  0.088 0.088  0.089 0.089  0.089  d=0.2 g, t=4 h  0.095 0.102  0.099 0.101  0.097 0.102  0.099  d=0.8 g, t=24 h  0.080  0.080  0.080  d=0.8 g, t=24 h  0.074  0.074  0.074  d=0.5 g, t=24 h  0.077  0.077  0.077  d=0.5 g, 1=24 h d=0.2g,t=24h  0.081 0.088  0.081  0.081  0.089  0.089  d=0.2 g, t=24 h  0.089  0.088  0.089  0.089  d=0,1=24 h  0.124  0.126  d=0,1=24 h  0.121  0.121  0.125 0.121  0.123  d=0.5 g, t=4 h d=0.5 g, t=4 h d=0.2 g, t=4 h  0.103  0.106 0.075  0.077 0.079  114  Table A.33 Test run 2 COD measurement mixing study 300 mL flask at 125 rpm Sample Blank Std. 100  Absorbance 0 0.025 0.025  Value  Std. 300 Std. 500 Std. 800  0.115 0.195 0.311  0.116 0.194 0.312  0.116 0.195 0.312  d=0, t=0 d=0, t=0 d=0.8 g, t=0.25 h d=0.8 g, t=0.25 h d=0.5 g, t=0.25 h d=0.5 g,t=0.25h d=0.2 g, t=0.25 h  0.114 0.112 0.156 0.164  0.114 0.112 0.157 0.162  0.114 0.112 0.157 0.163  0.145 0.149 0.124  0.014 0.149 0.123  0.080 0.149  d=0.2 g, t=0.25 h d=0.8 g, t=l h  0.117 0.161  d=0.8 g, t=l h d=0.5 g, t=l h d=0.5 g, t=l h d=0.2 g, t=l h d=0.2 g, t=l h d=0.8 g, t=4 h d=0.8 g, t=4 h d=0.5 g, t=4 h d=0.5 g, t=4 h  0.161 0.143 0.150 0.121 0.120 0.187 0.180  0.116 0.163 0.162 0.143 0.149 0.122 0.119 0.181 0.182  Average  0 0.025  0.124 0.117 0.162 0.162 0.143 0.150 0.122 0.120 0.184 0.181  0.113 0.160 0.114 0.120 0.162 0.146 0.121 0.183  0.155 0.165 0.122  0.156 0.163  0.156 0.164  0.118  0.120  0.119 0.205 0.214 0.164  0.120 0.207 0.213 0.163  0.120  0.120  0.206 0.214 0.164  0.210  d=0.5 g, t=24 h d=0.2 g, t=24 h d=0.2 g, t=24 h d=0, t=24 h  0.175 0.127 0.121  0.175  0.175  0.169  0.128 0.122  0.125  0.122  0.128 0.123 0.122  d=0, t=24 h  0.123  0.123  0.123  d=0.2 g, t=4 h d=0.2 g, t=4 h d=0.8g,t=24h d=0.8 g, t=24 h d=0.5 g, 1=24 h  0.160  0.122 0.123  115  Table A.34 Test run 2 D O C measurement mixing study 300 m L flask at 125 r p m Sample  DOC (mg/L)  d=0, t=0  283  d=0,t=0  239  d=0.8 g, 1=0.25 h  289  d=0.8 g, 1=0.25 h  275  d=0.5 g, t=0.25 h  265  d=0.5 g, t=0.25 h d=0.2 g, t=0.25 h d=0.2 g, t=0.25 h  264  d=0.8 g, t=l h  291  d=0.8 g, t=l h  285  d=0.5 g, t=l h  270  d=0.5g,t=lh  267  d=0.2 g, t=l h d=0.2 g, t=l h  226  d=0.8 g, t=4 h  282  d=0.8g,t=4h  267  d=0.5 g,t=4h  264  d=0.5 g, t=4 h d=0.2 g, t=4 h  254  d=0.2 g, t=4 h  207 293  210  d=0.8 g, 1=24 h d=0.8 g, t=24 h  295  294  d=0.5g,t=24h  292  d=0.5 g, t=24 h  284  d=0.2 g, t=24 h  219  d=0.2 g, t=24 h  211  d=0, t=24 h  189  d=0, t=24 h  206  Average (mg/L) 261 282 265  217 218  207  218 288 269 217 275 259  212  288 215 198  116  Table A.35 Test run 1 colour measurement mixing study 300 mLflaskat 200 rpm Sample Blank  Absorbance 0  Value  Average  0  Std. 50  0.017  0.018  0.018  Std. 100  0.042  0.040  0.041  Std. 300  0.105  0.103  0.104  Std. 500  0.178  0.178  0.178  d=0.8 g, t=0.25 h  0.087  0.088  d=0.8 g, 1=0.25 h d=0.5 g, 1=0.25 h d=0.5 g, t=0.25 h  0.084 0.113  0.083 0.116  0.088 0.084  0.117  0.117  0.117  d=0.2 g, t=0.25 h  0.113  0.112  d=0.2 g, t=0.25 h  0.107  0.110 0.111  d=0.8 g, 1=1 h  0.095  0.096  0.096  d=0.8 g, 1=1 h d=0.5 g, t=l h  0.097 0.105  0.094  0.096  0.104  0.105  d=0.5 g, t=l h d=0.2 g, t=l h  0.095 0.108  0.097 0.105  0.096 0.107  0.100  d=0.2 g, 1=1 h  0.107  0.109  0.108  0.107  d=0.8g,t=4h  0.092 0.088  0.095 0.089  0.094  d=0.8 g, 1=4 h d=0.5 g, 1=4 h d=0.5 g, 1=4 h  0.082 0.088  d=0.2 g, t=4 h  0.086  0.115  0.109  0.116 0.110 0.096  0.091  0.083  0.089 0.083  0.087  0.088  0.085  0.104  0.104  0.104  d=0.2 g, t=4 h  0.104  0.103  0.104  d=0.8 g, t=24 h  0.070  0.071  0.071  d=0.8 g, t=24 h  0.069  0.069  0.069  d=0.5 g, t=24 h  0.075  0.073  0.074  d=0.5 g, 1=24 h  0.075  0.071  0.073  d=0.2 g, 1=24 h  0.084  0.085  0.085  d=0.2 g, 1=24 h  0.084  0.085  0.085  d=0, t=24 h  0.121  0.120  0.121  d=0, t=24 h  0.124  0.121  0.123  0.104 0.070 0.074 0.085 0.122  117  Table A.36 Test r u n 1 C O D measurement mixing study 300 m L flask at 200 r p m Sample Blank  Absorbance 0  Value  Average  0  Std. 100  0.024  0.023  0.024  Std. 300  0.114  0.114  0.114  Std. 500  0.193  0.193  0.193  Std. 800  0.308  0.309  0.309  d=0, t=0 d=0, t=0  0.110  0.110  0.110  0.111  0.111  d=0.8 g, 1=0.25 h  0.110 0.174  0.172  0.173  d=0.8 g, t=0.25 h  0.168  0.168  0.168  d=0.5 g, t=0.25 h d=0.5 g, t=0.25 h  0.152  0.150  0.151  0.148  0.149  0.149  d=0.2 g, t=0.25 h  0.123  0.125  0.124  d=0.2 g, t=0.25 h d=0.8 g, t=l h  0.119 0.164  0.118  0.119 0.165  0.121  d=0.8 g, t=l h d=0.5 g, t=l h  0.171  0.170 0.142  0.171 0.142  0.168  0.141  d=0.5 g, 1=1 h  0.137  0.137  0.137  0.139  d=0.2 g, 1=1 h  0.127  0.125  0.126  d=0.2 g, t=l h  0.123  0.126  0.125  d=0.8 g, t=4 h  0.144  0.144  0.144  d=0.8 g, t=4 h  0.146  0.146  0.146  0.145  d=0.5 g, t=4 h d=0.5 g, t=4 h  0.137 0.124  0.136 0.128  0.137 0.126  0.131  d=0.2 g, t=4 h  0.114  0.112  0.113  d=0.2 g, t=4 h  0.113  0.112  0.113  d=0.8 g, t=24 h  0.212  0.215  0.214  d=0.8 g, t=24 h  0.205  0.206  0.206  d=0.5 g, t=24 h  0.162  0.161  0.162  d=0.5 g, t=24 h  0.165  0.158  0.157  0.158  d=0.2 g, t=24 h  0.171  0.171  0.171  d=0.2 g, t=24 h  0.134  0.134  0.134  d=0,1=24 h  0.128  0.127  0.128  d=0, t=24 h  0.120  0.120  0.120  0.110 0.171 0.150  0.125  0.113 0.210 0.160 0.153 0.124  118  Table A.37 Test run 1 DOC measurement mixing study 300 mL flask at 200 rpm Sample  DOC (mg/L)  Average (mg/L)  d=0.8 g, t=0.25 h  293  278  d=0.8 g, 1=0.25 h  265  250  279  d=0.5 g, t=0.25 h  272  d=0.5 g, t=0.25 h  257  257 242  265  d=0.2 g, t=0.25 h  217  202  d=0.2 g, t=0.25 h  210  d=0.8 g, t=l h d=0.8 g, t=l h d=0.5 g, t=l h  236 265 270  195 221 250  d=0.5 g, t=l h d=0.2 g, t=l h d=0.2 g, t=l h  271 232  256 217  271  208  228  d=0.8 g, 1=4 h d=0.8 g, t=4 h  223 251 251  236 236  251  d=0.5 g, 1=4 h d=0.5 g, t=4 h  268 251  253 236  260  d=0.2 g, t=4 h d=0.2 g, t=4 h  222  207 210 215  224  d=0.8 g, t=24 h  225 230  d=0.8 g, t=24 h d=0.5 g, t=24 h  251 230  236 215  241  d=0.5 g, 1=24 h d=0.2 g, 1=24 h d=0.2 g, t=24 h  251 204  236 189  241  211  196  208  d=0, t=24 h  202  187  d=0, t=24 h  179  164  214 251  255  191  119  Table A.38 Test run 2 colour measurement mixing study 300 mL flask at 200 rpm Sample  Absorbance  Value  Average  Blank Std. 50  0 0.015  0.015  Std. 