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A study of anaerobic treatment of CTMP/TMP effluents Vipat, Vasudha 1988

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A Study of Anaerobic Treatment of C T M P / T M P Effluents by Vasudha Vipat B.Sc, University of Zambia, Zambia, 1985. A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF APPLIED SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Chemical Engineering We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1988 © Vasudha Vipat, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Chemical Engineering The University of British Columbia Vancouver, Canada Date Sept 30, 1988.  DE-6 (2/88) Abstract Prior to any extensive anaerobic treatment studies, it is essential to assess the impact of the components of the wastewater on the anaerobic microorganisms. Therefore, a simple, effective technique to assess the toxicity and degradability of various components of the wastewater is necessary. In this thesis, a technique is developed for measuring the biodegradability (Bio-chemical Methane Potential — BMP) and toxicity (Anaerobic Toxicity Assay — ATA) of the effluent components subjected to anaerobic treatment. The ATA mea-sures the adverse effect of a compound on the rate of the total gas production from an easily-utilized methanogenic substrate (acetate and propionate). This method was used to determine the toxicity of sulphur compounds. Sulphate had no effect on anaerobic microorganisms, even at concentration of 1000 mg/L. Sulphide was found to be inhibitory above concentrations of 200 mg/L. Sulphite, on the other hand, was found to be extremely toxic even at a concentration of 100 mg/L. BMP is a measure of the substrate biodegradability determined by monitoring cumulative biogas production from a sample which is anaerobically incubated at constant temperature in a chemically defined medium. CTMP effluent was found to be more degradable and less toxic than TMP effluent. The degradability of CTMP effluent was maximum when it was diluted to between 50 and 25%, while that of the TMP effluent was maximum at 10% dilution. i Contents A B S T R A C T i LIST OF TABLES v LIST OF FIGURES vi LIST OF A C R O N Y M S viii A C K N O W L E D G E M E N T S ix 1 Introduction 1 1.1 Statement of Problem 3 1.2 Organization of Thesis 4 2 Literature Review 5 2.1 Overview 5 2.2 General Background 5 2.3 Importance of Assay Tests 8 2.4 Critique of Present Assay Techniques 8 2.5 Anaerobic Toxicity Assay (ATA) 9 2.6 Biochemical Methane Potential (BMP) 10 3 Methodology 11 3.1 Overview 11 3.2 Assay Bottles 11 3.3 Source of Inocula 12 ii 3.4 Mineral Nutrient Supplement 13 3.5 Growth Medium (Substrate) Composition 14 3.6 Substances Tested 14 3.6.1 Anaerobic Toxicity Assay (ATA) 14 3.6.2 Biological Methane Potential (BMP) 15 3.6.3 Control Experiments 16 3.6.4 Hydrogen Peroxide Degradation in CTMP Effluent 17 3.7 Start-Up Procedure 17 3.7.1 ATA 17 3.7.2 BMP 17 3.7.3 Peroxide Degradation " 18 3.8 Incubation Times and Sampling Frequencies 18 3.9 Analysis Program 21 3.9.1 Gas Volume Measurement 21 3.9.2 Gas Composition Analysis 21 3.9.3 Substrate Analysis 22 3.9.4 Resin Acid Analysis 22 3.9.5 Hydrogen Peroxide Analysis 22 4 Results and Discussion 23 4.1 Overview 23 4.2 Commentary on the Assay Technique 23 4.2.1 Design of Assay Flask 23 4.2.2 Sludge Characteristics 25 4.2.3 Nutrient Addition and Substrate Effects 26 4.3 Degradability Studies 33 4.3.1 CTMP Effluent 33 4.3.2 TMP Effluent 48 4.3.3 Degradability Studies of Peroxide in CTMP Effluent 65 4.4 Toxicity Studies 67 4.4.1 Sulphite Toxicity 67 iii 4.4.2 Sulphate Toxicity 75 4.4.3 Sulphide Toxicity 84 4.4.4 Resin Acids 88 5 Conclusion and Suggestions For Further Research 92 5.1 Conclusion 92 5.1.1 The Anaerobic Bioassay Technique 92 5.1.2 Biodegradability Studies 93 5.1.3 Toxicity Studies 94 5.1.4 Suggestions for Further Research 94 iv List of Tables 1.1 Typical Wastewater Parameters For TMP/CTMP Effluent 2 2.1 Effluent Quality Objectives for Mechanical Pulping Processes in British Columbia 6 3.1 Characteristics of Effluent from Quesnel River Pulp Ltd 16 3.2 Table of Tests Conducted For ATA 19 3.3 Table of Tests Conducted For Degradability Studies 20 3.4 Table of Sampling Frequencies 20 4.1 Table of Cumulative Gas Produced (CTMP Effluent) (ml) 37 4.2 Cumulative Gas Produced For CTMP Effluent Expressed as a Frac-tion of the Maximum Produced 37 4.3 Gas Potential of CTMP Effluent (ml biogas/ml sample) 40 4.4 Soluble COD of CTMP Effluent 44 4.5 Table of COD Removal Compared with the Theoretical and Actual Methane Production (CTMP Effluent) 47 4.6 Cumulative Gas Produced (TMP Effluent) (ml) 51 4.7 Cumulative Gas Produced for TMP Effluent Expressed as a Fraction of the Maximum Produced 51 4.8 Biogas Potential of TMP Effluent (ml biogas/ml effluent) 57 4.9 Soluble COD of TMP effluent 60 4.10 Table of COD Removal Compared with the Theoretical and Actual Methane Production (TMP Effluent) 63 4.11 Total Gas Produced (ATA of Sodium Sulphite) 67 4.12 MRR Values (ATA of Sodium Sulphite) 67 v List of Figures 3.1 Schematic Diagram of the Culture Flask 12 4.1 Gas Composition for Control Set: Seed Sludge in Tap Water . . . . 27 4.2 Cumulative Gas Produced by Control Set: Seed Sludge in Tap Water 28 4.3 Cumulative Gas Produced by Control Set: Substrate and Seed Sludge in Tap Water 29 4.4 Cumulative Gas Produced by Control Set: Substrate and Seed Sludge in Nutrient Solution 30 4.5 Gas Composition of Control Set: Substrate and Seed Sludge in Tap Water 31 4.6 Gas Composition of Control Set: Substrate and Seed Sludge in Nu-trient Solution 32 4.7 Cumulative Gas Produced (CTMP Effluent): Set 1 34 4.8 Cumulative Gas Produced (CTMP Effluent): Set 2 35 4.9 Cumulative Gas Produced (CTMP Effluent): Set 3 36 4.10 Normalized Cumulative Gas Produced (CTMP Effluent): Set 1. . . 38 4.11 Normalized Cumulative Gas Produced (CTMP Effluent): Set 2. . . 39 4.12 Rate of Gas Production (CTMP Effluent): Set 1 41 4.13 Rate of Gas Production (CTMP Effluent): Set 2 42 4.14 Cumulative Gas Produced (CTMP Effluent) Seeded with 2% Sludge. 45 4.15 Change in Soluble COD with time (CTMP Effluent) 46 4.16 Maximum Rate Ratios (MRR) for CTMP Effluent 49 4.17 Gas Composition (CTMP Effluent) 50 4.18 Cumulative Gas Produced (TMP Effluent): Set 1 52 4.19 Cumulative Gas Produced (TMP Effluent): Set 2 53 vi 4.20 Cumulative Gas Produced (TMP Effluent): Set 3 54 4.21 Normalized Cumulative Gas Produced (TMP Effluent): Set 1. . . . 55 4.22 Normalized Cumulative Gas Produced (TMP Effluent): Set 2. . . . 56 4.23 Rate of Gas Production (TMP Effluent): Set 1 58 4.24 Rate of Gas Production (TMP Effluent): Set 2 59 4.25 Cumulative Gas Produced (TMP Effluent) with 2% Sludge 61 4.26 Change in Soluble COD with Time (TMP Effluent) 62 4.27 Maximum Rate Ratios (MRR) for TMP Effluent 64 4.28 Gas Composition (TMP Effluent) 66 4.29 Cumulative Gas Produced (Sulphite ATA): Set 1 69 4.30 Cumulative Gas Produced (Sulphite ATA): Set 2. " 70 4.31 Normalized Cumulative Gas Produced (Sulphite ATA): Set 1. . . . 71 4.32 Normalized Cumulative Gas Produced (Sulphite ATA): Set 2. . . . 72 4.33 Rate of Gas Production (Sulphite ATA): Set 1 73 4.34 Rate of Gas Production (Sulphite ATA): Set 3 74 4.35 Daily Rate Ratios (Sulphite ATA) 76 4.36 Maximum Rate Ratios (Sulphite ATA) 77 4.37 Gas Composition (Sulphite ATA) 78 4.38 Cumulative Gas Produced (Sulphate ATA) 80 4.39 Rate of Gas Production (Sulphate ATA) 81 4.40 Daily Rate Ratios (Sulphate ATA) 82 4.41 Maximum Rate Ratios (Sulphate ATA) 83 4.42 Gas Composition (Sulphate ATA) 85 4.43 Cumulative Gas Produced (Sulphide ATA) 86 4.44 Rate of Gas Production (Sulphide ATA) 87 4.45 Daily Rate Ratios (Sulphide ATA) 89 4.46 Maximum Rate Ratios (Sulphide ATA) 90 4.47 Gas Composition (Sulphide ATA) 91 vii List of Acronyms ADtPD Air Dried tons Per Day (of pulp) ATA Anaerobic Toxciity Assay BMP Biochemical Methane Potential BODs Biochemical Oxygen Demand (5 day) COD Chemical Oxygen Demand CTMP Chemithermomechanical Pulp HRT Hydraulic Retention Time MRR Maximum Rate Ratio RAMM Revised Anaerobic Mineral Medium RR Rate Ratio sCOD soluble Chemical Oxygen Demand SRT Solids Retention Time TMP Thermomechanical Pulp TS Total Solids VFA Volatile Fatty Acids viii Acknowledgements I am very grateful to my advisor, Dr. R. M. R. Branion, for his continual help, support, and advice. I appreciate the considerable laboratory support of Steve Allen, Ken Wong, Paula Parkinson, Timothy Mah, Horace Lam and Susan Liptak. I am thankful towards Dr. Dennis Ouchi and Annette Langman of PAPRICAN for their help in resin acids analysis. I would like to thank Dr. K.C. Teo and Rob Stephenson for their advice. My heartfelt thanks to Lisa Brandly for her help in preparing this manuscript. Most of all, I am grateful for the caring support of my parents, and Pannu, Hari, Prem and Jyoti. I thank you all. ix Chapter 1 Introduction Canada is one of the world's leading producers of pulp and paper products. Its pulp and paper industry stands first in employment and is the largest manufacturing industry in Canada. In the pulping process, from the debarking of logs prior to chipping right up to the finished paper products, water is necessary in copious quantities for the various process stages. This large water usage leads to large quantities of wastewater being generated which contain dissolved and suspended solids. Such wastewater must be treated prior to discharge into receiving waters. The quality of this wastewater is regulated by legislation which requires, amongst other things, measurement of suspended solids (SS), biochemical oxygen demand (BOD), and toxicity to fish. The most common pulping process in British Columbia is the kraft process wherein wood chips are cooked in a pressure vessel in a solution of sodium hydroxide and sodium sulphide. The dissolution and removal of lignin in the solution leaves behind the cellulosic wood fibres. Often a subsequent bleaching process is used to remove even more of the wood lignin. The dissolved lignin and other wood and chemical residues are mostly recycled but some end up in the wastewater from the mill. Typical effluent discharge levels are, 10 to 40 Kg of SS and 12 to 20 Kg of BOD 5 per ton of pulp produced, and toxicity (96 hr LCso) of 15 to 50 (% v/v) [11]. In the last decade, some other pulping processes have caught the industry's attention. Thermomechanical pulp (TMP) and, more recently, chemithermome-chanical pulp (CTMP) are two such processes. In the TMP process, the pulp is made by presteaming chips and reducing them to fibres by an initial mechanical treatment in refiners at elevated temperature and pressure, with subsequent re-fining done at atmospheric pressure. In the CTMP process, the wood chips are pretreated with a chemical, usually sodium sulphite, prior to mechanical process-ing as in the TMP process. Because of the differences in the pulping processes, the wastewater characteristics are different. Some typical values of some common 1 Units CTMP TMP ss mg/L 221 209 BODs mg/L 3300 1587 Toxicity 96-hr L C S 0 (%V/V) 0.37-1.80 0.66-2.40 Table 1.1: Typical Wastewater Parameters For TMP/CTMP Effluent parameters are given in Table 1.1 [48] — The mill wastewaters are usually treated in clarifiers where the solids are allowed to settle. Then, the dissolved substances contributing to BOD are removed by aerobic treatment in lagoons where the effluents are microbially treated to oxidize organic matter to CO2 and H2O. The lagoon is aerated by mechanical means to provide 0 2 for this microbial oxidation process. Removal of BOD with aerobic processes often requires nutrient addition to satisfy the growth requirements of the aerobic microorganisms. Moreover, their oxygen requirements are high, resulting in high power consumption by the aeration devices and hence high operating costs. Since aeration costs are a major expense of wastewater treatment, an alternative is desired which would reduce these costs. Anaerobic digestion is the breakdown of organic matter by bacteria in the ab-sence of oxygen and it is a major pathway in the decomposition of organic wastes in nature. Anaerobic treatment of wastewaters has long been practiced in mu-nicipal sewage treatment and in the agricultural and food processing industries. The anaerobic process is regarded as a promising alternative for the treatment of high-strength pulp mill effluents [6,4,57,10]. Pulp and paper effluents have several characteristics which make them suitable for anaerobic treatment. Many in-plant streams and final effluents are discharged at elevated temperatures, and therefore, would require only minimum energy input for treatment at mesophilic temperatures (30-40°C) where the anaerobic process works best. In addition, the most troublesome streams contain relatively high con-centrations of BOD. Biological removal of these organics using an aerobic process would be expensive in terms of aeration costs and would probably require nutrient addition [e.g. N and P) to satisfy the requirements for the extensive growth of the aerobic microorganisms. Anaerobic