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A comparison study of agricultural materials as carbon sources for sulphate reducing bacteria in passive… Brown, Amber 2007

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A COMPARISON STUDY OF AGRICULTURAL MATERIALS AS CARBON SOURCES FOR SULPHATE REDUCING BACTERIA IN PASSIVE TREATMENT OF HIGH SULPHATE WATER by AMBER BROWN B.Sc , Queen's University, 2002 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF APPLIED SCIENCE in THE FACULTY OF GRADUATE STUDIES (Chemical and Biological Engineering) THE UNIVERSITY OF BRITISH COLUMBIA April 2007 ©AmberBrown, 2007 A b s t r a c t Several natural and agricultural organic materials were assessed as carbon sources for a potential passive sulphate reduction treatment system to treat lake and pond water containing high concentrations of sulphate for the purpose of creating drinking water for cattle. Of primary concern was that the system be low cost and simple to operate, s ince the end users would be farmers. Sediment removed from a low sulphate lake located near Kamloops, which contained a potentially highly active sulphate reducing bacteria (SRB) population was used in a mesocosm experiment to determine its effectiveness at supporting sulphate reduction. Originally operated as a continuous system, a flowrate of 6 mL min" 1 f lushed the nutrients out of the system. While operating in batch mode, sulphate reduction was observed within the sediment, but not above in the water layer due to mass transfer limitations. Thus, recirculation of water through the sediment was required to increase the overall rate of sulphate-reduction. Four different agricultural materials; barley, molasses, hay, and silage, were supplied to a S R B inoculum and concentrated S 0 4 2 " water to test their effectiveness as nutrient sources. A 1:1 (wt) mixture of hay and silage showed the highest drop in S 0 4 2 " and achieved a maximum S 0 4 2 " reduction rate of 14.4 mg S 0 4 2 ~ L: 1d" 1 . A second study, which tested the leachates of barley and orchard grass si lages, found similar rates (19.4 ± 1.6 mg S 0 4 2 " L"1d"1). Based on this work, a pair of mesocosms were built and supplied with a mixture of si lage and hay. These systems achieved initial maximum rates of 33.8 mg S O 4 2 L" 1d" 1. After a second sulphate addition the maximum rate increased to 132.4 mg S 0 4 2 " L" 1d" 1. The A S 0 4 2 " / A s C O D ratios were determined to be 1.10 and 0.91 g g" 1 for mesocosms A and B. A halt in sulphate reduction was concomitant with an increase in sulphide concentration to 323.5 mg L' 1. It is hypothesized that sulphide inhibition of the S R B occurred. Recycl ing the water through an aerobic treatment system containing sulphur-oxidizing bacteria, to oxidize the sulphide to sulphur, is one possible method to remediate these high sulphide levels. iii Table of Contents Abstract ii Tab le of Contents iv List of Tab les vi List of F igures vii G lossa ry x List of Units xii Acknowledgements xiii 1.0 Introduction 1 1.1 Rat ionale for Work 1 1.2 R e s e a r c h Object ives 3 1.3 Thes is Layout 4 2.0 Background Information and Literature Rev iew 5 2.1 The Effect on Cattle of Sa l ine Drinking Water 5 2.2 Sulphate 6 2.3 Treatment Technologies for Remov ing Sulphate from Water 10 2.4 Exper imental Des ign Cons idera t ions B a s e d Upon the Literature Rev iew 35 2.5 Background on the Field Study Si tes 43 3.0 Mater ia ls and Methods 4 5 3.1 L a c du Bois Grass lands Park Lakes Sampl ing ...45 3.2 Laboratory S R B Inoculum Creat ion and Maintenance .48 3.3 Lake 52 Sampl ing and In Si tu Se t -Up 50 3.4 Batch Bottle Exper iments Test ing Different Agricultural Mater ials 54 3.5 M e s o c o s m Experiment Set -up and Operat ion 65 3.6 Analyt ical Methods 73 4.0 Resul ts and Discuss ion 84 4.1 Character izat ion of High and Low Sulphate Lakes 84 4.2 The Suitability of Agricultural Mater ials as Substrates for Biological Sulphate Reduct ion 100 iv 4.3 Mesocosm Experiments 137 4.4 In Situ Experiment 167 5.0 Conclusions and Recommendat ions for Future Work 180 5.1 Conclusions 180 5.2 Future Work 183 Bibliography 185 Appendix 1 - Raw Data 195 Appendix 2 - Norwest Analysis of Lac du Bois and Lac du Bois Twin 281 V List of Tables Table 1 - Act ive system operating parameters 22 Tab le 2 - P a s s i v e system operating parameters 28 Tab le 3 - Organ ic substrates used in batch reaction mixtures as dry weight composit ion (wt %) 34 Tab le 4 - In-situ treatment set-up 53 Tab le 5 - Final vo lumes in leachate exper iments .....62 Tab le 6 - Sod ium sulphate added to ach ieve 1500 mg L"1 SO42" 63 Tab le 7 - C h a n g e s made to the agricultural m e s o c o s m s 72 Tab le 8 - Ave rage overall SO4 2 " change and maximum sulphate reduction rates ( S R R ) of non-pH adjusted agricultural bottle experiment 103 Tab le 9 - C h a n g e and S R R s of pH adjusted agricultural bottle experiments.. . . 116 Tab le 10 - Overal l change in SO4 2 " in leachate bottles over 71 days and maximum S R R s 131 Tab le 11 - pH of leachate 132 Tab le 12 - A S 0 4 2 7 A s C O D ratio for leachate experiment 136 Tab le 13 - Ammonia-ni t rogen and o r t h o - P 0 4 3 " concentrat ion in natural treatment system 145 Tab le 14 - Sulphate reduction when a lgae-sediment w a s inoculated into Postgate B medium 147 Tab le 15 - Compar i son of SO4 2 " levels in water co lumn and sediment of natural system 148 Tab le 16 - C h a n g e s made to the agricultural m e s o c o s m s 161 Tab le 17 - V isua l observat ions of in-situ buckets after 311 days. . . . 169 Tab le 18 - pH within Lake 52 170 List of Figures Figure 1 - Simplified sulphur cycle 9 Figure 2 - U A S B reactor 19 Figure 3 - Sampling sites for Lac du Bois 46 Figure 4 - Sampling locations for Lake 52 50 Figure 5 - In-situ treatment set-up 52 Figure 6 - Mesocosm design 65 Figure 7 - Sampling sites for natural system materials 67 Figure 8 - Natural system sampling locations 70 Figure 9 - Lac du Bois Twin shoreline 85 Figure 10 - S 0 4 2 ~ concentration in Lac du Bois versus depth 88 Figure 11 - S 0 4 2 " concentration in Lac du Bois Twin versus depth 89 Figure 12 - Total sulphide concentration in Lac du Bois versus depth 90 Figure 13 - Total sulphide concentration in Lac du Bois Twin versus depth 90 Figure 14 - Nitrate and ammonia-nitrogen concentrations in Lac du Bois and Lac du Bois Twin 91 Figure 15 - O r t ho -P0 4 3 " concentration in Lac du Bois and Lac du Bois Twin 92 Figure 16 - Soluble C O D concentration in Lac du Bois and Lac du Bois Twin... 92 Figure 17 - S 0 4 2 " concentration versus time for the non-pH-adjusted batch experiments 102 Figure 18 - Final total sulphide concentration in the non-pH-adjusted batch experiments 104 Figure 19 - Final soluble C O D concentration in the non-pH-adjusted batch experiments 105 Figure 20 - Final ammonia-nitrogen concentration in the non-pH-adjusted batch experiments 106 Figure 21 - Or tho-P0 4 3 ~ concentration in the non-pH-adjusted batch experiments 107 Figure 22 - pH in the non-pH-adjusted batch experiments 108 Figure 23 - pH versus time in the pH-adjusted batch experiments 112 Figure 24 - S 0 4 2 ~ concentration versus time in the pH-adjusted batch experiments 114 Figure 25 - S 0 4 2 " concentration versus time in the pH-adjusted batch experiments containing mixtures of hay and si lage 115 Figure 26 - Initial and final soluble C O D concentrations in pH-adjusted batch experiments 118 Figure 27 - Ammonia-nitrogen concentrations in the pH-adjusted batch experiments 119 Figure 28 - Or tho-P0 4 3 ~ concentrations in the pH adjusted batch experiments 120 Figure 29 - S 0 4 2 " concentration in leachate bottles 128 Figure 30 - Soluble C O D in leachate bottles 132 Figure 31 - pH in leachate bottles 133 Figure 32 - Natural treatment mesocosms 139 Figure 33 - pH in natural system mesocosms 141 Figure 34 - S 0 4 2 " concentration versus time in natural continuous system ...... 143 Figure 35 - Total sulphide versus time in natural continuous system 143 Figure 36 - S 0 4 2 " concentration in the water column... 148 Figure 37 - S 0 4 2 " concentration in natural system sediment under batch conditions 150 Figure 38 - Total sulphide in natural system sediment under batch condition 151 Figure 39 - pH in natural system's sediment 152 V l l l Figure 40 - Average agricultural mesocosm pH versus time 154 Figure 41 - Average ammonium-nitrogen concentration versus time in mesocosms 155 Figure 42 - Average ortho-PGy 3" concentration in mesocosms versus time 155 Figure 43 - Average S 0 4 2 " and total sulphide concentrations in agricultural mesocosms versus time 156 Figure 44 - Average soluble C O D concentration in agricultural mesocosms versus time 158 Figure 45 - Average S 0 4 2 " and total sulphide concentrations versus time in agricultural mesocosms 160 Figure 46 - Average soluble C O D versus time in agricultural mesocosms 160 Figure 47 - S R B presence in agricultural mesocosms 162 Figure 48 - S 0 4 2 " concentration in Lake 52 173 Figure 49 - Total sulphide concentration in Lake 52 173 Figure 50 - Or tho -P0 4 3 " concentration in Lake 52 175 Figure 51 - Ammonium-nitrogen concentration in Lake 52 175 Figure 52 - Soluble C O D concentrations in Lake 52 176 ix Glossary 1+1 a A g 2 S 0 4 A M D B a 2 + BaCI B a S 0 4 B.C. C a S 0 4 C H 3 C O O H C H 3 C O O N a - 3 H 2 0 c r C 0 2 C 0 3 2 " C O D C W DO DW E h E P A F e 3 + FeCI 3 F e S F e S 0 4 • 7 H 2 0 FISH FT HCO3-H 2 0 H 2 S H 2 S 0 4 K 2 C r 2 0 7 K H P K H 2 P 0 4 K N O 3 L A B L D B MgCI 2 • 6 H 2 0 M n 4 + M O N 2 N a + N a O H N a 2 S • 9 H 2 0 equal volumes signif icance level silver sulphate acid mine drainage barium ion barium chloride barium sulphate (Barite) British Columbia calcium sulphate (gypsum) acetic acid sodium acetate chlorine ion carbon dioxide carbonate ion chemical oxygen demand constructed wetlands dissolved oxygen distilled water symbol for oxidation-reduction potential environmental protection agency iron ion ferric chloride iron sulphide iron sulphate heptahydrate f luorescence in-situ hybridization proton bicarbonate ion water hydrogen sulphide sulphuric acid potassium dichromate potassium phthalate potassium phosphate potassium nitrate lactic acid bacteria Lac du Bois magnesium chloride hexahydrate manganese ion microorganisms nitrogen gas sodium ion sodium hydroxide sodium sulphide nonahydrate N a 2 S 0 4 N H 4 + N H 3 - N N N 0 3 N 0 3 0 2 O.D. O R C P P C R P O 4 3 -P F R P R B rDNA rRNA S° s2-s C O D S M S S 0 2 S 0 4 2 " S O B S R B S R R T TAD TBD T E A T S U A S B Z n S + sodium sulphate ammonium ion ammonia nitrogen or total ammonia. The summed weight of nitrogen in both the ionized (ammonium, N H 4 + ) and molecular ( N H 3 ) forms nitrate ion ammonia-nitrogen oxygen optical density orchard grass silage the probability of obtaining, by random chance, a value equal or greater than that observed polymerase chain reaction phosphate plug flow reactor permeable reactive barrier ribosomal deoxyribonucleic acid ribosomal ribonucleic acid elemental sulphur sulphide ion soluble chemical oxygen demand spent mushroom substrate sulphur dioxide sulphate sulphur oxidizing bacteria sulphate reducing bacteria sulphate reduction rate time (days) barley silage bio-ethanol waste terminal electron acceptor total sulphide upflow anaerobic sludge blanket zinc sulphide standard deviation of 3 or more measurements (or difference between two points if specified) XI List of Units °C degrees Cels ius cm centimetres cm d" 1 centimetres per day d day g grams g L"1 grams per litre g L" 1d" 1 grams per litre per day hr hour kg kilograms kg d" 1 kilograms per day kg d" 1 m"3 kilograms per day per cubic metre L litres L hr"1 litres per hour m metres m 2 square metres m s " 1 metres per second mg d" 1g" 1 milligram per day per gram mg L"1 milligram per litre mg L ' V milligram per litre per day mg L"1d~1g"1 milligram per litre per day per gram mL min" 1 millilitre per minute mm millimetre um micrometre mol mole mmol L"1 millimoles per litre mmol L"1 a" 1 millimoles per litre per annum mV millivolt N normal nm nanometre ppm parts per million x g times gravity Acknowledgements I'd like to thank Dr. Susan Baldwin for accepting me as her graduate student and allowing me to gain experience and knowledge in the field of bioremediation. Her guidance, energy and emphasis on independent learning and design were greatly appreciated. I would like to give special thanks to Doug Veira, Lavona Liggins and Barb Wheatley of Agriculture and Agri-Food Canada for all their help in my sampling trips to Kamloops as well as in the construction of my mesocosms and in situ treatment systems. They all put in long days with grace and cheerfulness. I received strong support from UBC faculty, staff and students. Thanks go to the guys in the workshop: Doug, Peter and Graham. I'd also like to thank Jana Schmidtova and Michael Lee for helping me in the lab and in setting up my mesocosms and in situ treatment system. Funding for this study was provided by the Canadian BC Water Expansion Program and the University of British Columbia. I dedicate this thesis to my family who have never faltered in their support and encouragement. X l l l 1.0 Introduction 1.1 Rat ionale for Work One major requirement for raising livestock is having suitable drinking water available. For those livestock that require large areas to graze, this drinking water usually comes from natural water reservoirs, such as lakes or ponds. However, natural water reservoirs can become unsuitable as livestock drinking water due to high salinity, which can be caused by the concentration of ions such as chloride, carbonate and sulphate ( S 0 4 2 ) . While these substances could have their origins from mankind's actions, these ions can also find their way into water reservoirs through the weathering and erosion of soil and rock. For SO42", bedrock deposits of shale, coal and sandstone are the prime donators (Blinn 1993). Water reservoirs with salinity levels over 3 g L"1 (total salts) are considered saline and have low biodiversity (Hammer 1986). Good quality aquifers for livestock drinking water are becoming scarcer in arid regions of Canada , such as the areas surrounding Kamloops and Merrit, B .C. and in central Saskatchewan. In the Kamloops area the cattle herds graze in the hills surrounding the town, where the only drinking water available comes from naturally occurring lakes and ponds. However, a good number of these lakes are highly saline due to S 0 4 2 " and the cattle either refuse to drink from them, or risk becoming sick if they do. This appears to be a problem not only for this particular cattle station but with many farmers in the B .C . interior, as well as in arid locations in the prairies 1 where saline and hypersaline lakes are often the only surface waters present (Beke and Hironaka 1991). The lakes in this arid region are unique since no where else in the world is there such a concentration and variety of salinities in the surface water (Last and Ginn 2005). In saline lakes north of the 47° latitude, S 0 4 2 " is typically the dominant anion and the prime cause of the salinity (Blinn 1993). To address this problem of high sulphate in cattle drinking water, we proposed to design and test a passive biological sulphate reduction system as part of a B .C . Canada Water Supply Expansion Program grant. With that goal in mind, the focus of this study was on utilizing a passive system to treat the S 0 4 2 ~ in these lakes, which relied on the activity of a naturally occurring group of microorganisms, special ized in reducing S 0 4 2 " , known as sulphate reducing bacteria. Considering the target user of these systems, it was decided that any system created must also meet certain requirements: • cheap and readily available nutrient source • easily constructed • low cost • low maintenance • built using materials that most farmers would have at their farm • final water quality in terms of sulphide and nutrients (C, N and P) must meet water quality guidelines for livestock. 2 To satisfy the last requirement, an additional downstream process for sulphide removal is needed. However, this thesis focuses only on the initial step of sulphate reduction to sulphide. 1.2 Research Object ives Overall objective To design a biological system to reduce levels of SO4 2" to below 1000 mg L"1 in a way that could be implemented by farmers. One thousand milligrams per litre is the maximum concentration for livestock drinking water recommended by H.G. Peterson of Agricultural and Agr i-Food Canada (Peterson 1999). Sub-obiectives 1. Conduct a field survey of high/low SO4 2" lakes in the Kamloops region to see if there is anything obviously different about these lakes, which would account for their different salinities. 2. Propose passive treatment configurations and test suitable nutrient sources for biological sulphate reduction. 3. Determine the batch kinetics of sulphate reduction in open mesocosms with natural and agricultural substrates. 4. Identify important parameters to control. The initial intention was to run a continuous system but, due to time constraints, this was not done. 3 1.3 Thes is Layout Four more chapters follow in this thesis. Chapter 2 gives essential background and a critical literature review on passive treatment systems. Chapter 3 presents all the materials and methods undertaken in the experimental work. Chapter 4 presents the results and offers a discussion on each individual experiment and how the sub-objectives have been met. Conclusions and recommendations for future work are given in Chapter 5. 4 2.0 Background Information and Literature Review 2.1 The Effect on Cattle of Sal ine Dr ink ing Water Having a steady supply of potable water is one of the most critical concerns for farmers raising a herd of cattle (Subcommittee-on-Dairy-Cattle-Nutrition 2001). Dairy cows, in particular, daily require a large quantity of fresh water to replenish the amounts lost through milking. When the daily milk production is between 33-35 kg d"1, the fresh water intake was 2.3 kg (Dado and Allen 1994), 3 kg (Murphy, Davis et al. 1983), or 2.6 kg (Holter and Urban Jr. 1992) per kg of milk produced. A typical prairie farm operation in Canada requires 1000 imperial gallons of livestock drinking water per day (Corkal and Braul 2003). In areas, such as B.C. interior or arid regions of Saskatchewan, where saline waters are prevalent, finding potable drinking water can pose a problem to cattle farmers. Studies performed by Weeth and Hunter (1971), and Weeth and Capps (1972), found that their cattle in Nevada would tolerate water with a 2500 mg L"1 SO4 2" concentration for 90 days with no ill effects. However, after this period the cows underwent increased renal filtration of sulphate by 37% compared to those cattle, which were supplied water with a lower sulphate concentration of 110 mg L"1. In addition, if water with lower SO4 2" concentrations were available, the cattle would reject the 2500 mg L"1 water. Smart, Cohen et al. (1986) found that cattle, which were supplied water with a SO4 2" concentration of 500 mg L"1, had lower levels of copper in their plasma and liver. While this did not have any health related effects to 5 the cattle, it was observed that their calves had lower weaning weights than those calves whose parents were fed lower S 0 4 2 " concentration water. From a medical standpoint, Beke and Hironaka (1991) found that a saline well with a total dissolved solids concentration of 3875 mg L"1 (of which S 0 4 2 " and Na were the predominant ions) was responsible for polioencephalomalacia disorders in yearling Holstein heifers. Studies on beef calves drinking saline well water by Hibbs and Thilsted (1983) also found that the calves were more likely to have the pol ioencephalomalacia disorder. With this disorder, the S 0 4 2 " ingested by the cattle is reduced to H2S by ruminal microbes, and the build-up of this toxic gas affects cellular metabolism (Merck 2006). Due to these adverse health effects, Canadian water quality guidelines for livestock drinking water limit sulphate concentration to less than 1000 mg L*1 (Peterson 1999). 2.2 Sulphate Considering that the primary salt ion affecting the salinity of the lakes in the prairies and interior B .C. is sulphate, a brief literature review is presented concerning the source of sulphate in these waters. 2.2.1 General Background Sulphate is an inorganic ion that is widely distributed in nature, particularly in the Earth's lithosphere and hydrosphere. Consist ing of a central sulphur atom single bonded to four tetrahedrally oriented oxygen atoms, this anion has a net negative 6 two electric charge. Sulphate exists as salts or esters of sulphuric acid ( H 2 S 0 4 ) and is formed by replacing one or both of the hydrogen atoms with a metal or a radical, such as sodium or ammonium. In the earth's crust, it is primarily found bound to metals to form mineral compounds such as gypsum ( C a S 0 4 ) or barite ( B a S 0 4 ) . The majority of sulphate compounds are water soluble, with the exception of lead, barium and strontium, and are easily eroded from the soil. Once it has entered the hydrosphere, it can remain as sulphate anions and form a large reservoir of biologically useful sulphur. 2.2.2 Sulphate in the Sulphur Cycle The majority of the earth's sulphur is concentrated in rocks and salts or buried deep within oceanic sediments. However, sulphur can enter the atmosphere through either natural or human actions. Naturally, sulphur is released to the atmosphere by volcanic eruptions as H 2 S gas or by the reduction of oxidized sulphur compounds by S R B which are released as H 2 S gas into the atmosphere. In aquatic environments, sulphide can be oxidized to elemental sulphur aerobically by such sulphur-oxidizing bacteria species as Thiothrix and Beggiatoa, while in anaerobic environments by phototrophic green and purple sulphur bacteria. Equations 1 and 2, demonstrate how, in an aerobic environment, these organisms can oxidize reduced sulphur to S 0 4 2 " . H 2 S + 1 / 2 0 2 ^ S° + H 2 0 (1) S° + 1 1 / 2 0 2 + H 2 0 ^  S 0 4 2 " + 2 H + (2) 7 If both reactions are completed, then sulphuric acid is produced, which can then enter the local surface and groundwater and reduce the pH. The actions of these bacteria are the source of acid rock drainage, although in those cases the H 2 S is generally replaced by a metal sulphide ore such as iron sulphide (FeS). Humans impact the sulphur cycle primarily in the production of sulphur dioxide (SO2) from industrial activities, such as burning coal. The S 0 2 can precipitate and then be oxidized to sulphate, reduced to sulphide in the atmosphere, or oxidized to sulphate in the atmosphere as sulphuric acid. The oxidation of SO2 to sulphuric acid in the atmosphere is a principal contributor to acid rain. The majority of sulphur available to the biosphere exists in an oxidized form, either as sulphates in the lithosphere and hydrosphere or as sulphur oxides in the atmosphere. However the majority of life-forms on earth require sulphur to be in a reduced form before it can be utilized. Sulphate can be reduced in two different ways (Figure 1). The first and most frequent method is known as assimilatory sulphate reduction. Plants and most bacteria and archaea assimilate sulphate as a source of sulphur for biosynthesis, where it is most often used to produce sulphur containing amino acids or co-factors (Postgate 1979). 8 2- S O 2 SO4 reduction 4 (assimilatory) SO 4 reduction Organic S (dissimilatory) s o \ . I "yT s Oxidation by v l > / sulfur oxidizing S bacteria Mineralization Figure 1 - Simplified sulphur cycle In the second method, organisms utilize sulphate as a terminal electron acceptor in the oxidation of organic matter. This process is known as dissimilatory sulphate reduction and is accomplished by S R B and some archaea. Sulphate reducing bacteria couple the oxidation of carbon substrates to the reduction of sulphate for the production of energy and growth. 2.2.3 Sulphate effects Sulphate is known for having a laxative effect when digested. A study by Chien, Robertson et al. (1968) found that infants who were given formulas prepared with water containing 630 to 1150 mg L"1 S 0 4 2 " developed diarrhoea shortly after ingestion. The Canadian guideline for the maximum acceptable concentration of S 0 4 2 " in drinking water is 500 mg L"1 while in the U.S. the E P A has specified the maximum contaminant level goal of drinking water to be 400 mg L"1 ( U . S . - E P A 1990). Sulphate is known to add an undesirable taste to water with a detectable taste threshold of 300-400 mg L~1 (National-Academy-of-Sciences 1977). Increasing levels of salinity have been shown to have a negative effect on plant-life and wildlife. Hammer (1986), Herbst and Blinn (1998) and Campbel l and Prepas (1986) report that as levels of salinity increase in aquatic ecosystems, the species diversity decreases. Campbel l and Prepas (1986) also found with their findings that low levels of chlorophyll a also tend to be indicative of saline lakes in North Amer ica. This resulted in lower than expected algal productivity, despite the presence of high nutrient levels. Runoff from mining and agricultural areas can also increase the levels of S 0 4 2 ~ in groundwater and surface water. In addition, sulphate levels can also be increased from pyrite containing sediments undergoing desiccation, due to oxygen intrusion (Schuurkes, Kempers et al . 1988, Lamers, van Roozendaal et al. 1998). 2.3 Treatment Techno log ies for Remov ing Sulphate from Water There are two different branches of treatment systems that can be used to remove sulphate from water. These are chemical and biological treatments. Some of the 10 most popular physico-chemical treatments include reverse osmosis, ion exchange and distillation. With reverse osmosis, water-containing sulphate is forced through a semi-permeable membrane. These units are used both for small-scale operations such as for home use, as well as for industrial purposes. A household unit typically can release 3 gallons of treated water per day and produces 1 gallon of treated water for every 4 to 10 gallons of water it is supplied (Minnesota-Department-of-Health 2006). Large scale industrial units in Florida used to treat the salinity in sea water can have a capital cost anywhere between $1341 to 2379 m" 3day" 1 per unit of daily capacity and an operation and maintenance cost between $1.02 to 1.54 rrf3 per unit of production (United-Nations-Environment-Programme 1997). With an ion exchange unit, sulphate-containing water is passed through the unit where it comes into contact with a resin. The sulphate ions in the water exchange places with other ions, usually chloride, which is on a resin. When the resin is full to capacity with sulphate, it must be regenerated with a salt solution. This is the most common system for removing sulphate in water for industrial purposes. However, the unit requires proper operation and maintenance to ensure that it continues to function properly. An ion exchange system designed to treat house water (max 12 gpm) can remove up to 2500 mg L"1 S 0 4 2 " and costs $2350 U.S.D (RainDance-Water-Systems 2006). 11 In a distillation system, the sulphate concentrated water is boiled and the steam is collected and condensed in another container, leaving the sulphate behind. Units typically require about four hours to produce 1 gallon of treated water, which requires a considerable amount of energy, making distillation impractical for treating large quantities of lake water (Minnesota-Department-of-Health 2006). While chemical treatments typically have high efficiencies of removing sulphate, they require considerable capital to set-up and maintain. In addition, they require high inputs of energy to operate which farmers are unlikely to have near the water reservoirs that require treatment. For this reason, the focus of this literature review is on biological sulphate reduction treatments. 2.3.1 Biological Sulphate Removal Processes Biological sulphate reduction is performed by microorganisms known as sulphate reducing bacteria (SRB) . Before reviewing the different treatment systems that utilize these bacteria, these key organisms are discussed further. 2.3.1.1 SRB - Growth Conditions Sulphate reducing bacteria are a ubiquitous group of microorganisms that are unique in their ability to take S 0 4 2 " and use it as a terminal electron acceptor (TEA) in the consumption of carbonaceous materials. Although S R B are a morphologically and phylogenetically diverse group, they are viewed as being physiologically unified because of this ability (Fauque 1995; Huycke and Gask ins 2004). Sulphate reducing 12 bacteria are generally considered strict anaerobes and the presence of oxygen inhibits their growth and SO4 2" reduction activity. The presence of oxygen does not kill S R B but instead places them into a dormant state. A recent study utilizing fluorescent in situ hybridization (FISH) and microelectrode probes found S R B distributed evenly throughout a biofilm, but SO4 2" reducing activity was limited to the anoxic zone (Okabe, Itoh et al . 1999). Sulphate reducing bacteria, as a group, are able to tolerate a range of environments with varying temperatures and pH. Psychrophil ic species have been observed in environments with temperatures at -1.7 °C (Knoblauch, Sahm et al. 1999) as well as thermophilic species in temperatures of 71 °C (Nakagawa, Hanada et al . 2002). In general, S R B can proliferate in a pH environment ranging from 5.5-9.0 with 7.5 being considered the ideal pH for most S R B strains (Postgate 1979; Fauque 1995; IWATaskGroup 2002). Sulphate reduction has been recorded from acid mine drainage areas with much lower pH values. Tuttle, P.R. et al . (1969) found that S R B were able to survive in an environment with a pH of 4 while removing approximately 700 mg L"1 S 0 4 2 ~ . It is thought that the S R B are able to form biofilms around alkaline substrates, which allow them to survive these otherwise harsh environments. A strong reducing environment is required for S R B to proliferate/with E h values below -150 mV being ideal (Postgate 1979; Fenchel , King et al. 1998). Several studies have indicated that S R B are typically found only within the top 10 cm of sediment in aquatic environments. Within rice paddy soil, Stubner (2002) 13 located S R B at depths of 5-10 cm using polymerase chain reaction ( P C R ) technology which targeted the 16s rDNA of Desulfotomaculum l ineage. Li , Purdy et al . (1999) used oligonucleotide probes complementary to the 16S rRNA of the major phylogenetic groups of S R B to search for S R B within the sediments of a freshwater lake in Japan. There results showed the presence of 3 genera of S R B (Desulfobulbus, Desulfobacterium and Desulfovibrio) within the first 6 cm of sediment. Below this depth, the amount of rRNA recovered was lower by more than a factor of 4. Within marine arctic sediments, 73% of all the S R B detected using FISH and rRNA slot blot hybridization were located at a depth of 2.25 cm. Below a depth of 10 cm no S R B were detected (Ravenschlag, Sahm et al . 2000). This information is later used in the design of the mesocosm experiments. The primary nutritional requirements of S R B , excluding carbon/energy sources which will be discussed later, are the need for nitrogen and phosphorous. These elements are important for both cellular growth and energy production. The amounts required depend both on the S R B density at their location and on their physiological state. Nitrogen can be supplied in the form of NH4 + and NO3" but many S R B have the ability to fix nitrogen (Postgate 1982). Phosphorus is generally assimilated as P 0 4 3 " . The limiting C :N ratio ranges between 45:1 -120:1 (Okabe, Nielsen et al. 1992) while the C : P ratio is between 400:1 - 800:1 (Okabe and Charackl is 1992). 14 2.3.1.2 SRB - Sulphate Reduction Reaction Sulphate reducing bacteria utilize a variety of organic carbon substrates to provide carbon for growth and a source of electrons for the production of energy. Using lactate as an example (Equation 3, Benner, Blowes et a l . 1999) in a simplified SO4 2 " reduction reaction, the S R B oxidize the carbon substrate to produce bicarbonate (HCO3) and reduce the S 0 4 2 " to sulphide (S2~), which then combines with hydrogen ions to form H2S gas. 2CH3CHOHCOO" + 3 S 0 4 2 " + 2 H + -> 6HCO3" + 3 H 2 S (3) The S 0 4 2 " reduction reaction has a direct effect on the pH of its local environment. Equation 3 indicates that the reaction removes protons to form H2S, which can then escape as a gas, thus permanently removing the protons from the environment. This results in an increase in the pH. In addition, the bicarbonate formed can serve as a buffer to the system and can also bind with additional hydrogen ions to form H 2 C 0 3 . A s a result, the activity of S R B results in an increase in the alkalinity of the S R B ' s local environment. While S R B can assimilate a small amount of the sulphur, the vast majority is released as either H 2 S gas or remains dissolved in the local aquatic environment as S 2 " , HS" or H 2 S species. This produces a distinct odour, which can act as preliminary indicator to the presence of S R B . Once released, the H 2 S can either escape into the atmosphere or undergo further reactions. If the local environment contains metal 15 cations, then the H 2 S can react to form metal sulphides. This results in giving the sediment a characteristic black colouring. This coloration is often used as an indicator to the presence and activity of S R B . Alternatively, as the H 2 S gas rises through the sediment or aquatic environment, it enters oxic zones. Within these zones sulphur oxidizing bacteria can be found which can oxidize the H 2 S to elemental sulphur, or even further to S 0 4 2 ~ (Odom and Singleton 1993; Barton 1995). 