100  0.040  0.040  Std. 300  0.101  0.102  0.040 0.102  Std. 500  0.174  0.175  0.175  d=0, t=0  0.121 0.121 0.091  0.120 0.120 0.092  0.120  0.088  0.119 0.119 0.093 0.087  0.088  0.090  d=0.5 g, t=0.25 h d=0.5 g, t=0.25 h  0.105 0.104  0.104 0.105  0.105 0.105  0.105  d=0.2 g, 1=0.25 h d=0.2 g, t=0.25 h d=0.8 g, t=l h  0.107  0.106  0.107  0.108 0.086  0.106 0.084  0.107 0.085  0.107  d=0.8 g, t=l h  0.082 0.092  0.084  0.084  0.094  0.090 0.094  0.083 0.091 0.094  d=0.2 g, t=l h d=0.2 g, t=l h  0.112 0.106  0.112 0.104  0.112 0.105  0.109  d=0.8 g, t=4 h d=0.8 g, 1=4 h  0.083 0.081  0.081 0.081  0.082 0.081  0.082  d=0.5 g, t=4 h d=0.5 g, t=4 h  0.100 0.103  0.101 0.103  0.101 0.103  0.102  d=0.2 g, t=4h  0.089  0.089  0.089  d=0.2 g, t=4 h  0.098 0.062  0.097 0.062  0.093  d=0.8 g, t=24 h d=0.8 g,t=24h  0.095 0.062  0.064  0.060  0.062  0.062  d=0.5 g, t=24 h  0.076  0.078  0.077  d=0.5 g, t=24 h  0.076  0.076  0.076  d=0.2 g, 1=24 h  0.085  0.084  0.085  d=0.2 g, t=24 h  0.086  0.086  0.086  d=0, t=24 h  0.126  0.123  0.125  d=0, t=24 h  0.120  0.120  0.120  d=0, t=0 d=0.8 g, t=0.25 h d=0.8 g, t=0.25 h  d=0.5 g, t=l h d=0.5 g, 1=1 h  0 0.015  0.093  0.077 0.085 0.122  120  Table A . 3 9 Test run 2 C O D measurement mixing study 300 m L flask at 200 r p m Sample Blank Std. 100  0.023  Value 0 0.024  0.114 0.193 0.308  0.114 0.193  0.114 0.193  0.309  0.309  d=0, t=0 d=0, t=0 d=0.8 g, t=0.25 h d=0.8 g, t=0.25 h d=0.5 g, t=0.25 h d=0.5 g, t=0.25 h d=0.2 g, t=0.25 h  0.110 0.110 0.174 0.168  0.110 0.111 0.172 0.168  0.110 0.111 0.173 0.168  0.152 0.148 0.123  0.150 0.149  0.151 0.149 0.124  d=0.2 d=0.8 d=0.8 d=0.5 d=0.5 d=0.2 d=0.2 d=0.8 d=0.8  0.119 0.164  Std. 300 Std. 500 Std. 800  Absorbance 0 0.024  Average  0.110 0.171 0.150  0.171 0.141  0.125 0.118 0.165 0.170 0.142  0.137 0.127 0.123 0.144 0.146 0.137 0.124 0.114  0.137 0.125 0.126 0.144 0.146 0.136 0.128 0.112  0.113 0.212 0.205 0.162  0.112 0.215 0.206 0.161  0.126 0.125 0.144 0.146 0.137 0.126 0.113 0.113 0.214 0.206 0.162  d=0.5 g, t=24 h d=0.2 g, t=24 h d=0.2 g, t=24 h  0.158  0.157  0.158  0.160  0.171 0.134  0.171 0.134  0.171 0.134  0.153  d=0, t=24 h  0.128  0.127  0.128  d=0, t=24 h  0.120  0.120  0.120  g, t=0.25 h g, t=l h g, t=l h g, t=l h g, t=l h g, t=l h g, t=l h g, t=4 h g, t=4 h  d=0.5 g, t=4 h d=0.5 g, t=4 h d=0.2 g, t=4 h d=0.2 g, t=4 h d=0.8 g, t=24 h d=0.8 g, t=24 h d=0.5 g, t=24 h  0.119 0.165 0.171 0.142 0.137  0.121 0.168 0.139 0.125 0.145 0.131 0.113 0.210  0.124  121  Table A.40 Test run 2 D O C measurement mixing study 300 mL flask at 200 rpm Sample  DOC (mg/L)  Average  d=0, t=0 d=0, t=0  200  d=0.8 g, t=0.25 h d=0.8 g, t=0.25 h d=0.5 g, t=0.25 h  275 215 236 222 231 232 278 247 234  261 201 222  241  d=0.5 g, t=0.25 h d=0.2 g, t=0.25 h d=0.2 g, t=0.25 h d=0.8 g, t=l h d=0.8 g, t=l h d=0.5g,t=lh d=0.5 g, t=l h  216  d=0.2 g, t=l h d=0.2 g, t=l h d=0.8 g, t=4 h d=0.8 g, t=4 h d=0.5 g, t=4 h d=0.5 g, t=4 h d=0.2 g, t=4 h d=0.2 g, t=4 h d=0.8 g, t=24 h d=0.8 g, t=24 h d=0.5 g, t=24 h d=0.5 g, t=24 h d=0.2 g, t=24 h  207 197 234 218 242 217 215 204 240 187 228 218 214  d=0.2 g, t=24 h d=0,t=24h  191  d=0, t=24 h  186 202  208 245  208 217 218 264  229  233 220  263  227 193 183 229 204 237 203 201 190 235 173 214 204  238  232  202 226 230 210 214 223 203  168  200 177 154  154  140  161  122  APPENDIX 8. KINETIC MINI-STUDY PART 1 Table A.41 Test run 1 colour measurement kinetic mini-study on Western Pulp effluent Sample Blank Std. 50  Absorbance  Value  0  Average  0  0.016  0.017  0.017  Std. 100  0.039  0.037  0.038  Std. 300 Std. 500  0.100 0.172  0.102 0.172  0.101 0.172  t=0  0.116  0.115  0.116  t=0  0.119  0.117  0.118  t=0.08 h  0.115  0.117  0.116  t=0.08 h t=0.17h  0.114  0.111  0.113  0.108  0.108  0.108  t=0.17h  0.109  0.110  0.110  t=0.25 h  0.110  0.110  0.110  t=0.25 h  0.115  0.116  0.116  t=0.5 h  0.106  0.108-  0.107  t=0.5 h  0.114  0.115  0.115  t=0.75 h  0.106  0.106  0.106  t=0.75 h  0.107  0.108  0.108  t=l h  0.102  0.102  0.102  t=l h  o.ioo-  0.101  0.101  t=4h t=4h  0.098  0.097  0.098  0.101  0.100  0.101  t=8h  0.089  0.089  0.089  t=8h  0.086  0.087  0.087  t=15 h  0.095  0.096  0.096  t=15h  0.094  0.095  0.095  t=19h  0.088  0.087  0.088  t=19h  0.088  0.087  0.088  t=24 h  0.089  0.089  0.089  t=24 h  0.089  0.089  0.089  t=32h  0.086  0.083  0.085  t=32h  0.084  0.085  0.085  t=40h  0.063  0.063  0.063  t=40h  0.061  0.063  0.062  t=44h  0.076  0.077  0.077  t=44h  0.075  0.075  0.075  t=48h  0.069  0.069  0.069  t=48 h  0.073  0.072  0.073  Blank t=48h  0.121  0.122  0.122  Blank t=48h  0.121  0.124  0.123  t=52 h  0.090  0.089  0.090  t=52 h  0.091  0.095  0.093  0.117 0.114 0.109 0.113 0.111 0.107 0.101 0.099 0.088 0.095 0.088 0.089 0.085 0.063 0.076 0.071 0.122 0.091  123  Table A.42 Test r u n 1 C O D measurement kinetic mini-study on Western P u l p effluent Sample Blank  Absorbance  Value  0  Average  0  Std. 100  0.023  0.023  0.023  Std. 300  0.114  0.114  0.114  Std. 500  0.193  0.192  0.193  Std. 800  0.306  0.306  0.306  t=0  0.113  0.113  0.113  t=0  0.112  0.113  0.113  t=0.08 h  0.116  0.116  0.116  t=0.08 h t=0.17 h  0.117 0.118  0.117 0.117  0.117 0.118  0.117  t=0.17h t=0.25 h  0.119  0.120  0.120  0.119  0.115 0.120  0.114  0.115  0.119 0.110  0.120  0.110  t=0.5 h  0.118  0.122  0.120  t=0.75 h t=0.75 h  0.122 0.121  0.122  0.122  0.122  0.122  t=l h  0.122  0.120  0.121  t=l h  0.116  0.117  0.117  t=4h  0.116  0.115  0.116  t=4h  0.118 0.112  0.118 0.112  0.117  t=8h t=8h '  0.118 0.112  0.120  0.120  0.120  0.116  t=15 h  0.112  0.112  0.112  t=15h t=19h  0.122  0.123  0.123  0.117  0.117  0.117  t=19h  0.116 0.114  0.117  0.117  0.112  0.113  t=24 h t=32 h  0.113  0.113  0.113  0.111  0.112  0.112  t=32 h t=40h t=40h  0.105  0.104  0.105  0.095  0.095  0.095 0.1.00 0.101  0.098  t=0.25 h t=0.5h  t=24h  0.113  0.117  0.110 0.115 0.122 0.119  0.117 0.117 0.113 0.108  0.100  0.100  t=44h  0.101  0.101  t=44h t=48h  0.103 0.105  0.103 0.105  0.102  .0.104  t=48h  0.115  0.116  0.116  0.110  Blank t=48h  0.125  0.123  0.124  Blank t=48 h  0.116  0.116  0.116  0.120  t=52h t=52h  0.123 0.126  0.123 0.126  0.123 0.126  0.125  0.102  124  Table A.43 Test run 1 D O C measurement kinetic mini-study on Western Pulp effluent Sample Blank Std. 50 Std. 500  t=0 t=0 t=0.08 h t=0.08 h t=0.17h t=0.17h t=0.25 h t=0.25 h  r=0.5 h t=0.5 h t=0.75 h t=0.75 h t=l h 1=1 h t=4h t=4h t=8h  t=8h t=15h 1=15 h t=19h t=19h t=24h t=24h  5 16 158 67 63 74 72 70 63 68 66 66 71 67 71 76 74 74 70 68 64 71 72 63 64  Blank 1=48h t=52h  66 68 59 66 52 56 53 51 48 56 62 63  t=52 h  71  1=32 h t=32h t=40h  t=40 h t=44h  t=44 h t=48 h  Blank t=48 h  Height of peak (mm) 5 8. 