treatment produces an effluent that is usually unacceptable for direct discharge but may allow significant BOD removal to be achieved without aeration and without nutrient supplementation. Subsequent aerobic treatment would pro-2 duce a dischargeable effluent at a lower overall cost. Other inherent advantages of anaerobic processes include low production of biological sludge, low capital costs, no oxygen requirement, and methane production (a potential source of fuel) [7,44]. Conventional digesters as used historically in sewage treatment include the plug flow, complete-mix digesters and anaerobic lagoons. They are characterized by equal solids retention time (SRT) and hydraulic retention time (HRT) of 10 to 30 days. Advanced high-rate reactors differ in that the biomass is retained in the process unit. Thus, the HRT is reduced while maintaining a longer SRT. Van den Berg and Kennedy [53] have discussed the design and treatment characteristics of high rate reactors. They fall into three groups: the fixed-film filters, the sludge blanket reactors, and the fluidized/expanded bed reactors. In the fixed-film reactors, such as the anaerobic filter and the downflow station-ary fixed-film (DSFF) reactor, fixed media are assembled on which bacteria grow. The feed streams are passed upward and downward respectively in the two types of reactors. In the Upflow Anaerobic Sludge Bed (UASB) reactor, the biomass is suspended and retained as a blanket which is kept in suspension by controlling the upward flow velocity of the feedstream. It contains a device to separate the gas, ef-fluent, and suspended solids at the surface of the reactor. In the fluidized/expanded bed reactors, bacteria are grown on particles of a medium, usually sand. A rapid, even flow of liquid keeps the particles in suspension. All of these various processes can be used for treatment of pulp mill wastewaters. However, pulping effluents have properties which make them difficult to treat using biological processes. Pulp and paper effluents often contain compounds which can be toxic or inhibitory to both the treatment process microorganisms and to organisms, such as fish, in the receiving waters. Typical characteristics of untreated pulp and paper mill effluents are available in the literature, classified according to manufacturing process [36], Resin acids, plant hormones, chlorinated organics and reduced sulphur compounds are several of the toxic constituents of concern [14,20]. Significant proportions of the organic matter in pulp and paper effluents are also not amenable to biological degradation [21]. A high COD:BOD ratio is typical of pulping effluents. When treated either aerobically or anaerobically, even com-plete BOD removal will leave a non-biodegradable COD residue. In spite of these difficulties, many researchers feel that anaerobic systems will be useful in treating the high strength wastes associated with TMP and CTMP production. 1.1 Statement of Problem 1. To develop a modification of the serum-bottle technique of Hungate [24] such 3 that it can be used for treatability studies of wastewaters. 2. To use this technique in assessing the toxicity of various substances found in CTMP and TMP effluents on an anaerobic mixed culture. 3. To use this technique to evaluate the biodegradability of the raw CTMP and TMP effluents. Also, to assess the effect of dilution on degradability. 1.2 Organization of Thesis Chapter 2 deals with a review of the assay techniques used to date and presents methods used for evaluating toxicity and degradability. Chapter 3 covers the experimental procedures and the substances tested. The analysis parameters are also detailed here. Chapter 4 presents the data collected, its analysis and discussion in order to assess the toxicity and degradability of the wastewaters. Chapter 5 summarizes the information gathered, the conclusions drawn and the recommendations for future work. 4 Chapter 2 Literature Review 2.1 Overview Section 2 presents a general background on the effluent discharge regulations and the pollutants of concern is presented here. Section 3 outlines the need for routine bench-scale testing of effluents and their effects on anaerobes. Section 4 reviews the techniques used to date in assessment of effluent character-istics for anaerobic treatment. Section 5 describes the Anaerobic Toxicity Assay test. Section 6 describes the Biochemical Methane Potential and its importance to anaerobic treatment processes. 2.2 General Background The Canadian pulp and paper industry uses a wide variety of wood sources and manufacturing technologies. As a result, mill effluent characteristics vary sub-stantially, depending on the process used, the production rate, and the degree of in-plant effluent reduction measures. In fact, the variability of effluents is both within a given plant and among plants [22]. Considering this situation, it is nec-essary to have guidelines on effluent discharge. Review of effluent regulations and toxicity studies have been presented by Walden et. al [55] and others. The fed-eral government has direct authority over the discharges to watercourses via the Fisheries Act and has legislation specific to pulp and paper effluent discharges (see 5 Characteristic Units Level A B 1. Total Suspended Solids kg/t 10 17.5 2. BODs kg/t 7.5 20 3. Temperature °C 35 35 4. pH Range — 6.5-8.0 6.5-8.0 5. Dissolved Oxygen mg/L 2.0 — 6. Zinc kg/t — 1 7. Toxicity 96-hr L C 5 0 (% v/v) 100 30 Table 2.1: Effluent Quality Objectives for Mechanical Pulping Processes in British Columbia. EPS l-WP-72-1 and EPS l-WP-72-2 [13,14]). The present pollution control ob-jectives for the forest products industry of British Columbia were established in 1971 [2] and revised in 1977. The B.C. effluent quality objectives for mechanical pulping processes are presented in Table 2.1. Pollutants from the pulp and paper industry can be classified in terms of oxy-gen demanding substances (BOD and COD), solids (total, dissolved, and suspended solids), synthetic organic compounds (oils and greases, detergents, pesticides, and other chemicals produced in the pulping and bleaching processes or extracted from the wood), thermal pollution, and inorganic chemicals and mineral substances (acids, alkalis and heavy metals) [51]. In the manufacture of thermomechanical pulp, the dissolved organic compounds consist of equal amounts of lignin and carbohydrates, about 40% each, the rest of the compounds being extractives. In the CTMP process, pretreatment of chips with chemicals and steam increases the dissolution of compounds. Some typical effluent characteristics are presented in Table 1.1. The principal pollution parameter of concern in mechanical pulping effluents is toxicity to fish and associated sublethal effects due to their high levels of wood extractives [15,14,8,29,30,37]. These extractives include resin acids (abietic, de-hydroabietic, pimaric, sandaracopimaric, levopimaric and neoabietic acids); fatty acids (oleic, linoleic and linolenic), and insect juvenile hormones (juvabione and juvabiol) [15,16]. No chlorophenols are present since chlorine is not used in bleach-ing the pulp. The toxicity is expressed as the concentration of test substance causing the death of 50% of the test fish over a period of 96 hours (96 hr LC50). Howard and Walden have discussed the bioassay techniques used [23,56] for toxi-6 city studies. The effluents from TMP and CTMP mills have been found to have no mutagenic properties [31,41], although some stress has been observed in fish exposed to it. Aerobic biological treatment of kraft pulping effluents is usually successful in complying with emission regulations except for fish toxicity compliance which is erratic. BOD reduction is usually satisfactory and SS reduction, while not always meeting the guidelines, is mostly satisfactory. What is discharged as SS is usually bio-sludge from the aerobic treatment processes. There is not sufficient information available on aerobic biological treatment of mechanical pulping effluents to say whether or not they are successful in meeting toxicity discharge limits. Anaerobic treatment followed by an aerobic polishing stage has been suggested as a cheaper alternative to aerobic treatment. Anaerobic treatment is a sequential bacterial process in which inhibition of any one group of bacteria in the sequence will lead to process failure. Therefore, anaerobic systems are considered more sensitive to toxic substances than aerobic systems [28]. It is known that absence of free 02 is necessary for stable anaerobic treatment. It is now widely accepted that even chemically bound oxygen in the wastewaters interferes with the anaerobic process. Thus, peroxide, sulphate and sulphite, found in mechanical pulping effluent streams, can inhibit anaerobiosis. Influence of hydrogen peroxide on anaerobic treatment was investigated by We-lander and Hansson [58]. On a lab scale they found that the influent peroxide concentration could be increased sevenfold when a small pretreatment stage was installed. More work has also been done by Andersson et. al [l] on its effect on CTMP effluent treatment. The role of sulphate and sulphite is to divert electrons from methanogenesis, possibly leading to lowered methane production. Sulphite and sulphate are present in many pulp and paper industry wastewaters. Sulphur-reducing bacteria utilize sulphate as an electron acceptor and reduce it to hydrogen sulphide. It is presumed that the sulphate reducers may also utilize sulphite. The bacterially mediated reduction of sulphate can be represented by the following equations: 2CHsCHOHCOONa + H2S04 -> 2CH3COONa + H2S + 2COz + 2H20 (2.1) 4H2 + H2S04 -+ H2S + 4H20 (2.2) Since the sulphate reducing bacteria ultilize hydrogen and acetate (important 7 methane precursors), they may interfere with methanogenesis. Another effect is the production of H2S which inhibits acetoclastic methanogenesis [27,12]. Wood extractives are known to be toxic to fish but the effect of wood extractives on anaerobic bacteria is not known. 2.3 Importance of Assay Tests Anaerobic treatment efficiency is based, among other things, on the ability of microorganisms to remove matter that exerts oxygen demand, and on the removal of materials toxic to aquatic life from the wastewater. Prior to any extensive treatment studies, it is essential to assess the impact of the components of the wastewater on the anaerobic microorganisms. The cause of anaerobic system failures has been difficult to assess because of the complex mixtures being treated and because of the interactions between various groups of microorganisms which contribute to the overall process. Other difficulties include analysis for the great variety of potential inhibitors (many of which are not yet identified), and the lack of understanding of the interactions between inhibitors, other constituents in the digesting mixture, and the methanogenic bacteria. It has also been difficult to distinguish between failures due to toxic materials and those due to improper design or operation. Therefore, a simple, effective technique to assess the toxicity and the degradability of various components of the wastewater would be useful. Anaerobic bioassay techniques for measuring the presence or absence of in-hibitory substances offer the most promise for resolving anaerobic treatment prob-lems because they are relatively simple and inexpensive, and, also do not require knowledge of what the specific inhibitor substances are nor their concentrations. Also, bioassay techniques are essential for determining biodegradability since no chemical procedure is available which distinguishes between biodegradable and non-biodegradable organics. 2.4 Critique of Present Assay Techniques Anaerobic bioassays are normally carried out using the serum-bottle technique which was described by Miller and Wolin [40] and further detailed by Daniels and Zeikus [9]; Owen et. al [42]; Benjamin tt. al [4]; Fedorak and Hrudey [17,18]; and Shelton and Tiedje [49]. In this general technique, serum bottles are filled with a microbial culture, a growth medium, and the substance to be tested. The head-8 space in the bottle is filled with a mixture of JV2 and C02 gas. The biogas produced is measured either by collection in a syringe (attached to a needle which is inserted through the rubber stopper and into the head-space [40,34]) or by measuring the pressure exerted by the excess gas using a pressure transducer [49,59,19]. These techniques are modifications of the original technique as described by Hungate [24] for the cultivation of anaerobic bacteria. Some of the problems of these methods include: • the complex and laborious method of anaerobic culture transfer. • inadvertent gas leakage through the bottle cover due to excessive pressure. • difficulty in detecting gas leakages. • contamination of head-space gas by atmospheric oxygen. • decrease in accuracy of gas volume measurement when low gas production is encountered. • the expenses involved in providing pressure transducers for each bottle. • manual dexterity and training needed. • unwieldy and top-heavy systems with dead volume spaces. 