2.3.1.3 SRB - Community Structure In addition to requiring an anaerobic, reduced environment as well as a supply of suitable nutrients, S R B also have to contend with competition for carbon and energy sources. S R B compete for carbon substrates and micronutrients with both aerobic bacteria and other anaerobic bacteria. The major competitors use the following as terminal electron acceptors; 0 2 (aerobes), N 0 3 " (denitrifiers), M n 4 + (manganese reducers), F e 3 + (iron reducers) and C 0 2 (methanogens). Of these, only methanogenesis is less thermodynamically favourable than sulphate reduction. In the production of H 2 S from S 0 4 2 \ S R B are able to create conditions that encourage S R B proliferation, while discouraging the growth of other competing organisms. H 2 S , in an environment with a pH of 7 and a temperature of 15°C, has an equilibrium E h of approximately -320 mV. This condition favours the activity of anaerobic organisms as free oxygen is quickly depleted. In addition, H 2 S is toxic to 16 many organisms and this also works to make conditions favourable for S R B . Although H 2 S has been shown to have an inhibitory effect on S R B growth and activity, noticeable at total sulphide (TS) levels of 200 mg L"1, the effects are known to be reversible if the sulphide is removed (Choi and Rim 1991; Okabe, Nielsen et al . 1992; Reis , Almeida et al. 1992). Depending on the system being used to grow S R B , the sulphide can be removed through sparging with nitrogen (Reis, Almeida et al . 1992) or by precipitating the sulphide out as metal sulphides (Mitsch and Wise 1998). While H 2 S is the more toxic form of sulphide, this species is most prevalent at pHs less than 7. By keeping the pH greater than 7, it is possible to reduce the toxic effects of sulphide. 2.3.1.4 SRB - Carbon sources One of the most important tasks when utilizing S R B for bioremediation purposes is to select an appropriate carbon source. S R B were initially found to be able to utilize low molecular weight molecules like C 0 2 , H 2 , lactate, ethanol, methanol, propionate, pyruvate, fumarate, acetate, and butyrate (Postgate 1979). More recently, S R B species have been shown to be able to utilize more complex petroleum hydrocarbons such as naphthalene, 1,3,5-trimethylbenzene, and heating oil (Kleikemper, Schroth et al. 2004) as well as aliphatic and aromatic hydrocarbons like n-alkanes and alkylbenzenes (Rueter, Rabus et al . 1994). S R B do not use natural biopolymers like starch, glycogen and proteins or lipids, and must depend on the 17 activities of other microorganisms (MO) to provide them with fermentation and degradation products. 2.3.2 Treatment Systems Although S R B were first discovered in 1895 by Beijerinck (Postgate 1979), it was not until a paper was released in 1969 which suggested that S R B could be used to treat acid mine drainage, that large number of studies were performed to characterize the S R B and their abilities (Tuttle, P.R. et al. 1969). However, the focus of these studies has not been on their ability to reduce S 0 4 2 " but primarily for their ability to produce sulphide. The sulphide then reacts with metal ions in solution and precipitates them as well as increases the pH of the local environment. The decrease of S 0 4 2 ~ is thus seen as more of a side effect rather than the goal. For this reason, there has been minimal effort undertaken to study how to maximize S 0 4 2 " reduction when heavy metals are not present in the solution and in several studies the S 0 4 2 " reduction rates are not even measured. Biological treatment systems are typically grouped as either active or passive treatments. In active systems, energy intensive equipment is required to transport fluids and materials within the systems, as well as to monitor conditions within the system and make changes as required. Pass ive systems, while they sometimes contain pumps and piping to ensure transport within the system, typically rely upon position/gravity to transport water through the system and energy is supplied by the 18 natural components within the system to maintain the desired biological reactions. Pass ive systems are generally more simplistic in design and operation. A brief overview of the different types of systems available is described further. 2.3.2.1 Active systems Upflow anaerobic sludge blanket reactor (UASB) : U A S B reactors are a combination of both physical and biological processes. The physical component is the separation of solids and gases from the liquid and the biological component is the degradation of decomposable organic matter under anaerobic conditions in the sludge blanket. The influent to be treated is pumped into the bottom of the reactor, where it comes into contact with the sludge blanket which contains active bacteria (Figure 2). At high organic loading rates, the biogas production guarantees that there is sufficient contact between the substrate and the biomass. In the liquid phase of the reactor, a U A S B approaches a completely mixed reactor design. Sludge Blanket t t 7 1 Influent Figure 2 - UASB reactor 19 Packed bed reactor (PBR) : These reactors ideally follow a plug flow design and typically consist of a column containing immobilized microbial cells. The inlet can either be at the top or bottom of the column, depending on whether downwards or upwards flow is preferred. In cases where S R B are utilized, H 2 S gas is one of the products, so upwards flow is preferred. G a s lift reactor: A gas lift reactor has a similar design to that of the packed bed reactor, in that the bacterial cells are immobilized. However, in this design, the cells are maintained in suspension in the reactor through the use of a gas stream, rather than settling to the bottom of the reactor, thus avoiding a steep pressure drop. In almost all treatment systems, both passive and active, the S R B that are used are generally not pure cultures. Instead, the S R B are obtained from local natural sources, such as sediment, sewage waste, or animal manure. Some of the advantages to using these natural sources are that there is usually a consortium of bacteria present that can utilize a wider range of carbon sources, and also it is very inexpensive to obtain the inoculum. Pure cultures would not survive in these systems, as they would rapidly be out-competed by natural organisms. Both active and passive systems have been created which utilize the S R B ' s unique dissimilatory SO4 2" reduction ability. Active systems typically require a higher cost to set-up and operate but in turn generally have faster sulphate reduction rates (SRR) . This higher cost is primarily due to the carbon source supplied to the bacteria, 20 usually a defined compound, as well as the running cost of energy to keep the system active. Stoichiometrically, an S R B bioreactor that was fed ethanol would cost approximately $800 per tonne of sulphate reduced based on a commercial ethanol price of 40 cents per litre. Using a gas mixture of C 0 2 and H 2 to support S R B gives an estimated cost of $500 per tonne of sulphate reduced, assuming the stoichiometric ratio of 4 moles of H 2 are consumed per mole of sulphate reduced. Several types of active bioreactors which bioremediate SO4 2" are described below. 2.3.2.2 Active system applications in treating SO4 2 Several different types of active systems designed to treat S 0 4 2 ~ , while utilizing S R B , have been developed over the years. While they may vary in design and nutrients supplied, they do share the same overall purpose. All of the systems discussed here were developed for the removal of heavy metals from water. No reports were found in the literature of active systems developed solely for the purpose of removing sulphate from high sulphate concentrated waters. The Paques Company (T. de Boerstraat 24, 8561 Ab Balk, The Netherlands ) creates the most well known commercial S R B bioreactors. These systems were successfully put into practice in 1992 at zinc refinery in Budelco (The Netherlands), for the treatment of groundwater polluted with zinc (Scheeren, Koch et al . 1993). Based upon work completed by Barnes, Janssen et al . (1991), these THIOPAQ™ 21 reactors utilize an upflow anaerobic sludge blanket (UASB) in order to treat groundwater contaminated with sulphate and heavy metals at metal refining sites. The sludge, which contains the S R B , resides at the base of the tank, while the influent ( S 0 4 2 " contaminated ground water) flows upwards through the sludge. After passing through the U A S B reactor, the influent then enters a submerged fixed film reactor that converts the sulphide to elemental sulphur aerobically. The system has a loading rate of 3000 L hr"1 and is maintained at a pH of 7 and a temperature of 20°C. Further design details for this system are detailed in Table 1. Table 1 - Active system operating parameters Reference Temperatur e and pH Treatmen t System Carbon Source Retention Time Sulphate Reduction Rate Barnes, Janssen e ta l . 1991 20 °C pH = 7 U A S B Ethanol 4 hours 7.7 g L V 1 Yamaguchi, Harada et al. 1999 35 °C pH = 7 U A S B Wastewater (2000 mg C O D 9.9 days 70.7 mg L"1 d 1 Jong and Parry 2003 25 °C pH = 4.5-7.5 Packed Bed Reactor Lactate 14.4 hours 3.04 g, L"1 d" van Houten, Yun eta l . 1997 55 °C ph = 7 Gas lift reactor 80 % H 2 and 20 % C 0 2 3.3 L hf1 4.5 hours 7.5 g L-'d"1 Annachhatre and Suktrakoolvait 2001 29.7 °C pH = 7.2-7.8 U A S B Molasses 0.019 hours 80% SO/-removal After their success with the U A S B reactor in Budelco, Paques developed a gas lift reactor which utilized a mixture C 0 2 and H 2 as an electron source. This system was developed to treat acid rock drainage at Kennecitt 's Bingham Canyon (Utah) copper mine, van Houten, Yun et al . 's (1997) design consisted of an internal draft tube, 22 used immobil ized S R B in pumice stones, which are maintained in suspens ion in the reactor, to treat SO42". To maintain the pumice s tones in suspens ion , a gas recycle f low rate of 350 L hr"1 w a s needed. The reactor was maintained at 55°C and a pH 7.0 and a S 0 4 2 " loading rate of 18 g L" 1d" 1 was used . Yamaguch i , Harada et a l . (1999) utilized a 14.5 L U A S B to treat the high levels of C O D in sulphate contaminated wastewater (1000 mg S 0 4 2 " - S L"1). Instead of adding a speci f ic carbon source to support S R B activity, the sys tem relied solely on the wastewater supplying all the necessary nutrients. The sys tem had lower S R R s than the other U A S B s ment ioned here. In order to keep sulphide levels within the column low, a recycle sys tem was used to pass the liquid in the reactor through a sulphide adsorpt ion co lumn, which was packed with ferrous oxide pellets. Annachhat re and Suktrakoolvait (2001) operated a 5.7 L U A S B simply to observe whether mo lasses could support sulphate reduction by S R B , even in the presence of methane producing bacter ia, which acts as a competitor to S R B for nutrients. The purpose of this reactor was to treat wastewater from a tapioca/starch plant. The effluent was sent to a settling tank where the washed out b iomass was removed. A synthetic wastewater was provided to the sys tem, which included F e C b to convert the H2S to F e S . The influent had a very short res idence time within the system (0.019 hrs), but low concentrat ions of S 0 4 2 " were provided (450 mg L"1 S 0 4 2 " ) . Eighty per cent S 0 4 2 " removal was ach ieved when the C O D : S ratio was 10. S ludge from an anaerobic lagoon was used to seed the reactor. 23 Jong and Parry (2003) designed a bioreactor to treat acid rock drainage containing metals such as copper, nickel, zinc and arsenic. Their system used a 4.78 L packed bed bioreactor to remove sulphate and heavy metals. The bioreactor was filled with the >2 mm fraction of commercially available coarse pool filter sand and fed a solution with lactate, heavy metals and S 0 4 2 " (2500 mg L"1) at a rate of 2.61 mL min" 1. The influent was initially pumped into a reservoir, which was continuously gassed with N 2 in order to reduce the levels of oxygen. With a loading rate of 3.71 kg d" 1 m"3 of SO42", the reactor was able to remove more than 82% of the SO42". While these active treatment systems show high levels of SO4 2" reduction and have short residence times, these applications are not suitable for farmers for use in the field. These types of systems require a high capital cost to construct and operate, primarily due to the cost of the carbon sources used, as well as due to the cost of power consumption and additional reagents required. For this reason, various passive treatment systems were investigated. 2.3.2.3 Passive Systems The main advantages to using passive systems to treat S 0 4 2 " are their low cost to set-up and operate as well as the low levels of maintenance required. Their primary disadvantage is that they are not as easily controlled as active systems. In addition they usually require a large amount of land on which to be constructed and also require longer retention times. Finally, as these passive treatments are generally 24 operated outside, they are affected by changing environmental conditions such as temperature. These passive treatment systems are usually described as either wetlands or treatment ponds. Most of these systems to date are designed almost solely treating wastewater or acid mine drainage. In the latter case, they are utilized primarily for metal removal, and in some cases the sulphate levels are not even monitored. Several different types of passive systems are briefly d iscussed below followed by a literature review of how they have been applied to treat SO42". Wetlands: Wetlands are complex biological systems that are home to many interconnected nutrient cycles and biotic associations. To be considered a wetland system, the water table must be at or above ground level sufficiently during the year so that the soil remains saturated (Gelt 1997). Within the system is vegetation adapted for life in saturated soil conditions. This vegetation aids in effectively filtering sediment and bioavailable nutrients from runoff waters as well as helping to control runoff volumes. They also provide a source of carbon to microorganisms when they decay. Pollutants are removed by infiltration, sedimentation, physical filtering, and biological uptake and conversion. Another prime advantage to using wetlands is that they can be visually appealing and provide habitat for migratory waterfowl. Natural wetlands are difficult to use as treatment systems, primarily because regulatory agencies consider them to be part of the receiving water, which means that the influent water must meet certain standards before it can be released into the 25 system which often means pre-treatment is required. In addition, they must be located close to the water which is to be treated to be of any practical use. Constructed wetlands: A constructed wetland is designed to simulate the natural system. Their prime advantages are that they can be built c lose to the water requiring treatment, rather than depending on a natural wetland being present, and they can receive more heavily polluted water, as the influent does not have to meet regulatory standards. Constructed wetlands typically have a liner to prevent leaks and are filled with local soils, gravel, and sand and are planted with native wetland plants, such as cattails or bulrush. Constructed wetlands fall under two main categories, depending on the type of hydraulic flow in the system. In free surface flow systems, which mimic natural marsh systems, the water's surface is exposed to the air and the hydraulic flow is above the sediment. In sub-surface flow the influent moves through a permeable medium and the water level is maintained below this material (Kent 1994). The advantage to using sub-surface flow is that it requires less land area for water treatment than free surface flow. Free surface flow systems typically have a depth between 0.1-0.6 m and a hydraulic retention time of 7-15 days. Sub-surface flow systems typically have a depth between 0.3-0.6 m and a hydraulic retention time between 3-14 days (Reed, Crites et al . 1998). Pond/Lagoon systems: These natural treatment systems, primarily used to treat wastewater, consist of a shallow body of water, and are characterized by the dominant biological reaction which takes place within them. Aerobic ponds are very shallow (between 30-45 cm) which allows light to reach the entire depth of the pond 26 to enhance the growth of oxygen producing algae. They rely solely on the activities of aerobic digestion and oxidation to treat the water entering the system. A facultative pond achieves treatment through a combination of aerobic and anaerobic digestion. They are typically between 1.2-2.5 m in depth and contain two major zones of microbial activity. The upper zone is aerobic with oxygen being supplied by photosynthetic algae, while the lower zone is anaerobic. The decay of algae results in deposition and subsequently anaerobic decay. Anaerobic ponds receive a heavier organic load than aerobic or facultative ponds, in order to ensure anaerobic conditions. They also possess the deepest depths (between 2.5-5 m) (Reed, Crites e ta l . 1998; Shilton 2005). In Situ Treatment Systems: An in situ system involves the remediation of a site by using the natural processes already present at that site, contrary to an ex situ system where the contaminated matter is removed and cleaned off site. These treatment systems can be accelerated by adding substrate or nutrients to the site to stimulate the growth of a target consortium of bacteria. These target bacteria are usually indigenous to the site; however enriched cultures of bacteria (from other sites) that are highly efficient at degrading a particular contaminant can be introduced into the aquifer. 27 2.3.2.4 Passive system applications in treating S 0 4 2 Fortin, Goulet et al. (2000) observed the cycling of Fe and S in a constructed wetland situated in Kanata, Canada . This was a young wetland, built in 1995, and was provided no additional carbon source, so that it had yet to accumulate much organic material in its sediment. The system was colonized primarily by Typha latifolia and only surface flow was observed. The flow in the wetlands was less than 0.5 m s'\ After observing the S 0 4 2 ~ in the water column over the course of 9 months, a total drop of 74 mg L"1 was observed from 132 mg L"1 S 0 4 2 " in Dec. 97 to 58 mg L 1 S 0 4 2 " in Aug , 98. Design features for this and the other passive systems discussed further are listed in Table 2. Table 2 - Passive system operating parameters Reference S ize Total depth/depth of organic substrate (cm) Carbon Source Sulphate Reduction Rate (mg L 1 d- 1 ) Mitsch and Wise 1998 2 cells 927/868 m 2 140/46 Animal waste/grain 0 Fortin, Goulet et al. 2000 6.37 x 1 0 b m 2 150/0 Nothing 0.3/0.2 Mclntire, Edenborn et al. 1990 27.4 m 2 75/46 Compost 0.19-57.6 Benner, Blowes et al. 2002 60 mz 360 Decaying leafs, wood chips 15.3-10.5 Stark, Will iams et al. 1995 0.4 mz 15/15 SMS/whey N/A 28 Mitsch and Wise (1998) demonstrated that even if you provide a carbon source, to a passive treatment system containing S R B , it does not guarantee that S 0 4 2 " reduction will occur. The system was designed to treat acid mine drainage and remove dissolved iron. Nine different cells, the first 7 being aerobic systems while the final two were anaerobic, treated acid mine drainage to remove dissolved iron and increase the pH of the water. The anaerobic cells received a 46 cm layer of fermway (animal manure mixed with offal). Instead of allowing the water to enter the anaerobic cells within the water column, or on the surface, the inlet water was released into the sediment through subsurface infusion pipes. This was to increase the retention time in the anaerobic cells, as well as ensure the contaminated water had as much contact with the sediment as possible. With an average hydraulic conductivity of 4.6 cm d" 1 the anaerobic cells received water with a pH of 2.82 and a S 0 4 2 " concentration of 1216 mg L"1. Unfortunately, the SO4 2" concentration increased within the anaerobic cells (1256 and 1286 mg L"1 S 0 4 2 ~ , cells 1 and 2 respectively), rather than decreased. It was suspected that the sulphur compounds within the water were re-oxidized back into S 0 4 2 " and that the carbon source was not utilizable by the S R B . Mclntire, Edenborn et al. (2000) achieved success in using a smaller constructed wetland to treat acid mine drainage when they used compost (straw/manure/corncobs) as a substrate. When the influent was directed to pass through the sediment by subsurface pipes, the pH of the effluent was observed to 29 rise from 2.5 to 6.5. The S R R s were highly variable, and the heterogeneous nature of the compost suggested as the cause of this variation. The most recent treatment method developed to treat S 0 4 2 " contaminated water in situ is the permeable reactive barrier (PRB) , which was first suggested by Blowes and Ptacek (1992). These barriers are installed into a stream and consist of a physical support, which immobilizes the S R B , and a solid organic substrate matter to provide nutrients. These treatments rely on the natural flow of contaminated groundwater through the P R B with S 0 4 2 " reduction occurring within the barrier. One of the advantages to this treatment system is that the organic substrates do not have to be replenished continuously but instead are degraded slowly over an extended period of time. Similarly to the natural and constructed wetland treatments, the S R B inoculum source is most often sediment removed from the local area. A P R B installed in 1995 near Sudbury, Ontario to treat acid mine drainage was operated for 3 years using an organic substrate mixture of municipal compost, leaf compost, wood chips and limestone. With an estimated residence time of 90 days and an inlet concentration of 2592 mg L"1 S 0 4 2 \ the overall rate of S 0 4 2 " reduction was found to decline from 15.3-10.5 mg L"1d 1 (Benner, Blowes et al. 2002). Instead of using a large constructed wetlands or P R B to treat high sulphate water, Stark, Will iams et al . (1995) developed a small scale mesocosm to simulate a constructed wetlands treatment. P laced in a greenhouse, each mesocosm (160x25x15 LxWxD) was filled with spent mushroom substrate as a carbon source, 30 followed by a weekly injection of 600 mL of whey. The influent (simulated acid mine drainage water) into the mesocosm was held constant at 30 mL min" 1 with a residence time of approximately 33 hours. Each mesocosm had an inlet concentration of 50 mg L"1 Fe for the first 60 days, and 150 mg L"1 Fe for the next 60 days. Unfortunately, the purpose of this system was to remove iron from the influent, rather than remove SO42", so no SO4 2" concentrations were provided, except to say that the simulated mine water contained ferrous sulphate and sulphuric acid. The mesocosm was successful at removing 6 5 % of the iron, which Stark suggests was due to the S R B actively producing sulphide to bind the iron. 2.3.2.5 Carbon Sources Used in Treatment Systems Lactate is a utilizable carbon source and electron donor for S R B and has been utilized frequently in small scale experiments such as an upflow anaerobic fixed film reactors (El Bayoumy, Bewtra et al . 1999), or in laboratory experiments with S R B (Mitsch and Wise 1998; Drury 1999; Edenborn and Brickett 2001). Some S R B strains have been found which are capable of using glucose or sucrose but often these materials are first fermented by other organisms and it is their by-products which the S R B use (Lloyd, K lessa et al . 2004). Molasses is another substrate that has been used as a carbon source in S R B remediation. Considered an inexpensive and easily obtained carbon substrate, its primary constituent is sucrose. While it has been used successfully, particularly in upflow anaerobic sludge blanket reactors (described in detail above) it can also be utilized by many other M O which can then 31 out-compete the S R B (Maree and Strydom 1987; Annachhatre and Suktrakoolvait 2001). While it is common to see simple defined compounds, such as sodium lactate or sodium acetate, supplied to S R B for small laboratory scale operations (White and Gadd 1996; Utgikar, Harmon et al . 2002) or when enriching S R B cultures for later use (Hauser and Holder 1986; Lloyd, K lessa et al . 2004) when a large scale passive treatment system is utilized, a natural complex organic material is more often supplied. This is due to the fact that these materials are usually inexpensive and quick to obtain, while the use of pure compounds is more costly. Ideally the material is rich in an organic matter that the S R B are immediately able to utilize or that is easily fermented by other M O in the community into forms that the S R B can use. Very often researchers in this field simply use whatever natural organic materials are available on hand. This can lead to situations where very little SO4 2" reduction is obtained or even increases in the SO4 2" levels. This was observed in the Mitsch and Wise (1998) study, previously mentioned above, where animal waste mixed with grain that had been fed to livestock and then recollected, was used as the carbon substrate and no sulphate reduction was observed. Thus there is a need to test the suitability of available complex carbon sources for supporting S R B before using them in a large-scale system. In some cases , the natural materials provided within lakes are more capable of supporting S R B activity than an added substrate. Webb, McGinness et al. (1998) 32 compared the metal removal efficiency of natural wetland sediments that contained mainly grass and reed vegetation, to constructed wetland sediments that consisted of an anaerobic cell containing straw and manure. Overall the natural wetland sediments were able to produce higher concentrations of sulphide (12 mmol L"1 sulphide) than the constructed wetland sediments (4.5 mmol L"1 sulphide) after 7.5 days. Hay and manure are common substrates provided to passive treatment systems. Barton and Karathanasis (1999) found that they could remediate a failed surface flow wetland, designed using spent mushroom compost as a carbon substrate, by replacing the substrate with a 50 cm layer of hay and manure and turning the system into a series of subsurface flow anaerobic cells and surface flow aerobic cells. These alterations enhanced the mass S 0 4 2 ~ reduction by 5 5 % (influent 3034 mg L"1 SO42", effluent 1352 mg L"1 S0 4 2 ~) . Dairy whey has also been used in several treatment systems. It is considered a suitable carbon source for S R B because lactose is a major component within whey. In a column experiment, designed to be used in situ within contaminated mines, 1250 mL of whey (50 g of lactose per litre) and 500 mL of cow manure was added to 19.6 L of acid mine water. After 203 days S R B were responsible for a S 0 4 2 " drop of 893 mg L"1 or a rate of 4.4 mg L" 1d" 1 S 0 4 2 " (Christensen, Laake et al . 1996). Dairy whey was also used as a carbon amendment in the Stark, Will iams et al. (1995) study, mentioned above. 33 Another limitation in choosing an acceptable carbon source for large-scale treatments is that there are very few studies to date that compare the effectiveness of complex carbon sources under the same operating conditions. In the majority of studies, experimenters simply use a material that has been used previously with success. However, since their experimental designs are usually different with varying loading rates and dissimilar inoculums, there is no consistency with the rates obtained. This makes determining the effectiveness of a particular carbon material difficult. One study which did test the effectiveness of a variety of different complex carbon sources was conducted by Waybrant, Blowes et al . (1998). In 1 L glass reaction flasks, 8 different mixtures of organic material were added, as described in Table 3 and the flasks were then filled with simulated mine drainage water with a S 0 4 2 " concentration of 3620 mg L"1. Table 3 - Organic substrates used in batch reaction mixtures as dry weight composition (wt %) (Waybrant, Blowes et al. 1998) Batch Sewage Leaf Wood Sheep Sawdust Cejlulose Mixture Sludge Mulch Chips Manure 1 100 2 100 3 100 4 10 65 25 5 20 10 25 20 25 6 60 40 7 15 60 15 10 8 100 34 Over a period of 60 days mixture #5, containing 5 different organic sources, had the highest sulphate reduction rate (4.23 mg L"1d"1g"1), while mixture #1, which contained only sewage sludge, had the lowest (0.14 mg L" 1d" 1g" 1). Total combustible carbon measurements revealed that those mixtures with the slowest S R R s (mixtures 1 and 2) also had the lowest carbon content, while those with the highest carbon content (mixtures 3, 5, 6, and 7) had the highest S R R s (2.15, 4.23, 1.52, 1.69 mg L" 1 d" 1 g"\ mixtures 3, 5, 6, and 7, respectively). The overall conclusion of this study was that a mixture of different organic materials was preferable to using a single carbon substrate in supporting S R B activity. 2.4 Exper imental Des ign Cons idera t ions Based Upon the Literature Review After completing this literature review of different passive systems and the carbon substrates provided, it was decided that a study, similar to that of Waybrant, Blowes et al . (1998) was required, as there needs to be further study into comparing the effectiveness of different organic substrates, especially agricultural waste materials, at supporting S R B activity. This initial study would be performed as a bench scale experiment. Four different agricultural materials: hay, barley, silage and molasses, commonly found on farms throughout Canada and the U.S.A. ( U S D A - N A S S 2002) and personal communication with Veira (2004) were tested for their effectiveness as nutrient sources for growth of S R B and reduction of S 0 4 2 \ A s the overall goal of this 35 research was to try to build a passive treatment system that could be built and implemented by farmers, it was imperative to select materials that the average farmer would have on hand or could obtain with ease. One of the difficulties in designing passive treatment systems is that a small fraction of the organic matter placed in passive treatment systems are likely to be immediately available to S R B (Benner, Blowes et al. 1999) because S R B typically require simpler molecules (organic acids, alcohols, or H 2) for energy (Widdel 1988). Anaerobic degradation of the complex material to simpler compounds by fermentative microbes is often required and may limit the rate at which utilizable substrates become available to S R B . For this reason, 4 different agricultural materials were tested for their ability to support sulfate reduction by S R B . Hay: Hay has been used previously in constructed wetlands treating acid mine drainage (AMD) (Barton and Karathanasis 1999), although its use was primarily to add structural support and surface area upon which the S R B can grow, which is also an important function. However, hay can undergo anaerobic fermentation and have its soluble carbohydrates converted into lactate, a known carbon source for S R B . This fermented hay is otherwise known as silage. Silage: Si lage is used as a feedstock primarily for the use of cattle, sheep and other cud-chewing animals. In the creation of si lage there are 4 distinct stages. These are aerobic, fermentation, stable and feedout stages. 