15 17 138 130 66 67 60 63 70 72 74 71 70 71 63 61 64 68 62 66 69 67 68 65 68 61 75 75 74 69 72 70 68 68 78 68 70 65 61 60 74 78 71 70 64 63 61 61 64 64 68 68 59 56 65 62 54 58 54 58 54 52 52 50 50 49 62 67 63 63 67 66 69  72  5 17 127  76  79 74  68 74  Value 6 16 138 67 62 73 72 70 62 67 65 67 68 65 74 75 73 70 72 68 62 74 71 63 62 65 68 58 64 55 56 53 51 49 62 63 65  74  72  Average  64 73 66 66 68 70 74 71 65 73 63 66 61 55 52 49 62 68  125  Table A.44 Test run 1 p H measurement kinetic mini-study on Western Pulp effluent Sample t=0 t=0.08 h t=0.17h  PH 8.15 8.38  t=0.25 h  8.46 8.20  t=0.50 h  8.32  1=0.75 h t=l h t=4h t=8h  8.49 8.55 8.41 8.12  t=15h t=19h  8.92 9.03  t=24h t=32h t=40h  8.89 8.63 8.36  1=44 h  8.56 8.49  1=48 h Blank 1=48 h  9.07  t=52hr  8.52  126  Table A.45 Test run 2 colour measurement kinetic mini-study on Western Pulp effluent Sample Blank  Absorbance 0  Value  Average  0  Std. 50  0.015  0.015  0.015  Std. 100  0.037  0.037  0.037  Std. 300  0.100  0.101  0.101  Std. 500  0.176  0.176  0.176  t=0  0.110  0.110  0.110  t=0  0.112  0.111  0.112  t=0.08 h  0.106  0.105  0.106  t=0.08 h t=0.17h  0.106  0.107  0.107  0.105  0.103  0.104  t=0.17h  0.103  0.104  0.104  t=0.25 h  0.106  0.106  0.106  t=0.25 h  0.104  0.104  0.104  t=0.5 h  0.105  0.107  0.106  t=0.5 h  0.106  0.106  0.106  t=0.75 h  0.099  0.098  0.099  t=0.75 h  0.100  0.100  0.100  t=l h  0.097  0.099  0.098  t=l h  0.099  0.100  0.100  t=4h  0.091  0.093  0.092  t=4h  0.091  0.091  0.091  t=8h t=8h  0.093  0.091  0.092  0.092  0.092  0.092  t=15 h  0.084  0.084  0.084  t=15 h  0.085  0.086  0.086  t=19h t=19h  0.085  0.087  0.086  0.084  0.081  0.083  t=24h  0.082  0.080  0.081  t=24h  0.077  0.078  0.078  t=32h  0.067  0.068  0.068  t=32h  0.067  0.066  0.067  t=40 h  0.072  0.072  0.072  t=40 h  0.073  0.074  0.074  t=44h  0.053  0.055  0.054  t=44h  0.053  0.052  0.053  t=48h  0.064  0.065  0.065  t=48h  0.065  0.066  0.066  Blank t=48 h  0.115  0.114  0.115  Blank t=48 h  0.115  0.112  0.114  t=52 h t=52h  0.048  0.049  0.049  0.047  0.049  0.048  0.111 0.106 0.104 0.105 0.106 0.099 0.099 0.092 0.092 0.085 0.084 0.079 0.067 0.073 0.053 0.065 0.114 0.048  127  Table A . 4 6 Test r u n 2 C O D measurement kinetic mini-study on Western P u l p effluent Sample Blank  Absorbance  Value  0  Average  0  Std. 100  0.024  0.025  0.025  Std. 300  0.115  0.115  0.115  Std. 500  0.196  0.196  0.196  Std. 800  0.310  0.310  0.310  t=0  0.109  0.109  0.109  t=0  0.115  0.115  0.115  t=0.08 h t=0.08 h  0.111  0.112  0.112  0.109 0.117  0.110  0.110  t=0.17h  0.118  0.118  t=0.17h  0.108  0.109  0.109  t=0.25 h  0.110  0.112  0.111  t=0.25 h t=0.5h .  0.113 0.114  0.113  0.113 0.114  0.112  0.113  t=0.5 h  0.118 0.116  0.119  0.119  0.116  0.118 0.112  0.117 0.112  0.115  0.119 0.112  0.118  0.119  0.112  0.112  0.115  0.115  0.115  t=4h t=8h  0.109  0.109 0.119  0.109 0.120  0.112  0.120  t=8h  0.119  0.119  0.119  0.119  t=15 h t=15h  0.120  0.120  0.120  0.112  0.111  0.112  0.116  t=19 h t=19h  0.110 0.115  0.109 0.114  0.110 0.115  0.112  t=24h  0.104 0.104  0.104  t=24 h  0.104 0.102  t=32 h  0.204  0.203  0.204  t=32 h t=40h  0.202  0.200  0.201  0.202  0.111  t=40h  0.115  0.111 0.114  0.111 0.115  0.113  t=44h  0.109  0.109  0.109  t=44h t=48 h  0.119 0.116  0.118 0.116  0.119 0.116  0.114 0.118  t=0.75 h t=0.75 h t=l h t=l h t=4h  0.112  0.103  t=48h  0.119  0.120  0.120  Blank t=48 h  0.123  0.121  0.122  Blank t=48 h  0.122  0.121  0.122  t=52h  0.091  0.090  0.091  t=52 h  0.093  0.093  0.093  0.112 0.111 0.113  0.115  0.104  0.122 0.092  128  Table A.47 Test r u n 2 D O C measurement kinetic mini-study on Western P u l p effluent Sample  Height of peak (mm)  Blank  4  5  Value  5  5  Std. 50  12  11  11  Std. 500  102  96  90  t=0  28  32  29  t=0  37  36  36  35  36  1=0.08 h  26  36  34  35  33  t=0.08 h t=0.17h t=0.17h  36  29  34  36  34  40 41  39 40  39  t=0.25 h  41  40  39  t=0.25 h t=0.5h  39  26 38  38  1=0.5 h 1=0.75 h  39 38  33 39  38 22  t=0.75 h  36  37  38  t=l h t=l h  37 37  35  37  38  37  38  36  38  37  1=4 h  37  36  36  36  1=4 h  33 34  18  36  18  27  t=8h  37  37  36  t=8 h  22  36  35  19  28  t=15h t=15h  34  29  35  35  33  32  26  34  28  t=19h  36  25  18 34  t=19h  35  35  12  27 •  t=24h  34  33  32  33  t=24h  31  31  32  31  t=32 h  31  31  42  35  t=32h  31  38  34  t=40h  34  32  34  t=40h  34  33  33  t=44h  39  35  35  37  37 •  t=44h  37  38  37  37  37  35  24  38  Average  11 95  96 30 33 33  39 41  37  38  40 39 37  36 37  38  40  38 34  37  37  36  38  37 31  37 32 30  32  34 43  32 35  36 33  t=48h  33  31  31  32  t=48 h  31  32  30  31  Blank 1=48 h  32  31  32  32  Blank 1=48 h  32  31  33  t=52h  32  27  32  t=52h  45  46  47  32 32  30  35 37 31 32  31 46  38  129  Table A.48 Test run 2 pH measurement kinetic mini-study on Western Pulp effluent Sample t=0  pH 7.83  1=0.08 h  8.28  1=0.17 h  8.34  1=0.25 h t=0.50 h  8.16  1=0.75 h t=l h t=4h t=8h  8.39 8.24 8.29 8.81  t=15 h t=19h  8.96 9.02  t=24h t=32h t=40h  8.61 8.54 8.40  t=44h t=48 h  8.45 8.56  Blank t=48 h  9.05  t=52hr  8.63  8.36  130  Table A.49 Colour measurement kinetic mini-study on Howe Sound effluent Sample Blank  Absorbance  Value  0  Average  0  Std. 50  0.016  0.018  0.017  Std. 100  0.038  0.038  0.038  Std. 300  0.104  0.103  0.104  Std. 500  0.178  0.175  0.177  t=0  0.073  0.074  0.074  t=0 t=0.08 h  0.074  0.073  0.074  0.061  0.061  0.061  t=0.08 h  0.060  0.061  0.061  t=0.17h  0.062  0.061  0.062  t=0.17h  0.064  0.064  0.064  t=0.25 h  0.060  0.060  0.060  t=0.25h  0.059  0.061  0.060  t=0.5 h  0.067  0.066  0.067  t=0.5 h  0.074  0.073  0.074  t=0.75 h  0.055  0.055  