2.5 Anaerobic Toxicity Assay (ATA) This is a batch procedure intended to determine the toxicity of a substrate to methanogens and/or hydrogen-forming bacteria. It can also determine the concen-tration at which a compound becomes toxic and the length of time required for acclimation. This is accomplished by feeding easily degradable substrates such as acetate/propionate solutions with various concentrations of the potential toxicant, to an active anaerobic microbial culture. Toxicity is indicated by a decreased rate of gas production relative to an active control in which tap water has replaced the toxicant solution. The presence of inhibition can be expressed by means of a Maximum Rate Ra-tio (MRR), measured during the same time interval, where: . sample gas production MRR -: ;— control gas production 9 Generally, an MRR of less than 0.95 suggests possible inhibition. A value of 0.90 suggests significant inhibition. Interference with the methanogenic bacterial metabolism can be manifested in a number of ways: 1. If a compound is severely toxic, it may kill all the organisms responsible for a step in methanogenesis, hence no gas production. 2. In a less extreme case the compound may inhibit metabolism resulting in lower levels of gas production. 3. Bacterial activity may continue at a reduced rate if only partial inhibition occurs. The compound may be detoxified either by the culture itself or by chemical means (e.g. sorption). 4. Some compounds can serve as substrates themselves, resulting in a larger cumulative gas production than that of the control. This may or may not proceed with a lag time for acclimation to the substance. 5. Finally, fermentation of the acetate/propionate spike may proceed without any effects from the test compound. 2.6 Biochemical Methane Potential (BMP) The BMP of a wastewater is determined in much the same way as the ATA but the assay lacks an easily degradable substrate such as acetate. Therefore, any methane produced originates from the degradation of the carbonaceous components of the wastewater. In short, it is a direct measure of a mixed microbial culture's (methanogenic and non-methanogenic organisms) ability to utilize the wastewater as a sole source of carbon for the ultimate production of methane (Benjamin et. al [4]). Therefore, BMP may be considered the anaerobic analogue of BOD. The BMP can be quantitatively referenced to • sample volume, as m3 CEU/m3 sample • sample mass, as m3 CH^kg sample • sample organic content, as m3 CEU/kg COD The last method is preferred as it permits direct transfer of results into percent organic matter converted to methane using the theoretical 0.35 m3 CH4 at STP produced per kilogram COD converted (McCarty [35]). 10 Chapter 3 Methodology 3.1 Overview Section 2 describes the assay bottles, their specifications and design criteria. Section 3 presents the source of inocula its characteristics, storage and use. Section 4 outlines the Mineral Nutrient Supplement provided to the microorgan-isms for optimum growth conditions. Section 5 describes the growth medium (substrate) composition, the choice of substrate and dosing levels. Section 6 presents the substances tested in both the ATA and BMP tests along with the control experiments. Section 7 explains the start-up procedure in detail and demonstrates the ease in setting up this technique. Section 8 presents a summary of the incubation times and sampling frequencies used to collect the data for various tests. Section 9 summarizes the analysis program, various tests carried out and the measurements taken. 3.2 Assay Bottles The BMP and ATA tests were performed in 250 ml Erlenmeyer flasks. The flasks were modified slightly by blowing a small side tube (1.75 cm x 0.6 cm ID) on the wall of each flask near its base. The mouth of this side-tube was sealed with a 11 Figure 3.1: Schematic Diagram of the Culture Flask sleeved GC septum (Wheaton, 7 x 13 mm natural rubber). This system enabled easy sampling of the gases produced. The flask's mouth was sealed using a one inch thick rubber stopper (Neoprene No. 6) that had a narrow inch ID), 10 inch long, rigid plastic tube inserted in it. One end of this tube was halfway into the flask while the other end was attached to a 5 ml disposable serological pipet with interval markings of 0.1 ml. The tube was rigid enough to stand upright in the flask but flexible enough to bend and be attached to the pipet (see Figure 3.1). The flasks were placed upside-down in specially constructed racks made of | inch thick plywood. Each rack held 6 flasks supported at their necks, the pipets being held vertically along a support on one side of each rack with the tubing attached to the tip of each pipet. These pipets measured the volume of gases produced by displacing equivalent volumes of liquid from the flasks. 3.3 Source of Inocula Fresh sludge from a 15 to 30 day retention time, primary, anaerobic digester with an organic content (TS) of 1 to 2% was obtained from the pilot plant, operated by Environmental Engineering UBC, at the B.C. Research site. This sludge originated from treatment of domestic sewage from a residential community. The sludge was filtered through one layer of cheese cloth and used the same day. When this was not 12 possible, it was stored at 4°C and allowed to warm up to room temperature before use. Lettinga and Stellema [32] have shown that well adapted anaerobic sludge can be stored, unfed, for about 8 months without any significant deterioration in activity by storing it below 15°C. The seed sludge was diluted to 10% (V/V) with the nutrient solution and used for the assays. 3.4 Mineral Nutrient Supplement Shelton and Tiedje [49] concluded that there is no strong basis for selecting a particular anaerobic medium and felt that 10% sludge would likely supply all min-eral and metal nutrients with the possible exception of potassium, ammonium and cobalt. They did, however, suggest a more complete medium as insurance. The re-vised anaerobic mineral medium (RAMM) consists of (per litre): Phosphate buffer, adjusted to pH 7.0 — 0.27 g KH2POA 0.35 g K2HP04 Mineral salts — 0.53 g NH4Cl 75 mg CaCl2 • 2H20 100 mg MgCl2 • 6H20 20 mg FeCl2 • 4H20 Trace metals, modified from Zehnder and Wuhrman [60] — 0.5 mg MnCl2 •4H20 0.05 mg H3BOs 0.05 mg ZnCl2 0.03 mg CuCl2 0.01 mg NaMo4 • 2H20 0.5 mg CoCl2 • 6H20 0.05 mg NiCl2 • 6H20 0.05 mg Na2Se03 For pH adjustment — 1.29 g NaHC03 The nutrient solution was prepared in batches of 20 litres. Tap water was boiled for 10 minutes to drive off the air and then the RAMM salts, except the sodium bicarbonate were added. The solution was cooled down to room temperature while sparging with nitrogen gas. On cooling, the NaHC03 was added and the pH was found to be at 7.0 ± 0.2. The oxygen concentration was measured using a D.O. 13 probe (YSI model 54ARC) and was found to be around 0.1 mg/L. 3.5 Growth Medium (Substrate) Composition This growth medium was needed only for the ATA test, where the effect of various toxicants on the ability of the microbial culture to convert an easily degradable substrate was observed. As a result of separate studies conducted by Jeris and McCarty [25], Smith and Mah [50], and Cappenberg and Prins [5], it was reported that approximately 70% of the methane generated anaerobically from sewage sludge was derived from acetate. Therefore, acetate was used as one of the substrates. The other substrate, propionate, was used to assess the impact upon both the hydrogen forming acetogens and the hydrogen utilizing methanogenic bacteria. To each bottle, 1 ml of solution containing 150 mg sodium acetate and 53 mg sodium propionate was added. The theoretical COD of this mixture was calculated to be 0.32 g/L. Owen et. al [42] recommended that the estimated degradable COD not exceed 2 g/L in the assay liquid. This is to avoid producing excessive amounts of methane gas since the theoretical maximum yield of methane (in the absence of any net growth in biomass) is 0.35 L CH^/g COD removed at STP. 3.6 Substances Tested 3.6.1 Anaerobic Toxicity Assay (ATA) The toxicity of the following compounds to the anaerobic culture was tested. • Sodium Sulphite • Sodium Sulphate • Sodium Sulphide • Abietic Acid Sodium Sulphite TMP pulp is sometimes brightened using the Borol sodium hydrosulphite process which leaves some residual sulphite in the effluent. Sodium sulphite is found at concentrations of about 200 mg/L in TMP effluents [3]. Sulphite has been found to be inhibitory to anaerobic bacteria [39]. Also, for every mole of sulphite reduced, 14 0.75 mole of potential methane is lost since organic COD is converted to insoluble COD in the form of H2S. Since H2S is more soluble than methane, more COD remains dissolved in solution and therefore COD removal is decreased [12]. Sodium sulphite liquor is also used in the chip steaming and impregnation stage during CTMP production. It has been found in the CTMP effluents at concentration of about 300 mg/L [3]. Sulphite being an oxidizing agent, is toxic to anaerobic bacteria. The toxicity of sulphite was tested at concentrations of 100, 200, 500, and 1000 mg/L. Sodium Sulphate Since sulphate is an oxidising agent, it is expected to be toxic-to anearobic bacteria. Sulphate at concentrations of 100, 200, 500, and 1000 mg/L was added to test flasks to verify this effect. Sodium Sulphide Sulphide is not used in either CTMP or TMP production, nevertheless, its effect on anaerobic bacteria could be important since sulphide was found to be the product of sulfur reduction in anaerobic systems [12], The ATA of the sodium salt was carried out for concentrations ranging from 0.1 to 1.0 g/L. Abietic Acid (a resin acid) Resin acids, found in significant quantities in softwood species, are soluble under most pulping conditions and have been shown to be toxic to a variety of fish [29,55]. Abietic acid is one of the major resin acids and has been reported to be present in raw CTMP and TMP effluent from Quesnel River Pulp at mean concentrations of 8.86 mg/L and 4.21 mg/L respectively [48]. In the present study, the toxicity of abietic acid was tested at concentrations between 0.5 mg/L and 16 mg/L. The abietic acid sample was obtained from Sigma Chemical Supply Co. 3.6.2 Biological Methane Potential (BMP) The degradability of the following effluents was studied: 15 Parameter Units TMP CTMP 1. Total Flow m 3/d 8000 10100 2. BOD 6 mg/L 1550 2929 3. COD mg/L 4300 8180 4. Hydrogen Peroxide mg/L — 50-100 5. Sulphite-S mg/L 200 300 6. Temperature 0 C 35 35 7. pH — 6 8 Table 3.1: Characteristics of Effluent from Quesnel River Pulp Ltd. • CTMP effluent • TMP effluent The samples were obtained from Quesnel River Pulp Ltd's TMP/CTMP pulp mill located at Quesnel, BC. The mill nominally produces 500 ADtPD of CTMP and TMP pulp on a bi-weekly basis. Table 3.1 summarizes some of the relevant wastewater characteristics [13]. 3.6.3 Control Experiments For ATA A group of controls was set up with the acetate-propionate mix in nutrient solution with 10% (V/V) seed sludge to compare the gas production with that of the samples that had the toxic materials added to them. This information was used to calculate the MRR. A second set of samples was set up to assess the effect of the nutrient solution by using the seed culture and acetate/propionate spike in tap water with no added nutrient solution. For B M P No controls were necessary for the BMP test as it is a measure of the anaerobic microbial culture's ability to utilize the wastewater as a sole carbon source for methane production. However, the BMP of the seed sludge alone was determined by setting up a control that had the seed in tap water (as in the ATA test above). 16 3.6.4 Hydrogen Peroxide Degradation in C T M P Effluent In some mills, including Quesnel River Pulp, CTMP effluent is bleached using a MoDo-Chemetics medium consistency hydrogen peroxide system which results in a discharged effluent containing between 50 to 100 mg/L of H2Oi [3]. As peroxide is a powerful oxidizing agent which releases 0 2 gas (inhibitory to anaerobes), it is necessary to assess how fast it is naturally degraded in CTMP effluent. Therefore, peroxide was added to CTMP effluent and the concentration remaining in solution was measured every half hour. Controls were set up to see the degradation of H2O2 in tap water instead of CTMP effluent. 3.7 Start-Up Procedure 3.7.1 A T A The modified Erlenmeyer flasks were labelled and rubber septa fitted across the side-tubes. These septa were replaced with new septa for every run. The flasks were partially filled with the nutrient solution and 30 ml of seed sludge was added to each flask in order to achieve an ultimate 10% dilution with the nutrient solution. The flasks were then filled to the brim with more nutrient solution, and then immediately stoppered firmly the excess solution being forced to rise in the plastic tube. To ensure that no air bubbles were trapped in the side-arms of the flasks, they were inverted and checked. The flasks were then gently shaken and placed upside-down in the specially made racks. The pipets attached to the plastic tubes were secured vertically. The acetate-propionate spike was injected into each flask and the flasks were placed in a constant temperature and humidity room maintained at 22° C and 50% humidity. They were allowed to equilibrate for one hour, followed by injection of appropriate test material into each flask through the septum. The water-level was then noted on the pipet; this was the initial reading. 