36 Aerobic Stage In the aerobic stage, the chopped forage is placed in a silo and the breakdown of plant sugars to CO2 and water occurs. Plant proteases become active and begin the breakdown of the proteins to produce amino acids, ammonia and peptides. The aerobic stage can be halted by either adding additional crop or by placing a polyethylene tarp over the silage pile. It is important to minimize the time in the aerobic stage since the supply of fermentable carbohydrates is constantly decreasing during this stage. Once all the oxygen has been consumed by these aerobic reactions then the aerobic stage is over and the fermentation stage begins (Barnett 1954; McDonald 1981). Fermentation stage During the fermentation stage, a group of M O known as lactic acid bacteria (LAB) become the most important microflora. Lactic acid bacteria convert plant sugars into lactic acid, which in turn is responsible for preserving the silage. Homofermentative L A B follow a glycolytic pathway which produces 2 moles of lactate for every mol of sugar fermented. In contrast, heterofermentative LAB produce a wide variety of materials such as organic acids, ethanol, acetate mannitol and C 0 2 , in addition to lactate. The products of fermentation using heterofermentative L A B depend upon the starting sugar substrate (McDonald 1981). By producing lactic acid, the pH of the silage is decreased which prevents other organisms such as Clostridia, 37 Enterobacteriaceae, yeasts and molds from forming (Woolford 1984). All of these organisms compete with the L A B for the plant sugars, but in contrast to the L A B they do not aid in preserving the silage. The fermentation stage typically lasts between 7-21 days (McDonald 1981). Stable stage The stable stage begins after the activity of the LAB has finished, either because the pH has dropped to below 4 or no more sugar remains (McCullough 1978). If the silo has been properly sealed then little biological activity is expected, due to the low pH. Feedout stage At this point the silo is opened and the silage removed to feed to the animals. The greatest amount of dry matter loss occurs in this stage as the introduction of oxygen to the silage pile stimulates the growth of yeasts and molds. Barley: Barley, as a cereal grain, contains high levels of starch which can be broken down into simple sugars, such as sucrose, which are utilizable by the S R B (Lloyd, K lessa et al. 2004). It is also one of the highest grain stocks present in Canada with 3489 x 10 6 tonnes in July 2005 (Canada 2005). 38 Molasses: Molasses has been used in many previous studies as a suitable carbon source for SRB (Annachhatre and Suktrakoolvait 2001, Maree and Strydom 1987, Lebel, do Nascimento et al. 1985). Sucrose is easily biodegradable and the fermentation products it provides, low chain fatty acids, are also utilizable by SRB. After determining which of these organic substrates are effective at supporting SRB, these materials would be tested on a larger scale. It was decided that a passive system, due to their lower cost and simpler design, would be the best choice for farmers to use in the field to treat SO4 2" concentrated water. Therefore, a simulated passive system, similar to what would be expected to be used by farmers in the field, would be constructed to test the effectiveness of the organic materials at supporting SO*42~ reduction by SRB. In addition to testing agricultural organic materials, a second system would be supplied with materials removed from a natural lake, similar to the Webb, McGinness et al. (1998) experiment where they tested sediments removed from natural and constructed wetlands and found that the natural wetland promoted the highest SRB activity. The Lac du Bois Grasslands Provincial Park was the region selected for obtaining these natural materials. Each system would be operated in duplicate and referred to as a mesocosm experiment. In designing the passive system experiment, several parameters had to be examined. First, the system would need to be anaerobic, particularly in the sediment, which is where the SRB would be introduced into the system. In contrast to a wetlands treatment system, it was decided not to grow plants (cattails or 39 bulrushes) within the sediment, as this would introduce oxygen. In the mesocosm which received natural materials removed from a lake, it was decided to seed the water's surface with biotic matter (algae/diatoms/microbes) which was observed floating on the surface of the lake, as this would supply the sediment with a continual carbon and electron source as this matter died and decayed. In addition, it would introduce oxygen only into the upper layers of the water which would hopefully allow anaerobic conditions to remain within the sediment. To encourage anaerobic conditions within the sediment, each mesocosm received a water cap of 18 cm. Dissolved oxygen measurements were taken to ensure that this water layer was sufficient to ensure the sediment was anaerobic. In addition, the water to be treated entered the system through the anaerobic sediment first, by passing through sub-surface pipes which lay upon the bottom of the sediment. While the addition of a tarp over the system would help to encourage anaerobic conditions, it was decided that this addition should not be made for two reasons. First of all it is possible that the actual system in the field would be too large to place a tarp over. Second, because this would not allow the growth of algae in the natural treatment system, which is needed to supply the system with a continual source of nutrients. A s mentioned previously in Section 2.3.1.1, S R B have been located primarily within the first 10 cm of sediment. For this reason, each mesocosm received a 10 cm layer of sediment within which S R B were found to exist, as an inoculum. 40 When determining a treatment pond's dimensions, 4 different modelling approaches are available: loading rate, empirical, reactor theory, and mathematical modelling. With the loading rate and empirical design models, the system is assumed to be a black box and either one parameter (MO population, flow, or B O D , for loading rate model) or numerous variables (solar radiation, flowrate, wind, for empirical model) are used to determine the pond's volume and area. These simplified approaches are commonly used to design facultative ponds, however Finney and Middlebrooks (1997) found that using the loading rate method did not provide consistent pond performance due to this model neglecting to consider how different hydraulic flows through the system could affect the pond's performance. Empirical design equations are derived from regressions of pond performance data and not from actual treatment mechanisms and do not provide accurate predictions for new systems being constructed (Shilton 2005). Mathematical modelling provides more accurate assessments of various pond designs, but requires a complex development of non-steady state equations for a multitude of variables (DO, algal cell mass, inorganic/organic carbon, pH, alkalinity, etc) which is too complex for the scope of this work. It was decided to utilize reactor theory, which applies process engineering to pond design, in order to determine the dimensions of the passive treatment system. Using reactor theory, it is assumed that the reactions in the system will undergo first order kinetics, and under these circumstances, the most effective hydraulic design is 41 plug flow. While it is impossible to achieve ideal plug flow characteristics in the system, several techniques exist which can enhance plug flow characteristics. First, instead of having one large treatment system, several small treatments should be placed in series. This is what is envisioned for use in the field. Second , the systems should be designed to be long and narrow, with length to width ratios greater than 4:1 (Shilton 2005). The mesocosms constructed in this study had length to width ratios of 7.88:1 and are similar to the mesocosms used by Stark, Wil l iams et al. (1995) to simulate a wetland treatment system for the remediation of acid mine drainage. Finally, the influent should enter the system in such a way as to dissipate inflow momentum in order to reduce short circuiting, which means that the influent passes through the system in a shorter than expected time, which results in less contact with the S R B . In order to prevent this, the influent in these mesocosms enters the system vertically through the sub-surface pipes. In addition to the two sets of mesocosms designed to simulate a passive treatment system, a second experiment was developed which would simulate an in-situ passive treatment, by amendment of a high sulphate lake with organic material. The effectiveness of the in-situ system can then be compared to that of the mesocosm systems. 42 2.5 Backg round on the Fie ld S tudy Si tes Lac du Bois, Lac du Bois Twin and Lake 52 (which was the lake utilized for the in-situ treatment, discussed further on in this thesis) are situated in the Lac Du Bois Grass lands Provincial Park. This park is located approximately 6 km northwest of Kamloops, B C , near where the North Thompson and Thompson rivers converge. It is one of the largest publicly owned grasslands in British Columbia, and encompasses over 15,000 hectares of protected wilderness. For the past 150 years the grasslands in the park have been used extensively for livestock grazing. This park contains a complex geology with a mixture of 3 different types of grasslands and several varied forest types. Beyond the grasslands are ponderosa pines, groves of aspens, and open Douglas-fir forests. Some of the different fauna, which inhabit this region, include: California bighorn sheep, mule deer, moose and waterfowl. There are many parcels of private land within the boundaries of and adjacent to the park. In order to perform any activities on lakes within these privately owned areas, permission must first be obtained from landowners. The soil in this area is primarily composed of chernozem, or black earth. This black-coloured soil contains a very high percentage of humus (3% to 15%), rich in phosphoric acids, phosphorus and ammonia. The bedrock geology contains a mix of 43 volcanic and sedimentary rock with glacial deposits due to the departure of the Pleistocene ice Sheet, more than 10 000 years ago. There are numerous small lakes and ponds spread throughout the park. Some of these water bodies are quite shallow and can dry up annually, or be dry for several years. According to the Ministry of Environment (2000), nearly all of the water bodies in this park are identified as C lass 3 under the Canada Land Inventory, Land Capability for Wildlife - waterfowl classification system. Water bodies, which have this classification, are reported to have slight limitations to the production of waterfowl. These limitations are due to either the climate or to other land characteristics that can affect the quality or quantity of the habitat. The productivity of life in these water bodies can be hindered during some years due to droughts. 44 3.0 3.1 Materials and Methods Lac du Bois Grasslands Park Lakes Sampling Two sampling visits were made to Lac du Bois and Lac du Bois Twin in 2004. The first occurred on July 6 t h and the second on October 4 t h . Based upon measurements of a map provided in Ministry of Environment (2000) it is estimated that Lac du Bois has a surface area of 3.75 x 105 m 2 while Lac du Bois Twin's area is 8 x 104 m2. Two sampling locations for each lake were selected at a distance of 6 feet from the shore. The two sampling locations for Lac du Bois are designated as LDB Rocky, located on the southern side of the lake, and LDB Algae, which was located on the eastern portion of the lake (towards the south). These locations were selected, not only because they were the most easily accessible, but also because they represented two distinct regions within the lake. LDB Rocky had little algae present within the water and the clarity within the water was high. In contrast, LDB Algae was thickly surrounded with Typha latifolia and a very thick mat of green/yellow organic matter (which was suspected to consist of algae) was observed floating on the water. Underneath this mat, a dense layer of degrading material was present. A wooden plank was needed for sampling LDB Algae, as this mat prevented wading into the lake. 45 a) b) Lac du Bois Twin, which has an oval shape, had sampling points at the northern and southern ends of the lake, which will be referred to as Site 1 and Site 2 respectively. During the July 6 t h sampling trip, samples of organic plant-like matter, suspected to be algae, were removed from both lakes. From Lac du Bois, samples were removed from both the floating mat at LDB Algae and also from the rocks on the lake floor at L D B Rocky. From Lac du Bois Twin, organic matter was removed from Site #1 both at the lake floor where it was attached to rocks and the middle of the water column where it were found suspended. The samples were placed into 50 mL tubes containing water already removed from the surrounding area. To this container, 2 drops of Lugol's solution was added to preserve the organic matter, as per instructions from Stockner (2004). Upon returning to Vancouver, these algae samples were sent to him for identification purposes. At each location, the depth to the lake floor was measured with measuring tape. Two probes were used, one to measure DO (dissolved oxygen), and a second to measure temperature and pH. The pH and temperature were measured using a 46 Mettler Toledo 1140 pH meter, while the DO was measured using a V W R Scientific Products S P 5 0 D Symphony probe. These probes were recalibrated at the beginning of the day. The pH calibration was confirmed with standard stock solutions of pH 7 and 9 before sampling at each location, while a wet cloth was used to calibrate the DO probe before sampling at each location. On the July 6 t h trip, the pH and temperature were measured 15 cm below the surface of the water, while on the second trip these measurements were made at 5 cm intervals. Unfortunately the DO probe was inoperable during the July 6 t h trip, but measurements were obtained during the Oct 4 t h sampling trip. Water was removed from the water column in 5 cm intervals for S 0 4 2 " and T S tests through the use of a syringe attached to a hose. At each depth the syringe was rinsed with water from that location and depth twice before adding 40 mL of water to the 50 mL tubes containing 8 mL of 2 0 % (w/v) zinc acetate. This was performed to preserve any sulphide present in the water as Z n S . After capping, the tubes were inverted several times to ensure mixing. The samples were stored in a cooler filled with ice, until returned to the Vancouver laboratory for analysis. In addition, at a depth of 20 cm, water was removed and placed into 250 mL containers to be analyzed back in the laboratory for ammonia, nitrate, phosphate, and soluble chemical oxygen demand (sCOD). 47 3.2 Laboratory S R B Inoculum Creat ion and Maintenance Sediment from Lac du Bois was removed from a location between L D B Rocky and L D B Algae where there was a distinct odour of H 2 S gas produced and gas bubbles released whenever the sediment was agitated. It is suspected that these gas bubbles contained H 2 S , or other gaseous products of anaerobic degradation such as methane, C 0 2 or H 2 . These two factors strongly suggested the presence of S R B . A 500 mL plastic container was filled to the brim with the sediment, brought back to the laboratory in Vancouver and placed in a refrigerator maintained at 4°C. This sediment was used as a starting inoculum in creating a laboratory culture of S R B . The protocol for creating and maintaining a laboratory culture of S R B is as follows. 1. Create a 10X concentrated S R B stock solution for Postgate B media (Postgate 1979). P lace 750 mL of distilled water in a 1 L flask, then add the following salts. K H 2 P 0 4 5 g M g S 0 4 • 7 H 2 0 20 g NH 4 CI 1 0 g F e S 0 4 • 7 H 2 0 5 g C a S 0 4 1 0 g Add a magnetic stir bar to the flask and let stir for 10-15 minutes. Fill the flask with distilled water so that the total volume is 1 L. 2. Using a clean 1 L flask, add 100 mL of the 10 times concentrated S R B stock solution and a magnetic stir bar. 3. Add 1 g of yeast extract and fill the flask to the 880 mL mark with distilled water. 48 4. Autoclave (Sanyo MLS-3780) the media containing flask, 200 mL of distilled water, a 160 mL bottle and cap and pipette tips for 10 mL and 5 mL pipettes at 120 °C for 15 minutes. The number and size of bottles can vary depending on much S R B are needed to be cultivated. 5. Adjust the pH of the media to 7.5 using 10N N a O H . 6. Remove oxygen from the media by bubbling N 2 through the solution for 8-10 min. 7. Add 2.5 mL of ethanol and 0.200 mL of methanol using a micropipette. 8. Add 10 ml of stock ascorbic acid solution (0.01 g mL" 1) and 10 mL of stock thioglycolic acid solution (0.01 g mL" 1) to the media. Using the autoclaved distilled water, fill the media flask until the total volume is 1 L. 9. In the fume hood, add the media to the autoclaved bottles. Leave about 20% of the total volume in the headspace. 10. Using the autoclaved pipettes, add 10% of the bottle's volume of Lac du Bois sediment into the bottom of the bottle. This is done to minimize the inoculum's exposure to oxygen. 11. Fill the bottles to the brim with growth media and cap tightly. 12. The bottles are incubated at 30°C. 13. In order to maintain an active inoculum of S R B , when the supply of SO4 2" in the bottles has been depleted, the S R B can be transferred to fresh media by repeating the above steps, with the inoculum being the cells removed from the bottom of the spent bottle instead of the sediment taken from Lac du Bois. 49 3.3 Lake 52 Sampl ing and In Si tu Set-Up Lake 52 was the site selected for the in situ treatment experiment. This lake was selected because it was reported to have high levels of S 0 4 2 " . In addition, because it was situated on privately owned land, and not the publicly owned park region, it was possible to get permission to conduct experiments within the lake. On the October 4, 2004 sampling expedition to Kamloops, Lake 52 was visited and sampled for the first time. The size of this lake is estimated as 10 x 10 4 m 2 based from the map provided in Ministry of Environment (2000). Two locations, Site 1 and Site 2, on the south shore of the lake were selected as sampling points (Figure 4). These sites were selected not only because of their accessibil ity, but also because Site 1 appeared to provide a good representation of the rest of the lake, and Site 2 provided some isolation which would aid in allowing the in-situ experiment to operate uninterrupted from outside human or wildlife activities. Figure 4 - Sampling locations for Lake 52. Left picture is Site 1 and right picture is Site 2. 50 At each site, at a distance of 6 feet from the shore, water was removed and tested for S 0 4 2 " , T S , s C O D , P 0 4 3 ~ , N 0 3 " - N , N H 3 - N , pH and DO. A s was the case with the sampling from Lac du Bois and Lac du Bois Twin, the water was removed using a syringe attached to a firm plastic tube to get water samples at varying depths. Water was removed at 5 cm intervals for measurements of S 0 4 2 " and T S , and from a depth of 20 cm for all other measurements. Samples for P 0 4 3 ~ , N 0 3 " - N , and N H 3 - N tests were analyzed at the site using a S M A R T portable colorimeter and LaMotte tests Phosphate - Low range (3653-SC), Nitrate-Nitrogen (3649-SC), and Ammonia-Nitrogen (3642-SC). Soluble chemical oxygen demand samples were taken back to laboratory in Vancouver for analysis using the standard method: 5220D closed reflux colorimetric method (American-Water-Works-Association 1975) which is described later in this section. Water samples for S 0 4 2 ~ and T S testing were immediately placed into tubes containing 0.2 mL (10% w/v) Zn-acetate per 1 mL of sample in order to preserve the T S and then brought back to Vancouver for analysis using the barium chloride ( S 0 4 2 ~ measurement) and methylene blue (TS measurement) methods, respectively, which are described later in this section. The pH and temperature were measured using a Mettler Toledo 1140 pH meter, while the DO was measured using a V W R Scientific Products S P 5 0 D Symphony probe. Site 2 was selected as the location for the in-situ set-up, as it was the more isolated area due to its position as an inlet along the shore. There was also some forest debris in the water, which helped to partially cordon off the area. This would protect the experiment more from outside interferences such as snowmobiles. 51 Four feet from the shore, 6 plastic buckets, with a diameter of 35.5 cm and a total height of 57 cm, were placed into the sediment of Lake 52. The bottoms of the buckets were open to the lake floor. Each bucket was screwed into the sediment and then anchored by tying the buckets to metal fence posts, which were hammered into the lake floor. Each bucket was wired to two metal posts, on opposite s ides of the bucket (Figure 5). Tying highly visible tape around the perimeter cordoned off the area. Figure 5 - In-situ treatment set-up The buckets, numbered from left to right 1-6 received the following treatments (Table 4). 52 Table 4 - In-situ treatment set-up 1 2 3 4 5 6 Control Inoculated and Amended Inoculated and Amended Amended Control Amended Depth of sediment/la ke side water/s hore side water in buckets (cm) 6.5/23.6/21.5 5/25.1/21 3/27.1/22.5 4.5/28.8/22.5 6/27/21 6/26/21 The buckets, specified as Control, received no additions through the course of the experiment. The bucket specified as Amended received 4 handfuls, approximately 1 kg, of a 1:1 hay:silage mixture. The hay was a 65:35 alfalfa.orchard grass mixture, and the silage was fermented alfalfa crop. In addition, these buckets also received 6.6 g of SmartCote controlled release fertilizer (Total Nitrogen 12%, Phosphoric acid 14%, Potash 12%) in order to increase levels of nitrogen and phosphate. The buckets specified as Inoculated received exactly the same materials and quantities as the Amended buckets. However, they also received a 3 cm layer (2.96 L) of sediment removed from L D B . This was to act as an inoculum source of S R B . After making these additions, a metal mesh was placed over the top of each bucket and tied using plastic zips. No further additions or sampling was made to the in-situ treatment system until August 11, 2005 (311 days later) when the system was re-visited to assess whether the amendments or inoculation had had any effect on reducing the SO4 2" levels. It was assumed that the lake sampling of Site 2 outside the buckets would serve as a suitable time (t)=0 for S 0 4 2 " and T S . 53 During the August visit, all water removed from the lake for sampling purposes was immediately filtered using a hand pump and 11 urn pore sized filter paper. For the S 0 4 2 " tests, water was removed every 5 cm from all the buckets and from within the lake at both Site 1 and 2. These samples were then placed in tubes containing zinc acetate, at a ratio of 0.2 mL (10% w/v) Zn-acetate per 1 mL of sample in order to preserve the TS and then brought back to Vancouver for analysis. Water was removed from the buckets and Sites l a n d 2 from three depths for D O , and nutrient tests. The depths tested were 0-5 cm, 10-11 cm, and the water/sediment interface. Dissolved oxygen samples were fixed at the lake to be analyzed using the La Motte Winkler Colorimetric method (3688-SC), described later. All the other samples were preserved with 2 mL L"1 of concentrated sulphuric acid. The samples were then placed in a cooler filled with ice and taken back to the laboratory in Vancouver for analysis of N H 3 - N , N 0 3 " - N and P 0 4 3 " . 3.4 Batch Bottle Exper iments Test ing Different Agr icu l tura l Mater ials 3.4.1 Agricultural Experiment without pH adjustment set-up Hay, si lage, barley and molasses were bagged from the Agricultural and Agri Food Canada cattle research station in Kamloops and brought to the laboratory in Vancouver. The hay was a mixed crop of alfalfa and orchard grass (65:35 mixture), the silage was fermented alfalfa crop and the molasses was Dried molasses on Sugar Beet Pulp Sweet 45 and was manufactured by Westway Feed Products 54 Division. The materials were placed in a blender and cut up to reduce their s ize and increase homogeneity. In duplicate, 3 grams of each material was individually placed in a 160 mL glass bottle. In addition 3 sets of duplicate bottles were made which contained 3 g of hay, si lage or barley as well as an addition of 0.3 g of molasses. A solution of 1750 mg L"1 SO42" was created using N a S G v The SO42" solution was de-oxygenated for 10 minutes using N2 gas. Each bottle was filled % with the SO42" solution and then received 15 ml of inoculum of S R B , which was injected into the bottom of the sample bottles to reduce the S R B from encountering oxygen. The S R B inoculum was created by placing sediment from Lac du Bois in modified Postgate B media (Postgate 1979) and transferring the bacteria into fresh media when the SO4 2" levels had dropped. The protocol followed for creating this media and performing S R B transfers was described previously. The inoculum for this experiment was taken from the 7 t h transfer and the activity of the S R B was ascertained by monitoring the SO4 2" levels in the inoculum bottles. In 17 days the S R B of the inoculum had reduced 679.4 mg L"1 SO42", which indicates that the inoculum did contain active S R B . After the addition of inoculum, the bottles were filled to the top with the S 0 4 2 " solution, and then capped with butyl rubber inserts. 55 For a positive control, 10% of the same inoculum was placed in Postgate B media and the S 0 4 2 ~ concentration measured at both the start of this experiment and 12 days later. A decrease in S 0 4 2 ~ would confirm that the S R B inoculum was active. For negative controls, 3 g of each agricultural material was blended and then placed in a 160 mL bottle. The bottles were placed in a Sanyo MLS-3780 autoclave for 20 minutes at 121°C in order to kill any bacteria remaining on the agricultural material. The bottles were then filled with 1500 mg L"1 S 0 4 2 " water. All of the bottles were placed in an incubator set at 30°C. Tinfoil was placed around the bottles to keep the contents of the bottles in darkness to prevent the growth of phototropic sulphur oxidizing bacteria. These purple and green sulphur bacteria grow in anoxic environments but require the presence of light to grow. Their presence is not desired since they oxidize sulphide back into sulphate. Sampling and Maintenance When sampling for S 0 4 2 " concentrations over time, the bottle was inverted several times to homogenize the liquid, and then 5 mL of liquid was removed and placed in 1 mL of 2% (w/v) Zn-acetate to preserve the sulphide as Zn-sulphide and prevent re-oxygenation of sulphide into sulphate. The samples were stored in the fridge at 4°C and analyzed either that same day or the next. If the samples were needed to be stored for longer, then they were placed in a -20°C freezer. Initially the bottles were sampled at roughly 2 week intervals, which was the time observed for the S R B 56 grown in Postgate B media in the laboratory transfers to undergo measurable SO4 2" reduction. Samples were taken at t=0, 16 and 33 days. However, as little S 0 4 2 " reduction was observed during this time, it was decided to increase the time between sampling and the next two samples were taken at t=75 and 170 days. The SO4 2" analysis followed the method listed in the sulphate analysis protocol described later. After sampling, the amount removed was replenished with distilled, deoxygenated water in order to maintain an anaerobic environment. The dilution effect of the make-up water was taken into account when calculating the sulphate reduction due to S R B activity. The pH was measured both at t=0 days and t=170 days using a Mettler Toledo 1140 pH meter. At t=170 days, the bottles were sacrificed so that the levels of S 0 4 2 " , T S , s C O D , P O 4 3 " , and N H 3 - N could be measured. Each analysis protocol is described later in this section. The liquid in the bottles were filtered using 11 um pore filter paper to remove large precipitates. To measure SO4 2" and T S , 10 mL of liquid was removed and placed into tubes containing 2 mL of (10% w/v) Zn-acetate. After mixing these tubes to ensure the Z n S particulates were homogeneous in the liquid, 5 mL for the S 0 4 2 " tests was removed and filtered through 0.45 urn pore filter paper to remove the Z n S particulates while the remaining suspension was used in the T S test. 57 3.4.2 Agricultural Experiment with pH adjustment set-up This experiment design was based upon the previous experiment with several modifications and improvements. Using N a 2 S 0 4 , 4500 mL of a 1500 mg L"1 S 0 4 2 " solution was created and degassed using N 2 for 15 minutes. Barley, hay, silage and molasses were again chopped in a blender to reduce their s ize and increase homogeneity. Three grams of each material was placed in a 160 mL bottle, in duplicates. In addition 3 more sets of duplicate bottles were created to hold varying mixtures of silage and hay in mass ratios of 1:3, 1:1 and 3:1 all having a total mass of 3 g. In this experiment, S R B cells were washed with distilled water to remove any vestiges of Postgate B medium. Into three 300 mL centrifuge tubes, 200 mL of distilled and deoxygenated water was added. Fifty millilitres of S R B inoculum was added to the 200 mL of distilled and deoxygenated water. The centrifuge tubes were spun at a relative centrifugal force of 2759 x g for 8 minutes using an Eppendorf 581 OR centrifuge. Two thirds of the distilled water in the centrifuge tubes were removed and replaced with fresh wash-water (distilled and deoxygenated). The tubes were centrifuged again at 2759 x g for 8 minutes. Twenty mL of the cell pellet from each centrifuge bottle was removed and combined 58 with 140 mL of distilled deoxygenated water for a total volume of 200 mL, which was later used as the inoculum in this experiment. In this way, any remaining Postgate B media would be removed from the inoculum, thus ensuring that only the nutrients provided by the agricultural material is available to the S R B . Each experiment bottle containing agricultural material received 130 mL of deoxygenated 1500 mg L"1 S 0 4 2 ~ water. The bottles and inoculum were moved into a Coy Laboratory Products Type A anaerobic chamber. The pH of each bottle was measured using pH strips, and then the pH was adjusted to 7.5 through the use of a 10 N N a O H solution. The contents of each bottle were well mixed and the pH was checked again after 10 minutes to ensure pH stability. Ten mL of the washed S R B cells inoculum was added to each bottle and then the bottles were filled to the top with the 1500 mg L"1 S 0 4 2 " solution. The bottles were capped and inverted several times to mix the contents. All of the bottles were wrapped with tinfoil to keep out the light. For the positive controls, three 22 mL glass vials were moved into a Coy Laboratory Products Type A Anaerobic Chamber, and each received 1.5 mL of the same inoculum as above into 15 mL of Postgate B media and then filled to the top with media. Some of the media was saved for S 0 4 2 " measurements. The vials were wrapped with tin foil to keep bacteria in darkness. 59 For each agricultural material, negative controls consisted of 2.25 g of the material, autoclaved (Sanyo MLS-3780) for 2 hours at 121°C to which 120 mL of a sterile 1500 mg L"1 S 0 4 2 " solution was added. The pHs were adjusted to 7.5 with the 10N N a O H . Negative controls were prepared in duplicate. Sampling and Maintenance Sampling occurred in the same manner as the previous experiment. However, because the experiment was isolated in the anaerobic chamber it was not necessary to replenish the liquid levels after every sampling to maintain an anaerobic system. The bottles had their levels replenished on t=29 days using distilled and deoxygenated water which had been spiked with N a 2 S 0 4 to the same levels as was measured on the previous day. In addition, 0.08 g of K H 2 P 0 4 was added to each molasses bottle on this day since a P 0 4 3 " measurement taken at t=28 days showed that the molasses bottles contained very low levels of phosphate. This amount is the equivalent amount which would be used in the Postgate B media recipe. On t=117 days and all sampling points afterwards, instead of removing 5 mL of sample into 1 mL of 2% zinc-acetate to measure S 0 4 2 " , 1.5 mL of sample was placed into 12.5 mL of distilled water and 1 mL of 2% zinc acetate. This would dilute the sample by a factor of 10 immediately, and would reduce the amount of liquid removed from the bottles. The higher percentage of zinc acetate to sample would also provide greater reassurance that all the sulphide would be preserved. In 60 addition, on t=126 days, prior to sampling, 0.5 mL of a 17.5 g L"1 solution of FeC I 3 in water was added to each bottle as well as 10 mL of inoculum in order to provide a fresh source of S R B to the system. 