0.055  t=0.75 h  0.059  0.058  0.059  t=l h  0.050  0.053  0.052  t=l ti  0.050  0.052  0.051  t=4h  0.047  0.050  0.049  t=4h  0.045  0.046  0.046  t=8h  0.045  0.044  0.045  t=8h  0.045  0.046  0.046  t=15h  0.053  0.052  t=15 h  0.052  0.051 0.052  t=19 h  0.054  0.054  0.054  t=19h  0.052  0.053  0.053  t=24h  0.055  0.057  0.056  t=24 h  0.053  0.052  0.053  t=32h  0.043  0.043  0.043  t=32 h  0.045  0.044  0.045  t=40h  0.037  0.038  0.038  t=40 h  0.038  0.038  0.038  t=44h  0.041  0.040  0.041  t=44h  0.043  0.045  0.044  t=48h  0.034  0.033  0.034  t=48h  0.037  0.037  0.037  Blank t=48 h  0.073  0.072  0.073  Blank t=48 h  0.074  0.075  0.075  t=52h  0.040  0.042  0.041  t=52 h  0.043  0.043  0.043  0.052  0.074 0.061 0.063 0.060 0.070 0.057 0.051 0.047 0.045 0.052 0.053 0.054 0.044 0.038 0.042 0.035 0.074 0.042  131  Table A.50 C O D measurement kinetic mini-study on Howe Sound effluent Sample Blank Std. 100  Absorbance  Value  0  Average  0  0.040  0.038  0.039  Std. 300  0.117  0.118  0.118  Std. 500  0.202  0.202  0.202  Std. 800  0.317  0.317  0.317  t=0 t=0 t=0.08 h  0.099  0.096  0.098  0.098  0.096  0.097  0.097  0.091 0.089  0.090 0.089  0.091 0.089  0.090  0.097  0.097  0.097  0.097 0.087  0.097  0.097  0.097  t=0.25h  0.088  t=0.25 h  0.091  0.092  0.088 0.092  0.090  t=0.5 h t=0.5 h  0.109  0.109  0.109  0.140  0.140  0.140  t=0.75 h  0.099  0.099  0.099  t=0.75 h  0.128  0.128  0.128  0.114  t=l h t=l h  0.096  0.095 0.089  0.096 0.089  0.092  t=4 h  0.093 0.092  0.094  0.094  0.090 0.077  0.091 0.078  0.092  0.078  t=8h  0.077  0.077  0.077  0.077  t=15h  0.095  0.095  0.095  t=15h  0.097  0.096  0.097  t=19h  0.100  0.100  0.100  t=19h  0.111  0.111  0.111  t=24h  0.102  0.100  0.101  t=24h  0.129  0.126  0.128  t=32h  0.090  0.089  0.090  t=32 h  0.106  0.106  0.106  t=40 h t=40h  0.086  0.085  0.086  0.082  0.082  0.082  0.084  t=44h t=44h  0.093 0.081  0.093 0.082  0.093 0.082  0.087  t=48h  0.081  0.082  0.082  t=48h  0.082  0.082  0.082  Blank t=48 h  0.102  0.101  0.102  Blank t=48 h  0.112  0.114  0.113  t=52 h  0.096  0.096  0.096  t=52h  0.099  0.099  0.099  t=0.08 h t=0.17h t=0.17h  t=4h t=8h  0.088  0.125  0.096 0.106 0.114 0.098  0.082 0.107 0.098  132  Table A.51 D O C measurement kinetic mini-study on Howe Sound effluent Standard Blank Std. 50  4 19  Std. 500 t=0  184 54  179 51  163 52  t=0 t=0.08 h 1=0.08 h  44  46  45  45  52 51 51 52 52 54  50 52 56 53  49 50 54 54  51 52  51 54  50 51 54 53 51 53  56 60 54  54 64 51  51 49 47 48 47 47 47 54 51 52  51 50 47 47 49 48 45 52 52 51  55 62 52 54 49 46 47 46 47 47  55 62 52 52 49 47 47 47 47 46  53 50 52  53 51 52  t=19h t=24 h t=24 h t=32 h  48  49 50 55 47  48 49 52 50  46 52  48 49 52  t=32h t=40h t=40h  45 47 48  49 49 44  47  49 47  48  48 46  47  t=44h  48 48 45 41  40  1=44 h t=48h t=48 h  38 41 44  40 42  39  40 45  40 40 44  40 40 44  46  47 40  40  46  40 47  46  46  52  53  52  52  t=0.17h t=0.17h  t=0.25 h t=0.25 h  1=0.5 h t=0.5 h t=0.75 h  1=0.75 h t=l h t=l h t=4h t=4h t=8h t=8h t=15h t=15h t=19h  Blank t=48 h Blank t=48 h t=52h t=52h  49 50 51  46 41  Height of peak (mm) 4 4 18 16  Value  Average  4 18 171  47  174 52 49 51 53 52 59 52 48 47 47 52 50 51  40 42  47 43 49  133  Table A.52 p H measurement kinetic mini-study on Howe Sound effluent Sample  pH  1=0  7.59  t=0.08 h t=0.17h  8.35  1=0.25 h t=0.50 h  8.26  t=0.75 h 1=1 h t=4h 1=8 h t=15 h t=19h  8.33 8.57 8.33 8.35 8.92 8.88  t=24 h  8.75  t=32h  8.51 8.41  1=40 h t=44h  8.31 8.28  8.57 8.60  t=48 h Blank t=48 h  9.06  t=52hr  8.60  APPENDIX 9. INHIBITION MINI-STUDY AND KINETIC MINI-STUDY PART 2 Table A.53 Test run 1 colour measurement kinetic rate at 4°C on Western Pulp effluent Sample Blank  Absorbance 0 0.014 0.014 0.038 0.038 0.101 0.102 0.175 0.177 0.124 0.124 0.121 0.120 0.105 0.105 0.107 0.106 0.102 0.103 0.103 0.101 0.095 0.096 0.095 0.093 0.096 0.095 0.093 0.094 0.092 0.095 0.093 0.095 0.090 0.089 0.089 0.084 0.088 0.087  Value 0 0.014 0.038 0.102 0.176 0.124  Average  0.121 0.105 0.107 0.103 0.102 0.096 0.094  0.122  t=48 h Blank t=48 h Blank t=48 h  0.089 0.121  0.089 0.120  0.088  0.091  0.117 0.091  0.118  t=52 h  0.089 0.118 0.117 0.090  t=52h  0.093  0.093  0.093  0.092  Std. Std. Std. Std.  50 100 300 500  t=0 t=0 t=l h t=l h t=8h t=8h t=19h 1=19 h t=24h t=24h t=32 h t=32h t=40 h  1=40 h t=48h  0.117  0.096 0.094 0.094 0.094 0.090 0.087 0.088  0.106 0.102 0.095 0.095 0.094 0.088  135  Table A.54 Test r u n 1 C O D measurement kinetic rate at 4 ° C on Western Pulp effluent Sample Blank  Absorbance 0  Std. 100 Std. 300  0.014 0.038  Std. 500 Std. 800  0.101 0.175  0.038 0.102 0.177  t=0  0.124  0.124  t=0 t=l h t=l h t=8h  0.121 0.105 0.107 0.102  0.120 0.105 0.106 0.103  t=8h t=19h t=19h t=24h  0.103 0.095  0.101 0.096 0.093 0.095 0.094  0.121 0.105 0.107 0.103 0.102 0.096 0.094 0.096 0.094  0.095 0.095  0.094 0.094  0.089 0.084 0.087 0.089 0.118 0.117 0.090  0.090 0.087 0.088 0.089 0.120 0.117 0.091  0.093  0.093  t=40h t=48h t=48 h Blank t=48 h Blank t=48 h 1=52 h  0.095 0.096 0.093 0.092 0.093 0.09 0.089 0.088 0.089 0.121 0.117 0.091  t=52h  0.093  1=24 h 1=32 h  t=32 h 1=40 h  0.014  Value 0 0.014 0.038  Average  0.102 0.176 0.124 0.122 0.106 0.102 0.095 0.095 0.094 0.088 0.088 0.118 0.092  136  Table A.55 Test run 1 DOC measurement kinetic rate at 4°C on Western Pulp effluent Sample Blank  Height of peak (mm) 6 6 2  Std. 50 Std. 500 t=0 t=0 t=l h  19 178 44  18 187 74  73  73 89  76 78 74  t=l h t=8h t=8h  75 68  .1=19 h  73  Average  18 180 74  66 73  76  79  70  77 77 73 65  78 77 77 69  78 76 75 67  78  70 62  70 57  71 59  69  65 61 56 57 64  65 59 56 57 63 60 55 61 64 57  62  57  56  57  t=19h t=24 h t=24 h t=32h t=32h t=40h t=40 h  73 60 62  t=48 h t=48 h Blank t=48 h Blank t=48 h t=52h  48 56 62 69 58  64 57 55 56 59 62 56 58 54 57  t=52 h  56  55  58 56 58 62  5  18 174 72 74  Value 5  58 68  67 67  64 54 62 66 57  66  76  57 60 58 62  Table A.56 Test run 1 pH measurement kinetic rate at 4°C on Western Pulp effluent Sample t=0 t=l h  PH 7.55 8.38  t=8h t=19h  8.46  t=24 h  8.48 8.52  t=32h t=40h t=48h Blank t=48 h  8.59 8.61 8.66 8.68  t=52h  8.58  137  Table A.57 Test run 2 colour measurement