3.7.2 B M P Samples of TMP and CTMP effluent were collected in 5 gallon carboys and sent by courier to UBC. The samples were stored at 4°C while not in use. When needed, about 2 L of each well mixed sample was transferred to a smaller jar and N2 gas was bubbled through while the samples returned to room temperature. Similarly, N2 gas was bubbled through about 2 L of tap water to be used for dilution of the 17 effluents. The dissolved oxygen levels were measured after gassing and were found to be less than 0.5 mg/L for the TMP and CTMP effluent, and less than 0.2 mg/L for the tap water. Some effluent was placed in each flask, the seed sludge added, and then the flasks were topped up with either more effluent or the dilution water depending on what concentration was desired. The flasks were then sealed, placed in racks, allowed to equilibrate, and the initial readings noted. See Table 3.2 for a summary of the testing strategy. 3.7.3 Peroxide Degradation A series of flasks was set up with 150 ml of CTMP effluent (or 150 ml water for controls). The peroxide solutions of various concentrations were added to all and a set of samples was analyzed for peroxide content every half hour. The peroxide was added at concentrations of 50 and 100 mg/L. 3.8 Incubation Times and Sampling Frequencies All the samples were run either in duplicate or in triplicate and each set was incubated for at least 4 weeks. Gas production was monitored volumetrically by the change in water level in the pipets. When the water level rose above the graduated portion of the pipet, the pipet was drained back into the flask by wasting gas via the side-tube in the flask. The gas volumes were measured on a daily basis. Some gas was withdrawn and analyzed on a G.C. once a week for the composition. COD analysis was also performed once a week. Table 3.2 and Table 3.3 show the various tests conducted while Table 3.4 explains the sampling frequencies. COD analysis was carried out every 3 to 4 days for one set each of ATA and BMP samples in order to evaluate the changes in COD over the period of a month. Resin acid analysis was carried out every 4 days by setting up a series of assay flasks with the same concentrations of resin acids and using a sample on every 4th day for GC analysis. The peroxide analysis was done every half hour after start-up and periodically thereafter for up to 3 hours because beyond that time it was found that there was no more peroxide remaining in the control set. 18 Test Material Concentration Total No. of Samples No. of Sets 1. Resin Acids 0.5 mg/L 5 + (2 x 9)* 3 1.0 mg/L 5 + (2 x 9) 3 2.0 mg/L 5 + (2 x 9) 3 4.0 mg/L 5 + (2 x 9) 3 8.0 mg/L 5 + (2 x 9) 3 12.0 mg/L 5 + (2 x 9) 3 16.0 mg/L 5 + (2 x 9) - 3 2. Sodium Sulphide 0.1 g/L 3 1 0.2 g/L 3 1 0.5 g/L 3 1 1.0 g/L 3 1 3. Sodium Sulphite 0.1 g/L 5 + (2 x 9) 3 0.2 g/L 5 + (2 x 9) 3 0.5 g/L 5 + (2 x 9) 3 1.0 g/L 5 + (2 x 9) 3 4. Sodium Sulphate 0.1 g/L 2 1 0.2 g/L 2 1 0.5 g/L 2 1 1.0 g/L 2 1 5. Control 1. Nutrient Solution — 5 + (2 x 9) 4 and Substrate 6. Control 2. Distilled Water — 5 + (2 x 9) 4 and Substrate (No Nutrients) *For soluble COD analyses. Table 3.2: Table of Tests Conducted For ATA 19 Effluent Concentration No. of Samples No. of Runs 1. TMP 100% 7 + (2 x 9)* 4 50 7 + (2 x 9) 4 25 7 + (2 x 9) 4 10 7 + (2 x 9) 4 2. CTMP 100% 7 + (2 x 9) 4 50 7 + (2 x 9) 4 25 7 + (2 x 9) 4 10 7 + (2 x 9) 4 3. Controls for TMP/ (2 x 9) 1 CTMP Using Substrate — (2 x 9) 1 (2 x 9) 1 (2 x 9) 1 4. H202 Degradation 50 mg/L (2 x 6) 1 in CTMP Effluent 100 mg/L (2 x 6) 1 5. H202 Degradation 50 mg/L (2 x 6) 1 in Tap Water 100 mg/L (2 x 6) 1 *For soluble COD analyses. Table 3.3: Table of Tests Conducted For Degradability Studies Test Sampling/Testing Frequency 1. Gas Volume Measurement 2. Gas Composition 3. Substrate Analysis (COD) 4. Resin Acid Analysis 5. Hydrogen Peroxide Analysis Daily Weekly Every 3 to 4 days* Every 10 days Intervals of \ hr. up to 3 hrs. * Carried out for a limited number of runs. Table 3.4: Table of Sampling Frequencies 20 3.9 Analysis Program 3.9.1 Gas Volume Measurement Gas volume was measured by displacing water upwards in the pipets. The pipets had a total capacity of 5 ml and changes in volumes could be measured to within 0.025 ml, thereby allowing small amounts of gas production to be monitored quite accurately. The volume of gas produced corresponded to the change in volume of liquid in the pipet. The volume measured was slightly less than the actual volume for various reasons: a) the head of the water rising in the pipet; b) the gases produced were soluble in water leading to lower readings, and c) evaporation of liquid from the pipets. All these factors were not important because corrections could be made for head effect and, the absolute gas measurements were not critical. It was only the ratio of total gas produced by the tests as compared with those of the controls that was important in this study. In order to check the effectiveness of this gas measuring system, various mea-sured amounts of the gas mix, withdrawn from another flask, were injected into a flask and the resulting changes in volume noted. The gas volumes registered were within ±5% of the amounts injected. 3.9.2 Gas Composition Analysis Gas was extracted for chromatographic analysis using a gas-tight, lockable needle and syringe system (Supelco Pressure-Lok Gas Syringe). The needle was inserted through the septum and when the tip of the needle was inside the gas bubble, some of the gas was withdrawn into the syringe. The septa seal hermetically so that gases cannot escape across them. Samples of gas (1 ml) were analyzed by a Varian Vista 6000 Gas Chromatograph using a 2 m long Porapak column. The flow rate of argon carrier gas was 25 ml/min. The GC was programmed for 2 minutes at 50°C followed by raising the temperature at a rate of 20°C/min up to 135°C where it was held for 1.5 min. The GC was equipped with both a flame ionization detector and a thermal conductivity detector. Sulfur compounds were analyzed on a Varian 6500 GC with a FPD detector and a Carbopack B-HT-100 column. The operation was isothermal at 50°C and the flow rate of argon was 30 ml/min. 21 3.9.3 Substrate Analysis Prior to start-up and later, at intervals of 3 to 4 days, the contents of the ATA and BMP flasks were analyzed for soluble COD. Analysis was carried out according to the procedures outlined in "The Standard Methods for the Examination of Water and Wastewater" [52]. COD is a meausure of the amount of oxygen required to oxidize organic and oxidizable inorganic compounds in water. 3.9.4 Resin Acid Analysis The concentration of resin acids in solution was determined every 4 days. Knowing the amount of resin acids added initially, and the final concentrations, the amount utilized or degraded could be determined. The samples were filtered through Whatman No. 4 filter paper and 100 ml aliquots of the samples were extracted into 1 ml of methyl-tert-butyl ether (MTBE) and methylated before being analyzed on a GC. The extraction and methylation procedures were adapted from Schlenk et. al, Saltsman et. al, and McMahon [47,46,38] and obtained from unpublished work at the PAPRICAN Vancouver lab-oratory [43]. A Hewlett Packard 5890A gas chromatograph with a FID detector and split ratio of 48:1 was used for the resin acid analysis using a 2 m, DB-1 (J & H Scientific Co.) 0.25 mm ID capillary column. The initial temperature was 140°C for 3 minutes; then it was increased at a rate of 5°C/min to 160°C and further increased to 350°C at 10°C/min where it was held for 6 minutes. The carrier gas (nitrogen) flow rate was 30 ml/min while that of the hydrogen was 1.8 ml/min. 3.9.5 Hydrogen Peroxide Analysis The amount of H202 remaining in solution at various times after addition was determined by titrating liberated iodine with thiosulphate. The iodine is released after reaction of H202 with potassium iodide. The procedure followed was as outlined in Vogel's Textbook of Quantitative Inorganic Analysis (4th edition) [54]. 22 Chapter 4 Results and Discussion 4.1 Overview Section 2 Commentary on Assay Technique Section 3 Degradability Studies • CTMP effluent • TMP effluent • H 2 0 2 Section 4 Toxicity Studies • sulphite • sulphide • sulphate • resin acids 4.2 Commentary on the Assay Technique 4.2.1 Design of Assay Flask Simple Start-Up Procedure In the techniques used previously, addition of oxygen free gas to the head-space made the start-up procedure laborious and complex [40,4,9,42,49]. In this tech-nique, the flasks were completely filled with the liquid medium. This eliminated the need for 0% free head-space gas. Nevertheless, some oxygen (from dissolved 23 air) was present in the liquid. Bubbling nitrogen through the solution prior to fill-ing the flasks ensured adequate stripping of oxygen. It is acknowledged that some dissolved oxygen might remain in solution even after gassing with nitrogen for 1 to 2 hours, but this 0% is present in very low concentrations (less than 0.5 mg/L) if at all, and normally gets absorbed by the sludge. While the other techniques might provide absolutely 02-free environments for the anaerobic culture, because of its simplicity, this technique has been found to be adequate for anaerobic assays of industrial effluents which do contain some dissolved oxygen. Efficient Gas Volume Measurement The most important parameter measured for the ATA and BMP tests is the volume of gas produced. While using a pressure transducer for gas volume measurement gives good accuracy in pressure changes, errors can result while converting pres-sure changes to changes in volume. Besides, providing pressure transducers with the associated plotters and other paraphernalia for each bottle can be very expen-sive. The pressure build-up can lead to gas leakages. The syringe method is more economical, but it is prone to gas leakages too. Gas volumes are measured by the movement of the pistons in the syringes and thus, the pressure in the bottles has to be sufficiently high to move the piston and register a change in volume. Sensitivity to small volume changes is low, this is especially true for initial measurements of gas volume. The small amounts of gas produced initially at the start of the experi-ment build up pressure in the flasks but no change in volume is registered. On the other hand, when large amounts of gas are produced, leakages can occur due to excessive pressure. It is difficult to detect leaks and erroneous readings can result. All these problems are eliminated by the present technique. Since the gas pro-duced displaces a similar volume of liquid, volume of gas produced is measured directly. Small or large volumes of gas produced can be measured accurately by attaching pipets of the desired sizes. This allows for greater precision in measure-ment irrespective of the variations in volume produced. The precision of volume measurement can be controlled simply by attaching pipets of various sizes. Gas leakages do not occur because the gas collects at the top of the upturned flasks from where gas cannot escape. Also, if any gas leaks out via the septum, it is detected by a drop in water level in the pipet, there are no indicators of gas leakage in the other techniques. Finally, the gas measuring system (the pipet) is cheap. Other Design Features • The tube leading from the flask to the pipet was inserted half way into the 24 flask (see Figure 3.1) so that the liquid displaced did not contain any sus-pended material which could block the narrow tube. This ensured no loss of sludge. Also, if any oxygen from the atmosphere was being transferred into the flask it would be through the narrow tube and would enter half-way up the flask. Thus, the oxygen would take a longer time in reaching the bacteria at the bottom of the setup. • The side-tube for injecting samples and removing gas was angled such that it allowed easy access to the gas bubble formed and allowed collection of up to 30 ml of gas in the flask before the gas was in contact with the septum. This eliminated gas leakages via the septa. • The septa were sleeved GC septa that were easy to slip on and they sealed hermetically so that no gas could leak out from the needle punctures. If any gas had leaked out, a fall in water level in the pipet would have been observed. • The experiment was conducted in a constant temperature and humidity room so that there were no fluctuations which could have altered the volume of gas produced, or the rate of evaporation of water from the pipets. The rate of evaporation was minimal since the pipets had narrow bores and any evaporation was cancelled out as it was the same for the controls as well. The water was found to evaporate at the rate of 0.025 ml per 2 to 3 weeks. • The use of conical flasks allowed the bacteria to settle to the bottom of the apparatus. This prevented the loss of bacteria via the manometer type tube and ensured a constant SRT. 4.2.2 Sludge Characteristics The same sludge was used for sets 1, 2 and 3 and it was stored at low temperatures while not in use. The sludge used for set 4 was obtained at a later date and hence the results were different. This confirms Lettinga's finding [32] that storage does not have a significant effect on sludge characteristics. A variation in sludge can result in different results as seen from set 4. This is in agreement with the results of Shelton and Tiedje [49]. They found a wide range of treatment efficiencies when different sources of sludge were tested for the same substrate. Along with each set, control experiments (containing the sludge in tap water) were conducted to measure the gas potential of the sludge alone. The total gas 25 collected was between 0.15 to 0.7 ml of which about 28% was methane (see Fig-ure 4.1). The rest of the gas was nitrogen which had been stripped from solution. The gas potential of the seed sludge was negligible, such that correction for gas potential of the seed when evaluating gas potentials of CTMP and TMP effluents was not necessary (see Figure 4.2). The gas volumes did not add up to 100% in some cases because the GC was not calibrated over a wide-range of nitrogen content of the gas. Also some drift in the detector calibration resulted in such errors. 