3.4.3 Agricultural Experiment Using Leachate from Different Silage Materials The previous experiments used silage that was produced through the fermentation of alfalfa crop. In this experiment, S R B would be inoculated in leachate obtained from two different types of si lages, barley silage (TAD), and orchard grass silage (ORC) . The barley silage was obtained from Pound Maker Agventures (Moellenbeck 2006), while the orchard grass was taken from Pacif ic Agri-Food Research Centre (Veira 2004). In addition, leachate obtained from washing spent material obtained from a bio-ethanol plant that used corn (TBD), was also tested. Thirty-five g of each silage material was measured and placed into 273 mL of distilled water. The bottles were repeatedly shaken and allowed to soak over night. The leachate was filtered out using #1 Whatman 11 um pore filter paper and a water vacuum. The pH of the leachate was measured using a Mettler Toledo 1140 pH meter. Half of this liquid was saved to be inoculated as raw leachate, while the other half underwent additional treatment to floe the small particles still remaining. To remove the solid material, a method similar to that for s C O D analysis was used. These bottles received 1 mL of a 100 g L"1 Z n S 0 4 solution per 100 mL of sample. The bottles were then shaken vigorously for 1 minute and then had their pH adjusted 61 to 10.5 using 6M N a O H . They were then mixed gently for 10 minutes. They were left over night in order to let the precipitate that formed time to settle completely. These flocced bottles were then filtered using 0.45 um pore filter paper and a water vacuum. All of the bottles had 15 mL removed for s C O D tests. The final volumes obtained are described in Table 5. Table 5 - Final volumes of leachate experiment Material Raw Flocced Volume (mL) Volume (mL) T A D 1 120 105 T A D 2 120 105 T B D 1 100 120 T B D 2 78 120 O R C 1 100 105 0 R C 2 96 105 All of the raw and flocced leachate had their pHs adjusted to 7.5, using 6M N a O H for the raw bottles and 1+1 HCI for the flocced bottles. Each bottle was gassed with N 2 for 5 minutes to remove any oxygen and then was moved into the anaerobic chamber. Each bottle received N a 2 S 0 4 to bring up their concentration to 1500 mg L"1 S 0 4 2 ~ (Table 6). The volumes used to calculate the amount of N a 2 S 0 4 needed also included the volume of the 10% inoculum. 62 Table 6 - Sodium sulphate added to achieve 1500 mg L"1 SQ 4 Leachate Inoculum Mass of Volume added N a 2 S 0 4 Raw (ml) (ml) (a) T A D 1 120 12 0.293 T A D 2 120 12 0.293 T B D 1 100 10 0.244 T B D 2 78 7.8 0.190 O R C 1 100 10 0.244 O R C 2 96 9.6 0.234 Flocced T A D 1 105 10.5 0.256 T A D 2 105 10.5 0.256 T B D 1 120 12 0.293 T B D 2 120 12 0.293 O R C 1 105 10.5 0.256 O R C 2 105 10.5 0.256 Each bottle received an inoculation of S R B that was 10% of their volume: The inoculum was obtained in the same manner as in the previous experiment, in order to concentrate the S R B and remove any nutrients present in the remaining media. The inoculum was taken from the 2 1 s t transfer of S R B into new media derived from the LDB sediment. Three positive controls were created where 1.5 mL of the same inoculum was placed in glass vials containing 13.5 mL of Postgate B media. These bottles were observed for the formation of F e S , a black precipitate, to indicate the presence of S R B activity. 63 Sampling and Maintenance This experiment was monitored for S 0 4 2 ~ and C O D . Before sampling, the liquid in the bottles was homogenized by hand shaking for 2 min, followed by 1 hour of standing to settle the solids (bacteria). When sampled, 2 mL was removed from the T A D and O R C bottles and placed into 18 mL of distilled water, while 3 mL was removed from the T B D bottles and placed in 3 mL of distilled water. These volumes were then analyzed for their S 0 4 2 " and C O D levels as described by the protocols listed later in this section. On t=17 days, all of the bottles received, 0.5 mL of a 17.5 g L"1 F e C b solution. This was added to precipitate any sulphide produced and prevent it from becoming inhibitory to the activity of the S R B . This amount was determined using the Postgate B recipe and adding an adjusted amount to obtain the same concentration. At this point, due to the presence of FeS precipitate, the samples removed were filtered first using 11 pm filter paper. Samples were taken for S 0 4 2 " on t=28 days and C O D on t=30 days 64 3.5 Mesocosm Experiment Set-up and Operation Four mesocosm boxes were constructed out of exterior grade plywood to the specifications provided in Figure 6. T 29.2 cm 1 Inside dimensions 200 x 25.4 x 36 cm (LxWxD) bulkhead Top View - 203.8 cm r - A - Q -Side View — A |«—36.6 c m -T 28 cm Crossbar to support equipment (loose) Pump ISQ... Influent Reservoir 210L End View Section A - A Figure 6 - Mesocosm design The mesocosm design was based primarily on the design used by Stark, Will iams et al . (1995), which had dimensions of 160x25x15 cm (LxWxD). This design was selected, with some modifications. Justification for the dimensions and configuration chosen are given at the end of Chapter 2. The mesocosms were placed in a greenhouse located on U B C campus. Two of the mesocosms used natural sediment and algae removed from Lac du Bois as a 65 carbon substrate, while the second set utilized a 1:1 (wt) of si lage and hay. Each box was lined with 2 layers of black polyethylene liner to prevent leaks. Most of the time, the room temperature was between 15-20°C without the need for cooling or heating, except from Nov. 04 - Feb. 05 during 6 pm - 8am when an electrical heater was required. The room temperature was monitored daily. In the natural treatment system, influent was introduced through a rigid polyethylene 0.5 cm diameter tube, perforated at 5 cm intervals, buried beneath the sediment and extending half the length of the mesocosm. Each mesocosm had a 3/8" bulkhead connectors attached at one end, 28 cm from the bottom of the mesocosm, to allow effluent to leave the system. Underneath each connector was a 30 L bucket to capture any effluent. The buckets were emptied into the sewer every 2-3 days. 3.5.1 Natural Treatment System The treatment approach for the natural system was to determine if the introduction of natural organic debris and a S R B inoculum to a high sulphate pond would result in sulphate reduction. For the two mesocosms simulating the natural treatment system, water, sediment and algae were collected from the Lac du Bois (LDB) on July 6, 2004. The water was removed from the designated rocky site, while the algae/sediment was removed from the algae site (Figure 7). 66 Figure 7 - Sampling sites for natural system materials. Left picture is where water was removed, right picture is where algae/sediment was removed. Water was removed from the lake and placed into a 210 L barrel. In addition, 125 L of sediment containing decomposing algae material was removed from the lake and placed into six 30 L barrels. These barrels were filled to the top with lake water and sealed, in order to keep this material as anaerobic as possible. Finally, yellow and green filamentous algae were removed from the lake's surface and placed into 30 L buckets containing lake water. The algae buckets were covered with translucent plastic bags in order to allow light to penetrate, so as to keep the algae alive for the trip back to Vancouver. A botanist later identified which algae spec ies were present (Stockner 2004). These two mesocosms are subsequently referred to as natural treatment mesocosms. The experiment began on July 7 t h 2004, when each of the natural treatment mesocosms received a 10 cm layer of sediment/decomposing algae material. A s mentioned in the Section 2.3.1.1., S R B are primarily located within the first 10 cm of sediment. This layer was immediately covered, with a 15 cm layer of L D B water to act as a water cap to maintain anaerobic conditions within the sediment. Finally, the 67 LDB algae were added to form a 0.6 cm thick layer on top of the water. In 11 days, the water level was observed to have dropped due to evaporation, thus 15 L distilled water (DW) was added to restore the same water height. Subsequently, the water levels were restored to their original height at two-week intervals by adding DW. The mesocosms were left as a batch system to acclimate before introducing high sulphate concentration water continuously. 3.5.2 Natural Treatment System: Continuous Simulation On September 2 8 t h (t=0), water containing 2500 mg L"1 S 0 4 2 ~ , as N a 2 S 0 4 , was pumped at a rate of 6 mL min" 1 into the two mesocosms through the subsurface infusion pipe. This was the slowest possible pumping rate. At this rate, the residence time of the water flowing through the mesocosm was estimated as 15.8 days. This residence time was tentatively selected since residence times vary greatly in passive treatment systems and the kinetics of this system was, as yet, unknown. The Stark, Will iams (1995) design, on which my system is based, had a residence time of 33 hours. However, studies in the laboratory with sediment removed from Lac du Bois and placed in Postgate B media, found that in 3 trials, S R B within this sediment required at least 10 days in order to reduce S 0 4 2 " by at least 800 mg L"1 (see Appendix A-1). S ince it was assumed that the S R R would be slower in the mesocosm, which would be open to the air and not using a growth medium to support S R B activity, a longer residence time was required. 68 Water samples were taken at 3 locations along the length of the mesocosm (50, 100, 150 cm) and at two depths (10 and 20 centimetres below the water surface) for testing for S 0 4 2 \ In addition, 15 mL of water was taken at the 100 cm length and 15 cm depth for ammonia-nitrogen and phosphate analysis. Sampling was repeated on t=15 days. After this point, water samples were taken only of the influent and the outlet. On t=43 days, the sulphate concentration in the feed reservoir was reduced from 2500 mg L"1 to 1500 mg L"1. The inlet concentration was lowered since reduction of 2500 mg L"1 in one step is unlikely due to product inhibition from sulphide. For every 1000 mg L"1 sulphate reduced, 333 mg L"1 sulphide is produced. At concentrations above this, sulphide becomes inhibitory to sulphate reduction. The natural treatment mesocosms were run continuously from September 2 8 t h to December 6 t h 2004. Due to poor performance, the continuous system was stopped and it was decided to run both the natural treatment mesocosms and the agricultural material mesocosms as batch systems, rather than continuous, so as to determine the sulphate reduction kinetics. An amendment of additional nutrients was made to the LDB mesocosm as no ammonia could be measured in the system, and the levels of phosphate were low. It was believed that the continuous water flow was flushing the nutrients out of the system faster than the degrading algae could replenish them. Using the recommended ratio of C : N : P for anaerobic waste water bacterial nutrient feed of 250:5:1 (Metcalf and Eddy 1991), the mesocosms were 69 amended with 250 mL of lactic acid (8.65 mol C) , 9.26 g of NH 4 CI (0.17 mol N) and 3.36 g of K H 2 P 0 4 in mesocosm A and 3.69 g of K H 2 P 0 4 in mesocosm B which gives a total phosphorus level of 0.03 mol. The amount of lactate added was 2.19 g L"1 which is slightly less than the 2.78 g L~1 suggested by (Postgate 1963) for the growth of S R B from pure cultures and natural samples. 3.5.3 Natural Treatment System: Batch Simulation The natural treatment system was run as a batch process for 175 days. During that time, sulphate concentrations at the sediment-water interface and within the sediment were monitored at the sampling locations shown in Figure 8. On day 63, high S 0 4 2 " water was recycled from the water column into the sediment using a peristaltic pump at 2 mL min" 1. This was done so as to introduce high S 0 4 2 " into the sediment layer. At t=91 days, sulphate concentrations were monitored weekly for an additional 84 days (this was considered t=0 for this new trial). Inlet 50 cm 100 cm 150 cm Figure 8 - Natural system sampling locations. + represents the initial sampling locations when the mesocosm was run as a continuous system. * indicates the sampling location at t=32 when the S0 4 2" concentration in the sediment and water column were compared. O represents the sampling location when the SO4 2 was measured in the sediment, after running the recycle system to increase the SO4 2 ' concentration in the sediment. 70 3.5.4 Agricultural Material Treatment System A mixture of alfalfa and orchard grass hay and alfalfa-derived si lage was removed from the Kamloops Agricultural and Agri Food Canada Research station on October 4 t h and placed in large black garbage bags for transportation back to Kamloops. On October 20 t h , a 13 cm layer of hay and silage (1:1 ratio), weighing approximately 5 kg, was placed along the bottom of the second pair of mesocosms, henceforth referred to as the agricultural mesocosms. Tap water, previously allowed to sit for 2 days to remove any residual chlorine present, was used to fill the mesocosms to a height of 25.5 cm. The chlorine levels were measured using free and total chlorine test strips (2745050 Hach Company) which have a range of 0-10 mg L~1 and measure chlorine concentrations at increments of 0, 0.5, 1, 2, 4 and 10 mg L"1. Prior to the addition of the inoculum, the pH in the mesocosms was adjusted from 5.5 to 7.5 using 10N N a O H . On November 2 n d , 16 L of L D B sediment, as an inoculum, was added along the length of each mesocosm. In addition 4 L of Postgate B media was added to each mesocosm. The S R B were allowed to acclimatize to the agricultural mesocosm environment for approximately 1 month prior to the initiation of the experiment. Two more pH adjustments were required on November 15 t h and December 10 t h as the pH had dropped to 6 in the mesocosms. 71 The SO4 2" concentrations in the agricultural mesocosms were increased to 1500 mg L"1 by adding 1200 mL of water containing 304.21 g of N a 2 S 0 4 to each mesocosm. Sampl ing began on December 10 t h (t=0 days) One sample was taken from each mesocosm weekly at 100 cm down the length of the mesocosm and at a depth of 15 cm in the water column for S 0 4 2 ~ , T S , s C O D , N H 3 - N and P 0 4 3 ~ . Table 7 below is a record of all the changes made to the agricultural mesocosms over the course of this experiment. Table 7 - Changes made to the agricultural mesocosms Event # Date of Event Event 1 t=82d Added 1200 mL of water containing 304.21 mg of N a 2 S 0 4 . A lso added a 13 cm layer of pre-soaked si lage and hay 2 t=109d Added 2 L of water containing 304.21 mg of N a 2 S 0 4 3 t=159 Add a 13 cm layer of silage and hay 4 t=174d Placed a tarp over the system to prevent air from entering 5 t=222 d Washed si lage with tap water and added this wash-water to the system 3.5.5 S R B Activity In order to determine if the S R B bacteria were still active, samples of sediment were removed from the two mesocosms and placed in Postgate B media. The formation of black precipitate over time would indicate the presence of active S R B reducing S 0 4 2 " to sulphide, which would then form the black precipitate, F e S . At two different locations within the mesocosms, sediment was removed at two depths. Five mL samples were taken 50 cm away from the two mesocosm ends within the bottom 0-3 72 cm of sediment and within the bottom 9-11 cm of sediment. The sediment was filtered through 0.22 um pore filter paper and the paper was placed within 50 mL tubes each filled with Postgate B media. The tubes were placed in an incubator set at 30°C and monitored visually for the appearance of black precipitate every day for 3 weeks. 3.6 Analy t ica l Methods 3.6.1 Sulphate - Turbidimetric Sulphate determination used the barium sulphate turbidimetric method (4500-SO 4 2 ~ E, American-Water-Works-Associat ion 1975). Barium ions, added as barium chloride, react with sulphate ions to produce insoluble barium sulphate (Equation 4). B a 2 + + SO4 2" -» B a S 0 4 (4) The barium sulphate forms a milky precipitate that affects the turbidity. The amount of turbidity is proportional to the amount of sulphate present. The turbidity can be measured using a spectrophotometer because the cloudiness reduces the amount of light that will pass through the sample. The minimum detection limit using the buffer A was 20 mg L"1 and the maximum detection limit was 100 mg L"1. Dilutions were used to achieve measurements within this range. Positive interferences can arise due to colour and turbidity present in the sample matrix. This can be negated by blanking the spectrophotometer with sample water of the same dilution as that being analyzed. Blanking is the process of subtracting the 73 absorbencies of the non-analyte compounds from the absorbance of the mixture to yield the absorbance of the analyte. All samples underwent filtration through 0.45 pm filter paper. If the sample was to be analyzed over the next two days, the filtered sample was stored in the fridge, otherwise it was frozen. 1. The spectrophotometer (Biochrom Ultrospec 1000) was turned on at least 0.5 hours before the analysis was to be undertaken. This time was necessary to allow the lamp time to warm up. The wavelength was set to 450 nm. 2. The samples were allowed to warm up to room temperature and then diluted, if necessary, using distilled water. The final volume size was 10 mL and each sample was placed in identical 50 mL vials with magnetic stir bars, also of the same shape and size. 3. A standard S 0 4 2 solution of 100 mg L 1 was created using N a 2 S 0 4 and this was used to create a calibration curve from 20-100 mg L"\ as well as used to check the calibration curve during experimentation. 4. Buffer A solution is prepared: Dissolve 30 g of magnesium chloride (MgCI 2 -6H 2 0) , 5 g of sodium acetate ( C H 3 C O O N a - 3 H 2 0 ) , 1.0 g of potassium nitrate ( K N 0 3 ) and 20 mL of acetic acid ( C H 3 C O O H ) in 500 mL distilled water and make up to 1000 mL. 5. Each sample of 10 mL received 2 mL of Buffer A . 74 This solution was temporarily poured into a cuvette in order to zero the spectrophotometer. The sample was placed on a magnetic stirrer and received 0.25 g of B a C b . The sample was stirred for 60 ± 2 s and then immediately removed and poured into a cuvette. After 5 ± 0.5 min, the sample was placed in the spectrophotometer and the optical density was recorded. 3.6.2 Sulphide - Methylene Blue This determination of total sulphide is based on the standard methylene blue method (428C, American-Water-Works-Associat ion 1975). This method is based on the ability of hydrogen sulphide and acid soluble sulphides to convert N,N-dimethyl-p-phenylenediamine directly to methylene blue, a well known dye, in the presence of a mild oxidizing agent such as potassium dichromate. The accuracy of this test is reported to be about ± 10% and have a minimum detection limit of 0.1 mg L"1 Reagents 1. Amine sulphuric acid stock solution. Dissolve 27 g of N, N-dimethyl-p-phenylenediamine oxalate in a cold mixture of 50 mL concentrated H 2 S O 4 and 20 mL of distilled water. Cool the mixture and dilute to 100 mL with distilled water. Store in a dark glass bottle. The amine oxalate must be fresh as an old supply can discolour to such a degree that the colour interferes with 75 the test. When the stock is diluted and used in the procedure, it must yield a colourless solution. 2. Amine sulphuric acid reagent. Dilute 25 mL of the amine sulphuric acid stock solution in 975 mL 1+1 H 2 S 0 4 and distilled water. 3. Ferric chloride solution. Dissolve 100g F e C I 3 - 6 H 2 0 in 40 mL of water. 4. Sulphuric acid solution. 1+1 H 2 S 0 4 . Add 1 mL of concentrated H 2 S 0 4 to 1 mL of distilled water. 5. Diammonium hydrogen phosphate solution: Dissolve 400 g of ( N H 4 ) 2 H P 0 4 in 800 mL of distilled water. 6. N a 2 S - 9 H 2 0 stock solution. Place 5 g of crushed N a 2 S - 9 H 2 0 crystals in 2.5 mL of distilled water and maintain at 30°C overnight. This will create a saturated sulphide solution with a concentration of 9.2434 x 10 4 mg L"1 T S (Perry 1997). This reagent is used to create a calibration curve. Procedure 1. The spectrophotometer is turned on and the wavelength is set to 625 nm 2. Water samples are filtered with 0.45 pm pore paper and then 2 % (w/v) Zn-Acetate is immediately added to preserve the sulphide as Z n S . 1 mL of Zn-Acetate is added for every 5 mL of sample. 3. If the samples are to be measured in a few days, then they are stored in a fridge, otherwise they are frozen. 4. 2.5 mL of sample is transferred into 4 mL cuvettes. An additional 2.5 mL is transferred to be a blanking solution. 76 5. To the blanking solution add 0.167 mL of 1+1 H 2 S 0 4 solution and to the test samples add 0.167 mL of the amine sulphuric acid reagent. 6. Immediately add 1 drop of the FeC I 3 solution to the samples. Cover the cuvette with parafilm and invert once, slowly. A blue colour should emerge if sulphide is present. 7. Wait 12 minutes and then add 0.533 mL of the ( N H 4 ) 2 H P 0 4 solution. Shake the cuvette to ensure mixing. 8. Wait 10 minutes to give the ammonium phosphate time to remove the colour caused by ferric chloride and then take the O.D. measurement. 3.6.3 Soluble Chemical Oxygen Demand The total soluble Chemical Oxygen Demand (sCOD) is a measure of the oxygen required to oxidize all soluble compounds, both organic and inorganic, in water. This procedure is based upon the C O D Standard Methods 5220D, c losed reflux, colorimetric method (American-Water-Works-Association 1975). To determine only the soluble C O D , the small particles in the water samples must first be floccuated and then filtered with a 0.45 um filter, and then measuring the C O D of the filtrate. To measure the C O D , the sample is refluxed in strongly acid solution with a known excess of potassium dichromate ( K 2 C r 2 0 7 ) . After digestion, the remaining unreduced K 2 C r 2 0 7 is titrated with ferrous ammonium sulfate to determine the amount of K 2 C r 2 0 7 consumed and the oxidizable matter is calculated in terms of the oxygen equivalent. This test analyzes both biodegradable and non-biodegradable (refractory) organic matter. The results of this test are expressed as mg L"1 0 2 . 77 Procedure 1. Samples are filtered using #1 Whatman filter paper (11 pm pore size). If the sample is not going to be analyzed immediately, then the pH is lowered to <2 with H 2 S O 4 and the sample is stored in the fridge at 4°C. Samples can be stored for up to 28 days under these conditions. 2. Add 1 ml of a 100 g L"1 z inc sulphate solution to a 100 ml of sample and mix vigorously for 1 minute. 3. The pH of the sample is adjusted to 10.5 using 6 M N a O H solution and the samples receive 10 min of gentle mixing. The samples then remain stationary for 15 minutes to allow the precipitate to settle. 4. The supernatant is removed a filtered through a 0.45 pm filter. 5. The supernatant is placed into high range C O D digestion vials, from the Hach company, at various dilutions. 6. Two reagents are prepared for the C O D test. a. Digestion Solution: Add 10.216 g K 2 C r 2 0 7 primary grade previously dried at 103°C for 2hr, 167 mL concentrated H 2 S 0 4 and 33.3 g H g S 0 4 to 500 mL of distilled water. Dissolve, cool to room temperature then dilute to 1000 mL. b. Sulphuric acid reagent: Add silver sulphate ( A g 2 S 0 4 ) , crystals or powder, to concentrated H 2 S 0 4 at a rate of 5.5 g A g 2 S 0 4 per kg H 2 S 0 4 . Then let stand for 1 to 2 days to dissolve the A g 2 S 0 4 . 78 7. To the C O D sample vial, add 1.2 mL of the digestion solution and 2.8 mL of the sulphuric acid reagent. Make sure to add the same reagents to a vial containing DW to act as a blank to zero the spectrophotometer. 8. Use an agitator (Thermolyne Maxi Mix II type 37600) to mix the vials for 15 seconds. 9. P lace sample vial into the heating block (COD reactor, Hach Company) for 2 hours at 150 °C. Upon completion, allow to cool and then use agitator to mix the solution for another 15 seconds. 10. P lace the C O D vial into a spectrophotometer (DR/2000, Hach Company) set at 600 nm and record the O.D. 11. A standard calibration curve is developed using potassium hydrogen phthalate. (KHP) has a theoretical C O D of 1.176 mg 0 2 per mg K H P . Dissolve 425 mg of K H P in distilled water and bring up to 500 mL and the stock standard solution will have a C O D of 1000 mg L"1. 3.6.4 Ammonia Nitrogen - Nesslerization Ammonia nitrogen is the summed weight of nitrogen in both the ionized (ammonium, N H 4 + ) and un-ionized (NH 3 ) forms. In these aquatic samples the ammonium species is generally the more prevalent. This protocol utilizes pre-calibrated reagents and a colour imeterforthe measurement (Nesslerization method, without preliminary distillation step) of ammonia-nitrogen as modified from Standard Methods for the Examination of Water and Wastewater, method 4 5 0 0 - N H 3 C (18 t h Ed . , A P H A , 79 A W W A , W E F , 1992). The reagents were purchased as part of the La Motte Smart Ammonia Nitrogen - High Range kit code 3642-SC. In this method, Nessler 's reagent, a solution of mercury (II) iodide (Hgl 2) in potassium iodide and potassium hydroxide, forms a brown colouration or precipitate in proportion to the amount of ammonia present in the sample. Rochel le salt is added to prevent precipitation of calcium or magnesium in un-distilled samples which could interfere with the colourimeter readings. The range of this test is between 0-3.0 ppm N H 3 - N . 1. After sampling, the water is filtered using #1 Whatman filter paper (11 pm pore size). Preservation is achieved through the addition of 2 mL of concentrated H2SO4 per litre of sample, and then refrigerated at 4°C. The maximum length of time samples can be preserved in this manner is 28 days. 2. The water sample to be analyzed is diluted, if necessary, and 10 mL placed in a clean colorimeter tube (code 0967 from La Motte). This sample is used to blank the colorimeter which has previously been set to Sequence #3 Ammonia-N High test. 3. Each sample vial receives 8 drops of Ammonia Nitrogen Reagent #1 (V-4797) and then the vial is capped and mixed. 4. Using the 1.0 mL pipet (Code 0354), 1.0 mL of Ammonia Nitrogen Reagent #2 (V-4798) is added. Again the colorimeter vial is capped and mixed. 5. After waiting 5 minutes to allow for full colour development, the tube is inserted into the colorimeter chamber and scanned. 80 3.6.5 Phosphate - Ascorbic Acid This protocol utilizes pre-calibrated reagents and a colorimeter for the measurement of ortho-phosphate in water samples as outlined from (American-Water-Works-Associat ion 1975) method 4500-P E. In this test, ammonium molybdate and antimony potassium tartrate react in an acid medium with dilute solutions of orthophosphate to form a complex which is reduced in an intense blue coloured complex by ascorbic acid. The intensity of the colour is proportional to the amount of phosphate present in the sample. The range of this test is between 0-3.0 ppm. 1. After sampling, the water is filtered using #1 Whatman filter paper (11 um pore size). Preservation is achieved through the addition of 2 mL of concentrated H 2 S 0 4 per litre of sample, and then refrigerated at 4°C. 2. The water sample to be analyzed is diluted, if necessary, and 10 mL placed in a clean colorimeter tube (code 0967 from La Motte). This sample is used to blank the colorimeter which has previously been set to Sequence #33 Phosphate-Low. 3. Each sample vial receives 1.0 mL of Phosphate Acid Reagent (V-6282) and then the vial is capped and mixed. 4. Using the 0.1 g spoon (code 0699), 1 measure of Phosphate Reducing Reagent (V-6283) is added. The vial is capped and shaken until the powder dissolves. 81 5. After waiting 5 minutes for full colour development, the vial is placed into the colorimeter chamber and scanned. 3.6.6 Dissolved Oxygen - Winkler Colorimetric Method This method (LaMotte 3688-SC) is a modification of the Winkler colorimetric protocol which uses a colourmetric determination of yellow iodine, which is produced from a reaction with dissolved oxygen (American-Water-Works-Associat ion 1975) (method 4500-OC). In this test, a manganese sulphate solution is added to the sample water and the manganese ions are liberated and loosely bound with excess hydroxide. In the presence of a strong base, the manganese is oxidized from (II) to (III) and binds the dissolved oxygen. Upon the addition of a strong acid (sulphuric acid) and an iodide solution, free iodide is produced at a rate of one l 2 molecule for each atom of oxygen. This results in a yellow/brown colour, whose intensity can be measured using a colorimeter and which is proportional to the amount of DO present in the water. After this stage, the sample is fixed and can be examined at a later time. The range of this test is between 0-12.5 ppm. 1. Thoroughly rinse the sample tubes with sample water 2. Tightly cap the sample tube (LaMotte #29180) and submerge to the desired depth. Remove the cap and allow the tube to fill. 3. Tap the side of the tubes to dislodge any air bubbles and then tightly replace the cap while still submerged. 82 4. Add two drops of manganese sulphate solution (LaMotte #4167G) and two drops of alakaline potassium iodide azide (LaMotte #7166G). C a p and invert 10 times. Allow the precipitate to form and settle below the shoulder of the sample tube. 5. Add two drops of sulphuric acid 1:1 (6141WT-G). C a p and gently shake the vial until the precipitate has fully dissolved. Now the sample is f ixed. 6. Set the colorimeter to test 27. Oxygen and place a clean colorimeter tube (LaMotte 0967) filled with sample water into the colorimeter chamber and scan blank. 7. Remove the blank and replace with the colorimeter tube containing the fixed water for analysis. Scan the sample and record the result. 83 4.0 Results and Discussion 4.1 Character izat ion of High and Low Sulphate Lakes In order to gain a better understanding of what factors contribute to high salinity in natural water reservoirs, two lakes with different salinities, situated less than 500 m apart in the Lac du Bois Grasslands Park in Kamloops, were examined. This section descr ibes site visits to Lac du Bois and Lac du Bois Twin and d iscusses the similarities and differences in the lakes' physical, chemical and biological makeup. Two locations at each lake, Rocky and Algae (Lac du Bois) and Site 1 and 2 (Lac du Bois Twin) were sampled and analyzed in detail. 4.1.1 Physical Characteristics Lac Du Bois Lac du Bois is one of the larger lakes in the Lac du Bois Grass lands Park with a surface area estimated as 3.75 x 10 5 m 2 . L D B Rocky had a depth of 40 cm (6 ft from shore) with a very thin sediment layer on the lake floor. The lake floor was clearly visible and covered in rocks and wood debris. Many small water insects were observed swimming in the water. Some long green filamentous algae were observed attached to the rocks on the lake floor. 84 L D B Algae had a total depth of 33.5 cm (6ft from shore). On the surface of the water was a large yellow/green algae mat. Beneath this mat was a very thick layer of decaying algae matter which, at some points, extended to the lake floor. Whenever the mat was disturbed, trapped gases were able to escape and bubble up to the surface and the scent of sulphide was observed. Lac du Bois Twin LDB Twin was a considerably smaller lake, estimated at 8 x 10 4 m 2 , when compared to Lac du Bois. It was oval in shape and the entirety of the lake could be seen from any point around its surface, unlike L D B which was irregular in shape. Around the shore of the lake, a thick white precipitate coated the ground and rocks (Figure 9). Figure 9 - Lac du Bois Twin shoreline. Oct 4, 2004 85 LDB Twin was shallow and the lake floor could be observed at all times. It is estimated that the maximum depth within the lake was less than 1 m. The two sampling sites in L D B Twin had very little to distinguish them other than their location at opposite ends of the lake. The water column depth at both sites was 25 cm. The sediment layer on the floor of the lake was very thin, measuring 5-10 cm in places. Physically, these two lakes were very different. Of the two, Lac du Bois was considerably larger in surface area. While it was not possible to determine the depth of Lac du Bois at distances further than 183 cm (6 ft) from shore, the lake floor could not be observed at greater distances from shore, unlike Lac du Bois Twin, which indicates that Lac du Bois was a deeper lake. 4.1.2 Chemical Characteristics Temperature/pH/DO It was found that the two lakes differed greatly in their pH. Lac du Bois had the lower pH of 8.62 ± 0.04, (July 6, 2004) 8.63 ± 0.01 (Oct 4, 2004) while Lac du Bois Twin had a pH of 10.35 ± 0.05, (July 6, 2004) and 10.35 ± 0.35 (Oct 4, 2004). The temperature of Lac du Bois was 22.95 ± 0.55°C (July 6, 2004) and 15.45 ± 0.22°C (Oct 4, 2004) while Lac du Bois Twin was 25.85 ± 0.15°C (July 6, 2004) and 86 14.15 ± 0.05 °C (Oct 4, 2004). The errors are the difference between the measurements at the two sampling sites in each lake. The DO probe was only operable during the October visit to the lakes. Lac du Bois was found to have a DO level of 15-20% at the L D B Rocky site, with higher readings near the water's surface. At LDB Algae readings of 12-15% saturated were observed in the first 5 cm of the algae mat at L D B Algae site. A s the probe was dropped lower, this level quickly dropped until it was too low to measure (-15 cm into the mat). The D O meter reported that the levels in L D B Twin were higher than 100% saturation. 