kinetic rate at 4°C on Western Pulp effluent Sample Blank Std. 50 Std. 100 Std. 300 Std. 500  Absorbance 0 0.016 0.038 0.104 0.178  0.018 0.038 0.103 0.175  Value  Average  0 0.017 0.038 0.104 0.177  0.122  t=8h t=19h  0.123 0.123 0.101 0.100 0.091 0.092 0.088  t=19h t=24h 1=24 h 1=32 h t=32h t=40h t=40h t=48h  0.090 0.087 0.089 0.084 0.079 0.101 0.103 0.095  0.089 0.088 0.088 0.084 0.082 0.099 0.103 0.094  0.088  t=48 h Blank t=48hr Blank t=48hr  0.090 0.129 0.118 0.083  0.092  1=52 h  0.089 0.131 0.119 0.084  0.088 0.089 0.087 0.083 0.084 0.097 0.102 0.093 0.090 0.126 0.116 0.081  t=52h  0.081  0.079  0.080  0.081  t=0 t=0 t=l h t=l h  t=8h  0.124 0.103 0.100 0.093 0.092 0.087  0.123 0.124 0.102 0.100 0.092 0.092 0.088  0.123 0.101 0.092  0.088 0.083 0.101  0.123  138  Table A.58 Test r u n 2 C O D measurement kinetic rate at 4 ° C on Western Pulp effluent Sample Blank Std. 100  Absorbance 0  Value 0  Average  Std. 300 Std. 500 Std. 800  0.040 0.117 0.202 0.317  0.038 0.118 0.202 0.317  0.039 0.118 0.202 0.317  t=0  0.120  0.120  0.120  t=0  0.127 0.133 0.119 0.108 0.115 0.115  0.126 0.132  0.127 0.133 0.118 0.108 0.115 0.115  0.123  0.113 0.120 0.122 0.104 0.093 0.112 0.115 0.113 0.114 0.121 0.118 0.113  0.113 0.120 0.122 0.104 0.095 0.112 0.116 0.113 0.115 0.121 0.119 0.113  0.114  t=32 h t=32 h t=40 h t=40h t=48h t=48h Blank t=48 h Blank t=48 h t=52h  0.113 0.120 0.121 0.104 0.096 0.111 0.116 0.113 0.116 0.120 0.119 0.113  t=52 h  0.119  0.118  0.119  0.116  t=l h  t=l h t=8h t=8 h t=19h t=19h t=24 h t=24h  0.117 0.108 0.115 0.115  0.125 0.112  0.121 0.099 0.114 0.114 0.120  139  Table A.59 Test run 2 DOC measurement kinetic rate at 4°C on Western Pulp effluent Sample Blank  Height of peak (mm) 4 4 4  Value 4  Std. 50 Std. 500  19 184  18 179  16 163  t=0 t=0  64  65 66 70  63 63 73  71  71 71 66 58 57 70 57 47 53 54 55 49 52 52 56 57  71 71 67 58 57 70 57 49 52 52 55 51 52 51 55 54  71  53 52  71 73 66 57 58 69 58 50 52 54 55 50 54 50 55 52  48  47  48  48  51  69 70  t=l h t=l h t=8h t=8h  71 70 70 58 56 70 55 51 51 47 56 52 51 52  1=19 h t=19h t=24 h t=24h t=32 h t=32h t=40h t=40 h t=48 h t=48h Blank t=48 h Blank t=48 h t=52h t=52 h  171  18 174  67  64 66  Average  53 51  65  69 57 63 51 54 51 53  Table A.60 Test run 2 pH measurement kinetic rate at 4°C on Western Pulp effluent Sample t=0 t=l h t=8h t=19h t=24h  PH 7.50 8.27 8.39 8.57 8.67  t=32h t=40h  8.40 8.64  t=48h  8.70  Blank t=48 h  8.71  t=52h  8.60  140  Table A.61 Colour measurement kinetic rate at 4 ° C on Howe Sound effluent Sample Blank Std. 50  Absorbance 0 0.015 0.016  Value  Std. 100  0.039  0.038  0.039  Std. 300 Std. 500  0.101 0.173  0.102 0.172  0.102 0.173  t=0 t=0 t=l h 1=1 h t=8h  0.076 0.073 0.056 0.055 0.054 0.052 0.049 0.050 0.048  0.077 0.073 0.056 0.055 0.054 0.052 0.049 0.050 0.049  0.047 0.049 0.05 0.047 0.045 0.045 0.045 0.074 0.074 0.052  0.048 0.050 0.050 0.047 0.044 0.046 0.045 0.075 0.074 0.052  0.048  Blank t=48hr Blank t=48hr t=52 h  0.077 0.073 0.055 0.055 0.054 0.052 0.048 0.050 0.049 0.048 0.051 0.049 0.046 0.043 0.046 0.045 0.076 0.074 0.052  t=52 h  0.055  0.054  0.055  0.053  1=8 h  t=19h t=19h t=24 h t=24 h t=32h 1=32 h  t=40h t=40h t=48h 1=48 h  Average  0 0.016  0.075 0.055 0.053 0.049  0.050 0.045 0.045 0.075  141  Table A.62 C O D measurement kinetic rate at 4°C on Howe Sound effluent Sample Blank Std. 100  Absorbance 0 0.038  0.038  Value 0 0.038  Average  Std. 300  0.117  0.116  0.117  Std. 500 Std. 800  0.200 0.316  0.201 0.316  0.201 0.316  t=0 t=0 t=l h t=l h  0.088 0.089 0.084 0.089  0.089 0.090 0.085 0.089  0.089 0.090 0.085 0.089  t=8h t=8h t=19h t=19h t=24 h t=24h t=32h t=32h t=40h t=40h t=48h t=48h Blank t=48 h Blank t=48 h t=52 h  0.095 0.103 0.084  0.095 0.104 0.084  0.095 0.104 0.084  0.083 0.098  0.083 0.098  0.083 0.098  0.084  0.090 0.086 0.089 0.093 0.095 0.095 0.093 0.101 0.107 0.106  0.089 0.086 0.089 0.093 0.095 0.092 0.093 0.101 0.105 0.106  0.090 0.086 0.089 0.093 0.095 0.094 0.093 0.101 0.106 0.106  0.094  t=52 h  0.102  0.100  0.101  0.104  0.089 0.087 0.099  0.088 0.094 0.093 0.104  142  Table A.63 DOC measurement kinetic rate at 4°C on Howe Sound effluent Standard Blank  Height of peak (mm) 5 5 5 16 17 18 175 176 168  Std. 50 Std. 500 t=0 t=0  55 56  1=1 h t=l h t=8h t=8h  57 55 55 49  t=19h t=19h t=24h t=24h t=32h t=32h . t=40h t=40 h t=48h t=48h Blank t=48 h Blank t=48 h  45 53 43 51 47  Value 5 17 171  173  58 59 54 54 54 53  57 63  57 59  55 58 52 49  55 56 54 50  52 52 45  49 52 44 51 47  t=52 h  43 54 55 47 54 58 58 50  50 51 43 52 48 43 50 51 49 49 55 57 52  t=52 h  53  51  52  51 46 41 51 57 47 51 54 52 59  Average  45  58 56 52 51 48  42 52 54 48 51 56 56 54  44  52  53  53 50 56  Table A.64 pH measurement kinetic rate at 4°C on Howe Sound effluent Sample 1=0 t=l h  pH 7.57 8.21  1=8 h 1=19 h t=24h t=32h  8.50  t=40h t=48h  8.58 8.72  Blank t=48h  8.76  t=52h  8.52  8.78 8.50 8.52  143  Table A.65 Colour measurement kinetic rate with NaN addition at room temperature 3  Sample Blank Std. 50 Std. 100 Std. 300 Std. 500 t=0 t=0 t=l h t=l h t=8h t=8h t=24 h t=24h t=32 h t=32h t=40h  0 0.014 0.040 0.103 0.174 0.123 0.118 0.101 0.097 0.091 0.088 0.087 0.083 0.080 0.081 0.079 0.082 • 0.082  Absorbance  Value 0  0.017  0.016  0.039 0.103 0.174 0.124 0.120 0.102  0.040 0.103 0.174 0.124 0.119 0.102  0.099 0.091 0.088 0.085 0.085 0.081 0.080 0.080  t=48 h Blank t=48 h  0.0.76 0.121  0.083 0.077 0.077 0.120  Blank t=48 h  0.123  0.124  t=40 h t=48h  0.098 0.093 0.091  0.081  0.091 0.088 0.086 0.084 0.081 0.081 0.080  Average  0.121 0.100 0.090 0.085 0.081  0.083 0.080 0.077 0.121  0.081  0.124  0.122  0.078  Table A.66 C O D measurement kinetic rate with N a N addition at room temperature on Western Pulp effluent 3  Sample Blank  0  Std. 100  0.038  Std. 300 Std. 500 Std. 800  0.117 0.201  t=0 t=0  t=l h t=l h t=8 h t=8h t=24h t=24h t=32h t=32h t=40h  Absorbance  0.318 0.122 0.120 0.121 0.123 0.119 0.120  Average  0.038 0.118 0.201  0.318 0.122 0.120 0.122 0.124  0.318 0.122 0.120 0.122 0.124  0.123  0.120 0.120  0.120 0.120 0.121  0.120  t=48h t=48h Blank t=48 h  0.128 0.119 0.124 0.120 0.120 0.149  0.119 0.121 0.118 0.128 0.120 0.126 0.119 0.120 0.152  Blank t=48 h  0.137  0.137  t=40 h  0.122 0.120 0.119  0.038 0.118 0.201  Value 0  0.121  0,121 0.119 0.128 0.120 0.125 0.120  0.121  0.120 0.151  0.120  0.137  0.144  0.123 0.122  145  Table A.67 DOC measurement kinetic rate with NaN addition at room temperature on Western Pulp effluent 3  Sample Blank  Height of peak (mm) 5 5 5 16 16 15 169 164 161 75 74 76 74 79 72 82 79 80  Std. 50 Std. 500 t=0 1=0 t=l h 1=1 h t=8h  1=8 h .1=24 h 1=24 h t=32h t=32h t=40 h t=40h t=48h 1=48 h Blank t=48 h Blank t=48 h  Value  Average  5 16 165 . 