4.2.3 Nutrient Addition and Substrate Effects Mineral nutrients were added to the sewage sludge in order to study the effect of nutrient addition on total gas production. The total gas produced by controls with seed and substrate in tap water was compared with controls that had seed sludge and substrate in nutrient solution. From Figure 4.3 and Figure 4.4 no appreciable difference either in the rate or the total gas produced was noticed. Thus, nutrient supplementation did not increase gas production significantly. The substrate used (acetate and propionate) is soluble and easily degradable by anaerobic microorganisms and, therefore, it is an ideal food source for anaerobes. Some tests were conducted where the substrate and seed sludge were added to tap water along with other tests where the substrate and seed were added to nutrient solution. The total gas produced was almost the same, (see Figure 4.3 and Figure 4.4) and production of 50% of the gas took about 15 to 20 days. The gas was composed of 80% methane, about 10% nitrogen and about 5% C0 2 , see Figure 4.5 and Figure 4.6. Thus, the substrate was utilized slowly, this allows for observation of experiments over a long period of time. The high percentage of methane confirms methanogenic activity removing the acetate and propionate from the solution. 26 0.7 0.6 0.5 8 0.4 3 o l_ 0l <n 0.3 o o 4) > O 0.2 E 8 o.H 0.0 -0.1 X A - r -10 / // '/ i 'I 15 —I— 20 Oays — i — 25 —I— 30 35 Legend A Set 1: A X Set 1: 8 Legend A Set 2: A X Set 2: B_ O Set 2: C Figure 4.2: Cumulative Gas Produced by Control Set: Seed Sludge in Tap Water 28 25 20 ~u a> o 3 15 x> O in a o <a Z 10 ~o 3 3 O it' X ' / / I / r X 10 15 Days 20 — i — 25 —I— 30 35 Legend A Set 3 : A X Set 3 :6 25 20 a> o 3 15 "O O in O O O .£ 10 J} E o 5H o-HA-o / / // / < // / / 10 15 20 Days 25 — i — 30 Legend A Set 2 : A X Set 2 : B 35 Figure 4.3: Cumulative Gas Produced by Control Set: Substrate and Seed Sludge in Tap Water 29 (%) UOJIJSOdUJOQ SDQ Figure 4.5: Gas Composition of Control Set: Substrate and Seed Sludge in Tap Water 31 (%) uoi j i sodaioQ SD0 i Figure 4.6: Gas Composition of Control Set: Substrate and Seed Sludge in Nutrient Solution 32 4.3 Degradability Studies The study on degradability of CTMP and TMP effluents was carried out in four separate experiments (sets) with each sample being in duplicate or triplicate, see Table 3.3. In each set, samples of dilutions at 10%, 25%, 50%, and 100% (V/V) effluent were included along with two sets of controls. The effluent was diluted in order to observe changes in the patterns of degradability and toxicity. To establish a relationship between toxicity and concentration of the effluent and to determine whether the degradability was a linear function of concentration, these dilution experiments were carried out. One set of controls had the blanks for biochemical methane potential (BMP) determination. These controls contained the seed sludge (in tap water) for the determination of BMP of the seed sludge alone. The BMP of a given sample was calculated as follows: BMP of sample = total volume of gas produced by sample — total volume of gas produced by seed sludge The other control set was used to evaluate the MRR of the effluents. These contained the seed sludge and a easily degradable substrate — acetate and pro-pionate, in nutrient solution. The MRR compares the total gas produced by the sample with that by the control. 4.3,1 C T M P Effluent Cumulative Gas Production The values of cumulative gas produced were found to be comparable at any given concentration within a given set but, they were highly variable between sets, see Figure 4.7, Figure 4.8 and Figure 4.9. The effluent sample was divided in three parts. One part was used for set 1 and the rest was stored at 4°C and eventually used for sets 2 and 4 where set 2 was a duplicate of set 1 and set 4 was used to asses the effect of lower seed sludge concentrations. The sample for set 3 was obtained at a later date but unfortunately it had been left at room temperature for about 5 days during transit and it had started degrading aerobically. This was evident from a very high initial COD count of around 8000 mg/L as compared with 2500 mg/L for the other three sets. The graphs of cumulative gas produced over time show that dilution of the CTMP effluent by 50 to 75% gives maximum COD removal in the form of evolved gases. This is better illustrated in Table 4.1, which shows the total gas produced at various concentrations of CTMP effluent. The long storage period may have caused hydrolysis of suspended COD in set 4, resulting in a larger 33 Figure 4.7: Cumulative Gas Produced (CTMP Effluent): Set 1. 34 -t— CO o "O o Q_ GO o O > 15 3 3 o 2H 0 10 5^ 0 L - A : 15 10 5H 0 10 51 T 5 10 T " 15 20 Days A y X 'A y y X / y> 25 30 100% CTMP 50% CTMP 25% CTMP 10% CTMP Figure 4.8: Cumulative Gas Produced (CTMP Effluent): Set 2. 35 100 100% CTMP 50% CTMP 25% CTMP 10% CTMP 0 10 20 30 40 Days Figure 4.9: Cumulative Gas Produced (CTMP Effluent): Set 3. 36 Sample 100% CTMP 50% CTMP 25% CTMP 10% CTMP Set 1: A 8.65 23.65 26.08 17.70 Set 1: B 8.48 28.85 28.85 15.45 Set 2: A 3.75 6.80 4.22 4.15 Set 2: B 3.22 7.82 10.67 5.25 Table 4.1: Table of Cumulative Gas Produced (CTMP Effluent) (ml) Sample 100% CTMP 50% CTMP 25% CTMP 10% CTMP Set 1 0.31 0.96 1.00 0.60 Set 2 0.47 0.98 1.00 0.63 Table 4.2: Cumulative Gas Produced For CTMP Effluent Expressed as a Fraction of the Maximum Produced. total gas volume produced. Thus, when the CTMP effluent was diluted to between 50% and 25% with water, the total gas produced was at an optimum, the maximum being at 25%. When the effluent was diluted to 10%, the gas produced was about 60% of the total. The 100% undiluted effluent produced on average only about 40% of the maximum possible gas. This is better illustrated in Table 4.2 where the total gas collected for each dilution of effluent is expressed as a fraction of the maximum gas produced in that set. The low gas production observed at 10% dilution of the effluent can be at-tributed to low concentrations of substrate present in the dilute waters. On the other hand,the undiluted effluent's low gas production was probably due to toxic effects. The effluent probably contained high concentrations of substances toxic to anaerobic bacteria, and when the effluent was diluted, the proportional dilution of the inhibitory substance rendered the gas production more efficient. In order to compare the cumulative gases produced in the various experiments, the total gas collected up until any given day was expressed as a function of the total gas produced by day 30, see Figure 4.10 and Figure 4.11. That is, the data was normalized. This information can be used for estimation of the percent gas evolved over the given time period. The observations show that 50% of the total gas evolved by day 30 required about 20 to 24 days of treatment. 37 1.5 Q) N O CO <D O Z5 O 1-0 . 5 -0-1 1.5 1-0 .5 -0 J 1.5 1-00 O O 0.5 H _D ZJ E O o-J 1.5 0.5 0 --0 .5 A-0 10 20 Days -A i 30 100% CTMP 50% CTMP 25% CTMP 10% CTMP 40 Figure 4.10: Normalized Cumulative Gas Produced (CTMP Effluent): Set 1. 38 1.5-i H 0.5H 0 1.5 0.5 0 1.5 -A== H v— 0.5 o-L-A= 1.5 H 0.5 0-r 0 .A: T 5 - T -10 15 Days 20 100% CTMP 50% CTMP A / 25% CTMP 10% CTMP 25 30 Figure 4.11: Normalized Cumulative Gas Produced (CTMP Effluent): Set 2. 39 Sample 100% CTMP 50% CTMP 25% CTMP 10% CTMP Set 1 A Set 1 B 0.0181 0.0176 0.1211 0.1336 0.1618 0.1807 0.4370 0.3734 Set 2 A Set 2 B 0.0101 0.0086 0.0374 0.0432 0.0273 0.0713 0.1119 0.1431 Table 4.3: Gas Potential of CTMP Effluent (ml biogas/ml sample) Biochemical Methane Potential (BMP) The bio-gas potential is the total gas accumulated per millilitre of the undiluted sample minus the gas produced by the seed sludge. The gas produced by the seed sludge alone was negligible and therefore ignored (see Figure 4.13). Table 4.3 shows the BMP of the CTMP effluent. Thus, in fact, per unit volume of the original effluent, the 10% dilution had the maximum gas potential while the gas potentials at 50% and 25% dilution are comparable. This shows that the more dilute the effluent, the higher is its BMP. However, some obvious disadvantages of treatment at low concentrations are the large quantities of dilution waters needed, the consequent increase in reactor sizes and, low cumulative gas production within a reasonable time period (see Table 4.1 and Table 4.3). Rates of Gas Production The gas volumes, from the rise in water level in the pipets, were measured daily and this was plotted against time. The rates of gas production followed a distinctive cycle which was common to all the various dilutions of the effluent, see Figure 4.12 and Figure 4.13. Each cycle for the 100% and the 50% effluent lasted about 10 days where it took 9 days for the rate of gas production to reach its'peak followed by a drop in rate on the tenth day. For the 25% and 10% effluents, the cycle was completed every 5 days. Gas production is the final stage in anaerobiosis. The various processes, start-ing from the solubilization of suspended solids, are carried out by a consortium of microorganisms that work sequentially [28]. These processes are dependent on the rate of production, availability, and concentration of substrate from the preceding stage. When gas production falls rapidly, it could be due to an un-healthy environment for the methanogens. The acetogens produce volatile fatty 40 1 100% CTMP 50% CTMP 25% CTMP 10% CTMP i - j 1 1 1 i 0 10 20 30 40 Days Figure 4.12: Rate of Gas Production (CTMP Effluent): Set 1. 