87 Sulphate Even though the two lakes, Lac du Bois and Lac du Bois Twin are spatially very c lose to one another, the S 0 4 2 " concentrations in the two lakes vary greatly. L D B was found to have low levels of S 0 4 2 " (Figure 10) with averages of 54.8 ± 2.0 mg L"1 at the algae site and 60.1 ± 1.9 mg L"1 at the rocky site. The S 0 4 2 " concentration was significantly lower at the LDB Algae site during the July 6 t h visit when compared to the L D B Rocky site (Tukey test P<0.01 a=0.05, A N O V A P=0.002), but not significantly different during the October 4 t h visit. S0 4 2- (mg L"1) 40 45 50 55 60 65 70 0 H . . . . , , Figure 10 - S0 4 2" concentration (mg L'1) in Lac du Bois versus depth (cm). • represents S0 4 2" levels at LDB Rocky and • represents levels at LDB Algae. Solid line represents samples taken July 6th while dotted line represents Oct 4, 2004 samples. Error bars represent standard deviations of the S0 4 2 test. In contrast, LDB Twin had exceptionally high levels of S 0 4 2 " averaging 6500 mg L"1 S 0 4 2 " (Figure 11). 88 S0 4 2- (mg L"1) 5 8 0 0 6 0 0 0 6 2 0 0 6 4 0 0 6 6 0 0 6 8 0 0 7 0 0 0 7 2 0 0 0 -j , , . . , , , Figure 11 - S0 4 2 ' concentration (mg L"1) in Lac du Bois Twin versus depth (cm). • represents S0 4 2 levels at Site 1 and • represents levels at Site 2. Solid line represents samples taken July 6, 2004 while dotted line represents Oct 4, 2004 samples. Error bars represent standard deviations of the test. Sulphide The presence of sulphide in natural water reservoirs indicates the presence of S R B . Sulphide was present in both lakes with the concentrations increasing at the water/sediment interface, which indicates that the lakes are not well mixed (Figure 12, Figure 13). 89 0.60 Figure 12-Total sulphide concentration mg L'1 in Lac du Bois versus depth (cm). • represents TS levels at LDB Rocky and • represents levels at LDB Algae. Solid line represents samples taken July 6, 2004 while dotted line represents Oct 4, 2004 samples. Error bars represent standard deviation of the test. TS (mg L"1) 0.00 0.50 1.00 1.50 2.00 0 H ' ' ' • Figure 13 - Total sulphide concentration (mg L"1) in Lac du Bois Twin versus depth (cm). • represents TS levels at Site 1 and • represents levels at Site 2. Solid line represents samples taken July 6, 2004 while dotted line represents Oct 4, 2004 samples. Error bars represent standard deviation of the test. Nutrients The two lakes were examined for levels of ammonium, nitrate, phosphate and s C O D to obtain an idea of the amount of nutrients available in the lake to support S R B 90 growth and activity. What was observed was that while there were low levels of nitrate in both lakes, there were higher levels of ammonia, a form of nitrogen easily utilized by S R B (Figure 14). LDB Rocky LDB Algae LDB Twin Site 1 LDB Twin Site 2 Sample Location Figure 14 - (a) Nitrate and (b) ammonia-nitrogen concentrations (mg L"1) in Lac du Bois and Lac du Bois Twin. Solid bars represent July 6, 2004 sampling and the hatched bars October 4, 2004. Error bars represent standard deviation of the test. Phosphate levels were higher in L D B Twin than in L D B (Figure 15). In addition, the levels of P 0 4 3 " were higher at L D B Rocky than at L D B Algae. 91 6.00 -j 5.00 -Li 4.00 -3.00 -CO d 2.00 -CL 1.00 -0.00 -LDB Rocky LDB Algae LDB Twin Site 1 LDB Twin Site 2 Sample Location Figure 15 - Ortho-P043" concentration (mg L'1) Lac du Bois and Lac du Bois Twin. Solid bars represent July 6, 2004 sampling and the hatched bars October 4, 2004. Error bars represent standard deviation of the test. Soluble C O D levels were higher in L D B than in L D B Twin by almost a factor of two (Figure 16). 500 n _ 4 0 0 TO 300 E, § 200 o 100 0 LDB Rocky LDB Algae LDB Twin Site 1 LDB Twin Site 2 Sample Location Figure 16- Soluble COD concentration (mg L"1) in Lac du Bois and Lac du Bois Twin. Sampled October 4, 2004. Error bars represent standard deviation of the test. The higher levels of s C O D indicate higher levels of dissolved carbonaceous material present in LDB than in LDB Twin. A n assumption is being made that organic 92 material is the primary contributor to the chemical oxygen demand. An earlier chemical analysis of these lakes, sampled by Susan Baldwin and analyzed by Norwest labs, found that the dissolved organic carbon (DOC) content of the water in the algae site to be 283 mg L"1, while in LDB Twin the D O C was 113, 130 and 360 mg L"1 in 3 different locations. The complete Norwest chemical analysis of L D B and LDB Twin can be found in Appendix A. The chemical characteristics of the two lakes are very different, despite their close proximity. Lac du Bois Twin had higher levels of DO, pH and S 0 4 2 " when compared to LDB. Nutrient levels were similar in the two lakes with L D B Twin having higher levels of P 0 4 3 " but L D B having higher levels of s C O D . 4.1.3 Biological Characteristics Lac du Bois Lac du Bois is set in a very lush area, with healthy looking trees and bushes surrounding the lake. Atop the surface of the lake in several areas near the shore was a very thick layer of green/yellow filamentous plant-like organic matter. On the second visit to the lake, a large group of cattle was found near the shore, and hoof prints were observed in the mud next to the lake, indicating that it had likely been used as a watering hole. Ducks and other wildfowl were observed swimming in the lake on both site visits. At Lac du Bois Algae, a large population of cattails (Typha 93 latifolia) was observed growing around the shore. Sedge (Carex) was also observed growing around the lake. The plant-like matter of Lac du Bois was later identified by Dr. John Stockner of Eco-Logic Ltd. The samples removed from LDB were found to be primarily a green filamentous alga known as Cladophora cf. glomerata with some scattered filaments of Ulothrix sp. and Mougeotia sp. The Cladophora, in appearance, were generally coarse, with regular branching filaments that had cross walls separating multinucleate segments. Lacking a mucilage outer sheath, the filaments are a favourite substratum for attached epiphytic diatoms. According to Dr. Stockner, each filament from Lac du Bois was completely coated with a wide variety of attached diatoms, the most dominant one being Achnanthes minutissima. Other diatom genera found on the filaments were: Navicula, Nitzschia, Frustrulia, Cymbella, Fragilaria, Rhopalodia, Amphora, and Cocconeis. Dr. Stockner also observed a wide variety of small bacteria and protozoan grazers within the samples. The sediment of LDB was tested by Jenny Eng, an undergraduate researcher at U B C , for the presence of S R B . Over a period of 51 days, the level of SO4 2" was monitored in 300 mL glass jars containing Postgate B media and 30 mL aliquots of L D B sediment. The results indicate that S R B were present in the L D B sediment as S 0 4 2 " reduction was observed with an average drop of 1220.1 ± 49.5 mg L"1 S 0 4 2 " from sediment sample #1 and 1071.4 ± 8.2 mg L"1 SO4 2" from sediment sample #2. 94 This gives an overall average drop in S 0 4 2 " of 1145.8 ± 28.8 mg L"1 S 0 4 2 " for the L D B sediment. Lac du Bois Twin In contrast to Lac du Bois, L D B Twin was not set in a lush environment. A s mentioned previously, instead of cattails and sedge surrounding the lakeshore, rock and soil covered in a thick crusty white precipitate, likely sulphate salt, was observed. Within the lake itself, green organic matter was observed both on the sediment floor and suspended in the water. The organic matter did not look the same as the algae found in LDB and instead had a granular appearance. By visual inspection, the amount of material present was also smaller. After being analyzed by Dr. John Stockner it was determined that no Cladophora were present and the dark green mat was primarily a filamentous blue-green Cyanobacteria known as Nodularia spumigena. A lso present but not commonly found in the lake were the coccoid blue-greens Chroococcus sp. and Cyanothecae sp. Unlike Lac du Bois, this organic matter did not have a coating of attached diatoms, but did have a coating of small bacteria, picocyanobacteria and a host of protozoan grazers (ciliates, amoeba, Paramecium, and several species of rotifers). 95 The sediment of LDB Twin, as analyzed by Jenny Eng in the same manner as the sediment of LDB, found that S R B were present. An average decrease in S 0 4 2 ~ of 406.9 ± 266.8 mg L"1 S 0 4 2 " was obtained using sediment from location #1 and 1295.5 ± 555.0 mg L"1 S 0 4 2 " using sediment from location #2 giving an average overall S 0 4 2 " drop of 851.2 ± 410.9 mg L"1 S 0 4 2 " . Biologically, the two lakes are dissimilar. Lac du Bois has a healthy appearance with lush vegetation both surrounding and within the lake. In contrast, Lac du Bois Twin has very little vegetation surrounding it and the granular algae within the only form of vegetation observed. Testing the sediment for the presence of S R B indicated that both lakes had communities present, but those of LDB had a greater potential for reducing S 0 4 2 " in Postgate B media. 4.1.4 Discussion and Conclusions Lac du Bois and Lac du Bois Twin, although less than 500 m apart, had very different physical, chemical and biological attributes. Of the two, LDB was the larger lake, both in surface area, and in depth. Dissolved oxygen levels within Lac du Bois were low and decreased to below detection limits within the decaying algae/sediment. Dissolved oxygen will decrease when it is utilized to decay organic wastes. This creates an anaerobic environment that is more suitable for S R B . In contrast, LDB Twin was saturated with dissolved ,96 oxygen. Dissolved oxygen is introduced into natural water reservoirs either through the photosynthesis of algae and plants, or through the dissolving of atmospheric oxygen into the water facilitated by wind and mixing. It is likely that LDB Twin was saturated as little plant life was observed either surrounding or within LDB Twin to provide decaying material which would utilize oxygen. The presence of sulphide within the lakes could indicate that there are S R B present and actively reducing S 0 4 2 " within the lake. With the exception of the July 6 t h L D B Rocky sample, the sulphide concentration increased with depth to a maximum at the water/sediment interface. This suggests that S R B are present within the sediment. The work of Eng (2005), who inoculated Postgate B medium with sediment samples removed from these lakes and observed sulphate reduction in all samples, confirms the presence of S R B in the sediment of both lakes. The medium containing L D B sediment had a higher rate of S 0 4 2 ~ reduction, which suggests that either there were more S R B present in the LDB sediment, or the bacteria which were there were more efficient at utilizing the Postgate B medium. Both the s C O D results, as well as the findings of the Norwest Laboratory, indicate that L D B had higher levels of dissolved organic compounds than L D B Twin. This is likely due to the larger presence of plant life (Typha latifolia and sedges) growing around LDB, as well as organic mats of Cladophora, diatoms and other prokaryotes observed within the lake. No Typha latifolia, nor other macrophytes were observed growing around the LDB Twin waters. Cattails can survive in acidic, neutral and 97 basic pH, but Dyhr-Jensen and Brix (1996) found that growth is depressed when the plants have prolonged exposure to pH 8.0 and completely suppressed at pH 3.5. Lac du Bois Twin, with a pH greater than 10, is likely too basic to allow their growth. The cyanobacteria observed in LDB Twin, Nodularia spumigena, is well known for its toxic blooms throughout the world in brackish lakes and waters (Runnegar, Jackson et al . 1988; Sivonen, Kononen et al . 1989). It can live either as plankton, or attached to sediments, both of which were observed in LDB twin. The toxin produced is the cyclic pentapeptide nodularin which is known to be toxic to mammals, including humans. Karjalainen (2004) reported that nodularin was responsible for the deaths of 400 ducks in Jasmunder Bodden, Germany, in 1963, and the death of 16 young cattle in Stelasund, Germany, in 1983. This toxin could be another deterrent to cattle drinking from the lake, in addition to the high levels of S 0 4 2 " . Mazur-Marzec et al. (2005) found that the nodularin content of Nodularia spumigena increases with salinity. In contrast, the Cladophora group of algae, observed in LDB , is common to nutrient-rich lakes and ponds and has a very high tolerance for moderately saline lakes and even some coastal estuaries. They grow attached to rocks on lake bottoms, which are sufficiently shallow to provide exposure to light. Strong winds can detach large quantities of the algae to form floating masses which wash up to the shoreline and decay (Power 1990). Considered an important component of freshwater systems, Cladophora provide food and shelter for invertebrates and small fish. It is thought 98 that while living they may not be a preferred food source, but upon decomposit ion they can be utilized by detrital feeders (Dodds and Gudder 1992). Lac du Bois Twin, with its high pH and salinity, as well as the toxin producing Nodularia spumigena, is a toxic environment for most plants and animals. In addition, the aerobic environment in L D B Twin is detrimental to supporting S R B . However, since Eng (2005) observed sulphate reduction in Postgate B medium inoculated with Lac du Bois Twin sediment, S R B may be present in this lake. In contrast, L D B had a lower pH which was more suitable for sustaining plant life and an anoxic zone was observed in the decaying algae/sediment region of the lake. Sulphate reducing bacteria were shown to occupy this sediment and were effective at reducing S 0 4 2 " . It was shown that a potentially active population of S R B is present in L D B within sediment mixed with the decaying algae. Thus we hypothesized that this material could be used in a natural treatment system to remove sulphate from high sulphate water. It is envisaged that this treatment system would be a series of open ponds through which high sulphate water would flow. Algae growing on the pond surface would provide a carbon input to the system and anaerobic sediment on the pond bottom, containing decaying algae and microorganisms, would provide a habitat for S R B . Thus, a mesocosm-scale laboratory model of such a treatment system was constructed and tested. 99 Although evidence for the presence of S R B was found in L D B Twin, little sulphate reduction was assumed to be occurring due to the lack of anaerobic conditions, high pH and absence of utilizable electron donor carbon sources for S R B . The question arose that if an electron donor carbon source, proven to be suitable to support S R B , was supplied to LDB Twin, would sulphate reduction occur? If so, then in-situ remediation of high sulphate lakes could be another treatment approach. This was tested with in situ organic amendment of Lake 52, also a high sulphate lake, which was located on private land permitting experimentation. Since carbon source is usually the limiting nutrient in sulphate reduction, we decided to investigate what agricultural materials, available to cattle farmers, could be used as amendments to flow-through or in situ treatment systems. A selection of these waste materials was tested for their ability to support biological sulphate reduction. Initial batch test screenings of these materials is described next. 4.2 The Suitabi l i ty of Agr icu l tura l Materials as Subst ra tes for B io log ica l Sulphate Reduct ion Four different agricultural materials: hay, barley, silage and molasses, were tested for their effectiveness as nutrient sources for supporting S 0 4 2 " reduction activity by S R B . Initial experiments contained no additives other than the organic material and high sulphate water. However, it became clear that pH adjustment was required as well as sulphide removal and/or nutrient (PIN) addition in some cases . Thus, subsequent experiments were performed to compare the organic materials under 100 more ideal condit ions for biological sulphate reduction. 4.2.1 Sulphate Reduction Without pH Adjustment S ince the objective of my work was to develop a low-cost pass ive sulphate treatment, I dec ided to see if S R B cultures could be maintained on these C-sou rces without supplementat ion of addit ional chemica ls for nutrients or pH control. The materials were tested both individually and with a smal l addition of mo lasses to provide an immediate source of avai lable carbon. M o l a s s e s has been used to support S R B growth previously (Maree and Strydom 1987; Tare and S a b u m o n 1995). Of the four agricultural materials used , s i lage promoted the largest overal l dec rease in S 0 4 2 " when compared to the other materials (Figure 17, Tab le 8). 101 The bottles containing Hay and Bar ley did show sulphate reduction over the first 50 days of the experiment, but after this time their rates s lowed drastical ly. Only in the bottles containing si lage did the sulphate decrease almost linearly for the entire experiment. Figure 17 - (a)-(d) S0 4 2" concentration (mg L"1) versus time for the non-pH-adjusted batch experiments. Points represent the average S0 4 2" concentration in duplicate bottles. Error bars represent the difference in S0 4 2 between the two bottles in each pair. 102 Table 8 - Average overall S0 4 " change and maximum sulphate reduction rates (SRR) of non-Agricultural Material Overall Change in S 0 4 2 - ( m g L - 1 ) Maximum S R R Obtained mg L"1 d" 1 (mg d" 1 g" 1 substrate) Hay 523.5 ± 55.0 9.8 ± 0 . 9 R 2=0.98 (0.5 ±0 .0 ) Barley 548.6 ± 13.7 9.5 ± 2.1 R^=0.97 (0.5 ±0 .1 ) Si lage 1324.3 ±252.4 8.9 ± 2 . 1 R^=0.99 (0.5 ±0 .1 ) Molasses 286.2 ±21 .4 N/A Signif icance was determined by the Tukey test which performed multiple pairwise comparisons of the mean change in S 0 4 2 " for all agricultural materials. A pair were considered significantly different when P<0.05 and the level of significance was set to a=0.05. Except for the experiment with molasses, sulphate concentrations decreased linearly with respect to time for the first 33 days. Thus, maximum sulphate reduction rates were determined from the slope of the sulphate concentration versus time curve over the first 33 days. The maximum rate is derived from the linear curve fit to these points and the R 2 is provided. The bottles containing an addition of molasses showed no significant difference from their counterparts without the addition. Thus, results from these treatments are not shown here, but can be found in Appendix A -3 . When T S concentrations were measured at the end of the experiment, the concentration was highest in the bottle containing si lage (Figure 18). Since 333 mg L~1 sulphide is produced per 1000 mg L"1 S 0 4 2 " reduced, this result suggests 103 that the decrease in S 0 4 2 ~ as observed in the bottles containing s i lage was mainly due to chemical reduction of sulphate, likely carried out by S R B . However, the amount of sulphide measured at the end of the experiment in the bottle containing s i lage (172 mg L"1) only accounts for 4 0 % of the sulphide produced from 1324 mg L"1 sulphate reduced. 200 - 1 160 o> E V 120 f- 80 w 3 40 o 111 Hay Barley Silage Agricultural Materials Molasses Figure 18 - Final total sulphide concentration in the non-pH-adjusted batch experiments. Bars represent the average sulphide concentration in the pair of bottles. Error bars represent the difference in concentration of the pair The s C O D was observed to be high at the end of the experiment (Figure 19); however we cannot conc lude from this data whether the s C O D that remained was due to organic materials that are not util izable by the S R B . Detectable levels of N H 3 - N were observed in all of the bottles at the conclus ion of the experiment (Figure 20). The si lage had higher levels of ammon ia - nitrogen than the other materials but was still well under the levels obtained when using Postgate B med ia where the levels of ammonia present are 337 ppm. This is assuming that the Postgate B medium contains non-inhibiting concentrat ions of ammonium for S R B growth. 104 12000 -10000 -8000 D) 6000 Q O O in 4000 2000 81111 Hay HH IP Barley Silage Molasses Agricultural Materials Figure 19 - Final soluble COD concentration in the non-pH-adjusted batch experiments. Bars represent the average sCOD concentration in each pair of bottles. Error bars represent the difference in the pair's concentration 105 80 70 60 50 40 z • CO I 30 20 10 0 D) E • • Sift ijjj Hay Barley Silage Agricultural Materials Molasses Figure 20 - Final ammonia-nitrogen concentration in the non-pH-adjusted batch experiments. Bars represent the average concentration in each pair of bottles. Error bars represent the difference in the pair's concentration However, the orthophosphate levels were found to be low for all materials with the exception of silage (Figure 21). Ideally, for anaerobic digestion, a molar ratio of 250:5:1 is desired for C : N : P . 106 2.50 2.00 A O 0. • o E 1.50 A 1.00 A o 0.50 A 0.00 Hay Barley Silage Molasses Agricultural Material Figure 21 - Ortho-P043" concentration in the non-pH-adjusted batch experiments. Error bars represent the difference between the concentrations in the replicates The pH in each bottle remained largely unchanged over the course of the experiment, with slight increases in the hay and silage bottles, and decreases in the barley and molasses (Figure 22). 107 X a 10 8 6 4 2 0 mm V V 1^  p i ! .vs; \Yv-Hay Barley Silage Agricultural materials Molasses Figure 22 - pH in the non-pH-adjusted batch experiments. The filled bar represents the pH at the start of the experiment. The hatched bars represent the final pH. Error bars represent the difference between the pH found in the replicates 4.2.1.1 Discussion and Conclusions The largest reduction of sulphate occurred in the presence of si lage. While silage did not have the highest maximum S R R of all the agricultural materials used (8.9 ± 2 . 1 mg L"1 d~1), it was the only material that was able to sustain its rate throughout the course of the experiment. In contrast, the bottles containing the other agricultural materials were only able to sustain their high rates during the first 33 days of the experiment, after which the sulphate concentrations began to plateau. Of the four different materials, the bottles containing silage were the only ones, both at the start and at the end of the experiment, which had a pH within the tolerance r range (6-9) commonly used for growing S R B (Postgate 1979). It appears that the pH 108 did increase slightly for the si lage over time, which is not unusual since one of the products of S R B activity is carbonate which helps to increase the pH. For the other agricultural materials it appears the leachate was acidic from the beginning and little change was observed over time. In particular the pH of the barley and the molasses was very low for supporting S R B activity. It was also observed that the agricultural materials, other than si lage, had low levels of phosphate at the end of the experiment, and it is not known whether their s C O D contained any of the low molecular weight carbon sources required by S R B . These factors together likely contributed to the low sulphate reduction observed. One other likely reason why silage outperformed the other agricultural materials is that in the production of si lage, lactic acid bacteria (LAB) ferment the plant material and produce lactic acid, which is known to be an utilizable electron donor carbon source for S R B . Despite this, the maximum S R R obtained when silage was used is still much slower than S R R s measured with a defined carbon sources. An identical amount of inoculum from the same source grown in defined Postgate B medium was able to achieve S R R s of 40.0 mg L" 1d" 1 . It is possible that the silage introduced some compounds that are inhibitory to S R B . While homofermentative L A B turn the plant carbohydrates into lactic acid, there exists another group of L A B known as heterofermentative L A B that produce other compounds, in addition to lactic acid. One of the known compounds produced by heterofermentative L A B is acetate, which can act as an inhibitor to S R B activity. 109 Reis, Lemos et al. (1990) demonstrated that undissociated acetic acid inhibits S R B growth by 50% at a concentration of 54 mg L"1 acetic acid, by crossing the membrane of the S R B and interfering with the cells proton motive forces. Rzeczycka and Blaszczykm (2005) found that S R B inhibition was observed when acetate concentrations were greater than 10 mg L"1. The sulphide measured in the bottles containing si lage accounted for only 4 0 % of the total sulphide that would have been produced through the reduction of 1324 mg L"1 sulphate. It is hypothesized that the remaining 6 0 % escaped as H 2 S gas during sampling, when it was necessary to remove the cap to sample the fluid. A second set of experiments was performed to see if maintaining the pH at 7.5, which is optimal for the majority of S R B species, would improve the sulphate reduction for all the materials. There were some concerns that the inoculum of S R B could have had nutrients present due to residual Postgate B media in which is was grown, which may have contributed to the initial S R R s . To rectify this, the inoculum in the second experiment would be washed twice with distilled water before being used. Third, it was decided to carry out the experiment in an anaerobic chamber in order to maintain anoxic conditions. This would prevent introduction of 0 2 into the bottles during repeated sampling. Finally, the negative controls would undergo 2 hours of autoclaving in order to kill all bacteria upon it, as some of the negative controls did experience SO4 2" decrease. The negative controls were set-up to prove 110 that any drop in S 0 4 2 " was due solely to S R B activity and not by the absorption of SO4 2" on either the agricultural material or the interior of the bottle. I l l 4.2.2 Sulphate Reduction With pH Adjustment The pH in all the bottles of this new experiment dropped between sampling periods, particularly at the beginning of the experiment, and required further adjusting (Figure 23). 10 - Silage and Molasses 8 6 -j 4 * 2 0 50 100 150 Time (days) 200 0 50 100 150 200 Time (days) 10 Silage & Hay mixtures I Q. 4 -2 -0 , , , , 0 50 100 150 200 Time (days) Figure 23 -pH versus time in the pH-adjusted batch experiments. Error bars represent the difference between the pairs. • is Hay, A with the dotted line is Barley, • is Silage, x with the dotted line is Molasses, O is the 1:3 mixture, • is the 1:1 mixture and A is the 3:1 mixture. 112 In particular, the barley and the molasses proved the most difficult to stabilize at a pH suitable for S R B growth and activity, whereas the hay and the silage/hay mixtures required less pH adjustment. The sulphate removal in this experiment is different from what was observed in the previous experiment where no pH adjustment was made (Figure 24, Figure 25). In contrast to the previous batch experiment, where low S R B activityoccurred in the bottles that did not contain si lage, in this experiment, higher activities were observed in the bottles containing the other agricultural materials. For the first 50 days we can see that the bottles containing Hay and the Si lage and Hay mixtures had the highest overall decrease in S 0 4 2 " concentration. Statistically, the Hay and the Si lage/Hay mixtures 1:3 and 1:1, had significant overall decreases in their SO4 2 concentration (Table 9). 113 Replenish Inoculum H 2 0 lewis & FeCI 3 50 100 150 Time (days) 200 (b) Barley 1600 r- 1200 _i E CO 400 -I Replenish Inoculum H 2 0 levels & FeCI3 50 100 150 Time (days) 200 Figure 24 - (a)-(d) S0 4 2 ' concentration versus time in the pH-adjusted batch experiments. Lines represent the average S0 4 2 concentration of each pair. Error bars represent the difference in S0 4 2" between the two bottles in each pair. Stars indicate times when sulphide was measured in the bottles. 114 (a) Silage: Hay 1:3 Replenish H 2 0 levels Inoculum & FeCI 3 (b) Silage:Hay 1:1 Replenish H 2 0 levels | n o c u | u m & FeC I 3 1600 1200 o co 800 400 A 50 100 150 Time (days) 200 50 100 150 Time (days) 200 (c) Silage:Hay 3:1 1600 ^ 1200 O) E O co 800 400 Inoculum & Replenish FeC I 3 H 2 0 levels | 50 100 150 Time (days) 200 Figure 25 - (a)-(c) S0 4 " concentration versus time in the pH-adjusted batch experiments containing mixtures of hay and silage. Lines represent the average S0 4 2 concentration of each pair. Error bars represent the difference in S0 4 2" between the two bottles in each pair. Stars indicate times when sulphide was measured in the bottles. 115 Table 9 - Change and SRRs of pH adjusted agricultural bottle experiments. Results in brackets represent the mg d'1 g'1 substrate rates. Errors represent the difference between replicate bottles. Agricultural Material Overall Change in S 0 4 2 " (mg L"1) after 180 days Maximum S R R for first 50 days mg L ' 1 d- f (mg d" 1 g" 1 substrate) Maximum S R R after 126 days to day 150 mg L" 1d" 1 (mg d" 1 g 1 substrate) Hay 1099.6 ± 100.9 8.3 ± 0.6 R^=0.91 (0.5 ±0 .1 ) 10.4 ± 4 . 7 R^=0.97 (0.5 ±0 .1 ) Barley 378.7 ± 73.3 N/A 5.3 ± 1 . 5 R^=0.90 (0.3 ±0 .1 ) Si lage 589.9 ± 14.1 11.0 ± 2 . 5 R*=0.95 (0.6 ±0 .1 ) 8.1 ± 1 . 4 R^=0.89 (0.4 ±0 .1 ) Molasses 729.3 ± 26.0 N/A 9.5 ± 0 . 1 R^=0.93 (0.1 ±0 .1 ) Si lage/Hay 1:3 1045.5 ± 5 . 6 10.5 ± 1.0 R^=0.95 (0.6 ±0 .1 ) 8.7 ± 0 . 7 R*=0.95 (0.4 ± 0.0) Si lage/Hay 1:1 1124.517.5 14.4 ± 4 . 0 R^= 0.93 (0.8 ± 0.2) 8.5 ± 1 . 1 R 2=0.91 (0.5 ±0 .1 ) Si lage/Hay 3:1 930.7 ± 18.5 12.6 ± 0 . 2 R 2=0.92 (0.7 ± 0.0) 11.6 ± 1.8 R 2=0.90 (0.6 ±0 .1 ) The drop in S 0 4 2 " was biphasic in most of the bottles, with an initial steep decrease in SO4 2" followed by a period where the S 0 4 2 " levels began to plateau or increase. In an attempt to remedy this, fresh inoculum was added on day 126. After this addition, all of the bottles again underwent an increased rate of S 0 4 2 " reduction. At the time of the second inoculum addition, the pH in all the bottles remained stable and no further pH adjustments were made. For comparison purposes, two maximum sulphate reduction rates were calculated. A linear curve was fit through the first 5 data points from day 0 to day 57. However, during this period the pH was not always in the optimum range for S R B . Another sulphate reduction rate was calculated after 116 fresh inoculum was added and when the pH was constant, between day 126 and day 150. For the positive controls, black precipitate was observed throughout the vials, signifying that sulphide was being produced, which also suggests that the S R B in the original inoculum were active. After 50 days, one positive control vial was sacrificed and the SO4 2" concentration analyzed. All the readings were below the minimum detection limit for S 0 4 2 " , signifying that S R B had utilized the S 0 4 2 " present in the vial (Appendix A-4a). The negative controls were analyzed after 67 days and the average SO4 2" levels of the bottles were found to be unchanged from the starting SO4 2" concentrations. In addition the sulphide levels were measured and found to be too low to be detected. The exception was the 2 n d silage:hay mixture 3:1, which showed a sulphide increase which corresponds to a decrease in sulphate of 119.5 mg L"1. That particular bottle showed a decrease of 182 ± 254.7 mg L"1 S 0 4 2 " . Statistically there was no significant decrease in SO42". The results from this experiment can be found in Appendix A -4a . This signifies that without the addition of S R B to a SO4 2" solution the SO4 2" molecule is stable. This also indicates that any loss of S 0 4 2 " in my experiments is due to the presence of S R B . Sulphide was measured periodically in the bottles demonstrating the highest SO4 2" reduction rates and in all the bottles at the conclusion of the experiment 117 (Appendix A-4b). Throughout the course of this experiment, the analyzed total sulphide concentrations never rose above 100 mg L"1. A drop in the s C O D was observed for the bottles containing hay, si lage, and the hay/silage mixtures, while the barley and molasses either increased or remained at the same level (Figure 26). 12000 9000 O) E, Q 6000 O o 3000 • Hay Barley Silage Molasses S:H 1:3 S:H 1:1 S:H3:1 Agricultural Materials Figure 26 - Initial (solid bars) and final (hashed bars) soluble COD concentrations in pH-adjusted batch experiments. Bars represent the average sCOD concentration in each pair of bottles. Error bars represent the difference concentration. The ammonia-nitrogen and phosphate levels were measured 1 month into the experiment, and at the end of the experiment (Figure 27 and Figure 28). It was found, after the first sampling, that the phosphate levels in the bottles containing molasses were very low. It can be seen in the abiotic controls (Appendix A-4e) that bottles containing molasses as a sole carbon source have much lower levels of phosphate than the other materials. After observing that the molasses bottles had, up until this point, undergone no drop in SO42", it was concluded that the phosphate 118 was limiting and an addition of phosphate was made on t=29 days. Shortly afterwards, SO4 2" reduction was observed in the molasses bottles. With the exception of molasses, all of the bottles had increased levels of N H 3 - N over time. 250 -I — 200 -Li |> 150 -Z 100 to X Z 50 0 & Jt-% Agricultural Materials Figure 27 - Ammonia-nitrogen concentrations in the pH-adjusted batch experiments. The filled bar represents 28 days into the experiment. The hatched bar represents the final concentration after 180 days. Bars represent the average concentration in each pair of bottles. Error bars represent the difference in concentration between the two replicates. 