75 75 80 72 80 84  75  70 78 82  71 80 86  73 82  75 69 72 75 65 61 65 58 69  71 69 83 75 67 59 65 60 73  75 73 77 78 61 60 71 62 75  74 70 77 76 64 60 67 60 72  62  67  68  70  68  70  84  75  76 82 72 77  64  Table A.68 pH measurement kinetic rate with NaN addition at room temperature on Western Pulp effluent 3  Sample t=0  pH 7.72  1=1 h  8.35 8.88  1=8 ht=24h  8.93  t=32h t=40h  8.90 8.73  t=48h  8.79  Blank t=48 h  9.05  146  Table A.69 Colour measurement kinetic rate with NaN addition at room temperature on Howe Sound effluent 3  Sample Blank Std. 50  0 0.015  0.014  Value 0 0.015  Std. 100 Std. 300  0.037 0.102  0.039 0.103  0.038 0.103  Std. 500 t=0  0.173 0.071 0.071 0.051 0.050  0.173 0.071 0.072 0.051 0.051  0.050 0.053  0.051 0.053  0.053 0.054  t=48h Blank t=48 h  0.173 0.070 0.073 0.051 0.051 0.051 0.053 0.052 0.055 0.047 0.048 0.047 0.045 0.038 0.039 0.078  0.047 0.047 0.05 0.045 0.038 0.041 0.077  0.053 0.055 0.047 0.048 0.049 0.045 0.038 0.040 0.078  Blank t=48 h  0.076  0.078  0.077  t=0 t=l h t=l h t=8h t=8h t=24 h t=24h t=32h t=32h t=40h •1=40 h  1=48 h  Absorbance  Average  0.071 0.051 0.052 0.054 0.047 0.047 0.039 0.077  147  Table A.70 COD measurement kinetic rate with NaN addition at room temperature on Howe Sound effluent 3  Sample Blank Std. 100 Std. 300 Std. 500 Std. 800 t=0 t=0 t=l h t=l h t=8 h t=8 h t=24h t=24h t=32 h t=32h t=40 h t=40h t=48h t=48h Blank t=48 h Blank t = 4 8 h  Absorbance 0 0.038 0.117 0.199 0.316 0.091 0.092 0.084 0.086 0.100 0.098 0.105 0.106 0.098 0.099 0.100 0.099 0.085 0.087 0.104 0.107  0.038 0.116 0.200 0.315 0.091 0.093 0.083 0.085 0.1000.098 0.105 0.108 0.097 0.098 0.100 0.100 0.085 0.086 0.104 0.107  Value 0 0:038 0.117 0.200 0.316 0.091 0.093 0.084 0.086 0.100 0.098 0.105 0.107 0.098 0.099 0.100 0.100 0.085 0.087 0.104 0.107  Average  0.092 0.085 0.099 0.106 0.098 0.100 0.086 0.106  148  Table A.71 DOC measurement kinetic rate with NaN addition at room temperature on Howe Sound effluent 3  Standard Blank Std. 50 Std. 500 t=0 t=0 t=l h t=l h t=8h t=8h t=24 h t=24 h t=32h t=32 h t=40 h t=40h t=48h t=48 h Blank t=48 h Blank t=48 h  3 11 106 31 31 30 29 34 32 36 35 24 32 25 29 28 28 31 29  Height of peak (mm) 2 3 10 10 105 100 32 31 30 31 31 30 30 31 34 33 32 32 36 35 37 36 25 26 32 31 25 25 30 31 28 28 28 30 32 31 28 28  171 31 31 30 30 34 32 36 36 25 32 25 30 28 29 31 28  Average 3 10 121 31 30 33 36 28 28 28 30  Table A.72 pH measurement kinetic rate with NaN addition at room temperature on Howe Sound effluent 3  Sample 1=0  t=l h t=8h t=24h t=32h t=40h t=48h Blank t=48 h  PH 7.72 8.35 8.88 8.93 8.90 8.73 8.79 9.05  149  Table A.73 Colour measurement kinetic rate with NaF addition at room temperature on Western Pulp effluent Sample Blank Std. 50 Std. 100 Std. 300 Std. 500 t=0 t=0 t=l h t=l h t=8h •t=8h t=24h t=24h  Absorbance 0 0.014 0.040 0.103 0.174 0.120 0.124 0.103 0.103 0.091 0.090 0.083 0.084  0.017 0.039 0.103 0.174 0.123 0.120 0.101 0.101 0.089 0.089  t=32 h t=32h t=40 h t=40h t=48h t=48h Blank t=48 h  0.070 0.073 0.069 0.070 0.073 0.076 0.113  0.083 0.085 0.069 0.073 0.070 0.069 0.071 0.075 0.115  Blank t=48 h  0.118  0.118  Average 0 0.016 0.040 0.103 0.174 0.122 0.122 0.102  0.122  0.102 0.090 0.090 0.083 0.085 0.070  0.102  0.073 0.070 0.070 0.072 0.076 0.114  0.071  0.118  0.116  0.090 0.084  0.070 0.074  150  Table A.74 COD measurement kinetic rate with NaF addition at room temperature on ' Western Pulp effluent Sample Blank Std. 100 Std. 300 Std. 500 Std. 800 t=0 t=0 t=l h t=l h t=8 h t=8h t=24h t=24 h t=32h t=32h t=40h t=40 h t=48h t=48h Blank t=48 h Blank t=48 h  Absorbance 0 0.038 0.117 0.201 0.318 0.125 0.115 0.118 0.118 0.112 0.117 0.111 0.115 0.101 0.101 0.110 0.119 0.117 0.124 0.126 0.142  0.038 0.118 0.201 0.318 0.125 0.116 0.118 0.118 0.112 0.115 0.111 0.116 0.101 0.101 0.110 0.117 0.119 0.126 0.128 0.142  Value 0 0.038 0.118 0.201 0.318 0.125 0.116 0.118 0.118 0.112 0.116 0.111 0.116 0.101 0.101 0.110 0.118 0.118 0.125 0.127 0.142  Average  0.120 0.118 0.114 0.113 0.101 0.114 0.122 0.127  151  Table A.75 DOC measurement kinetic rate with NaF addition at room temperature on Western Pulp effluent Sample Blank  3  Height o f peak ( m m )  Average  3  2  3 10  Std. 50  11  10  10  Std. 500  106  105  100  171  t=0  31  31  32  31  t=0  31  30  31  31  t=l h  30  31  30  30  1=1 h  29  31  30  30  t=8h  34  34  33  34  t=8h  32  32  32  32  t=24 h  36  36  35  36  t=24 h  35  36  37  36  t=32 h  24  25  26  25  t=32h  32  32  31  32  t=40h  25  25  25  25  t=40h  29  30  31  30  t=48 h  28  28  28  28  t=48h  28  30  28  29  B l a n k t=48 h  31  32  31  31  B l a n k t=48 h  29  28  28  28  121 31 30 33 36 28 28 28 30  Table A.76 pH measurement kinetic rate with NaN addition at room temperature on Howe Sound effluent 3  Sample  PH  t=0  7.81  t=l h  8.49  t=8h  8.60  t=24h  8.98  t=32h  9.06  t=40h  8.59  t=48h  8.66  B l a n k t=48 h  9.05  152  APPENDIX 10. ISOTHERM MINI-STUDY  Table A.77 Colour measurement equilibrium isotherms with NaN addition at room temperature on Western Pulp effluent 3  Sample Blank Std. 50 Std. 100 Std. 300 Std. 500 d=0, t=0 d=0, t=0 d=0.1 g,t=32h d=0.1 g,t=32h d=0.5 g, t=32 h  Absorbance 0 0.015 0.037 0.099 0.167  d=0.5 g, t=32 h d=1.0g,t=32h d=1.0 g, t=32h d=2.0 g, t=32 h d=2.0 g, t=32 h d=3.0 g, t=32 h d=3.0g,t=32h d=0, t=32 h  0.118 0,118 0.093 0.089 0.063 0.061 0.060 0.054 0.039 0.043 0.045 0.045 0.107  d=0, t=32 h  0.110  0.014 0.036 0.100 0.169 0.118 0.115 0.091 0.088 0.061 0.061 0.058 0.050 0.038 0.041 0.045 0.044  Value  Average  0 0.015 0.037 0.100 0.168 0.118 0.117 0.092 0.089 0.062 0.061 0.059 0.052 0.039 0.042  0.117 0.090 0.062 