41 acids (VFAs) at a rate faster than the rate of removal of VFAs in the form of methane by methanogens. Therefore VFAs should build up, lower the pH and inhibit methanogenesis. It is hypothesised that some step prior to VFA formation may also be inhibited by high levels of VFAs or by the resultant drop in pH. This would stop the production of VFAs resulting in a stop in further pH drop, and thus allowing the methanogens (which were being inhibited by the lowering of pH) to slowly start increasing the rate of gas production. As the VFAs are being con-verted to gas their concentration depletes and the inhibition on the VFA precursors is lifted so VFA production can start up again. As the VFA concentration builds up, it inhibits methanogenesis as well as stops further accumulation of VFAs until gas production has removed the excess VFAs. Thus, this cycle keeps repeating itself. An implication of this cycle is that for optimal COD removal, the treatment would be more efficient if carried out in two reactors — one for the acedogenic phase and another for the methanogenic phase. The physical separation of the acedogenic phase which operates optimally at a lower pH than the methanogenic phase with its higher pH and longer HRT requirements would lead to more efficient treatment. Effect of Dilution of Inoculum A set of samples was seeded with 2% ( of the total flask volume ) seed sludge, as opposed to 10%, to assess the effect of low concentrations of inoculum. From Fig-ure 4.14 in comparison with Figures 4.7, 4.8 and 4.9 the cumulative gas production was observed to rise steadily and then level off. The initial rise in gas produced shows that the effluent was not toxic to the bacteria, if it were, the system would have needed some time for acclimation resulting in a more gradual rise in the cu-mulative gases evolved. On the other hand, the levelling off of the gas collected implies that no more gas was being produced. This could be because of increased concentrations of an inhibitory intermediate product or due to utilization of all the degradable substrate, or it could be a result of the characteristics of the particular seed sludge being used. The increase in concentration of an intermediate could be due to the differential rates of metabolism of the various types of bacteria that operate sequentially in the production of methane. As this waste is complex, the substances that are easily digestible are converted rapidly resulting in large initial gas production. Then, a lag period sets in when the rate limiting step is being overcome. Now, if the concentration of sludge were increased, it would mean that more microorganisms would be available to break down the same amount of sub-strate and keep the system producing gas on a more continuous basis. Thus, the 43 COD (mg/L) 100% CTMP 50% CTMP 25% CTMP 10% CTMP Initial COD 2480 1291 945 409 Maximum COD 8992 5445 4067 1622 Minimum COD 2480 1238 843 310 Table 4.4: Soluble COD of CTMP Effluent treatment efficiency is dependent on the concentration of seed sludge. Chemical Oxygen Demand (COD) Maximum gas production could be used to indicate the maximum COD removal since COD is converted to methane and C02. This was observed at concentrations between 50% and 25% of the CTMP effluent. The soluble COD (sCOD) values were determined at intervals of 3 days and plotted against time, see Figure 4.15. The sCOD depends on the total amount of solids solubilized by the bacteria so that they can ultimately be converted to methane and carbon dioxide. Thus the COD removal was estimated from the difference between the maximum sCOD and the minimum sCOD, see Table 4.4. The initial sCOD values did not coincide with the maximum sCOD since ini-tially not all the potentially soluble COD material was in the hydrolyzed form. Once bacterial action had hydrolyzed it, the concentration of soluble COD in-creased. Another reason for the increase in soluble COD is the dissolution of CH4 and C02 produced. While the methane remain in solution, they exert oxygen demand. From Figure 4.15, the maxima in soluble COD, irrespective of the concentra-tion of effluent were around day 6, ie, it took the bacteria 6 days to produce the maximum amount of soluble COD. This increase in soluble COD was also due to accumulation of sCOD because of the initial lag in conversion to methane and carbon dioxide (since methanogenesis is known to be a slow process) and, due to saturation of the water with CH4 and C02 formed. This high concentration of soluble COD continued for another 8 to 10 days and then the levels of sCOD dropped. It is interesting to note that the initial and the final sCOD values were almost the same. This could be because the COD being registered both initially and finally was not biodegradable. Some of the COD that was biodegradable was in the form of suspended solids (SS), so, after hydrolysis (which seemed to take about 6 days in this case), the soluble COD increased. Table 4.5 shows the COD 44 CO <D O 3 TD O s_ CL V) O o > • • -15 £ o 100% CTMP 50% CTMP 25% CTMP 10% CTMP Days Figure 4.14: Cumulative Gas Produced (CTMP Effluent) Seeded with 2% Sludge. 45 10000 5000 J I r 100% CTMP - A o 6000 4000-2000- J I 0 6000 Q O O _Q O 00 4000-2000 o i 2000 1000 A / 0 10 50% CTMP -A \ \ 25% CTMP -A 10% CTMP — A 20 30 Days Figure 4.15: Change in Soluble COD with time (CTMP Effluent). 46 Effluent COD removed (mg/L) Theoretical CH4 production (ml) Actual CH\ production (ml) 100% 6512 2.28 1.39 50% 4207 1.47 5.12 25% 3224 1.13 5.58 10% 1312 0.46 3.29 Table 4.5: Table of COD Removal Compared with the Theoretical and Actual Methane Production (CTMP Effluent). removal (COD maximum - COD minimum) and compares it with the actual and theoretical methane yield. The theoretical methane yield is-calculated from 0.35 L of methane produced per kg of COD removed at STP [35], The actual Cif 4 is determined by multiplying the volume of gas collected with the fraction of methane in the gas (from GC results). Thus, the actual methane yield is higher than that predicted from the amount of COD removed. This may be because of solubilization of substrate. Although some COD was being removed from solution, the solubilization process probably produced more soluble COD. These results show that gas measurement and its methane and CO2 content are a better indication of COD removal than the change in COD of the solution. This is true for effluents where COD from the insoluble state is quickly converted to sCOD. At 100% effluent concentration, the actual methane yield is lower than the theoretical. This could be because the substrate was not solubilized when at high concentration. This further proves that dilution of CTMP effluent results in better COD removal (as gas) than the whole effluent. Some soluble COD is also attributable to the seed, obtained by setting up a control that had seed sludge in tap water. The soluble COD for this control was found to be about 90 mg/L at its peak level on day 6. Maximum Rate Ratio (MRR) The MRR is the total gas produced by a test sample expressed as a fraction of the total gas produced by a control that had bacteria in a optimal environment. The MRR values were calculated to asses the toxicity of the CTMP effluent. Control 47 experiments containing the seed sludge under ideal growth conditions in terms of availability of nutrients and substrate were used to calculate the MRRs. Graphs of MRR against concentration are presented in Figure 4.16. The MRR values are low for the 10% CTMP, then a rise is observed for the 25% and 50% CTMP followed by a decline for the 100% CTMP effluent. The whole CTMP (100%) wastewater was found to be inhibitory to the anaerobic bacteria, but, as the effluent was diluted, the total gas production increased. In fact, where the MRR values have exceeded 1.0, more of the COD from the effluent had been converted to gas than that from the controls. The reason for this rise and subsequent fall in ratio of gas produced could be due to toxic effects and inhibition of the anaerobes at high concentrations of effluent. On the other hand, it could be because the effluent was too concentrated to allow sequential breakdown of complex substances to take place at a rate which main-tained a balance between the rates of formation and utilization of intermediates in the anaerobic pathway. Evidence attributing the lower gas formation to low degradability rather than to toxicity comes primarily from examination of the normalized cumulative gas pro-duction graphs, Figure 4.10 and Figure 4.11. In the graph for 100% CTMP effluent, an initial lag phase would have been observed due to inhibition and acclimation. Instead, the gas production rises steadily showing no toxic effects. Gas Composition From the histogram of gas composition versus concentration of CTMP effluent (see Figure 4.17, methane makes up 70% of the gas followed by between 5 to 15% C02 and between 10 to 20% N2. Trace quantities of H2, H2S and 02 have also been observed. The test effluent prior to start-up had been saturated with N2 gas. The N2 found in the head-space was that which had been stripped from the solution by the rising bubbles of CH4 and C02 formed. The percentage of methane seems fairly constant at 70% while that of C02 kept rising the more concentrated the effluent was. 4.3.2 T M P Effluent Cumulative Gas Production Toxic or inhibitory effects of the TMP effluent have been established from the rates of gas production. The cumulative gas produced reflects this, see Figure 4.18, 48 Figure 4.16: Maximum Rate Ratios (MRR) for CTMP Effluent. 49 <o 5 _ I "O >. i S: u -o I O Z 2 O I B I 0 Q B D V//////////, o o c <D o to V////////, I i 1 1 1 V o o o o o o o CT> CO <0 l O • * K> <_> c _o .CN £ "c a> o c o o (%) uo i j i sodujoo SDQ Figure 4.17: Gas Composition (CTMP Effluent). 50 Sample 100% TMP 50% TMP 25% TMP 10% TMP Set 1 A 2.45 5.60 8.22 11.72 Set 1 B 4.72 6.28 10.25 13.00 Set 4 A 0.30 1.79 1.75 5.00 Set 4 B 0.27 1.10 1.57 5.22 Set 4 C 0.30 1.78 1.58 4.97 Table 4.6: Cumulative Gas Produced (TMP Effluent) (ml) Sample 100% TMP 50% TMP 25% TMP 10% TMP Set 1 0.29 0.48 0.75 . 1.00 Set 4 0.06 0.31 0.32 1.00 Table 4.7: Cumulative Gas Produced for TMP Effluent Expressed as a Fraction of the Maximum Produced Figure 4.19 and Figure 4.20. Initially, there is very little gas produced during the acclimation stage but later, the volume of gas produced rises steadily. Maximum cumulative gas is produced when the TMP effluent is diluted to 10%, see Table 4.6. To compare the cumulative gas produced, the total gas collected for each con-centration of effluent is expressed as a fraction of the maximum value in that set, see Table 4.7. Optimum gas production is observed at 10% dilution of TMP efflu-ent. At this concentration, although the substrate available to anaerobes is low, the toxic effects are low too, allowing maximum gas production. The normalized graphs of cumulative gas produced are presented in Figure 4.21 and Figure 4.22. Fifty per cent of the total gasses evolved by day 30 takes 25 days of treatment. Biochemical Methane Potential (BMP) Table 4.8 shows the biogas potential of the TMP effluent. Again, the gas potential of the 10% TMP is higher than the more concentrated effluents. The drop in BMP when the concentration is increased from 10% to 25% is about 80%. This substantial increase indicates that treatment at 10% concentration is most efficient in terms of gas production but the large quantities of dilution water needed and the large reactor sizes associated with this would require some trade-off in terms 51 Days Figure 4.18: Cumulative Gas Produced (TMP Effluent): Set 1. 52 100% TMP A X / 7 50% TMP A" 25% TMP o 5 10 T -15 20 Days X 25 30 10% TMP Figure 4.19: Cumulative Gas Produced (TMP Effluent): Set 2. 53 100 Figure 4.20: Cumulative Gas Produced (TMP Effluent): Set 3. 54 1.5 <D N « — "O E 00 o 3 o D O > 15 3 E 3 O 0 .5 -A 0 J 1.5 0.5 -0-»A-1.5-1-0 .5 -0 --0.5 1.5 0.5 -0 - |A-0 10 - r -20 I 30 Days 100% TMP 50% TMP 25% TMP 10% TMP 40 Figure 4.21: Normalized Cumulative Gas Produced (TMP Effluent): Set 1. 55 1.5 1 0.5H 0 -A r / 100% TMP 50% TMP 25% TMP 10% TMP Days Figure 4.22: Normalized Cumulative Gas Produced (TMP Effluent): Set 2. 56 Sample 100% TMP 50% TMP 25% TMP 10% TMP Set 1 A Set 1 B 0.0067 0.0130 0.0308 0.0346 0.0543 0.0677 0.3227 0.3579 Table 4.8: Biogas Potential of TMP Effluent (ml biogas/ml effluent) of the total gas production. Rates of Gas Production The rise and fall observed in the rate of formation of gas for the TMP effluents is different from that observed for the CTMP effluent, see Figure 4.23 and Figure 4.24. Here, the initial rate of gas formation is negligible for nearly the first 25 days in the case of the 100% TMP effluent. This initial lag period keeps decreasing with the decrease in strength of the effluent such that, for the 10% effluent, the lag period lasts only 6 days. Another observation is that once the lag period is over, the rises in the rates of gas production increase progressively. Each cycle lasts approximately 5 days — thus, the cycle time is shorter. The initial lag can be attributed to toxicity of the wastewater to anaerobic bacteria. The more concentrated the effluent the more toxic it is and the longer the lag period. As the concentration is decreased, the anaerobic system takes less time in producing gas: compare 25 days at 100% concentration and 6 days at 10% concentration. The rise in rate of gas production is a result of the system getting acclimatized to the presence of inhibitory substances. As more time is allowed, the more the acclimation and the higher the rate of gas production. The shorter cycle could be a result of the greater digestibility of the wastes present in the TMP effluent. The CTMP pulping process involves presteaming and addition of sodium sulphite in order to produce pulp that is stronger and brighter than TMP pulp. This heat and chemical treatment results in the dissolution of substances not ordinarily dissolved during thermomechanical pulping, see Table 4.1. Therefore, the ease in biodegradability of the TMP waste can be explained over that of the CTMP waste resulting in a shorter cycle of gas formation. The higher degradability of the TMP effluent is also coupled with higher toxicity. The heat and chemical treatment in the CTMP process probably destroys some of these toxic substances rendering the effluent less toxic than the TMP effluent. 57 1 0.5 0 o •TO •0.5 1 0) CO _o T5 O Q_ if) D O s o ~S 0.5--0.5 1.5 1 0.5-0--0.5 2 A-0-/ A y A 0 10 20 Days 30 100% TMP 50% TMP 25% TMP A — J A 10% TMP 40 Figure 4.23: Rate of Gas Production (TMP Effluent): Set 1. 58 10 Days Figure 4.24: Rate of Gas Production (TMP Effluent): Set 2. 