119 350 — 250 Li L 150 O °- 50 -I 1 1 NSS -50 tf Agricultural Materials .N Figure 28 - Ortho-P043' concentrations in the pH adjusted batch experiments. The filled bar represents 28 days into the experiment. The hatched bar represents the final concentration after 180 days. Bars represent the average concentration in each pair of bottles. Error bars represent the difference in concentration between the two replicates. 4.2.2.1 Discussion and Conclusions The results from this experiment demonstrated that with closer monitoring and adjustment of pH, other materials besides silage, especially hay, could be used to support S R B activity. Without exception, all of the media containing agricultural materials experienced drops in pH, particularly at the beginning of the experiment. These drops in pH could be due to the decay of the plant biomass material over time, which releases organic acids. Clostridia, for example, are ubiquitous anaerobic organisms that can ferment grains to produce organic acids and are indigenous to si lage fodder (Barnett 1954). It is surprising that the silage and silage/hay mixtures have relatively high pHs since 120 it would be expected that their pH would be low due to lactic acid accumulated on the plant biomass as a result of the fermentation. However, it has been reported that maceration of si lage plant materials generally produce pHs of 6 or slightly higher (McDonald 1981). In all of the bottles an increase in SO4 2" was observed 84 days into the experiment. It is possible that this increase was due to the oxidation of sulphide back into S 0 4 2 " by other anoxic bacteria. While most sulphur oxidizing bacteria are obligate aerobes, strains of Acidithiobacillus have been found to operate in anoxic conditions by using nitrate as a T E A (Visscher and Taylor 1993). A s mentioned previously in Chapter 2, hydrogen sulphide, one of the products of S C V " reduction, is inhibitory to S R B . Its toxicity effect on S R B is directly proportional to its concentration and the effect is reversible if the sulphide is removed. Complete inhibition of S R B has been reported at H2S concentrations of 547 mg L"1 (Reis, Almeida et al . 1992), while others have found the S R B to have higher tolerances with only 50% inhibition observed with a H 2 S concentration of 1000 mg L"1 (Isa, Grusenmeyer et al . 1986). Throughout the course of this experiment, the analyzed total sulphide concentrations never rose above 100 mg L"1 and at a pH of 7.4-7.5 the H 2 S species would only account for 1/3 of the total sulphide while HS", not considered to be strongly inhibitory to S R B , accounts for the remaining. However, T S was only measured intermittently and higher levels of T S could have been 121 reached and inhibited S R B activity before being subsequently converted back into S 0 4 2 " by S O B . Ammonia-nitrogen and phosphate levels increased over time in all the bottles containing agricultural material, with the exception of the molasses. This could be due to the mineralization or decay of the organic plant material by indigenous bacteria found on the plant material. Through the actions of decomposer bacteria, the organic plant material is converted into ammonium and phosphate, forms suitable to support S R B growth. Molasses, as it is not organic plant material but instead composed primarily of sugars containing little or no nitrogen or phosphorus compounds, would not undergo decay and provide more N H 3 - N or P 0 4 3 " to the system. This would explain why its levels of N H 3 -N go down over the course of the experiment as its nutrients are utilized by the S R B . An addition of P 0 4 3 ~ was made to the molasses bottles at t=29 as measurements at the time indicated that PO4 3" levels were limited. Barley was still unable to support sustained S R B activity (overall drop of 378 mg L"1 S 0 4 2 " over 180 days), despite having the second highest levels of s C O D after molasses. This could be due to its electron providing carbonaceous material being maintained in the form of starch, which is not an utilizable form for S R B . Both experiments, both with and without a pH adjustment, demonstrate that barley is not a suitable nutrient source for S R B in a passive treatment system, while in its raw form. 122 Molasses did support higher S R B activity when the pH was adjusted; however it is clear that if it were to be used in a passive system, then the farmer would be required to make additions of phosphate. While molasses was able to achieve a maximum S R R of 9.5 ± 0.1 mg L' 1 d" 1 SO4 2" this was not the highest rate obtained in the experiment. Molasses has been used as a successful carbon source in bioreactors treating high sulphate water, but K H 2 P O 4 was provided to the S R B from the very beginning (Lebel, do Nascimento et al . 1985; Annachhatre and Suktrakoolvait2001). The s C O D concentration of the molasses bottles remained unchanged over the course of this experiment despite the activity of S R B . However, solid molasses was observed on the bottom of the bottle throughout the course of the experiment. It is likely that these solids continued to dissolve into the liquid over time, thus replacing s C O D that had been utilized by the S R B . Annachhatre and Suktrakoolvait (2001) found that there is a significant non-biodegradable component of molasses, and C O D : S ratios greater than 2 are needed to support effective S R B activity to compensate for this. Hay and the 1:1 silage:hay mixtures supported the highest reduction in S 0 4 2 " by S R B (1099.6 ± 100.9 mg L"1 S 0 4 2 " for hay and 1124.5 ± 7.5 mg L"1 S 0 4 2 ~ for 1:1 silage:hay) with high S R R s that were sustained for longer periods of time than in the bottles containing barley and molasses. (8.3 ± 0.6 mg L" 1d" 1 for hay and 123 14.4 ± 4.0 mg L" 1d" 1 for 1:1 s i lage:hay). S ince the 1:1 hay:silage mixture resulted in the largest drop in S 0 4 2 ~ , this mixture was tested in a larger scale (137 L) open-system, typical of a configuration likely to be used in the field (Section 3.5). Hay has not been used as a sole carbon source for S R B in other studies, however it has been used in combination with other materials, primarily manure (Barton and Karathanasis 1999), or in mushroom compost (Mclntire, Edenborn et al. 1990; Hammack and Edenborn 1992). In the first study, no S R B activity was observed, while in the second an addition of lactate was required to promote S R B activity. In the third study, the rates of S 0 4 2 " reduction varied between 0.192 - 57.6 mg L" 1d" 1. The hay and silage:hay S R R s compared well with those of other bench scale experiments which supplied S R B with non-defined carbon sources. Christensen, Laake et al . (1996) achieved S 0 4 2 " rates ranging between 3-4.4 mg L" 1d" 1 when using 1250 mL of whey, which has high levels of lactose. Waybrant, Blowes et al. (1998) achieved rates of 0.14 mg L"1 d~1 (g of organic substrate)" 1 using sewage sludge and 0.76 mg L 1 d" 1g" 1 while using leaf mulch, manure and sawdust. The 1:1 si lage:hay mixture performed better than these two substrates (0.8 ± 0.2 mg d"1g"1) however it did not achieve as high a S 0 4 2 " reduction rate as the mixture of sewage sludge, leaf mulch, wood chips, sheep manure and sawdust (4.23 mg L" 1d" 1g" 1). Although following a second addition of S 0 4 2 " , this rate dropped to 1.70 mg L"1 d" 1g" 1. 124 While the bottles containing only silage did not have the same degree of SO4 2" removal as in the previous experiment (1324.3 ± 252.4 mg L"1 without pH adjustment vs 589.9 ±14 .1 mg L"1 with pH adjustment), this could be due to the fact that the silage material used was from the same stock as the previous experiment, and was, therefore, not fresh. It is likely that, due to the time elapsed, the nutrients it would have provided in its leachate were consumed by microbes and the quality of the silage itself deteriorated. By opening the bag containing silage for the first experiment, oxygen was introduced into the silage. It has been reported that the aerobic organisms involved in silage deterioration utilize the lactic acid, acetic acid and soluble carbohydrates as their main sources of energy (McDonald 1981), thus reducing the amount of usable carbon for the S R B . It has also been found that the organisms responsible for this deterioration are indigenous to the silage itself (Beever 1980). This hypothesis is supported by the s C O D data. The measured final s C O D concentration for silage in the previous (no pH adjustment) experiment was 4814 ± 618 mg L"1 s C O D whereas the initial s C O D for this experiment (pH adjustment) was 2378 ± 148 mg L"1 s C O D . This is a 49% decrease in s C O D . Since the same mass of silage from the same stock was used in both trials, some of the s C O D must have been degraded during the storage of the silage between experiments. The silage must be stored without contact with air if it is to be used as a nutrient source. 125 With these first experiments, I wanted to determine how well each of these agricultural materials could support S R B activity with as little addition of other amendments as possible. The results revealed that si lage and hay performed the best as supporters of sulphate reduction and therefore these were used in a later mesocosm-scale experiment. Because the product of si lage fermentation is lactic acid, a known usable carbon source for S R B , one more experiment involving silage leachate was planned. The final batch experiment was designed using only silage leachate as a nutrient source. It was thought that using only the leachate in a treatment system could be beneficial by a) excluding solid material that would accumulate over time with repeated additions and potentially clog up the system, and b) prevent the solid sludge material from degrading further into unwanted organic compounds that might further contaminate the cattle's drinking water. 4.2.3 Sulphate Reduction Using Leachate from Different Silage Materials In the previous two experiments, the silage, derived from alfalfa, did demonstrate that it could support S R B growth and activity, particularly when it was fresh and had not come into much contact with oxygen. In this final batch experiment, two different types of silage, one derived from orchard grass and a second from barley, had their effectiveness at supporting S R B activity compared along with leachate obtained using waste material taken from a bio-ethanol plant which used corn as a substrate. In contrast to the two previous experiments, the agricultural materials were soaked with distilled water and filtered, and only the leachate was used as a nutrient source. 126 This was done in order to test the hypothesis that the S R B available carbon provided by the silage is due to the fermentation of the silage itself, and thus can be removed from the material by washing. Since it has been reported (McDonald 1981; Woolford 1984) that lactic acid is one of the prime carbon products produced through the fermentation of silage by lactic acid bacteria, and lactic acid is a known suitable carbon source for S R B , then the leachate from silage should act as an effective nutrient source. In this experiment the S 0 4 2 ~ and the s C O D were monitored at approximately 2 week intervals. The raw leachate still contained colloidal compounds, which had been removed from the flocced samples by zinc precipitation. A s can be seen in Figure 29 and Table 10, both the barley (TAD) and the orchard grass (ORC) based silages were able to support substantial S 0 4 2 ~ activity, although there was clearly batch variability present in the T A D Raw and O R C flocced bottles. The rates of sulphate reduction are given in both milligrams of S 0 4 2 " per litre per day, and milligrams of S 0 4 2 " per day per gram of substrate (the wet weight of solid silage material from which leachate was obtained). Maximum sulphate reduction rates were calculated from the slope of a straight line fitted through the first 4 data points where there was no lag, or through the 2 n d , 3 r d and 4 t h , data points in cases where there was a lag. 127 Figure 29 - (a)-(f) S0 4 2" concentration in leachate bottles. Solid line represents the first bottle in the pair, while the dotted line is the second. Error bars are the standard deviation of the analysis. 128 Table 10 - Overall change in SQ4 2" in leachate bottles over 71 days and maximum SRRs Barley Orchard Grass Bioethanol Waste A S 0 4 2 " (mg L-1) Raw 1227 .91325 .7 1026.6 ± 123.4 318.2 ±92 .3 Flocced 870.0 + 60.1 955.6 ±256 .1 290.4 ± 123.4 Maximum Sulphate Reduction Rates mg L V 1 mg cTV substrate mg L" 1d" 1 mg d~V substrate mg L/ 'd" 1 mg d ' g " 1 substrate Raw 17.2 ± 0.14 ± 13.6 ± 0.11 ± 11.4 ± 0.09 ± 0.2 0.0 0.8 0.0 1.5 0.0 R 2=0.97 R 2=0.86 R 2=0.99 Flocced 19.4 ± 0 .15± 18.5 ± 0.14 ± 4.4 ± 1.8 0.03 ± 1.6 0.0 3.0 0.0 R 2=0.90 0.0 R 2=0.96 R 2=0.98 In contrast to the silage experiments, the bioethanol waste (TBD) did not appear to strongly support S R B activity. When the s C O D is examined (Figure 30), it can be seen that the amount of s C O D provided by the materials did vary substantially; with the bioethanol waste having much lower levels of s C O D compared to the orchard grass and the barley si lages. 131 12000 — 9000 10 20 30 40 50 Time (days) 60 70 80 Figure 30 - Soluble COD in leachate bottles. Solid lines are the first bottle in the pair, while the dotted line is the second. Error bars represent standard deviation of the test • is barley silage, • is the orchard grass silage, and x is the bioethanol waste. Before adjusting the pH to 7.5 at the beginning of the experiment, the pH was measured and found to be too low to sustain S R B activity (Table 11). This is not surprising as the product of silage fermentation is lactic acid, which would cause the pH to be low. Table 11 - pH of leachate Poundmaker Orchard Grass Bioethanol 3.96 4.38 3.99 After adjusting the pH to 7.5 and inoculating, the pH was again monitored throughout the experiment. However, in contrast to the previous experiments, the pH remained more or less constant, requiring only one pH adjustment on day 14 (Figure 31). This is likely due to the absence of the solid agricultural material. Stirling and Whittenbury (1963) and Fenton (1987) showed that lactic acid bacteria, 132 responsible for creating acidic compounds during the fermentation of silage, reside on the surfaces of the silage material. By using only leachate for the biological sulphate reduction process, numbers of these acid-producing organisms are potentially reduced by excluding solid silage. (a) Orchard grass silage x a 4.0 2.0 0.0 20 40 60 Time (days) 80 (b) Barleysilage 8.0 6.0 X °- 4.0 2.0 0.0 20 40 60 Time (days) 80 Figure 31 - (a)-(c) pH in leachate bottles. Solid line represents the raw leachate. Dotted line represents the flocced leachate. Error bars represent the difference between replicate bottles. 133 (c) Bioethanol waste 8.0 6.0 X a 4.0 2.0 0.0 20 40 60 Time (days) 80 4.2.3.1 Discussion and Conclusions In this final experiment, it was demonstrated that silage leachate was able to support S R B activity. The barley and orchard grass silage leachates were both able to obtain high maximum sulphate reduction rates of 19.4 ± 1 . 6 and 18.5 ± 3.0 mg L"1 d" 1 S 0 4 2 " (flocced leachate), respectively. This is considerably higher than the rates obtained in the presence of the solid material. The maximum sulphate reduction rate measured when a 1:1 mixture of hay and silage was used was 14.4 mg S 0 4 2 ~ L"1 d" 1. While both the barley silage and barley grains had high concentrations of s C O D (82.1 and 557.8 mg s C O D g" 1 substrate, respectively) they had very different rates of sulphate reduction. The solid barley grains, although briefly able to achieve a maximum S R R of 21.8 ± 6.3 mg L"1 d" 1 S 0 4 2 ~ achieved a low overall change in the S 0 4 2 " concentration (378.7 ± 73.3 mg L~1 S 0 4 2 " ) . A s mentioned previously, the carbohydrates in barley grains are primarily composed of starch, a non-utilizable carbon source for S R B . In contrast the barley silage gave was able to maintain a high S R R throughout the experiment, most likely due to the ensi lage process which results in the production of lactic acid from the barley crop (leaves and stalks of the barley plant) (Barnett 1954; Woolford 1984), a utilizable form of carbon to act as an electron donor for S R B . 135 In order to calculate the stoichiometric A S 0 4 2 7 A C O D ratio for S R B grown on lactic acid, we see that 1 mol of lactate can convert 1.5 mol of sulphate (Eqn. 5) and 3 mol of C O D can oxidize 1 mol of lactate (Eqn. 6). 3S02~ + 2C3H603 -> 3HS~ + 6HCO~ + 3H+ (5) C3H603 + 302 -> 3 C 0 2 + 3H20 (6) This means that if lactate is fully oxidized by the bacteria then the theoretical ratio of A S 0 4 2 7 A C O D is 1.5 g g" 1. However, the average A S 0 4 2 7 A C O D ratios found for this experiment over 71 days are all well below this value (Table 12). Table 12 - AS0 4 VAsCOD ratio for leachate ex periment Tad Raw T A D Floe T B D Raw TBD Floe O R C Raw O R C Floe 0.32 0.29 0.34 0.25 0.21 0.24 What this means is that approximately 5 times more C O D was utilized than would be expected if lactate was accounting for all the C O D and S R B were the only bacteria present in the system. These numbers could mean that lactate is not the only bioavailable soluble carbon compound present, which is likely, especial ly if heterolactic bacteria instead of homolactic bacteria had fermented the si lage. Heterolactic bacteria produce ethanol, acetate and carbon dioxide in addition to lactate (McCullough 1978). Another possibility is that bacteria other than S R B are degrading the lactic acids, and other carbon compounds. Since S R B use only low molecular weight carbon compounds, we expect to find these in the soluble portion of the si lage. Lactic acid, produced in the ensi lage 136 process, is one of the carbon compounds found in silage wash water, and is readily utilizable by S R B . From our results it appears as if there is no benefit to having the solid portion of the silage present in the culture medium. Using leachate as the nutrient source has several advantages over using solid agricultural material. First, solid agricultural material can clog filters in a passive treatment system, whereas leachate would not. Secondly, using leachate would make sampling and analysis of the treatment system faster and more efficient since there would be less debris present in any samples taken. Based on these results, a passive treatment system (mesocosm experiment) could be supplied with silage leachate as the carbon source, rather than the whole solid si lage. Unfortunately the silage leachate experiment was undertaken after constructing and operating the mesocosms at the end of my studies, so this was not tested at the mesocosm level. 4.3 M e s o c o s m Exper iments While the previous microcosm bottle experiments were useful for determining what agricultural materials were effective at supporting S R B growth and what modifications or adjustments are needed (pH adjustment, monitoring of nutrients) to maintain activity, the S R R s obtained do not reflect what could be expected by farmers using these materials in a passive treatment system. Microcosm tests are useful as they are more space-efficient than larger-scale mesocosm or field studies, 137 and are easier to maintain under uniform conditions. Mesocosm experiments, simulated field studies conducted in controlled environments such as artificial ponds, streams or large outdoor tanks, are designed to more closely approximate actual operating conditions, such as would exist in a fully functional passive treatment system. Unlike the previous microcosm bottle experiments, the mesocosm experiments discussed here were operated under open air conditions, as it may be impractical for farmers to place a cover on a treatment pond. This section outlines the two sets of mesocosm experiments using different nutrient sources to stimulate S R B growth and activity. One pair of mesocosms used natural sediments and algae removed from Lac du Bois, and a second pair utilized a 1:1 si lage:hay mixture. 4.3.1 Natural Treatment System - Continuous A s mentioned previously in Section 4.1, Lac du Bois was found to have a potentially highly active population of S R B in a region of decaying algae and sediment. It was hypothesized that the S R B were utilizing low molecular weight carbon compounds, produced during the decay of the algae, as a nutrient source. This theory was tested by placing L D B sediment into a mesocosm with high sulphate concentration water, and monitoring the S 0 4 2 ~ levels within the system. The rate at which the S 0 4 2 ~ is reduced in the mesocosm would reveal how useful a system using natural lake sediment and algae would be in a passive treatment system for farmers to treat high S 0 4 2 " ponds. 138 Two mesocosms, containing high S 0 4 2 " water, decaying algae and sediment and seeded with live algae removed from Lac du Bois were initially operated as continuous systems. The mesocosms were fed a concentrated S 0 4 2 ~ (2500 mg L"1) solution at a rate of 6 mL min" 1, giving an estimated residence time of 15.8 days (residence time = Volume/Flow rate), assuming no short circuiting. The rational for this flowrate and retention time are outlined in Section 3.5. Both mesocosms had a thick layer of green algae from LDB covering the majority of the water's surface (Figure 32). The surface of this layer contained many bubbles, which were due to the release of gases from the sediment. This was visually ascertained whenever the sediment was moved in any way, such as when the rigid tubing was first placed in the bottom of the mesocosm. The pH in the mesocosms was initially found to be higher near the surface of the water and decreasing with depth. At depths of 10 and 20 cm below the water's 139 surface the pH was found on average to be 8.53 ± 0.8 and 7.89 ± 0.6 (st.dev. of 3 sampling points) in LDB A and 8.83 ± 0.2 and 7.74 ± 0.3 in LDB B. These measurements were made after the system had been left to equilibrate for a few months. It was decided that the pH did not require adjusting as it fell within the S R B pH tolerance levels of 6-9. After pumping concentrated sulphate water into the system, beginning September 2 8 t h (t=0), several changes were observed. First, the dense algae covering the water's surface immediately began to thin and large holes revealing the water's surface began to develop. By day 15 the green algae layer was restricted only to the effluent end of the mesocosms and by day 24, the water's surface was devoid of any apparent algae. Thus, the algae were not able to survive in the greenhouse under the conditions provided in the natural treatment system. Secondly, the pH in the mesocosms began to change. In particular, the pH at the 10 cm depth in the water column decreased as the algae layer disappeared. In contrast the pH 20 cm (water/sediment interface) below the water surface fluctuated but did not appear to undergo any overall change from the starting pH (Figure 33). The inlet water had a pH of 7.1 ± 0.1. 140 Mesocosm B £ 8.00 -I-10 20 30 40 50 Time (days) 60 Figure 33 - pH in natural system mesocosms. Solid line represents measurements at 10 cm depth, while the dotted line represents 20 cm depths. The D O measurements at the 20 cm depth (water/sediment interface) indicated that the oxygen levels in the system always remained below 0.4 ppm. If the DO probe was placed in the sediment layer itself, then the reading was too low to register, which indicates that the sediment was anaerobic. If it is assumed that plug flow characteristics were achieved in the mesocosms, then S 0 4 2 " reduction was observed. After a residence time of 15.8 days, all of the water in the mesocosms would have been replaced with the inlet concentrated S 0 4 2 ~ water, and any drop between the inlet and outlet concentrations would be due to the action of S R B . After 15.8 days the difference between the inlet and outlet S 0 4 2 " 141 concentration was 1677.3 ± 107.9 and 1645.9 ± 106.8 mg L"1 S 0 4 2 " for mesocosms A and B, respectively. If ideal plug flow behaviour were assumed, then this difference would be due to sulphate reduction by S R B in the sediment. However ideal plug flow behaviour in the mesocosm, where the residence time is the same for all fluid elements (Wilkes 2006), was unlikely due to the positioning of the inlet manifold, which extended half the length of the mesocosm. In addition, some axial mixing and short-circuiting might have been occurring. Since no tracer studies were performed, the actual flow characteristics of the mesocosm are unknown. If ideal plug flow characteristics are not present in the mesocosms then the estimated residence time of 15.8 days is incorrect. While the flow characteristics in the natural treatment mesocosms are unknown, it is strongly suspected that the S R B were briefly active as the breakthrough in sulphate concentration occurred much later than at the mean residence time. The levels of sulphide increased at t=15, which indicates that either some S 0 4 2 " reduction did occur, particularly in mesocosm B (Figure 35), or this sulphide was present in the sediment pore water, prior to t=0, and the actions of the pump caused it to be pushed out. An increase of 29.6 mg L"1 T S , as seen in mesocosm B, only corresponds with a decrease of 88.9 mg L"1 S 0 4 2 " . 142 o> E O cn 3000 2000 1000 Mesocosm A 20 40 Time (days) 60 3000 E O CO 2000 1000 \ Mesocosm B • * *\ ^ L Z ^ 20 40 Time (days) 60 Figure 34 - S0 4 " concentration versus time in natural continuous system. Dotted line represents outlet concentration; solid line is the inlet concentration. Stars represent the estimated residence time of 15.8 days. cn E 40 30 20 £ 10 20 40 60 Time (days) Figure 35 - Total sulphide versus time in natural continuous system. Solid line is mesocosm A, dotted line is mesocosm B. Stars represent the estimated residence time of 15.8 days. After checking the nutrient levels of ammonia and phosphate on day 48, no ammonium could be detected in the system and the phosphate levels, while still 143 detectable, had dropped to levels approximately 1/4 -1/5 of the starting values (Table 13). The initial levels of ammonium were similar to what was observed in Lac du Bois (0.67 and 0.39 mg L"1 N H 3 - N , Rocky and Algae locations in July 04, respectively). However, the P 0 4 3 ~ levels are high considering the levels in the lake were 1.72 and 0.52 mg L"1 P 0 4 3 ~ (Rocky and Algae locations in July 04, respectively). These high levels of PO4 3" are likely due to the addition of plant growth fertilizer, as mentioned previously in Section 3.5.1, which was added to encourage the growth of the algae. These levels of PO43", although higher than what was observed in the lakes, are lower than the levels present in the Postgate B media (349 mg L"1 P 0 4 3 " ) . Assuming that the Postgate B media does not have inhibitory levels of P 0 4 3 " , it is assumed that these levels of P 0 4 3 " in the mesocosms are not detrimental to S R B growth. 144 Table 13 - Ammonia-nitrogen and ortho-P043 concentration in natural treatment system. Errors are the difference between two test results. Initial After 48 days Mesocosm A mgL" 1 N H 3 - N 0.52 ± 0 . 1 2 0.00 ± 0.00 Mesocosm B mgL" 1 N H 3 - N 0.83 ± 0.25 0.00 ± 0.01 Mesocosm A mgL" 1 P 0 4 3 " 27.05 ± 0.45 7.43 ±0 .12 Mesocosm B mgL" 1 P 0 4 3 " 23.75 ± 1.15 4.92 ±0 .13 It is assumed that the continuous process washed out the nutrients from the mesocosm, faster than the degradation of algae could replenish them. In addition, as the algae growing on the water's surface was diminished, no fresh algae were provided to the sediment layer for decay. A s the fluid dynamics of the mesocosm system were unknown, and the kinetics of the algae decomposition and sulphate reduction were not available, it was decided to terminate the continuous process. Instead, the system would be operated as a batch reactor, so as to measure rates of sulphate reduction and nutrient depletion. This approach would allow us to determine some of the required kinetic information needed to design a future continuous system. 4.3.2 Natural Treatment System - Batch After determining that the continuous process had washed away the nutrients in the mesocosm, the sulphate pump was shut down and the system was allowed to operate as a batch instead. The mesocosms received an amendment of C : N : P in a 250:5:1 ratio, suggested as an appropriate nutrient feed to anaerobic reactors (Metcalf and Eddy 1991), using lactic acid, ammonium chloride and potassium phosphate in order to replace some of the nutrients lost and stimulate the re-growth 145 of the a lgae, which had all but d isappeared when the continuous system w a s running, and S R B . The carbon addit ion was intended to reach similar levels to what is suppl ied in Postgate B media which gives a concentrat ion of 1.03 g C L"1 but there was only sufficient quantities of lactic ac id avai lable to increase the concentrat ion by 0.76 g C L"1. The system was initially monitored, in one location, ha l fway down the length of the m e s o c o s m (100 cm) and at a depth of 15 cm from the surface, which was c lose to the water/sediment interface. Four days after the addition of nutrients, a lgae growth was observed on the water's surface, which deve loped into thick green a lgae completely cover ing the surface in one month's t ime. This a lgae was again sent out to an limnologist special ist for identification, with the results showing that what had initially been Cladophora glomerata a lgae (the spec ies found in the healthy L a c du Bois) , had been replaced by Nodularia spumegena (the spec ies found in L a c du Bo is Twin. Whi le Cladophora glomerata has been observed to occupy brackish environments with salinit ies of 17 g L"1 it is primarily a freshwater spec ies (van den Hoek 1963). It is suspected that the rate at which the salinity increased in the m e s o c o s m tanks was either too fast to al low the freshwater spec ies to adapt, or this strain of Cladophora was not capab le of adapting to this salinity. Thus, the introduction of this Cladophora sp. to sal ine waters is unlikely to succeed in a treatment sys tem. Nodularia, as ment ioned before, is a cyanobacter ia that demonstrates preference for sal ine environments. Horstmann (1975) found that the growth of nodularia strains in salinities ranging from 0-30 g,L" 1 , exhibited the highest 146 growth rates between 5-15 g L"1. Nordin and Stein 1980) found that the Uodularia strains showed no preference for the different cations (CI", C 0 3 2 " , or SO42"), and the growth rate was highest at salinities between 5-10 g L"1. Nodularia is an undesirable algae due to its toxicity. After monitoring the sulphate levels in the water column, it became apparent that SO42" reduction was not occurring. It was known that viable S R B were present in the sediment of the mesocosms as of day 57. A small microcosm experiment which placed sediment taken from the mesocosms into Postgate B media demonstrated that there were S R B present in the system (Table 14). Table 14 - Sulphate reduction when algae-sediment was inoculated into Postgate B medium Overall SO4 2" change (mg L"1) Inoculum sediment removed from different longitudinal locations and their culture time Mesocosm A Mesocosm B 50 cm/8 days 150 cm/12 days 50 cm/8 days 150 cm/12 days 684.9 1330.4 1051.3 1300.6 However, it appeared that even though there were S R B present in the sediment, they were unable to significantly affect the S 0 4 2 " present at the water-sediment interface (Figure 36). 147 2500 Z, 1500 -o> E. CN 6 1000 -to 500 -0 J , , , , , , , , , 0 10 20 30 40 50 60 70 80 90 Time (days) Figure 36 - S0 4 2" concentration in the water column. 