0.056 0.040 0.045  0.108  0.045 0.045 0.108  0.108  0.109  0.108  153  Table A.78 COD measurement equilibrium isotherms with NaN addition at room temperature on Western Pulp effluent 3  Sample Blank  Absorbance 0  Std. 100  0.035  0.036  0.036  Std. 300 Std. 500  0.121 0.190  0.122 0.192  0.122 0.191  Std. 800 d=0, t=0 d=0, t=0 d=0.1 g,t=32h  0.276 0.101 0.099 0.119  d=0.1 g, t=32h  0.274 0.101 0.100 0.120 0.120  d=0.5 g, t=32 h d=0.5 g, t=32 h  0.133 0.141  0.119 0.133 0.142  0.275 0.101 0.100 0.120 0.120 0.133 0.142  d=2.0 g, t=32 h d=2.0g,t=32h d=3.0 g, t=32 h  0.319 0.330 0.494  0.319 0.332 0.495  0.319 0.331 0.495  0.325  d=3.0 g, 1=32 h  0.481  0.478  0.480  0.487  Value 0  Average  0.100 0.120 0.137  Table A.79 DOC measurement equilibrium isotherms with NaN addition at room temperature on Western Pulp effluent 3  Sample Blank Std. 50 Std. 500 d=0, t=0 d=0.1 g,t=32h d=0.5 g, t=32 h d=1.0g,t=32h d=1.0 g,t=32h d=2.0 g, t=32 h d=3.0 g, t=32 h d=0,t=32h d=0,1=32 h  Height of peak (mm) 1 1 11 10 93 94 36 35 36 34 35 36 36 29 32  Value 1 10 98 36  105 36 35  Average 1 10 98 36 35  37 29  37 31  37 30  37  33 122 169 24  32 119 170 24  31 119 170  24  31 120 172 24  23  23  23  23  24  113 173  119 165  154  Table A.80 pH measurement equilibrium isotherms with NaN addition at room temperature on Western Pulp effluent 3  Sample d=0, t=0  '  p_H 8.07  d=0.1g,t=32h  8.79  d=0.5 g, t=32 h d=2.0 g, 1=32 h  8.60 8.34  d=3.0 g, 1=32 h  8.49  Table A.81 Colour measurement equilibrium isotherms with NaN addition at room temperature on Howe Sound effluent 3  Absorbance  Value 0  Average  Sample Blank  0  Std. 50 Std. 100  0.015 0.037  0.014 0.036  Std. 300 Std. 500  0.099  0.100  0.015 0.037 0.100  0.167 0.078  0.169  0.168  0.079  0.079  0.080 0.054  0.083 0.053  0.082 0.054  0.080  d=0.1 g, t=32h d=0.5 g, t=32 h d=0.5g,t=32h d=1.0 g,t=32h d=1.0 g,t=32h  0.059 0.036 0.034  0.058 0.036 0.036 0.024  0.059 0.036  0.056 0.036  d=2.0 g, 1=32 h d=2.0 g, 1=32 h  0.020 0.018  0.035 0.023 0.018 0.021 0.017  0.019  d=3.0 g, t=32 h d=3.0g,t=32h  0.022 0.019  0.020 0.020  0.021 0.020  0.020  d=0,1=32 h  0.081  0.080  0.081  d=0, t=32 h  0.082  0.081  0.082  d=0,1=0 d=0, t=0 d=0.1 g,t=32h  0.022 0.018  0.018 0.022 0.016  0.021  0.081  155  Table A.82 COD measurement equilibrium isotherms with NaN addition at room temperature on Howe Sound effluent 3  Sample Blank Std. 100 Std. 300 Std. 500 Std. 800 d=0, t=0 d=0, t=0 d=0.1 g,t=32h d=0.1 g,t=32h d=0.5 g, t=32 h d=0.5 g, t=32 h  Absorbance 0 0.035 0.121 0.190 0.274 0.116 0.114 0.093 0.093 0.095 0.098  Value  Average  0 0.036  0.036 0.122 0.191  0.122 0.192 0.276 0.115 0.114 0.092  d=2.0 g, t=32 h d=2.0 g, t=32 h d=3.0 g, t=32 h  0.296 0.264 0.317  0.093 0.095 0.098 0.297 0.266 0.316  d=3.0 g, t=32 h  0.319  0.32  0.275 0.116 0.114 0.093 0.093 0.095 0.098 0.297 0.265 0.317 0.320  0.115 0.093 0.097 0.281 0.318  Table A.83 DOC measurement equilibrium isotherms with NaN addition at room temperature on Howe Sound effluent 3  Sample Blank Std. 50 Std. 500 d=0, t=0 d=0.1 g,t=32h  Height of peak (mm) 1 1 1 11 10 10 93 94 98 24 24 23 27 27 29  Value 1  d=0.5 g, t=32 h d=1.0g,t=32h  26 15  27 15  26 15  26 15  26  d=1.0 g,t=32h  14  14  14  14  15  d=2.0 g, t=32 h d=3.0 g, t=32 h  74 139  76 135  75 138  75 137  75 137  d=0, t=32 h  30  29  31  30  d=0, t=32 h  34  34  35  34  105  134  10 98 24 28  Average  24 28  32  156  Table A.84 pH measurement equilibrium isotherms with NaN addition at room temperature on Howe Sound effluent 3  Sample d=0, t=0 d=0.1 g,t=32h d=0.5 g , t=32 h d=2.0 g , t=32 h d=3'.0 g , t=32 h  pH 7.81 8.56 8.50 8.20 8.30  157  APPENDIX 11. REMOVED FRACTION MINI-STUDY Table A.85 Colour measurement of lg dose of biomass for removed fraction mini-study on Western Pulp effluent Sample Blank Std. 50  Absorbance  Value  Std. 100 Std. 300 Std. 500 500 Daltons 500 Daltons . 3000 Daltons 3000 Daltons 10000 Daltons  0 0.015 0.037 0.099 0.167 0.005 0.003 0.006 0.006 0.008  0.014 0.036 0.100 0.169 0.005 0.003 0.006 0.006 0.008  10000 Daltons 100000 Daltons 100000 Daltons  0.012 0.019 01017  0.011 0.019 0.018  Total  0.027  0.025  0.005 0.003 0.006 0.006 0.008 0.012 0.019 0.018 0.026  Total  0.03  0.029  0.030  Average  0.015 0.037 0.100 0.168 0.004  0.008  0.006  0.012  0.010  0.02  0.018  0.036  0.028  0.056  Table A.86 TOC measurement of lg dose of biomass for removed fraction mini-study on Western Pulp effluent Sample 3000 Daltons 3000 Daltons 10000 Daltons 10000 Daltons 100000 Daltons 100000 Daltons Total Total  Peak hei ght  Average  6  6  6  6 9  6 8  6 9  6  9  9  9  9  12 14  13 14  13 13  13  28  29  28  29  29  29  29  158  Table A.87 Colour measurement of blank for removed fraction mini-study on Western Pulp effluent Sample Blank  Absorbance  Average  Std. 50  0 0.015  0.014  0 0.015  Std. 100 Std. 300  0.037 0.099  0.036 0.100  0.037 0.100  Std. 500  3000 Daltons  0.014  0.169 0.006 0.004 0.013 0.013  0.168  500 Daltons 500 Daltons 3000 Daltons  0.167 0.005 0.004 0.013  10000 Daltons 10000 Daltons 100000 Daltons 100000 Daltons Total  0.028 0.026  0.097 0.094 0.109  0.095  0.110  0.030 0.027 0.097 0.093 0.108  Total  0.107  0.108  0.108  0.108  0.096 0.094  0.006 0.004 0.013  0.005  0.014  0.013  0.029 0.027  0.028  Table A.88 COD measurement of blank for removed fraction mini-study on Western Pulp effluent Sample 500 Daltons 500 Daltons 3000 Daltons 3000 Daltons 10000 Daltons  8 8 12 12 17  Peak height 8 8 12 12 16  Average 8 8 12 12 17  10000 Daltons 100000 Daltons 100000 Daltons  16 28 30  16 28 29  17 29 29  Total  32  31  33  Total  29  29  31  8 12 17 29 31  159  Table A.89 Colour measurement of l g dose of biomass for removed fraction mini-study on Howe Sound effluent Adjusted as per Sample Absorbance Average dilution Blank 0 0 0.015 Std. 50 0.015 0.014 Std. 100  0.037  0.036  0.037  Std. 300  0.099  0.100  0.100  Std. 500  0.167  0.169  0.168  500 Daltons 500 Daltons  0.006 0.005  0.006 0.005  0.005  3000 Daltons  0.011  0.01  0.011  3000 Daltons  0.009  0.006  0.008  10000 Daltons  0.008  0.008  0.008  10000 Daltons  0.008  0.008  0.008  Daltons 100000 Daltons Total Total  0.009  0.009  0.009  100000  0.006  0.010  0.011  0.011  0.009  