59 COD (mg/L) 100% TMP 50% TMP 25% TMP 10% TMP Initial COD 1417 709 551 236 Maximum COD 7159 4101 2803 1395 Minimum COD 1643 912 843 278 Table 4.9: Soluble COD of TMP effluent Effect of Dilution of Inoculum Low concentrations of inoculum produce low gas volumes and the toxic effects are evident in the initial lag time, see Figure 4.25. The lag time was about 25 days for 100% TMP and it decreased with the diluted effluent such that at 10% concentration, the lag period was only 6 days. Comparison between these lag times with those in Figures 4.18, 4.19 and 4.20 show that the lag time is dependent on the concentration of effluent and independent of the concentration of seed sludge. There was no lag time for 100% CTMP effluent, in comparison, showing that the CTMP effluent was not toxic while the TMP effluent was. Chemical Oxygen Demand (COD) The differences in the pulping processes lead to different strengths of wastewater. The TMP effluent is generally lower in COD than the CTMP effluent for this reason. The soluble COD results are listed in Table 4.9. The changes in soluble COD with time are plotted in Figure 4.26. The charac-teristics of the change in COD for the TMP effluent were similar to those for the CTMP effluent. A rise in soluble COD was observed on day 6 which declined and by day 26, i.e. 10 days later, the COD levels fell to the initial values. It seemed to take the same amount of time to solubilize the TMP and CTMP suspended COD. This shows that the materials degraded were the same in both the effluents. Table 4.10 presents the COD removal and the actual and theoretical volumes of methane produced. As with the CTMP effluent, the COD removal does not give an accurate indication of gas produced. The reason for the higher actual yield of methane is the solubilization of more substrate thus registering a lower COD removal. 60 0.4 0.2-0.0 2 • A — 0 J 2 0 J 10-i 0 A^ • ..A X -A X 10 20 30 40 Days 100% TMP 50% TMP 25% TMP 10% TMP 50 Figure 4.25: Cumulative Gas Produced (TMP Effluent) with 2% Sludge. 61 10000 5000 6000 4000 Q O O _Q _D O 00 100% TMP 50% TMP 25% TMP 1500 1000 500-/ A / 10% TMP -A 0 + 0 10 20 30 Days Figure 4.26: Change in Soluble COD with Time (TMP Effluent). 62 Effluent COD removed (mg/L) Theoretical CH4 Production (ml) Actual CH4 Production (ml) 100% 5516 1.93 2.15 50% 3189 1.11 4.16 25% 1960 0.68 6.46 10% 1117 0.39 8.65 Table 4.10: Table of COD Removal Compared with the Theoretical and Actual Methane Production (TMP Effluent). Maximum Rate Ratio (MRR) Graphs of MRR against concentration are presented in Figure 4.27. The effluent being highly toxic, the rate ratios fell rapidly as the concentration was increased. Even at 10% dilution, the rate ratios show the effluent to be very toxic to anaerobic microorganisms. The CTMP effluent was much less toxic in comparison, the rate ratios were much higher and even exceeded 1.00 at times. The maximum was at 25 Gas Composition Approximately 65% of the gas was methane, see Figure 4.28. The carbon dioxide present in the gas increased with increasing content of TMP effluent. It is known that carbon dioxide formed during acetogenesis is later converted to methane by methanogens. In the 100% TMP effluent, due to inhibition, the lag time was greater and therefore, at the end of the 30 day period, not much of the CO2 was converted to CH4. In the case of the more dilute effluents, the recovery time had been shorter and so more of the CO2 had been converted to CH4. This is why the concentration of CO2 was higher as the concentration of effluent was increased. The two principal mechanisms for the biochemical formation of methane have been shown to be acetate cleavage (Equation 4.1) and carbon dioxide reduction (Equation 4.2) [25]. CH3COOH -> CH4 + C02 (4.1) 63 0.25 0.20 t 0.15 4 OH 2 Figure 4.27: Maximum Rate Ratios (MRR) for TMP Effluent. 64 C02 + 8H -• CHA + 2H20 (4.2) 4.3.3 Degradability Studies of Peroxide in C T M P Effluent Two sets of experiments were conducted to determine the rate of degradation of 50 and 100 mg/L of hydrogen peroxide in CTMP effluent. The concentration of peroxide was low and could not be assessed using the thiosulphate method [54]. No peroxide was observed in the effluent. It is not known whether it degraded rapidly or whether it was at concentrations too low to be detected in the effluent. For further studies, the use of some other method is recommended since the titration of liberated iodine requires letting the sample stand for 15 minutes (after addition of potassium iodide) before titration can commence/ This 15 minute delay alters the time allowed for degradation from every half hour to anywhere up to 45 minutes or more. Assessing the toxicity of peroxide using the ATA test was attempted but the large volumes of oxygen produced killed the bacteria. 65 3 era* 3 to 00 O O o B o o S3 H S3 (B S3 IS C _o o a E o o CO o O 80-70-60-50-40-30-20-10-I J 10 25 50 Concentration of TMP Effluent (%) Legend ZZ Hydrogen •I Oxygen CS Nitrogen • Methane ESS Carbon dioxide C2 Hydrogen Sulphide 100 Control 0.1 g/L 0.2 g/L 0.5 g/L 1.0 g/L Set 1: A 24.95 14.08 16.06 9.20 0.39 B 24.94 14.06 15.42 8.61 0.62 C 25.10 14.06 15.90 9.10 0.35 Set 2: A 25.28 26.97 24.65 15.37 1.65 B 22.83 25.37 28.85 13.90 3.40 Table 4.11: Total Gas Produced (ATA of Sodium Sulphite). Control 0.1 g/L 0.2 g/L 0.5 g/L 1.0 g/L Set 1 1.00 0.56 0.63 0.36 . 0.02 Set 2 1.00 1.09 1.11 0.61 0.10 Table 4.12: MRR Values (ATA of Sodium Sulphite). 4.4 Toxicity Studies 4.4.1 Sulphite Toxicity Sulphite, as its sodium salt, is found at concentrations between 100 and 200 mg/L in CTMP effluents [3]. When a spill occurs the concentration in the wastewater can increase up to 1000 mg/L [3]. Sulphite is known to be toxic to anaerobic bacteria [12,39]. In order to quantitatively assess its effect, varying concentrations of sodium sulphite were added to anaerobic culture flasks. Optimum growth conditions were provided for the bacteria. A control experiment containing no toxic substance was also conducted. Cumulative Gas Produced The total gas produced over the 30 day test period was plotted against time. This is shown in Figure 4.29 and Figure 4.30. A progressive decline in the total gas produced can be seen as a result of inhibition as the concentration of sulphite is increased. The final gas volumes are shown in Table 4.11. In order to compare the total gas produced, the values were expressed as frac-tions of the gas volume of the controls (i.e. MRR values) as shown in Table 4.12. 67 Tables 4.11 and 4.12 show that at 0.1 g/L of sulphite, less gas is produced than at 0.2 g/L. It can be seen from Figure 4.29 and Figure 4.30 that the gas production drops as the concentration of sulphide is increased above 0.2 g/L. Thus the gas production goes through a maximum at 0.2 g/L of sulphite. Another observation from Figure 4.29 and Figure 4.30 is that as the concentra-tion of sulphite is increased, the anaerobic bacteria take longer in getting acclima-tized before gas production commences. The increase in lag times shows that the more concentrated the sulphite the more the inhibition of anaerobiosis. The ac-climatization times in set 1 (Figure 4.29) are lower than those in set 2 (Figure 4.30) although they both follow similar patterns. This variation in lag time could be the result of two separate batches of seed sludge being used for the two experiments. The cumulative gas plots were normalized in order to compare separate ex-periments, see Figure 4.31 and Figure 4.32. The number of days taken for 50% of the gas to be produced increased as the concentration of sodium sulphite was increased. Rates of Gas Production A shift in the time period over which maximum rate of gas production occured is shown in Figure 4.33 and Figure 4.34. For the controls, the rate of gas production increased sharply to a peak of 5.5 ml/day and by day 10 the rate fell and stayed almost uniform around 0.5 ml/day (which was the rate of gas production of the seed sludge control). As higher concentrations of sodium sulphite were studied, the observations were a) with increasing concentration, the maximum rates fell and b) the time period of maximum gas production increased. Both these observations are the result of inhibition of the metabolic process of the anaerobes. In set 1 for 0.1 g/L Na2SOs, gas production almost ceased by day 26 while that for 0.2 g/L SO\~ ceased by day 40. This implies that in the presence of 0.1 g/L and 0.2 g/L sulphite, it takes 26 and 40 days respectively for complete removal of an easily digestible substrate, while in the control set most of the substrate had been removed by day 10, see Figure 4.33. Maximum Rate Ratio (MRR) Rate ratios (as opposed to the maximum rate ratio obtained from final cumulative gas produced) were calculated using the daily gas volumes of the controls and plotted against time, see Figure 4.35. These show the day to day rates. The higher the rate ratio (RR), the closer is the volume of gas produced to that of the controls. 68 1 0.5 g/L 0.2 g/L 0.1 g/L Control u 1 1 1 1 1 0 tO 20 30 40 50 Days Figure 4.29: Cumulative Gas Produced (Sulphite ATA): Set 1. 69 <D O D T> O v_ Q_ CO D O > £ CJ - 2 20 10 o-*A-40-20 0 J A-40-20 0--20 40 20 X / / . A A X X 'A - A i 10 20 30 Days 1.0 g/L 0.5 g/L 0.2 g/L 0.1 g/L Control 40 Figure 4.30: Cumulative Gas Produced (Sulphite ATA): Set 2. 70 1.5 1-0.5-1.5-1 Days Figure 4.31: Normalized Cumulative Gas Produced (Sulphite ATA): Set 1. 1.0 g/L 71 1.5 1.0 g/L 0.5 g/L 0.2 g/L 0.1g/L Control 0 10 20 30 40 Days Figure 4.32: Normalized Cumulative Gas Produced (Sulphite ATA): Set 2. 72 0.10 1.0 g/L 0.5 g/L 0.2 g/L 0.1 g/L Control Days Figure 4.33: Rate of Gas Production (Sulphite ATA): Set 1. 73 1 0.5-0 -1.0 g/L o C O "o 3 TJ O i_ Q_ (/) O o H— o -4— o -0.5 3 2 n 0 -1 4 4-2-0 - 2 10 V 10 20 Days 30 0.5 g/L A 0.2 g/L 0.1 g/L Control 40 Figure 4.34: Rate of Gas Production (Sulphite ATA): Set 2. 74 As the concentration of SO\~ was increased, the lag time was found to increase with it. This daily RR monitoring can detect variations from the control set that can't be seen in the final MRR values. The final MRR values are plotted against the concentration of the sulphite in Figure 4.36. The decrease in MRR with increasing concentrations of sodium sul-phite seems to be varying with different seed sludge samples — all other parameters remaining constant. Gas Composition The gas produced was analyzed and was found to contain between 70 to 80% methane for 0.1, 0.2 and 0.5 g/L of sulphite, see Figure 4.37. For 1.0 g/L of sulphite, the percentage of methane was lower due to the smaller total volume of gas produced while the amount of N2 being stripped from all the bottles remained almost constant — it being independent of the concentration of sulphite. Also, the amount of C02 produced was found to be very low in general. Summary Sulphite was found to be toxic to anaerobic bacteria even at low concentration of 100 mg/L. Gas produced by the controls was 50% greater than that produced by medium containing 100 mg/L sulphite. The medium containing 1000 mg/L sulphite produced 2% of the control experiment gas. The acclimation times were longer and the rates of gas production were lower as the concentration of sulphite was increased. These results are in agreement with the work carried out by Puhakka et al. [45]. They found sulphite at 100 mg/L to be toxic to anaerobes with increasing lag time and lower gas production as the sulphite concentration was increased. 4.4.2 Sulphate Toxicity Cumulative Gas Production Graphs of accumulation of gas over a given period of time (Figure 4.38) show the total gas produced at any given time to be the same for all the samples. For the controls as well as the test, the gas production increased steadily from day 1 onwards and on day 13 the slope started levelling off as most of the substrate had been converted to bio-gas. All the graphs were almost identical and this indicated 75 0.10 00 q "o c q 3 T5 •" O Q_ oo D O Q 1.0 g/L 0.5 g/L 0.2 g/L 0.1 g/L Days Figure 4.35: Daily Rate Ratios (Sulphite ATA). 76 A A OH 1 1 1 1 1 1 1 1 1 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 Concentration of Sodium Sulphite (g/L) Concentration of Sodium Sulphite (g/L) Figure 4.36: Maximum Rate Ratios (Sulphite ATA). 77 100-s s c _o 'in O CL E o o in O O 80-60-40-20-J ESL 0.1 0.2 0.5 Concentration of Sodium Sulphite (g/L) ) 1.0 Legend ZZ Hydrogen H Oxygon C S Nltrogtn C D Mtthon* Carbon dloxld« a HyoVog«n Sulphld* 0.1 0.2 0.5 Concentration of Sodium Sulphite (g/L) 1.0 Legend EZ) HydVogan M 0xyg«n C 3 Carbon dioxide 1 1 Hydrogen Sulphide Figure 4.37: Gas Composition (Sulphite ATA). 