100 cm down the length of the LDB mesocosms. • represents LDB A and • is LDB B. Error bars are the standard deviation of the analysis. Since sulphate reduction could not be measured at the water-sediment interface, we decided to measure sulphate concentrations within the sediment, where S R B were shown to reside. Sampling the sediment for S 0 4 2 " showed that the concentration in the sediment was considerably lower than what was present in the water column (Table .15). Table 15 - Comparison of SQ42" levels in water column and sediment of natural system Samples taken Jan 7, 2005 t=101 days Water Column (15 cm depth, 100 cm length) S 0 4 2 " mg L"1 Sediment (5 cm from sediment/water interface, 100 cm length) S 0 4 2 " mg L 1 Mesocosm A 2023.27 ±28 .1 676.07 ± 16.6 Mesocosm B 2074.91 ± 24.0 388.42 ±73 .1 148 Therefore there is a strong, vertical S 0 4 2 ~ concentration gradient within the mesocosm. The results indicate that the S R B were active within the sediment, considering the sulphate concentration in the sediment pore water would have been - 2 5 0 0 mg L"1 at the beginning of the batch experiment. The large S 0 4 2 ~ concentration gradient implies that vertical S 0 4 2 " diffusion is slow. This is why negligible sulphate reduction was observed at the sediment/water interface. Therefore, for a treatment system using sediment of this type, high sulphate water would have to be passed through the sediment where the S R B reside. To test the rate of sulphate reduction within the sediment, a recycle system was initiated which pumped water from the water column into the sediment through the rigid pipe system. This was continued until the S 0 4 2 " levels in the sediment had increased, at which point the recycle system was turned off and the sediment layer was monitored over time for S 0 4 2 " reduction at the inlet location (50 cm down the length of the mesocosm, 5 cm into the sediment from the water/sediment interface). Unlike the water column, which showed no decrease in S 0 4 2 " over time, there was an observable decrease in S 0 4 2 " in both mesocosms within the sediment at this location (Figure 37). Mesocosm A showed a decrease of 755.1 ± 100.5 mg L"1 S 0 4 2 " over 14 days within the pore water, while mesocosm B exhibited a drop of 848.5 ±152.1 mg L"1 S 0 4 2 " . After this drop, there was a slow slight increase in the sulphate concentration. Eventually, the sulphate reduction recommenced, albeit at a much slower rate than that measured initially. The increase in S 0 4 2 " could be due to several things. First, water from the water column could have accidentally mixed 149 with the sediment layer during sampling, thus increasing the S 0 4 2 " levels in the sediment, since the water column did not show SO4 2 " reduction and maintained its high concentration. Sampling the pore water required inserting a tube into the sediment, which always disturbed the sediment. Care was taken to insert the tube without an excess of axial movement in order to minimize this disturbance. Figure 37 - S0 4 2" concentration in natural system sediment under batch conditions. Solid line is mesocosm A, dotted line is mesocosm B 150 The total sulphide in the sys tem increased over time as the S 0 4 2 " dropped, which is a s expected when S R B are reducing SO42" (Figure 38). 250 -200 • A _l 150 TS (mg 100 -50 -0 -0 10 20 30 40 50 60 70 80 90 Time (days) Figure 38 - Total sulphide in natural system sediment under batch condition. Solid line represents mesocosm A and dotted line is mesocosm B. Error bars are the standard deviation of the test. The drop in sulphide during the middle of the run co inc ides with an increase in S 0 4 2 ~ and could be due to a re-oxidation of sulphide to S 0 4 2 " . Be tween days 15-49 the m e s o c o s m s exper ienced drops of 66.2 ± 5.9 and 39.2 ± 13.0 mg L"1 T S (mesocosms A and B, respectively). Assuming that the sulphide was re-oxidized completely back into S 0 4 2 " , an increase of 198.7 and 117.8 mg L"1 S 0 4 2 " (mesocosms A and B, respectively) would be expected in the pore water of the mesocosms . During this s a m e time, mesocosm A underwent a S 0 4 2 " increase of 264.1 ± 48.3 mg L"1 S 0 4 2 ~ , and m e s o c o s m B underwent a slight increase in S 0 4 2 " of 67.5 mg L"1 S 0 4 2 " between day 15-36 fol lowed by a drop of 462.86 ± 83.5 mg L"1 S 0 4 2 " . Within the error range, the A S 2 " cor responds to A S 0 4 2 " 151 The pH in the mesocosms' sediment (Figure 39), while it did fluctuate, always remained in the optimum range for S R B . 9.00 -, 7.00 -I , r- , , , , , , , 0 10 20 30 40 50 60 70 80 90 Time (days) Figure 39- pH in natural system's sediment. • represents LDB A and • LDB B A full discussion of the natural treatment system follows in Section 4.3.5. However some conclusions can be made here. First, sulphate reduction was observed within the sediment, over the first 15 days. However, after this time the SO4 2" slowly increased before undergoing a final drop, at a slower rate than previously observed. It is likely that this decrease in the S R R is due to depletion of nutrients, although this was not verified since nutrient concentrations were not measured. The algae supplied to the mesocosm were unable to survive at high salinities and were also insufficient for supplying nutrients to support continuous SO4 2" reduction. 4.3.3 Agricultural Material Treatment System In the previous experiment, natural sediment removed from L D B was used as a source of nutrients and S R B to promote sulphate reduction. There appears to be 152 limitations to using this approach to support sulphate reduction, as d iscussed in the previous section, A s mentioned in Section 2.3.2.5, many different agricultural materials have been used, with varying success , as nutrient sources in passive treatment systems. The results from this work (Section 4.2.2) indicated that using a 1:1 mixture of hay and silage resulted in the highest S R R out of all the materials tested. Thus this agricultural material was used in a scaled-up mesocosm. The mesocosm was operated as a batch so as to determine the sulphate reduction and C O D removal rates. Unfortunately, there was not enough time to run a continuous system. Two mesocosms were set-up that received sediment from Lac du Bois as an inoculum source. However, in contrast to the natural system which used algae, both alive and degrading, as a nutrient source, the agricultural system used a 1:1 (13 cm layer) mixture of silage and hay. Initially, the hay/silage mixture floated on the water's surface but gradually became water saturated over time. The system was run as a batch operation with close monitoring of the nutrient levels within the mesocosm. The pH within the mesocosms remained stable and well within the acceptable levels for S R B growth after being adjusted at the initiation of the experiment (Figure 40). The only time the pH dropped was after an addition of silage and hay was made to the mesocosms in order to increase the C O D levels. The pH in the mesocosm 153 dropped to 5.38 after this event. However, without making any additions of base to the systems, the pH re-stabilized to the previous levels within 2 days. 0 J , : r ~ , , , , 0 20 40 60 80 100 120 Time (days) Figure 40 - Average agricultural mesocosm pH versus time. Error bars represent the difference between the two replicate mesocosms. Utilizing silage and hay to supply the nutrients required for the S R B provided a substantial increase in the levels of ammonia and ortho-phosphate compared to those levels provided in the natural system mesocosms (Figure 41 and Figure 42). While the levels reached in the agricultural mesocosms were high, they were less than what is provided to the S R B in Postgate B media (349 mg L"1 P 0 4 3 " and 337 mg L"1 NH 4 CI) , a commonly used media for growing S R B , so it can be assumed that the levels are non-inhibitory. Unlike the ortho-PGv 3" which appeared to generally follow a downwards trend, the ammonia nitrogen levels remained fairly constant with a jump in the concentration after the addition of new agricultural materials. This suggests that the continued decay of the hay/silage material provided a steady release of ammonia which replaced that which was utilized by the S R B . 154 Time (Days) Figure 41 - Average ammonium-nitrogen concentration versus time in mesocosms. A is the agricultural system, • is the natural treatment system. Error bars represent the difference between the two replicate mesocosms. Figure 42 - Average ortho-P04 3 concentration in mesocosms versus time. A is the agricultural mesocosm, • is the natural treatment mesocosm. . Error bars represent the difference between the two replicate mesocosms. 155 In contrast to the natural mesocosm systems, where SO4 2" reduction was observed only in the sediment, in the agricultural treatment system SO4 2" reduction was observed in the water column. Samples taken halfway down the length of the mesocosm and at a depth of 15 cm demonstrated that the system was capable of reducing S 0 4 2 " (Figure 43) Time (days) Figure 43 - Average S0 4 2" and total sulphide concentrations in agricultural mesocosms versus time. • and • represent S0 4 2 and TS concentrations, respectively, Error bars represent the difference between the two replicate mesocosms. A s Figure 43 illustrates, SO4 2 " and T S concentrations in the two replicate mesocosms followed the same trends as evidenced by the relatively small error bars. The maximum S 0 4 2 " reduction rate after the first addition of S 0 4 2 " was 33.8 ± 2.3 mg L 1 d " 1 (R 2=0.99) calculated from the slope of a straight line fitted through data points for the first 19 days where the sulphate concentration drops approximately linearly with respect to time. Thereafter, the rate of sulphate reduction 156 slows, partly due to Monod kinetics with sulphate as the limiting substrate and potentially due to the build up of sulphide or other inhibitory compounds. However, sulphate was reduced eventually to levels less than 100 mg L"1. On day 82 a second addition of sulphate was made after which a much larger maximum S R R of 132.4 + 8.2 mg L" 1d" 1 (R 2=0.97) was measured using data points for the first 7 days. Another indicator that the S R B were successful in reducing the S 0 4 2 " present in the system is the increasing concentration of sulphide in the mesocosm. Over the course of the two additions of SO4 2" mg L"1 total sulphide in the two mesocosms followed an increasing trend to reach an average of 323.5 mg L"1 T S at the end of the experiment (day 101). The s C O D in the agricultural mesocosms followed a decreasing trend over time, which was expected, as the carbonaceous materials provided by the silage/hay would be utilized by the S R B and other organisms present in the system (Figure 44). 157 6000 5000 H 1000 -0 \ , , 1 , , , 0 20 40 60 80 100 120 Time (days) Figure 44-Average soluble COD concentration in agricultural mesocosms versus time. Error bars represent the difference between the replicate mesocosms. The second addition of silage and hay was soaked in H 2 0 prior to being added to the mesocosms with the intention of improving the wetting of the solid material. However, this resulted in unintentional washing off the s C O D compounds, thus the increase in s C O D was minimal. Nevertheless, nutrient levels were still sufficient to sustain sulphate reduction, as seen in Figure 43 where the second spike of sulphate was rapidly reduced. The average A S 0 4 2 7 A s C O D ratio was 1.005. 158 4.3.4 Agricultural Mesocosm System Failure After the second decrease in S 0 4 2 ' a third addition of S 0 4 2 " was made. However, instead of following the previous trend of decreasing over t ime, instead the S 0 4 2 " levels began to increase. Simultaneously, sulphide levels decreased. From day 109/110 to day 157, sulphate increased by 704.1 ± 136.0 and 768.1 ± 42.1 for mesocosms A and B, respectively (Figure 45). During this time, s C O D was still being consumed (Figure 46). Thus there was still bacterial activity in the system. Sulphide levels dropped 256.8 and 287.8 mg L"2 T S in mesocosms A and B, respectively, which corresponds to an increase in S 0 4 2 " of 778.2 and 872.3 mg L"1 S 0 4 2 " for mesocosms A and B, respectively. It is clear that re-oxidation of sulphide back into sulphate by S O B outweighed any sulphate reduction by any S R B still active. In fact, S O B were isolated from white slimy material found on the surface of the mesocosm (McLean 2005). It is hypothesized that the increasing sulphide concentrations over time led to this system failure. Subsequently, sulphate and sulphide cycling occurred in phase with each other, such that as sulphate concentrations increased, the sulphide concentrations decreased and vice versa. 159 S 0 4 2 ~ 100 150 200 250 300 Time (days) Figure 45 - Average S0 4 2 and total sulphide concentrations versus time in agricultural mesocosms. • represents sulphate and • is total sulphide. Error bars represent the difference between replicate mesocosms. Arrows indicate where an addition or alteration was made to the system. 0 -I , , , : , 100 150 200 250 300 Time (days) Figure 46 - Average soluble COD versus time in agricultural mesocosms. Error bars represent the difference between replicate mesocosms. 160 At this point several strategies were attempted to restore the system, as stated in Table 16. Table 16 - Changes made to the agricultural mesocosms Date of Event Event t= 159 days Add a 13 cm layer of silage and hay (1:1 wt%) t=174 days Placed a tarp over the system to restore anaerobic conditions and prevent S2' oxidation. t=222 days Added silage leachate to increase s C O D levels. A tarp was placed over the system to exclude air and block out sunlight, which could cause phototropic sulphur oxidizers to grow. The white slimy layer on the surface of the mesocosms visually disappeared over a 2-week period, but S 0 4 2 " concentrations continued to increase. Since s C O D levels remained low (Figure 46), si lage leachate was added on day 222. After performing all these amendments, samples of sediment and water were removed from the mesocosm, filtered, and then the filter paper was placed in Postgate B media to determine if S R B were still present. The presence of S R B would be determined through the blackening of the tubes containing the filter papers, due to the formation of F e S . However, as observed below, in Figure 47, no S R B activity was observed. The S R B in the mesocosm system appeared to have expired through the course of the experiment. Likely sulphide accumulation inhibited S R B activity and caused a shift in the microbial community. 161 Figure 47 - SRB presence in agricultural mesocosms. Each picture from a-d represents the SRB level at two depths in the mesocosm. The first pair was sampled at the bottom of the mesocosm, while the second pair is taken at a depth 9 cm from the bottom, a) is Agri A at the 50 cm location, b) is Agri A at 150 cm, c) is Agri B at 50 cm, d) is Agri B at 150 cm. e) is two negative controls (autoclaved sediment and inoculum SRB) followed by two positive controls (inoculum SRB) 4.3.5 Discussion of Mesocosm Experiments Within the pore water of the natural treatment mesocosms, located above the buried inlet pipes, S R R s of 53.9 and 60.6 mg L" 1d" 1 S 0 4 2 " were achieved for the first 14 days of operation (mesocosms A & B, respectively). However, these rates of sulfate 162 reduction decreased after 14 days , likely due to a lack of nutrients ( s C O D , N, P) and eighty-four days were required to achieve a total S 0 4 2 " drop of at least 1000 mg L"1 in both mesocosms in the batch sys tem. Assuming that a drop of 1000 mg L"1 SO42" is required in the final treatment sys tem, and assuming that the initial high rates of SO42" reduction could be maintained, a residence time of 19 days would be sufficient. However, the natural treatment system was unable to supply sufficient nutrients needed to cont inue the activity and the system became inactive. Al though sulfate reduction w a s occurr ing in the sediment, strong vertical SO4 2 " concentration gradients indicated that diffusion from the water column into the sediment was very s low. Thus in a treatment sys tem, high sulfate water would have to be directed through the sediment layer. It was observed in this exper iment that the subsurface infusion pipes occasional ly exper ienced clogging. A n alternative approach, utilized by Mclnt ire, Edenborn et al . (1990) who a lso exper ienced clogging with subsurface infusion pipes, involves placing a piece of p lexiglass in the mesocosm, situated so that the water is dammed behind the plexiglass and is forced to flow downwards, beneath the water co lumn and through the sediment. Another advantage to this sys tem is that the water would be introduced through the entire width of the sediment, rather than along one narrow region, as with the subsur face infusion pipe. S ince the sediment acts as a barrier to diffusion, it is necessary to introduce the water in a radial direction, rather than just 163 axially. If the subsurface infusion pipe system is utilized, then there would have to be several pipes situated in the system The maximum average SO4 2" reduction rate in the agricultural mesocosms after the first addition of SO4 2" was 33.8 mg L~ 1d"\ while after the second sulphate addition the rate increased to 132.4 mg L" 1d" 1. The rates obtained after the first SO4 2" addition are higher than what was obtained in the leachate experiments (19.4 mg L" 1d" 1 raw barley leachate), which was the highest S R R obtained in the previous experiments. However, the rates obtained after the second addition are considerably higher. Assuming that the maximum rate could be maintained, then a continuous system would only require a residence time of 8 days. The high second rate could be due to the S R B having acclimatized fully to their environment and nutrient source, or there could have been due to greater S R B numbers within the mesocosm. Sulphide is the product released by the dissimilatory sulphate reduction of sulphate by S R B . In the natural lake system of Lac du Bois, low levels of sulphide were observed in the water column. Sulphide in lakes is typically oxidized spontaneously by chemical oxidation in aerobic zones or by the actions of chemolithotrophic bacteria or phototrophic bacteria, otherwise known as sulphur oxidizing bacteria (SOB) into S° and S 0 4 2 " species (Thy, Fritz et al . 2000; Tonolla, Peduzzi et al . 2004). Sulphide can also be removed from the water column by mixing with a metal ion in solution, such as iron, which will precipitate the sulphide as a metal sulphide. 164 In the agricultural mesocosms, the sulphide levels increased rapidly until S 0 4 2 ~ reduction stopped. The sulphide concentration increased faster than it could be removed through natural methods such as sulphur oxidizing bacteria or volatilization of H 2 S gas. Hydrogen sulphide is well recognized as an inhibitor to S R B (Postgate 1979; Uberoi and Bhattacharya 1995), with the level of inhibition being directly proportional to the amount of sulphide present. The levels reached in the agricultural mesocosms (297.97 and 350.87 mg L"1 TS) are well above where inhibition has been observed for S R B . These increased levels of sulphide are one obvious reason why the S 0 4 2 ~ reduction stopped. In addition to inhibiting S R B and slowing down the rate of S 0 4 2 ~ reduction, it is believed that the high levels of sulphide also contributed to increasing the levels of S 0 4 2 " within the mesocosms. After 109 days, the S 0 4 2 " and the sulphide underwent cycling, whereby the S 0 4 2 " and sulphide concentrations would alternate increasing and decreasing. One obvious explanation for this is that S O B , demonstrated to be present in the mesocosm by McLean (2005), were oxidizing the sulphide in the system back into its S 0 4 2 " form. Once the sulphide levels had decreased, the S R B were no longer inhibited and could continue sulphate reduction until once again the levels of sulphide produced inhibited their activity. Oxidizing sulphide completely to sulphate is favourable for S O B , in terms of energy (Krishnakumar, Majumdar et al . 2005), so the easiest way to prevent the S 0 4 2 " 165 inhibition and cycling from occurring is to remove sulphide from the mesocosm as quickly as possible. In a continuous system, sulphide would not accumulate in the system. However, it would be necessary to remove sulphide from the effluent before the water could be released into the environment or fed to cattle. One way to treat high sulphide concentrations in the effluent of a SO4 2" reduction passive treatment system is to design a biological treatment system that oxidizes the sulphide, in a controlled manner, into solid sulphur. The sulphur will form aggregates that will settle in the system and can then be removed (Janssen, M a et al. 1997). One simple S O B reactor design was developed by Fox and Venkatasubbiah 1996) and consisted of a plexiglass ladder structure where a biofilms of Thiobacillus was cultivated. This system received water from an anaerobic reactor where S R B were actively reducing SO4 2" to sulphide and then recycled sulphide treated water back to the anaerobic system. By limiting the oxygen mass transfer by the surface area and flow-rate, they reduced the sulphide in the effluent of the anaerobic system from 212 to 20 mg L"1 sulphide. The A S 0 4 2 7 A s C O D ratio for the mesocosms were 1.10 and 0.91 for mesocosms A and B, respectively. These values are considerably higher than those obtained in the leachate experiment where the highest value obtained was 0.34 (bioethanol raw leachate) and also much closer to the theoretical A S 0 4 2 7 A C O D ratio of 1.5 gg" 1 for the reduction of sulphate by S R B grown on lactate. Either the S R B were able to utilize the carbonaceous materials provided in the agricultural mesocosms more 166 effectively than in the leachate experiments, or the degradation of the solid silage provided a continuous s C O D input Of the two treatment systems, the agricultural mesocosm design is more appropriate for use in the field by farmers. The agricultural mesocosm did not exhibit the same mass transfer limitations as the natural treatment system so additional equipment is not needed to recycle water through the sediment. With the agricultural treatment system, the hay/silage was mixed with the sediment, thus increasing the porosity of the sediment and allowing water to have greater access to the S R B . The agricultural material also provided more bioavailable nutrients. The agricultural mesocosms were also able to achieve the fastest rate of SO4 2" reduction, measured after the second addition of SO42". However, if such a system were to be used successful ly in the field, it would have to be continuous and include an additional downstream step to remove sulphide in order to prevent sulphide accumulation. 4.4 In Si tu Exper iment Due to the success in using hay and silage as a nutrient source for S R B in both the agricultural bottle and mesocosm experiments, one final experiment was undertaken which tested the effectiveness of this material at promoting S R B activity in an in-situ set-up located within a high SO4 2" concentrated lake, located in the Lac du Bois Grasslands Park. Unlike the bottle and mesocosm experiments, which were monitored frequently, this experiment would be monitored once during start-up and 167 311 days later. 4.4.1 Lake Description Lake 52, as seen on the map presented in the materials and methods section, is in the northern region of the Lac du Bois Grasslands Park. It is not easily accessible by road and is surrounded by sparse forests. Lake 52 is on private land and permission was obtained from the landowner to perform experiments within the lake. The lake's surface area is estimated to be 2.4 x 10 5 m 2 , which makes it smaller than Lac du Bois, but larger than Lac du Bois Twin. Unlike these two lakes, algae were not observed within the lake, although there was sparse growth of aquatic plant-life at the in-situ location. At a distance of 6 feet from the shore there was a 2.5 cm deep sediment layer followed by a 30.9 cm water column to the surface of the lake. Surrounding the lake, particularly at Site 1, was white precipitate on the shoreline, like that observed surrounding Lac du Bois Twin. Several dead trees were observed surrounding the shoreline. The buckets for the in-situ experiment were placed in a line along the shore of Site #2. After 311 days, the following observations were made for each bucket (Table 17). 168 Table 17 - Visual observations of in-situ buckets after 311 days #1 - Control Could see the sediment bottom. No signs of plant life, although a few small red insects were observed swimming. Same as was observed outside the buckets. #2 - Inoculated Hay/silage/fertilizer and decaying algae/sediment from Lac du Bois This bucket had unfortunately tipped over and was no longer useable for the experiment. #3 - Inoculated Hay/silage/fertilizer and decaying algae/sediment from Lac du Bois All of the hay and silage had sunk and was well mixed with the added L D B sediment #4 - Amended Hay/silage/fertilizer Significant red tint to the water. Likely caused by rust from a wire overhanging the bucket. 1 plant growing within. This plant appeared much healthier and larger than those observed in the lake outside the experiment. All of the hay and silage had sunk and was well mixed with the added L D B sediment #5 - Control Could see the sediment bottom. No signs of life. S a m e as was observed outside the buckets. #6 - Amended Hay/silage/fertilizer All of the hay and silage had sunk and was well mixed with the added L D B sediment 4.4.2 Chemical Analysis Temperature/pH/DO At the beginning of the experiment (October 4 , 2004), the average temperature at the sample sites within the lake was 11.9 and 13.9 °C at sites 1 and 2, respectively. The pH at each site was 9.67 and 9.32 at sites 1 and 2, respectively. After 311 days (August 11, 2005) the average temperature within the lakes was 23.6 and 21.2 °C. 169 The pH was measured at 3 different depths and the averages are presented here (Table 18). All had a standard deviation of ± 0.0. Table 18 - pH within Lake 52. Taken on day 311 (August 11, 2005) Site 1 Site 2 #1 Control #3 Inoculated #4 Amended #5 Control #6 Amended 9.33 9.39 9.40 9.40 8.91 9.33 9.34 Using A N O V A and Tukey tests to compare the pHs indicated that there were significant differences, not only between some of the buckets containing different treatments, but also within the replicates (P<0.0001 a=0.05). What was particularly interesting was that, with the exception of the #4 Amended bucket, buckets situated next to each other spatially were not significantly different. The pH of the lake water at site 2, the location for the in-situ experiment, was not significantly different from #1 Control, or #3 Inoculated. This suggests that the spatial location within the lake played an important role in the pH. All of the readings are considered well above the ideal pH for growing S R B and may pose a problem for an in-situ treatment. Although S R B thrive in a broad range of environments, they are typically grown in pHs between 6-9. Only recently have a few S R B species, Desulfonatronum cooperativum, Desulfotomaculum alkaliphilum and the Desulfovibrio alkalitolerans been discovered and identified as alkaliphilic (Pikuta, Lysenko et al . 2000; Zhil ina, Zavarzina et al. 2005; Abildgaard, Nielsen et al . 2006). Desulfonatronum cooperativumare is an obligate alkaliphile and requires a pH greater than 10, while Desulfovibrio alkalitolerans can tolerate lower pHs from 6-9.9, with an ideal pH range from 9-9.4. 170 The dissolved oxygen (DO) was measured within the lake at the two location sites at the start of the experiment in October 2004. The DO, averaged from 5 or 6 depths was 93.1 ± 2.5 % saturation and 93.4 ± 1.2 % saturation at sites 1 and 2, respectively. At the time, I believed these values to be suspect. At the time I was suspicious of the high DO readings found in Lake 52 and Lac du Bois Twin, and I wrongly believed that the high readings were due to interferences to the probe caused by the high salinity. I couldn't rationalize why these waters should be close to saturated, especial ly since I did not observe high levels of plant life within the lakes which could supply oxygen. It wasn't until much later that I found out that high levels of salinity decrease the maximum amount of oxygen that can be dissolved in water, which means that the water can become saturated at lower concentrations of dissolved oxygen. However, during the sampling trip on day 311,1 was still under the impression that the DO probe was not functioning correctly in highly saline lakes, so I instead used the Winkler colorimetric method to test the DO in the lakes and the buckets. One of the steps in the test is to add a sulphuric acid reagent to the water sample, which causes the manganese oxide precipitate (brown flakes) to dissolve. All of the flakes must dissolve before proceeding with the test, however, if the water has a high DO, this can take several minutes. When this test was performed on the lake 52 and bucket samples, the brown flakes did not dissolve, even after more than 20 minutes of mixing. For this reason, I could not determine the exact DO concentration within the experiment, except to report that the DO was high in every bucket, and in the lake at both site 1 and 2. This problem has been encountered 171 before by Morgan 1998 who found that the brown flakes did not dissolve even after 30 minutes of mixing, when testing marsh water in Bogue Sound, North Carol ina. Sulphate and Sulphide At the beginning of the experiment in October 2004, S 0 4 2 " levels were found to be 4188.7 ± 165.6 and 4156.0 ±179.5 mg L"1 and TS to be 0.14 ± 0.10 and 0.05 ± 0.02 mg L"1 at Sites 1 and 2, respectively. It was assumed that the levels of S 0 4 2 ~ and TS in the buckets would be similar and there was no need to test these levels individually in each bucket. After 311 days, the buckets were sampled for SO4 2" and TS (Figure 48, Figure 49). The results from sampling the lake and the in situ experiment indicate that the addition of agricultural material and slow release fertilizer had no positive effect on reducing S 0 4 2 " levels. The results from an A N O V A test indicate that there was at least one significantly different S 0 4 2 " mean (P=0.0001, a=0.05) while an additional Tukey test found that #4 Amended and #5 Control had significantly higher S 0 4 2 " means than the other buckets and the lake. 172 6000 — 4500 l i O ) E. 3000 o w 1500 of tr 6 T was* i i If "PA "mi "/''•>' M tr Figure 48 - S0 4 " concentration in Lake 52. Taken on day 311. Average and st. dev. from 5 or more samples. Sulphide levels in the in situ buckets, in contrast to the S 0 4 2 " levels, were all significantly higher than in the lake (ANOVA, p=0.0001 a=0.05, Tukey test). Amongst the buckets, #4 Amended was significantly different from all other buckets. CD E CO I - 1 0 0. 50 00 50 00 50 00 50 00 of tr 38.71 7.40 IT9 Figure 49 - Total sulphide concentration in Lake 52. Average and st. dev. from 5 or more samples. Text above #5 and #6 bars indicate high TS levels in the sediment/water interface. 173 Nutrients Initial sampl ing of Lake 52 found levels of N H 3 - N (0.39 ± 0.03 mg L"1) similar to those of L D B (0.44 ± 0.03 mg L"1) and L D B Twin (0.5 ± 0.02 mg L~1), N 0 3 " - N levels (0.05 ± 0.01 mg L"1) slightly higher than found in those lakes (Lac du Bo is 0.02 ± 0.02 mg L"1, L D B Twin 0.02 ± 0.0 mg L"1), and P 0 4 3 ~ levels (0.06 ± 0.03 mg L"1) which were considerably lower (LDB 0.56 ± 0.14 mg L"1, L D B Twin 5.00 ± 0.70 mg L"1). Al l the measurements were taken on the October 4, 2004 expedit ion. Consider ing that phosphate and ammon ia are both necessary nutrients for S R B growth, it was considered important that some nutrient addition be made to increase these levels in the in-situ treatment sys tem. For this reason, a slow re lease fertilizer was added to all buckets with the except ion of the controls. During the August 2005 trip, the levels of P 0 4 3 ~ and N H 3 - N were tested again (Figure 50, Figure 51). O n c e again it appears that spatial location within the lake has had an effect on nutrient levels. Al l the buckets situated left of the #4 A m e n d e d treatment do not differ significantly, while those to the right of the #3 Inoculated are significantly different from them. ( A N O V A P O . 0 0 0 1 , a = 0.05) 174 0.50 0.40 ro 0.30 E O CL 0.20 0.10 0.00 r i do Figure 50 - Ortho-P04 " concentration in Lake 52. Average and st. dev. of 3 depths. 1.50 _ 1.20 Li 0.90 E. 2 0.60 CO I Z 0.30 0.00 ¥0, of # -c> fe M Mr**! Figure 51 - Ammonium-nitrogen concentration in Lake 52. Average and st. dev. of 3 depths. Soluble C O D levels in the lake were 143.5 mg L"1 ± 2.5 (difference between Sites 1 and 2) in October 2004. These levels are lower than what was observed in either Lac du Bois (350.0 ± 23.5 mg L"1 s C O D ) or Lac du Bois Twin (170.2 ± 22.7 mg L"1 175 s C O D ) . It was hypothesized that an addition of hay and silage would provide sufficient s C O D to encourage S R B activity within the lake. The addition of hay and silage to Buckets 2, 3, 4, and 6 should have immediately increased the s C O D levels within by at least 1315 mg L"1 due to the silage material alone. The s C O D was analyzed in each bucket after 311 days (Figure 52). Once again buckets to the left of the #4 Amended do not differ significantly, while the #4 Amended and #5 Control are significantly different from the other buckets. The #6 Amended, while significantly different from the #4 Amended, is not significantly different from buckets #1 or #3 or from the lake water outside the experiment. 250 200 en 150 E. g 100 O W 50 of mm isp | | | Rtf fry . 0 / <f 4> i •hit:, 4? Figure 52 - Soluble COD concentrations in Lake 52. Average and st dev. of 3 depths. 