0.009  0.009  0.011  0.012  0.012  0.006  0.012  0.009  0.018  0.008  0.016  0.010  0.020  0.010  0.020  Table A.90 TOC measurement of l g dose of biomass for removed fraction mini-study on Howe Sound effluent Peak height (mm)  Sample  Value  3000 Daltons 3000 Daltons  8  8  8  8  8  8  10000 Daltons 10000 Daltons  9  8  9  9  9  9  Daltons 100000 Daltons Total Total  12  13  13  13  13  13  14  14  14  15  15  14  100000  8 9 13 14  160  Table A.91 Colour measurement of blank for removed fraction mini-study on Western Pulp effluent Sample Blank  Average  Absorbance  Std. 50  0.015  0.014  0 0.015  Std. 100 Std. 300  0.037 0.099  0.036 0.100  0.037 0.100  Std. 500 500 Daltons 500 Daltons 3000 Daltons 3000 Daltons  0.167 0.007 0.007 0.041  0.169 0.008 0.008 0.041  0.168 0.008 0.008 0.041  10000 Daltons 10000 Daltons  0.036 0.042 0.041  0.036 0.042 0.040  0.036 0.042 0.041  100000 Daltons 100000 Daltons Total  0.071 0.070 0.082  0.070 0.071 0.081  0.071 0.071 0.082  0.071  Total  0.081  0.080  0.081  0.081  0.008 0.039 0.041  Table A.92 COD measurement of blank for removed fraction mini-study on Western Pulp effluent Sample 3000 Daltons 3000 Daltons 10000 Daltons 10000 Daltons 100000 Daltons  28 28 28 29 32  Peak height 29 28 28 28 34  100000 Daltons Total  35 30  Total  33  Average 29 29 29 29 32  29  33 29  35 31  34  34  34  32  29  161  APPENDIX 12. T E M P E R A T U R E MINI-STUDY  Table A.93 Colour measurement equilibrium isotherms with NaN addition at 35°C on Western Pulp effluent 3  Sample Blank  Absorbance 0  Average 0  Std. 50 Std. 100  0.015 0.037  0.014 0.039  0.015 0.038  Std. 300  0.102  0.103  0.103  Std. 500  0.173  0.173  0.173  t=0 t=0  0.118 0.117  0.118 0.118  0.118 0.118  0.118  t=l h  0.098  0.097  0.098  t=l h t=8h  0.098 0.089  0.097 0.088  0.098 0.089  0.098  t=8h t=20hr t=20hr t=24h t=24 h  0.088 0.079 0.081 0.071 0.072  0.088 0.080 0.081 0.072 0.071  ,0.088 0.080 0.081 0.072 0.072  0.088  t=32h t=32 h  0.063 0.061  0.063 0.060  0.063 0.061  0.062  t=40h t=40h  0.061 0.061  0.060 0.059  0.061 0.060  . 0.060  t=48h  0.057  0.059  0.058  t=48h  0.057  0.057  0.057  Blank t=48 h  0.119  0.119  0.119  Blank t=48h  0.125  0.125  0.125  _  0.080 0.072  0.058 0.122  162  Table A.94 COD measurement equilibrium isotherms with NaN addition at 35°C on Western Pulp effluent 3  Sample Blank Std. 100  Absorbance 0  Value  Average  0  0.038  0.038  0.038  Std. 300 Std. 500  0.117  0.116 0.200  0.117  Std. 800  0.316 0.130  0.316 0.131 0.128  0.131  0.128  .. 0.315 0.132 0.128  t=l h  0.127  0.128  0.128  t=8h  0.132  0.140 0.133  t=20hr  0.132  0.140 0.133 0.132  0.136  t=20hr  0.133 0.140 0.133  0.128 0.133  0.132  0.133  t=24h t=24h  0.110 0.108 0.092 0.094  0.111 0.108  0.111 0.108  0.109  0.092 0.094  0.092 0.094  0.093  0.124  0.123 0.115  0.124 0.116  0.12  t=0 t=l h  1=8 h  1=32 h  t=32 h t=40h  0.199  0.200  •1=40 h.  0.116  t=48h  0.113  0.113  0.113  t=48h Blank t=48 h  0.116 0.120  0.114 0.121  0.115 0.121  0.114  Blank t=48 h  0.119  0.118  0.119  0.120  163  Table A.95 DOC measurement equilibrium isotherms with NaN addition at 35°C on Western Pulp effluent 3  Standard Blank Std. 50  Height of peak (mm) 2 3 3 11 10 10  Std. 500 t=0  106 39 41 42 41 50 51 50 52 43 42 32 33 36  Value  Average  3 10  105 37  100 39 42 46 39  36 35 35 42  35 35 34 43  35  t=48 h Blank t=48 h  35 36 34 43  43 46 39 52 50 53 51 44 43 35 34 35 34 35 33 43  Blank t=48 h  40  39  42  40  42  t=0 t=l h t=l h t=8h t=8h t=20hr t=20hr t=24 h t=24h t=32 h t=32 h t=40h t=40 h t=48h  51 51 53 49 44 42 33 34 35  171  121 38 42 45 40 51 51 53 51 44 42  50 55  33  33 34 35  40 42 51 52 43 33  35  Table A.96 pH measurement equilibrium isotherms with NaN addition at 35°C on Western Pulp effluent 3  Sample t=0 t=l h  pH 7.79 8.77  t=8h t=20h t=24h  8.92 9.02 8.82  t=32h t=40h t=48h  8.47 8.42 8.47  Blank t=48 h  9.17  164  A P P E N D I X 13. P R A C T I C A L A P P L I C A T I O N S T U D Y  Sample Blank  0  Std. 50 Std. 100 Std. 300  0.015 0.038 0.105  0.015 0.040 0.104  0.015 0.039 0.105  Std. 500 Blank l,t=32h Blank l,t=32h  0.178 0.076 0.071 0.042 0.041 0.032 0.032  0.178 0.076 0.072 0.042 0.043  0.025 0.022 0.069  0.178 0.076 0.073 0.042 0.044 0.031 0.029 0.027 0.023 0.067  0.069  0.068  0.069  Absorbance  d=0.8 g, t=32 h d=0.8 g, t=32 h d=4 g, 1=32 h d=4 g, t=32 h d=8 g, t=32 h d=8g, t=32h Blank 2, t=32 h Blank 2, t=32 h  Value 0  0.032 0.031 0.026 0.023 0.068  Average  0.074 0.042 0.031 0.02.4 0.068  Table A.98 pH measurement of batch activated sludge testing on Howe Sound effluent Sample Blank l,t=32h d=0.8 g, 1=32 h  PH 8.36 8.14  d=4 g, 1=32 h d=8 g, 1=32 h  7.82 7.75  Blank 2,1=32 h  8.53  165  Table A.99 T O C measurement of batch activated sludge testing on Howe Sound effluent Sample 0 50  1 18  500  168  Blank 1, t=32 h  146 118  136 128  188 180 100  d=0.8 g, t=32 h d=4 g, t=32 h d=8 g, t=32 h Blank 2, t=32 h  Peak height (mm) 1 1 16 16 164 170  Average 1 17 172  169  103 122  116  125 123  186 192*  186 192*  180 192*  185 189  95  90  115  100  *Maximum value  166  Table A.100 TSS measurement of batch activated sludge testing for Howe Sound effluent  Sample size (L)  Dish & filter (g)  Blank 1, t=32 h  0.015  1.1215  1.1218  0.0003  d=0.8 g, t=32 h  0.015  1.0884  1.0884  0.0000  0.0003  20  d=4 g, t=32 h  0.015  1.0978  1.0982  0.0004  0.0007  47  d=8 g, 1=32 h  0.005  1.1555  1.1563  0.0008  0.0011  220  Blank 2, t=32 h  0.015  1.1103  1.1101  -0.0002  0.0001  7  1.1074  1.1071  -0.0003  0.0000  Sample  Filter blank  After sample & drying (g)  Difference (g)  Blank adjust, (g)  TSS mg/L  0.0006  40  Table A.101 B O D measurement of batch activated sludge testing for Howe Sound effluent Volume of sample (mL)  Bottle number  Initial DO (mg/L)  Final DO (mg/L)  Value (mg/L)  With dilution water adjustment  7.0  44  9.74  5.96  32  31  2.0  67  9.84  7.78  62  60  d=0.8 g,t=32h  2.0  61  9.76  5.76  120  118  d=4 g, t=32 h  0.5  64  9.81  3.72  731  729  d=8 g, 1=32 h  0.5  69  9.78  0.36  1130  1129*  7.0  50  9.78  6.57  28  26  2.0  59  9.85  7.96  57  55  Sample Blank 1,1=32 h  Blank 2, t=32 h * Not a valid measurement  ON.  

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