78 that addition of sulphate to an aerobic mixed culture does not affect the total gas produced. Half of the total amount of gas was produced by day 8 and since no lag time was observed, it seems that sulphate has no toxic effects on anaerobes. As reported by Mehrotra et al. [39], sulphate has the least inhibitory effect of the sulphur compounds. The effect of sulphate by itself was observed at concen-trations exceeding 5000 mg/L of sulphate as reported by Lettinga and Rinzema [33]. The present experiments have shown that up to 1000 mg/L SO4 addition has not shown any significant change either in the rate or in the total gas produced (as compared with the control experiments). H2S, the product of reduction of sulphate, is known to be toxic to anerobes [39,33]. Therefore, the amount of hydrogen sulphide is directly related to the amount of sulphate removed. Since no toxicity is detected, the sulphate may not have been reduced to H2S. Rates of Gas Production The rates of gas production showed a rapid rise up to day 8 followed by a more gradual decline, see Figure 4.39. The similarity in the graphs shows that the test samples were behaving exactly like the controls. The maximum rate of gas production was 5.75 ml/day on day 8. Maximum Rate Ratio (MRR) Graphs of daily rate ratios against time are plotted in Figure 4.40. By the end of the test period the total gas produced was comparable with that of the controls. Except during the first few days, the rate ratios were much lower. As the concentration of sulphate was increased from 0.1 to 0.2 g/L, the rate ratios took longer times in reaching the gas production rates of the controls. Above this concentration, i.e. for 0.2, 0.5, and 1.0 g/L sodium sulphate, the time taken to reach a steady rate ratio was 8 days. This implies that it took 8 days to remove most of the substrate from solution. The final MRR values at various concentrations of sulphate are presented in Figure 4.41. The MRR values are generally higher than the controls i.e., the samples produced more gas than the controls. Sulphur reducing bacteria utilize the sulphate as an electron acceptor with hydrogen sulphide being the end product, see Equation 4.3. This hydrogen sulphide contributes to the excess final gas produced. 4H2 + SO\~ + H+ -+ HS~ + 4H20 (4.3) 79 X5 Q> O Z5 X> o CL to D O > ZJ ZJ O 10 O-^A* 20-10 0 - L A -20-10-0-f A s 0 5 10 15 Days 20 1-0 g/L 0.5 g/L 0.2 g/L 0.1 g/L Control 25 Figure 4.38: Cumulative Gas Produced (Sulphate ATA). 80 10 Days Figure 4.39: Rate of Gas Production (Sulphate ATA). 81 1.5 / f 0.5-X 0 1.5 -A 1.0 g/L i -0.5-/ 0-»-X-1.5 I 0.5-X 0 1.4 1.2-1-0.8 10 15 Days -A. A 20 0.5 g/L 0.2 g/L A ^ ' A 0.1 g/L 25 Figure 4.40: Daily Rate Ratios (Sulphate ATA). 82 Gas Composition The gas composition was almost identical between the various samples, see Fig-ure 4.42. The methane content was about 80%, the nitrogen content about 10%, COi about 4% and trace quantities of hydrogen sulphide and hydrogen were found in the test samples reconfirming the excess gas to be hydrogen sulphide. 4.4.3 Sulphide Toxicity Sulphide, although not present as such in either TMP or CTMP effluent, is a product of reduction of sulphate and sulphite [12,39]. Since sulphur reduction can be favoured over methanogenesis and since hydrogen sulphide is formed in the process, assessing the toxicity of sulphide to anaerobic bacteria seemed necessary [39]. Cumulative Gas Production The cumulative gas produced in the test samples was nearly as much as that produced in the controls, see Figure 4.43. There was no lag period, thus bacterial acclimation to the presence of sulphide was not necessary. Fifty percent of the gas was produced by day 5 in all the tests except those containing 1.0 g/L of sulphide, which took 7 days. Thus, it seems that the presence of sulphide does not have toxic effects on the anaerobic bacteria, it only retards the bacterial process. In the controls, 50% of the gas was produced by day 4 while that in the samples was produced by day 5 for 0.1, 0.2 and 0.5 g/L of sulphide. For 1.0 g/L of sulphide it took 7 days. This precipitation of sulphide with heavy metals from the nutrient solution (RAMM) may have been a reason why sulphide was not toxic to the bacteria. Theoretically, from the RAMM, from each flask containing 300 ml of solution, 35 mg of sulphide is precipitated by the heavy metals. Rates of Gas Production The rates of gas production were highest between days 1 and 5 with the peak rate of gas production at about 6 ml/day on day 4. After the 5th day, the rates were nearly constant at 1 ml/day. The rates of gas production are illustrated in Figure 4.44. At sulphide concentration of 1.0 g/L, the peak rate of gas production was about 3.5 ml/day and the time interval for maximum gas production extended over a 13 day period. After this, the rate dropped to 0.5 ml/day. 84 100 80-c _o ' m o a E o o o o 60-40-20-I 1 0.1 0.2 0.5 1.0 Concentration of Sodium Sulphate (g/L) Legend 22 Hydrogen • Oxygen C3 Nitrogen CD Methane ESS Carbon dioxide C2 Hydrogen Sulphide 40 20-0-40-1.0 g/L 20 0.5 g/L 40-A-— A 20 0.2 g/L o J A -40-— A 20- 0.1 g/L 0 J A . 40 20 Control A . i 10 —T -20 30 D a y s 40 Figure 4.43: Cumulative Gas Produced (Sulphide ATA). 86 6 1.0 g/L 0.5 g/L 0.2 g/L 0.1 g/L Control 0 10 20 30 40 Days Figure 4.44: Rate of Gas Production (Sulphide ATA). 87 Maximum Rate Ratio (MRR) From the plots of rate ratios, see Figure 4.45, the rates of gas production were initially low. As the bacteria became acclimatized to the presence of sulphide by about the 3rd day, the rate ratios started climbing and became nearly uniform by day 5. This information could not have been obtained from the plots of cumulative gas. Therefore, the acclimation time for sulphide is 5 days. As the concentration of sulphide was increased, the total gas produced de-creased. This is reflected in the decreasing values of MRR with increasing con-centrations, see Figure 4.46. Thus, at 0.1 g/L, the sulphide is not inhibitory to anaerobic microorganisms. As the concentration is increased the inhibition in-creases. Gas Composition The methane content of the gas was about 90%. The amount of C02 decreased as the concentration of sulphide increased because C02 get further reduced to methane as time elapses. Also, at low sulphide levels, the bacterial process is less inhibited resulting in a larger available time for conversion of C02 to CH±, see Figure 4.47. 4.4.4 Resin Acids The resin acids sample from Sigma Chemical Co. was found to contain very small amounts of abietic (or other resin acids). Also, the contents were not homogenous — some samples tested did not have any resin acids at all. Therefore, this test was abandoned.a? 88 0.5- 1.0 g /L o i 0.8-0.6-0.4 1.1 1-0.9 0.8 1.2 0.5 g/L A A 3l // A AT * -~ —. X • • 0.2 g/L A \ / / i / 0.8 0 10 20 Days : A i 30 0.1 g/L 40 Figure 4.45: Daily Rate Ratios (Sulphide ATA). 89 1 0.1 0.2 0.5 1.0 Concentration of Sodium Sulphide (g/L) Legend EZ3 Hydrogen • • Oxygen C 3 Nitrogen CD Methane 6SS Carbon dioxide I 1 1 1 1 1 Legend EZ3 Hydrogen • i Oxygen (SI Carbon dioxide 0.1 0.2 0.5 1.0 Concentration of Sodium Sulphide (g/L) Figure 4.47: Gas Composition (Sulphide ATA). 91 Chapter 5 Conclusion and Suggestions For Further Research 5.1 Conclusion The purpose of this study was to develop a practical method for assessing anaerobic treatability of industrial effluents. To this effect, a modification of the serum-bottle test was developed. The toxicity of sulphur compounds and degradability of CTMP and TMP effluents were demonstrated using this technique. A summary of the investigation is presented in this chapter along with some suggestions for future work. 5.1.1 The Anaerobic Bioassay Technique The techniques presently used for anaerobic assays require skilled personnel and extensive laboratory facilities thereby making transportation of the effluent neces-sary. The two important criteria for waste removal are degradability and toxicity — both of which can be sensitive to delays in testing after sample collection. Since the new technique can be setup with no need for extensive laboratory facilities on site, the monitoring of degradability, and toxicity can be carried out accurately and routinely at economical costs. The salient points regarding the modified technique as a test for assessing the effects of industrial wastewaters on anaerobic treatment are summarized below: • The apparatus is simple and economical. • The start-up procedure is not complicated. • Sampling of gas or liquid is possible without disturbing the setup. 92 • Good reproducibility in repeating the experiment with same sludge at the same time is obtained. • It is easy to measure gas. • The sludge characteristics affect the overall performance, therefore when pos-sible, the same seed sludge should be used. The sludge can be stored quite adequately at low temperatures. • Nutrient addition is not necessary. 5.1.2 Biodegradability Studies The removal of oxygen demanding substances from wastewaters by anaerobic bac-teria depends on the biodegradability of the wastes. No chemical test has yet been developed which distinguishes between biodegradable and non- biodegradable or-ganics. Hence, the first step in deciding whether a waste can be successfully treated anaerobically is to determine its degradability. In this study the biodegradability of CTMP and TMP effluents were investigated. Some salient points of the biodegradability study of CTMP effluent are: • CTMP effluent produced optimum gas when treated at dilutions between 50 and 25%. The undiluted effluent producing on average only 40% of the gas produced at 25% dilution. • The slow rate of hydrolysis of complex substrate coupled with toxic effects may have been the reason for dilution giving better COD removal. • The bio-gas potential was found to be higher the more the dilution of effluent. • The soluble COD was found to increase from the initial value by day 6 into the experiment. The reason for this could be the time lag in hydrolysis breaking down the insoluble substances to soluble COD. • The maximum rate ratios show that CTMP effluent is toxic to anaerobic systems and that the toxicity decreases with increasing dilution with water. Salient features of the degradability study of TMP effluent: • The TMP effluent was found to be more toxic to anaerobic systems than the CTMP effluent. • The maximum gas was produced when the effluent was diluted to 10%. 93 • Although the toxicity of TMP effluent was high, it was also found to be more biodegradable than CTMP effluent. • The sCOD levels were found to rise on day 6 as in the CTMP effluent. • From the MRR values, the TMP effluent was found to be toxic to anaerobes even at 10% dilution. 5.1.3 Toxicity Studies The toxicity of sulphite, sulphate and sulphide was determined on anaerobic sys-tems. Some salient points are: • Sulphite was found to be most toxic followed by sulphide while sulphate was not found to be toxic to anaerobic bacteria. • Sulphite was found to be toxic even at 0.1 g/L concentration. • Sulphite at concentrations below 0.2 g/L allowed about 60% of the gas pro-duction of the controls while 0.5 and 1.0 g/L gave very little gas. • The acclimation time to presence of sulphite increased with increasing con-centrations. • Sulphate was not found to be toxic to anaerobic bacteria even at concentra-tions of 1.0 g/L. This was evidenced from the almost identical results obtained for the control experiments. • Sulphide was found to be inhibitory above 0.5 g/L concentration where the MRR values were lower than 0.90. The toxicity of sulphide may have been lowered due to precipitation of sulphide as heavy metal salts. 5.1.4 Suggestions for Further Research The discussion in Chapter 4 suggests that the Anaerobic Bioassay Technique is a useful tool for analyzing toxicity and biodegradability of wastewaters. Its applica-tions are not limited to the pulp and paper industry but can be used for study of viability of anaerobic treatment for any industrial effluent. Some recommendations for future work include: • Improvements in the design of the flask, eg. adding a support media for the microorganisms and preventing evaporation of liquid from pipets by placing a thin film of oil at interface. 94 Monitoring parameters like VFAs and pH in order to gain a better under-standing of the anaerobic processes especially in the degradability studies. Allowing the system to reach steady state prior to addition of potentially inhibitory substance in order to better assess effects of shock leading. Use of this technique for routine on-line toxicity and degradability tests along-side full scale anaerobic treatment plants. Use of this technique to further research in mechanisms of degradation and kinetics of anaerobic reactions. Assessment of toxicity of various potential toxicants on an individual basis. To conduct experiments on BMP of TMP and CTMP effluent using sludge acclimated to these effluents. 95 Bibliography [1] Andersson, P-E, L. Gunnarsson, G. Olsson, T. Welander and A. Wikstrom, TAPPI Environmental Conference, pp. 11-16 (1985). [2] British Columbia Government, "Pollution Control Objectives for the Forest Products Industry of British Columbia", Victoria, B.C. (1977). [3] Beak Consultants Ltd., "Anaerobic Treatment of TMP/CTMP Wastewaters", Report for Environment Canada, WTC, Burlington, Ontario (1986). [4] Benjamin, M.M., S.L. Woods and J.F. Ferguson, "Anaerobic Toxicity and Biodegradability of Pulp Mill Waste Constituents", Water Res. 18(5):601-607 (1984). 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