4.4.3 Discussion of In Situ Experiment The results from these tests are surprising. There appears to be some inconsistency within the results obtained from the replicate buckets. The #1 Control bucket had nutrient levels, S 0 4 2 * and T S levels which mimic those of the lake outside the 176 experiment, but the #5 Control bucket had levels which were significantly higher. In addition, while the #3 Inoculated experiment results did not differ from what was observed in the lake, #4 and #6 Amended buckets had much higher levels of P 0 4 3 " and N H 3 . This is interesting considering that these buckets received exactly the same materials as the inoculated bucket, with the exception that no sediment from Lac du Bois was added as a source of S R B inoculation. Overal l , this experiment was poorly designed, which makes drawing conclusions difficult, if not impossible. If this experiment were to be tried a second time, the following alterations should be made. 1. The lake should have been visited and sampled prior to constructing any in situ treatment system. One of the major problems I encountered was that I had no physical or chemical information concerning Lake 52, before I arrived to set up the system. I did not know how deep the lake was, which impacted the size of the buckets used, nor did I know how hard the lake floor would be, which affected how I was to anchor the buckets within the lake. Finally, I didn't know the lake pH, which could have prompted me to bring chemicals to adjust the pH to within the tolerance levels for the majority of S R B species (6-9). At the time, it was decided not to adjust the pH within the buckets, since on a large scale this would not be feasible. 2. I pounded metal posts into the lake floor and wedged the buckets between two of these spikes in order to anchor the buckets within the system. I wrapped steel wire around the buckets and tied them to the metal posts in order to secure them. This proved insufficient as the #2 Inoculated bucket 177 tipped over during the course of the experiment. I would recommend using at least 3 metal posts to anchor the buckets more securely. 3. The steel wire which I used to anchor #4 Amended bucket to its metal posts had its ends suspended over the bucket. After 311 days, this wire had corroded and iron oxides were released into the bucket, contaminating the experiment. The water in this bucket had a red tint, and a large aquatic plant was observed growing within. The plant was larger than any of the other plants observed in the lake system outside the experiments. Whether the iron oxide enhanced the growth of this plant is unknown, but in the future, care should be taken to make sure that nothing is suspended over the buckets that can cause contamination. 4. The biggest mistake I made in this experiment, was making the assumption that sampling the lake at two locations was sufficient for determining the t=0 conditions within the buckets. Each of the buckets should have been sampled at t=0 for S 0 4 2 " , T S , ammonia, phosphate, nitrate, and s C O D . This would have enabled me to determine if the differences in the replicate buckets was present at the start of the experiment or caused by something happening during the 311 days of the experiment. 5. I should have brought along a DO meter during the experiment's final sampling, in addition to bringing the Winkler colourimetric test, even if I was suspicious of the results. S ince the high DO concentration within the 178 lake caused the Winkler test to fail, it would have been beneficial to have the DO meter present to take a reading. If this experiment were to be repeated, I would recommend having triplicate buckets, rather than duplicates. This would provide more security that the results can be compared with greater reliability and statistical confidence. 179 5.0 Conclusions and Recommendations for Future Work 5.1 Conc lus i ons 1. Lac du Bois and Lac du Bois Twin had several differences which may have contributed to their different salinities a. Lac du Bois Twin was saturated with dissolved oxygen while Lac du Bois had areas with low or no detectable dissolved oxygen present. S R B require anaerobic conditions in order to be active. b. While both lakes did have S R B present in the sediment, laboratory experiments suggest that those in Lac du Bois had a greater potential for reducing SO42". c. Lac du Bois Twin had a high pH and contained toxin producing Nodularia spumigena, creating a more toxic environment for plants and animals. Lac du Bois Twin had lower levels of dissolved organic compounds and s C O D thus providing less matter to provide electron donor compounds to support S R B . 2. a. After testing several agricultural materials for their effectiveness as a nutrient source for S R B , it was determined that si lage, whether prepared from orchard grass (18.5 mg SO4 2" L" 1d" 1), alfalfa (14.4 mg S 0 4 2 " L"1d"1), or barley (19.4 mg S 0 4 2 " L"1d~1), was an effective substrate. These materials are suitable for a treatment system with the following attributes 180 i. cheap and readily available carbon source ii. easily constructed iii. low cost iv. low maintenance v. built using materials that most farmer would have at their farm vi. capable of reducing S 0 4 2 " concentration in water to below 1000 mg I/1. The leachate alone was sufficient for supporting S R B . The physical mass of si lage was not necessary, which is advantageous in passive treatment systems, since there would be less debris to clog filters or pipes or interfere with sampling. Of the other agricultural materials tested, the following conclusions could be made: i. Molasses requires an addition of phosphate to initiate sulphate reduction. ii. Barley in its grain form was unable to support significant S R B activity; however barley silage (made from the fermentation of the entire barley plant) did provide suitable substrate in its leachate to support S R B activity. The agricultural mesocosms, containing 1:1 hay:silage, demonstrated two successful batch decreases of SO4 2" with maximum S R R s of 33.8 mg L" 1d" 1 after the first addition of S 0 4 2 " and 132.4 mg L / V after the second. If the second rate could be maintained, then a residence time of 8 days would be sufficient for lowering SO4 2" concentrations by 1000 mg L"1. b. In the natural treatment system, after raising the levels of S 0 4 2 ~ in the sediment, S R R s of 53.9 and 63.6 mg L" 1d" 1 SO4 2" were achieved in the first 14 days in mesocosms A and B, respectively. After this time, a brief period of sulphur oxidation was observed followed by sulphate reduction at a much slower rate than first observed. It was concluded that the natural treatment system was unable to supply sufficient nutrients to sustain active S R B activity. a. In the natural treatment mesocosm, operating as a continuous system, the rate at which new water flowed through the system (6 mL min' 1) washed the nutrients, provided into the water column by the decaying algae, out of the system. A much slower rate would be needed if this system were to be operated continuously. b. It is suspected that sulphide within the agricultural treatment mesocosms inhibited the S R B and halted the reduction of S 0 4 2 " . i. Sulphur oxidizing bacteria were observed within the mesocosms, and it is suspected that they are responsible for converting the sulphide within the mesocosm back into SO42". 182 ii. In a continuous system, the sulphide could be prevented from reaching lethal toxic levels, by limiting the amount of sulphate reduced. 5.2 Future Work The final application of this work is to help develop a continuous system that farmers can utilize to reduce sulphate in natural reservoirs to provide drinking water for their cattle. Therefore, the focus of the future work should be directed towards achieving this goal. 1. It is recommended that organic compounds in the silage/leachate be identified. This would indicate what concentrations of utilizable carbon compounds are provided, but more importantly, it would also reveal what inhibitory compounds are present and if there are any compounds that are toxic to cattle but which may not be toxic to the S R B . 2. Different dimensions should be investigated for the treatment system. Currently the L:W ratio was 7.8:1. This ratio could be varied to determine the effect on residence time distribution. According to literature, ponds with dimensions of 1:1 are more prone towards short circuiting, but those with high ratios can experience pressure drop problems (Shilton 2005). 3. Instead of the current manifold design, the system could be altered to mimic an upflow anaerobic sludge blanket reactor, by having a series of 183 subsurface pipes pumping the concentrated SO4 2" water throughout the sediment. 4. Tracer studies should be performed to determine the flow regime in the treatment system. This is important to determine if short circuiting is occurring. 5. Instead of using the hay/silage mixture, utilize only the leachate, in order to reduce the solid material going into the system. This makes sampling easier, as well as reduces clogging in pipes and filters, and improves the clarity of the water, making it more suitable for the cattle. 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Sulphate reduction in laboratory microcosms containing LDB sediment and Postgate B media Date Time (days) Transfer # O.D. SG-42" (mg L-1) O.D. S0 4 2" (mg L-1) Average S0 4 z" (mg L"1) 6-Mar 0 1 0.466 1710.08 0.463 1701.75 1705.91 11-Mar 5 1 0.267 1157.49 0.254 1121.40 1139.44 16-Mar 10 1 0.161 863.15 0.170 888.14 875.65 20-Mar 14 1 0.39 749.52 0.377 731.47 740.50 24-Mar 18 1 0.345 687.04 0.353 698.15 692.60 31-Mar 25 1 0.356 351.16 0.325 329.64 340.40 4-Apr 0 2 0.415 1568.46 0.421 1585.12 1576.79 13-Apr 9 2 0.379 1468.50 0.362 1421.29 1444.89 16-Apr 12 2 0.237 1074.19 0.233 1063.08 1068.64 18-Apr 0 3 0.414 1565.68 0.413 1562.91 1564.29 28-Apr 10 3 0.002 421.64 0.174 449.6256 435.63 29-Apr 0 4 0.458 1687.86 0.432 1615.67 1651.76 11-May 12 4 0.939 1511.75 0.376 1460.16 1485.96 17-May 18 4 0.577 1009.15 0.569 998.04 1003.60 20-May 0 . 5 0.53 1887.79 0.528 1882.24 1885.02 28-May 8 5 0.103 702.10 0.287 606.51 654.31 7-Jun 18 5 0.203 489.89 0.216 507.94 498.91 Transfer 6 data N/A 19-Jul 0 7 0.386 1655.30 0.267 1649.08 1652.19 5-Aug 17 7 0.139 957.81 0.148 983.22 970.52 A1-2a. S0 4 2 ' concentrations in Lac du Bois and Lac du Bois Twin 6-Jul-04 Lac du Bois Depth (cm) Sample Dil. O.D. S0 4 2 ' (mg L-1) Dil. O.D. S0 4 2" (mg L-1) Dil. O.D. S0 4 2 _ (mg L-1) Average S0 4 2" (mg L-1) St. Dev. 10 LDB Rocky 1.2 0.241 62.29 1.2 0.235 61.44 1.2 0.234 61.30 61.68 0.53 20 LDB Rocky 1.2 0.237 61.73 1.2 0.253 63.99 1.2 0.227 60.32 62.01 1.85 30 LDB Rocky 1.2 0.223 59.75 1.2 0.245 62.86 1.2 0.222 59.61 60.74 1.84 40 LDB Rocky 1.2 0.233 61.16 1.2 0.220 59.33 1.2 0.256 64.41 61.63 2.57 5 LDB Algae 1.2 0.201 56.64 1.2 0.181 53.82 1.2 0.172 52.55 54.34 2.10 10 LDB Algae 1.2 0.198 56.22 1.2 0.187 54.67 1.2 0.192 55.37 55.42 0.78 4-Oct-04 10 LDB Rocky 1.2 0.471 60.75 2 0.189 60.49 1.2 0.475 61.19 60.81 0.35 20 LDB Rocky 1.2 0.462 59.78 2 0.181 58.76 1.2 0.465 60.10 59.55 0.70 30 LDB Rocky 1.2 0.441 57.51 2 0.174 57.24 1.2 0.452 58.70 57.82 0.78 40 LDB Rocky 1.2 0.44 57.40 2 0.161 54.43 1.2 0.449 58.37 56.73 2.05 5 LDB Algae 1.2 0.43 56.32 2 0.163 54.86 1.2 0.426 55.89 55.69 0.75 10 LDB Algae 1.2 0.324 44.85 2 0.161 54.43 1.2 0.422 55.45 51.58 5.85 20 LDB Algae 1.2 0.442 57.62 2 0.168 55.94 1.2 0.437 57.08 56.88 0.85 6-Jul-04 Lac du Bois Twin Depth (cm) Sample Dil. O.D. S0 4 2" (mg L-1) Dil. O.D. so42-(mg L"1) Dil. O.D. S0 4 2 ' (mg L-1) Average S0 4 2" (mg L-1) St. Dev. 5 LDB Twin Site 1 95.17 0.396 6675.92 95.17 0.388 6586.34 190.33 0.101 6744.85 6669.04 79.48 10 LDB Twin Site 1 47.58 1.019 over 95.17 0.413 6866.29 95.17 0.401 6731.91 6799.10 15 LDB Twin Site 1 190.33 0.103 6789.64 95.17 0.381 6507.96 95.17 0.393 6642.33 6646.64 140.89 20 LDB Twin Site 1 95.17 0.391 6619.94 190.33 0.103 6789.64 95.17 0.398 6698.32 6702.63 84.93 25 LDB Twin Site 1 190.33 0.099 6700.06 95.17 0.365 6328.80 95.17 0.387 6575.14 6534.67 188.91 ON Depth (cm) Sample Dil. O.D. S0 4 2" (mg L-1) Dil. O.D. S0 4 2 ' (mg L-1) Dil. O.D. SO4 2 " (mg L-1) Average S0 4 2" (mg L'1) St. Dev. 5 LDB Twin Site 2 95.17 0.386 6563.95 95.17 0.391 6619.94 190.33 0.092 6543.30 6575.73 39.65 10 LDB Twin Site 2 95.17 0.381 6507.96 95.17 0.387 6575.14 190.33 0.098 6677.67 6586.92 85.47 15 LDB Twin Site 2 95.17 0.403 6754.31 95.17 0.412 6855.09 95.17 0.428 7034.25 6881.22 141.79 20 LDB Twin Site 2 190.33 0.101 6744.85 95.17 0.385 6552.75 95.17 0.37 6384.78 6560.79 180.17 25 LDB Twin Site 2 190.33 0.095 6610.48 95.17 0.384 6541.55 95.17 0.386 6563.95 6571.99 35.16 4-Oct-04 5 LDB Twin Site 1 80 0.635 6279.75 160 0.289 6570.06 80 0.635 6279.75 6376.52 167.61 10 LDB Twin Site 1 80 0.597 5950.85 160 0.303 6812.41 80 0.661 6504.79 6422.68 436.61 15 LDB Twin Site 1 80 0.641 6331.68 160 0.281 6431.57 80 0.653 6435.54 6399.60 58.85 20 LDB Twin Site 1 80 0.666 6548.06 160 0.295 6673.92 80 0.671 6591.34 6604.44 63.94 25 LDB Twin Site 1 80 0.672 6600.00 160 0.297 6708.54 80 0.662 6513.44 6607.33 97.76 5 LDB Twin Site 2 80 0.655 6452.86 160 0.287 6535.44 80 0.671 6591.34 6526.54 69.67 10 LDB Twin Site 2 80 0.676 6634.62 160 0.292 6621.99 80 0.673 6608.65 6621.75 12.98 15 LDB Twin Site 2 80 0.672 6600.00 160 0.284 6483.51 80 0.664 6530.75 6538.08 58.59 20 LDB Twin Site 2 80 0.62 6149.92 160 0.285 6500.82 80 0.651 6418.23 6356.32 183.46 25 LDB Twin Site 2 80 0.653 6435.54 160 0.289 6570.06 80 0.661 6504.79 6503.46 67.27 A1-2b. Total sulphide concentrations in Lac du Bois and Lac du Bois Twin 6-Jul-04 Lac du Bois Depth (cm) Sample Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg L-1) Average Sulphide (mg L-1) St. Dev. 10 LDB Rocky 1 0.007 0.04 2 0.001 0.04 5 -0.006 0.02 0.03 0.01 20 LDB Rocky 1 -0.004 0.01 2 0.004 0.06 5 -0.001 0.08 0.05 0.04 30 LDB Rocky 1 0.014 0.06 5 0.007 0.19 2 0.014 0.11 0.12 0.07 40 LDB Rocky 1 0.009 0.04 2 -0.004 0.02 5 -0.001 0.08 0.05 0.03 SO Depth (cm) Sample Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg L'1) Average Sulphide (mg L-1) St. Dev. 5 LDB Algae 1 0.074 0.22 2 0.037 0.24 5 0.013 0.27 0.24 0.03 10 LDB Algae 1 0.049 0.15 2 0.024 0.17 5 0.015 0.30 0.20 0.08 4-Oct-04 10 LDB Rocky 1 0.016 0.01 1 0.017 0.02 2 0.02 0.05 0.02 0.02 20 LDB Rocky 1 0.013 0.01 1 0.011 0.00 2 0.01 0.00 0.00 0.00 30 LDB Rocky 1 0.175 0.37 1 0.166 0.35 2 0.089 0.35 0.35 0.01 40 LDB Rocky 1 0.238 0.51 1 0.225 0.48 2 0.111 0.45 0.48 0.03 5 LDB Algae 1 0.096 0.19 1 0.121 0.25 2 0.062 0.23 0.22 0.03 10 LDB Algae 1 0.101 0.20 1 0.096 0.19 2 0.037 0.12 0.17 0.04 6-Jul-04 Lac du Bois Twin Depth (cm) Sample Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg L'1) Dil. O.D. Sulphide (mg L-1) Average Sulphide (mg L-1) St. Dev. 5 LDB Twin Site 1 1 0.094 0.27 2 0.07 0.41 1 0.112 0.32 0.33 0.07 10 LDB Twin Site 1 1 0.06 0.18 2 0.029 0.19 1 0.065 0.19 0.19 0.01 15 LDB Twin Site 1 1 0.762 2.05 2 0.442 2.40 5 0.171 2.38 2.28 0.19 20 LDB Twin Site 1 1 0.41 1.11 2 0.248 1.36 5 0.112 1.59 1.36 0.24 5 LDB Twin Site 2 1 0.077 0.22 2 0.097 0.56 1 0.123 0.35 0.38 0.17 10 LDB Twin Site 2 1 0.057 0.17 2 0.037 0.24 5 0.019 0.35 0.25 0.09 15 LDB Twin Site 2 1 0.036 0.12 2 0.018 0.13 5 0.01 0.23 0.16 0.06 20 LDB Twin Site 2 1 0.264 0.72 2 0.109 0.62 5 0.048 0.74 0.69 0.06 vo OO 4-Oct-04 Lac du Bois Twin Depth (cm) Sample Dil. O.D. Sulphide (mg L"1) Dil. O.D. Sulphide (mg L"1) Dil. O.D. Sulphide (mg L-1) Average Sulphide (mg L-1) St. Dev. 5 LDB Twin Site 1 1 0.101 0.20 2 0.054 0.20 1 0.086 0.17 0.19 0.02 10 LDB Twin Site 1 1 0.097 0.19 2 0.035 0.11 1 0.094 0.19 0.16 0.05 15 LDB Twin Site 1 1 0.031 0.05 2 0.017 0.03 1 0.033 0.05 0.04 0.01 20 LDB Twin Site 1 1 0.53 1.15 2 0.21 0.89 1 0.56 1.22 1.09 0.18 Depth (cm) Sample Dil. O.D. Sulphide (mg L"1) Dil. O.D. Sulphide (mg L'1) Dil. O.D. Sulphide (mg I."1) Average Sulphide (mg L-1) St. Dev. 5 LDB Twin Site 2 1 0.057 0.10 2 0.041 0.14 1 0.061 0.11 0.12 0.02 10 LDB Twin Site 2 1 0.042 0.07 2 0.022 0.05 1 0.037 0.06 0.06 0.01 15 LDB Twin Site 2 1 0.032 0.05 2 0.021 0.05 1 0.028 0.04 0.05 0.01 20 LDB Twin Site 2 1 0.087 0.17 1 0.079 0.15 2 0.051 0.18 0.17 0.01 A1-2c. Ammonia-nitrogen, nitrate-nitrogen, ortho-phosphate and sCOD concentrations in Lac du Bois and Lac du Bois Twin 6-Jul-04 Dil. O.D. NH3-N (mg L-1) Dil. O.D. NH3-N (mg L'1) Dil. O.D. NH3-N (mg L"1) Average NH3-N (mg L-1) St. Dev. LDB Rocky 1 0.72 0.72 2 0.31 0.62 1 0.68 0.68 0.67 0.05 LDB Algae 1 0.39 0.39 2 0.19 0.38 1 0.4 0.4 0.39 0.01 LDB Twin Site 1 1 0.55 0.55 2 0.3 0.6 1 0.54 0.54 0.56 0.03 LDB Twin Site 2 1 0.49 0.49 2 0.24 0.48 1 0.51 0.51 0.49 0.02 4-Oct-04 Dil. O.D. NH3-N (mg L-1) Dil. O.D. NH3-N (mg L-1) Dil. O.D. NH3-N (mg L-1) Average NH3-N (mg L-1) St. Dev. LDB Rocky 1 0.72 0.72 2 0.41 0.82 1 0.75 0.75 0.76 0.05 LDB Algae 1 0.46 0.46 2 0.22 0.44 1 0.41 0.41 0.44 0.03 LDB Twin Site 1 1 0.59 0.59 2 0.29 0.58 1 0.49 0.49 0.55 0.06 LDB Twin Site 2 1 0.48 0.48 2 0.26 0.52 1 0.49 0.49 0.50 0.02 6-Jul-04 Dil. O.D. N03"-N (mg L-1) Dil. O.D. N03-N (mg L-1) Dil. O.D. NO3-N (mg L-1) Average NO3-N (mg L"1) St. Dev. LDB Rocky 1 0.02 0.02 2 0 0 1 0.01 0.01 0.01 0.01 LDB Algae 1 0.04 0.04 2 0 0 1 0.01 0.01 0.02 0.02 LDB Twin Site 1 1 0.02 0.02 2 0 0 1 0 0 0.01 0.01 LDB Twin Site 2 1 0.02 0.02 2 0.01 0.02 1 0.02 0.02 0.02 0.00 O O 4-Oct-04 Dil. O.D. N03'-N (mg L-1) Dil. O.D. N03-N (mg L'1) Dil. O.D. NO3-N (mg L-1) Average NO3-N (mg L"1) St. Dev. LDB Rocky 1 0.03 0.03 2 0 0 1 0.02 0.02 0.02 0.02 LDB Algae 1 0.03 0.03 2 0.01 0.02 1 0.04 0.04 0.03 0.01 LDB Twin Site 1 1 0.01 0.01 2 0 0 1 0.01 0.01 0.01 0.01 LDB Twin Site 2 1 0.02 0.02 2 0 0 1 0.01 0.01 0.01 0.01 6-Jul-04 Dil. O.D. P0 4 3" (mg L-1) Dil. O.D. P0 4 3 ' (mg L-1) Dil. O.D. P0 4 3 ' (mg L'1) Average P0 4 3 ' (mg L"1) St. Dev. LDB Rocky 1 1.91 1.91 10 0.18 1.8 2 0.72 1.44 1.72 0.25 LDB Algae 1 0.51 0.51 10 0.06 0.6 2 0.22 0.44 0.52 0.08 LDB Twin Site 1 1 2.5 2.5 10 0.31 3.1 2 1.4 2.8 2.80 0.30 LDB Twin Site 2 1 over >3 10 0.41 4.1 5 0.88 4.4 4.25 4-Oct-04 LDB Rocky 10 0.24 2.4 5 0.49 2.45 1 2.6 2.6 2.48 0.10 LDB Algae 1 0.42 0.42 10 0.07 0.7 5 0.11 0.55 0.56 0.14 LDB Twin Site 1 10 0.28 2.8 5 0.64 3.2 1 over >3 3.00 LDB Twin Site 2 10 0.53 5.3 5 1.1 5.5 20 0.21 4.2 5.00 0.70 4-Oct-04 Dil. O.D. sCOD (mg L-1) Dil. O.D. sCOD (mg L-1) Dil. O.D. sCOD (mg L'1) Average sCOD (mg L-1) St. Dev. LDB Rocky 1 0.134 331.44 2 0.065 321.54 1 0.132 326.49 326.49 4.95 LDB Algae 1 0.146 361.12 2 0.078 385.85 1 0.151 373.48 373.48 12.37 LDB Twin Site 1 1 0.077 190.45 2 0.041 202.82 1 0.075 185.51 192.93 8.92 LDB Twin Site 2 1 0.064 158.30 2 0.026 128.62 1 0.063 155.82 147.58 16.47 O A1-2d. pH, temperature and DO of Lac du Bois and Lac du Bois Twin 6-Jul-04 4-Oct-04 6-Jul-04 4-Oct-04 4-Oct-04 PH pH Temperature (°C) Temperature (°C) DO (% sat) LDB Rocky 8.67 8.64 23.5 16.2 (0-10 cm) 15.8 (10-20 cm) 15.6 (20-35 cm) 15-20% LDB Algae 8.58 8.62 22.4 16.2 (0-10 cm) 14.8 (10-20 cm) 14 (20-35 cm) 12-15% 0% below 15 cm LDB Twin Site 1 10.3 10 26 14.4 (0-10 cm) 14.2(10-20 cm) 14.1 (20-35 cm) >100% LDB Twin Site 2 10.4 10.7 25.7 14.2 (0-10 cm) 14.1 (10-20 cm) 14.0 (20-35 cm) >100% A1-3a. S0 4 2" concentrations in SRB growth without pH adjustment experiment Hay 1 Date Time (Days) Dil. O.D. S0 4 2" (mg L 1) Dil. O.D. SO4 2' (mg L-1) Dil. O.D. S0 42" (mg L-1) Average S 0 4 ^ (mg L-1) St. Dev. 7-Aug 0 20 0.678 1662.98 20 0.68 1667.31 25 0.505 1610.80 1647.03 31.45 23-Aug 16 20 0.572 1433.62 20 0.594 1481.22 25 0.455 1475.56 1463.47 26.01 9-Sep 33 20 0.538 1357.85 25 0.396 1306.43 25 0.357 1210.50 1291.59 74.79 21-Oct 75 20 0.484 1235.54 40 0.196 1178.65 33 0.268 1259.39 1224.53 41.48 24-Jan 170 25 0.321 1087.25 25 0.305 1042.38 20 0.41 1075.97 1068.53 23.34 O Hay 2 Date Time (Days) Dil. O.D. S0 4 2" (mg L-1) Dil. O.D. S0 4 2 ' (mg L-1) Dil. O.D. S0 4 2" (mg L-1) Average S0 4 2 ' (mg I."1) St. Dev. 7-Aug 0 20 0.688 1684.62 20 0.719 1751.70 25 0.506 1613.51 1683.27 69.11 23-Aug 16 20 0.591 1474.73 20 0.598 1489.88 25 0.453 1470.15 1478.25 10.32 9-Sep 33 20 0.547 1377.67 25 0.432 1405.52 25 0.425 1386.26 1389.81 14.27 21-Oct 75 20 0.555 1394.85 40 0.216 1268.40 33 0.249 1190.35 1284.54 103.20 24-Jan 170 25 0.371 1227.49 20 0.47 1204.13 25 0.362 1212.84 1214.82 11.81 Hay + Molasses 1 7-Aug 0 20 0.688 1684.62 25 0.554 1743.34 20 0.682 1671.64 1699.86 38.20 23-Aug 16 20 0.624 1546.14 25 0.486 1559.41 20 0.639 1578.59 1561.38 16.32 9-Sep 33 20 0.569 1426.11 25 0.413 1353.22 25 0.39 1289.91 1356.42 68.16 21-Oct 75 20 0.549 1381.39 40 0.216 1268.40 33 0.273 1277.56 1309.12 62.76 24-Jan 170 25 0.36 1196.64 25 0.345 1154.57 20 0.42 1097.99 1149.73 49.50 Hay + Molasses 2 7-Aug 0 20 0.689 1686.78 20 0.706 1723.57 25 0.567 1778.50 1729.62 46.16 23-Aug 16 20 0.608 1511.51 25 0.484 1554.00 20 0.622 1541.81 1535.77 21.88 9-Sep 33 20 0.575 1439.33 25 0.441 1430.30 25 0.42 1372.49 1414.04 36.26 21-Oct 75 20 0.569 1426.27 40 0.22 1286.35 33 0.306 1397.47 1370.03 73.88 24-Jan 170 25 0.409 1334.07 25 0.395 1294.80 20 0.505 1285.18 1304.68 25.90 7-Aug 0 25 0.505 1610.80 20 0.673 1652.16 20 0.647 1595.90 1619.62 29.15 23-Aug 16 20 0.605 1505.02 20 0.609 1513.68 25 0.465 1502.61 1507.10 5.82 9-Sep 33 20 0.562 1410.70 25 0.425 1386.26 25 0.372 1240.36 1345.77 92.10 21-Oct 75 20 0.486 1240.03 40 0.18 1106.85 33 0.26 1230.32 1192.40 74.25 24-Jan 170 25 0.331 1115.30 25 0.332 1118.10 20 0.385 1020.92 1084.77 55.32 O Barley 2 Date Time (Days) Dil. O.D. S0 4 2" (mg L"1) Dil. O.D. S0 4 2" (mg L-1) Dil. O.D. so42-(mg L"1) Average S0 4 2" (mg L'1) St. Dev. 7-Aug 0 20 0.661 1626.20 20 0.643 1587.25 25 0.498 1591.87 1601.77 21.28 23-Aug 16 25 0.465 1502.61 20 0.616 1528.83 20 0.598 1489.88 1507.10 19.86 9-Sep 33 20 0.525 1329.22 25 0.36 1207.33 25 0.362 1212.84 1249.80 68.84 21-Oct 75 20 0.496 1262.47 40 0.176 1088.90 33 0.249 1190.35 1180.57 87.20 24-Jan 170 25 0.323 1092.86 25 0.319 1081.64 20 0.35 943.84 1039.45 82.99 Barley + Molasses 1 7-Aug 0 . 20 0.678 1662.98 25 0.498 1591.87 20 0.653 1608.89 1621.25 37.13 23-Aug 16 20 0.559 1405.49 25 0.451 1464.74 20 0.613 1522.33 1464.19 58.43 9-Sep 33 20 0.526 1331.42 25 0.399 1314.69 25 0.366 1223.85 1289.98 57.88 21-Oct 75 20 0.478 1222.08 40 0.192 1160.70 33 0.251 1197.62 1193.47 30.90 24-Jan 170 25 0.311 1059.20 25 0.32 1084.45 20 0.36 965.86 1036.51 62.47 Barley + Molasses 2 7-Aug 0 20 0.669 1643.51 20 0.691 1691.11 25 0.526 1667.60 1667.41 23.80 23-Aug 16 20 0.549 1383.85 20 0.553 1392.50 25 0.42 1380.90 1385.75 6.03 9-Sep 33 20 0.596 1485.57 25 0.415 1358.73 25 0.4 1317.44 1387.25 87.62 21-Oct' 75 20 0.56 1406.07 40 0.238 1367.13 33 0.341 1524.64 1432.61 82.04 24-Jan 170 25 0.36 1196.64 25 0.371 1227.49 20 0.48 1230.12 1218.08 18.62 7-Aug 0 25 0.622 1927.26 20 0.831 1994.05 20 0.812 1952.93 1958.08 33.69 23-Aug 16 20 0.724 1762.52 25 0.561 1762.27 20 0.726 1766.84 1763.88 2.57 9-Sep 33 20 0.658 1622.10 25 0.476 1526.64 25 0.513 1628.49 1592.41 57.05 21-Oct 75 20 0.414 1078.48 40 0.165 1039.54 33 0.229 1117.68 1078.56 39.07 24-Jan 170 25 0.258 910.55 25 0.257 907.75 20 0.303 840.34 886.21 39.75 O Silage 2 Date Time (Days) Dil. O.D. S0 4 2 ' (mg I-"1) Dil. O.D. S0 4 2" (mg L-1) Dil. O.D. S0 4 2 ' (mg L-1) Average S0 4 2" (mg I."1) St. Dev. 7-Aug 0 20 0.777 1877.20 20 0.777 1877.20 25 0.552 1737.93 1830.77 80.41 23-Aug 16 25 0.562 1764.97 20 0.72 1753.86 20 0.738 1792.81 1770.55 20.06 9-Sep 33 20 0.69 1692.57 25 0.507 1611.97 25 0.473 1518.38 1607.64 87.17 21-Oct 75 20 0.524 1325.29 40 0.217 1272.89 33 0.28 1302.99 1300.39 26.30 24-Jan 170 5 0.322 218.01 10 0.15 243.06 4 0.605 301.08 254.05 42.61 7-Aug 0 20 0786 1896.67 25 0.604 1878.57 20 0.781 1885.85 1887.03 9.11 23-Aug 16 25 0.455 1475.56 20 0.572 1433.62 20 0.603 1500.70 1469.96 33.89 9-Sep 33 20 0.627 1553.84 25 0.418 1366.99 25 0.43 1400.02 1440.28 99.72 21-Oct 75 20 0.419 1089.69 40 0.165 1039.54 33 0.248 1186.72 1105.32 74.83 24-Jan 170 25 0.198 742.27 25 0.202 753.49 20 0.31 855.76 783.84 62.54 Silage + Molasses 2 7-Aug 0 20 0.806 1939.95 25 0.633 1957.01 20 0.83 1991.88 1962.95 26.47 23-Aug 16 20 0.652 1606.72 20 0.645 1591.58 25 0.495 1583.75 1594.02 11.68 9-Sep 33 20 0.558 1401.89 25 0.44 1427.55 25 0.382 1267.89 1365.77 85.74 21-Oct 75 20 0.325 878.78 40 0.142 936.32 33 0.199 1008.68 941.26 65.09 24-Jan 170 25 0.317 1076.03 25 0.305 1042.38 20 0.38 1009.91 1042.77 33.06 Molasses 1 7-Aug 0 20 0.783 1890.18 20 0.773 1868.54 25 0.569 1783.91 1847.54 56.16 23-Aug 16 20 0.787 1898.84 25 0.614 1905.62 20 0.786 1896.67 1900.38 4.67 9-Sep 33 20 0.775 1879.75 25 0.508 1614.73 25 0.492 1570.68 1688.39 167.18 21-Oct 75 20 0.737 1803.23 40 0.271 1515.22 33 0.354 1571.88 1630.11 152.58 24-Jan 170 25 0.481 1536.01 25 0.5 1589.30 20 0.6 1494.38 1539.90 47.58 K> O Molasses 2 Date Time (Days) Dil. O.D. S0 42" (mg I."1) Dil. O.D. S0 42 ' (mg L'1) Dil. O.D. s o 42 -(mg L-1) Average S0 4 2" (mg I."1) St. Dev. 7-Aug 0 20 0.739 1794.97 20 0.726 1766.84 25 0.575 1800.14 1787.32 17.92 23-Aug 16 20 0.833 1998.37 20 0.786 1896.67 25 0.611 1897.51 1930.85 58.48 9-Sep 33 20 0.735 1791.67 25 0.508 1614.73 25 0.489 1562.43 1656.27 120.14 21-Oct 75 20 0.653 1614.75 40 0.232 1340.21 33 0.367 1619.11 1524.69 159.78 24-Jan 170 25 0.485 1547.23 25 0.468 1499.55 20 0.612 1520.80 1522.53 23.89 Negative Control Hay 9-Sep 0 20 0.644 1591.27 25 0.495 1578.94 25 0.451 1457.82 1542.68 73.75 21-Oct 42 25 0.407 1328.46 40 0.233 1344.69 33 0.326 1470.14 1381.10 77.54 24-Jan 137 25 0.418 1359.31 25 0.387 1272.37 20 0.485 1241.13 1290.94 61.24 Negative Control Barley 9-Sep 0 20 0.622 1542.83 25 0.477 1529.39 25 0.429 1397.27 1489.83 80.44 21-Oct 42 25 0.417 1356.51 40 0.199 1192.11 33 0.285 1321.16 1289.93 86.53 24-Jan 137 25 0.341 1143.35 25 0.35 1168.59 20 0.443 1148.64 1153.53 13.31 Negative Control Silage 9-Sep 0 20 0.654 1613.29 25 0.477 1529.39 25 0.454 1466.08 1536.26 73.85 21-Oct 42 25 0.503 1597.72 40 0.253 1434.45 33 0.336 1506.47 1512.88 81.82 24-Jan 137 25 0.468 1499.55 25 0.48 1533.21 20 0.58 1450.34 1494.37 41.68 Negative Control Molasses 9-Sep 0 20 0.633 1567.05 25 0.47 1510.13 25 0.459 1479.85 1519.01 44.28 21-Oct 42 25 0.511 1620.16 40 0.264 1483.81 33 0.329 1481.04 1528.33 79.53 24-Jan 137 25 0.469 1502.36 25 0.465 1491.14 20 0.574 1437.12 1476.87 34.88 O A1-3b. Total sulphide concentrations in SRB growth without pH adjustment experiment at T=170 Agricultural Material Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg I-"1) Average Sulphide (mg L-1) St. Dev. Hay 1 2.4 0.016 0.03 1.2 0.048 0.08 1.2 0.046 0.08 0.06 0.03 Hay 2 1.2 0.036 0.06 1.2 0.044 0.08 1.2 0.037 0.06 0.06 0.01 Hay + Molasses 1 1.2 0.088 0.17 1.2 0.078 0.15 1.2 0.077 0.15 0.16 0.01 Hay + Molasses 2 1.2 0.077 0.15 1.2 0.064 0.12 1.2 0.058 0.11 0.13 0.02 Barley 1 9.6 0.113 1.83 2.4 0.434 1.88 2.4 0.437 1.89 1.87 0.03 Barley 2 2.4 0.025 0.07 1.2 0.052 0.09 1.2 0.066 0.12 0.10 0.03 Barley + Molasses 1 2.4 0.023 0.06 1.2 0.05 0.09 1.2 0.049 0.09 0.08 0.02 Barley + Molasses 2 2.4 0.104 0.42 1.2 0.223 0.47 1.2 0.201 0.42 0.44 0.03 Silage 1 192 0.525 182.47 192 0.53 184.24 192 0.501 173.97 180.23 5.49 Silage 2 192 0.484 167.95 192 0.477 165.47 192 0.469 162.64 165.35 2.66 Silage + Molasses 1 192 0.087 27.34 96 0.193 32.44 96 0.201 33.86 31.21 3.43 Silage + Molasses 2 192 0.057 16.71 96 0.121 19.69 96 0.122 19.87 18.76 1.77 Molasses 1 2.4 0.03 0.09 1.2 0.054 0.10 1.2 0.055 0.10 0.10 0.01 Molasses 2 2.4 0.021 0.05 1.2 0.044 0.08 1.2 0.039 0.06 0.06 0.01 Control Hay 1.2 0.036 0.06 1.2 0.022 0.03 1.2 0.03 0.04 0.04 0.02 Control Barley 1.2 0.009 -0.00 1.2 0.011 0.00 1.2 0.015 0.01 0.00 0.01 Control Silage 1.2 0.01 0.00 1.2 0.008 -0.00 1.2 0.013 0.01 0.00 0.01 Control Molasses 1.2 0.005 -0.01 1.2 0.014 0.01 1.2 0.013 0.01 0.00 0.01 O A1-3c. pH in SRB growth without pH adjustment experiment at T=170 and at the beginning of the pH adjusted experiment Initial pH (second experiment) Agricultural Material Hay 1 Hay 2 Barley 1 Barley 2 Silage 1 Silage 2 Molasses 1 Molasses 2 PH 5 5 4 4 7 7 4 4 Final pH Agricultural Material Hay 1 Hay 2 Hay + Molasses 1 Hay + Molasses 2 Barley 1 Barley 2 Barley + Molasses 1 Barley + Molasses 2 Silage 1 Silage 2 PH 5.3 5.4 5.2 5.2 3.5 3.5 3.6 3.6 8 7.9 Agricultural Material Silage + Molasses 1 Silage + Molasses 2 Molasses 1 Molasses 2 Control Hay Control Barley Control Silage Control Molasses PH 7.3 7.8 3.5 3.5 5.2 3.5 7.3 3.4 A1-3d. Ammonia-nitrogen in SRB growth without pH adjustment experiment at T=170. Agricultural Material Dil. O.D. NH3-N (mg L-1) Dil. O.D. NH3-N (mg L"1) Dil. O.D. NH3-N (mg I-"1) Average NH3-N (mg L-1) St. Dev. Hay 1 11 0.18 1.98 11 0.16 1.76 5.5 0.32 1.76 1.83 0.13 Hay 2 5.5 0.34 1.87 2.2 0.83 1.83 2.2 0.84 1.85 1.85 0.02 Hay + Molasses 1 4.4 0.54 2.38 5.5 0.45 2.48 4.4 0.59 2.60 2.48 0.11 Hay + Molasses 2 4.4 0.66 2.90 4.4 0.59 2.60 5.5 0.47 2.59 2.70 0.18 Barley 1 5.5 0.85 4.68 2.2 2.33 5.13 2.2 2.21 4.86 4.89 0.23 Barley 2 2.2 2.48 5.46 4.4 1.2 5.28 2.2 2.4 5.28 5.34 0.10 Barley + Molasses 1 11 0.49 5.39 11 0.45 4.95 11 0.47 5.17 5.17 0.22 O OO Agricultural Material Dil. O.D. NH3-N (mg L-1) Dil. O.D. NH3-N (mg L'1) Dil. O.D. NH3-N (mg L'1) Average NH3-N (mg L"1) St. Dev. Barley + Molasses 2 2.2 2.08 4.58 11 0.4 4.40 2.2 2.06 4.53 4.50 0.09 Silage 1 55 1.34 73.70 55 1.32 72.60 55 1.28 70.40 72.23 1.68 Silage 2 55 1.37 75.35 55 1.37 75.35 55 1.35 74.25 74.98 0.64 Silage + Molasses 1 55 1.32 72.60 55 1.33 73.15 55 1.33 73.15 72.97 0.32 Silage + Molasses 2 55 1.3 71.50 55 1.29 70.95 55 1.32 72.60 71.68 0.84 Molasses 1 5.5 0.51 2.81 4.4 0.57 2.51 4.4 0.59 2.60 2.64 0.15 Molasses 2 4.4 0.61 2.68 4.4 0.48 2.11 5.5 0.45 2.48 2.42 0.29 Control Hay 2.2 0.58 1.28 2.2 0.49 1.08 2.2 0.55 1.21 1.19 0.10 Control Barley 4.4 1.2 5.28 5.5 0.95 5.23 4.4 1.2 5.28 5.26 0.03 Control Silage 55 0.72 39.60 55 0.77 42.35 55 0.76 41.80 41.25 1.46 Control Molasses 4.4 0.2 0.88 5.5 0.15 0.83 4.4 0.2 0.88 0.86 0.03 O V O A1-3e. Ortho-phosphate in SRB growth without pH adjustment experiment at T=170. Agricultural Material Dil. O.D. P0 4 3 ' (mg I."1) Dil. O.D. P0 4 3" (mg L-1) Average P0 4 3" (mg I-"1) Hay 1 5.50 0 0.00 1.1 0.01 0.01 0.01 Hay 2 2.75 0 0.00 1.1 0 0.00 0.00 Hay + Molasses 1 1.38 0 0.00 1.1 0.05 0.06 0.03 Hay + Molasses 2 1.38 0.04 0.06 1.1 0.08 0.09 0.07 Barley 1 2.75 0 0.00 1.1 0.02 0.02 0.01 Barley 2 2.75 0 0.00 1.1 0 0.00 0.00 Barley + Molasses 1 2.75 0 0.00 1.1 0 0.00 0.00 Barley + Molasses 2 2.75 0 0.00 1.1 0 0.00 0.00 Silage 1 11.00 0.22 2.42 0.21 1.16 1.79 Silage 2 1.83 1.14 2.09 1.1 1.8 1.98 2.04 Silage + Molasses 1 1.83 0 0.00 1.1 0 0.00 0.00 Silage + Molasses 2 1.83 0 0.00 1.1 0 0.00 0.00 Molasses 1 1.38 0 0.00 1.1 0 0.00 0.00 Molasses 2 1.38 0 0.00 1.1 0.06 0.07 0.03 Control Hay 1.38 0 0.00 1.1 0 0.00 0.00 Control Barley 1.38 0 0.00 1.1 0 0.00 0.00 Control Silage 1.38 0 0.00 1.1 0 0.00 0.00 Control Molasses 1.38 0 0.00 1.1 0 0.00 0.00 O A1-3f. Soluble COD in SRB growth without pH adjustment experiment at T=170. Agricultural Material Dil. O.D. sCOD (mg L"1) Dil. O.D. sCOD (mg L-1) Dil. O.D. sCOD (mg I-"1) Average sCOD (mg L-1) St. Dev. Hay 1 20 0.095 4699.46 10 0.195 4823.13 10 0.196 4847.86 4790.15 79.51 Hay 2 10 0.16 3957.44 20 0.079 3907.97 10 0.177 4377.92 4081.11 258.23 Hay + Molasses 1 10 0.23 5688.82 10 0.233 5763.02 20 0.111 5490.95 5647.60 140.64 Hay + Molasses 2 20 0.108 5342.54 10 0.221 5466.21 10 0.231 5713.55 5507.44 188.91 Barley 1 20 0.121 5985.63 10 0.222 5490.95 10 0.221 5466.21 5647.60 293.00 Barley 2 10 0.204 5045.74 10 0.198 4897.33 20 0.101 4996.27 4979.78 75.56 Barley + Molasses 1 10 0.345 8533.23 20 0.151 7469.67 10 0.311 7692.27 7898.39 560.94 Barley + Molasses 2 20 0.222 10981.90 20 0.215 10635.62 10808.76 Silage 1 20 0.09 4452.12 10 0.163 4031.64 10 0.166 4105.84 4196.54 224.43 Silage 2 10 0.222 5490.95 10 0.212 5243.61 10 0.225 5565.15 5433.24 168.36 Silage + Molasses 1 10 0.123 3042.28 10 0.143 3536.96 10 0.156 3858.50 3479.25 411.16 Silage + Molasses 2 10 0.149 3685.37 10 0.114 2819.68 10 0.151 3734.83 3413.29 514.68 Molasses 1 10 0.323 7989.08 10 0.333 8236.42 10 0.298 7370.73 7865.41 445.90 Molasses 2 10 0.315 7791.21 10 0.32 7914.88 10 0.341 8434.29 8046.79 341.23 Control Hay 10 0.318 7865.41 10 0.334 8261.16 10 0.317 7840.68 7989.08 235.95 Control Barley 20 0.195 9646.26 20 0.2 9893.60 20 0.205 10140.94 9893.60 247.34 Control Silage 5 0.119 1471.67 10 0.096 2374.46 10 0.057 1409.84 1751.99 539.96 Control Molasses 20 0.197 9745.20 20 0.205 10140.94 20 0.186 9201.05 9695.73 471.89 A1-4a. S0 4 2 ' concentrations in SRB growth with pH adjustment experiment Hay 1 Date Time (Days) Dil. O.D. S0 4 2" (mg L-1) Dil. O.D. S0 4 2" (mg L-1) Dil. O.D. s o 42 -(mg L"1) Average S0 4 (mg L-1) St. Dev. 24-Feb 0 33 0.308 1387.04 25 0.447 1440.65 20 0.564 1415.05 1414.25 26.81 10-Mar 14 33 0.311 1398.15 20 0.601 1498.07 25 0.453 1457.48 1451.23 50.25 24-Mar 28 33 0.241 1138.99 25 0.351 1171.39 20 0.451 1161.50 1157.29 16.61 7-Apr 42 25 0.375 1238.71 25 0.354 1179.81 20 0.465 1192.91 1203.81 30.93 22-Apr 57 33 0.185 931.66 25 0.297 1019.94 20 0.379 999.94 983.85 46.29 19-May 84 20 0.404 880.03 25 0.295 845.27 20 0.393 859.46 861.59 17.48 21-Jun 117 25 0.211 624.79 30 0.159 612.04 20 0.273 609.29 615.37 8.27 30-Jun 126 25 0.174 543.14 30 0.12 508.77 20 0.217 510.42 520.78 19.38 28-Jul 154 30 0.055 336.63 25 0.07 313.63 10 0.272 303.76 318.01 16.87 23-Aug 180 20 0.034 187.35 25 0.039 245.22 10 0.164 208.43 213.67 29.29 Date Time (Days) Dil. O.D. S0 42 _ (mg L-1) Dil. O.D. S0 42 ' (mg L'1) Dil. O.D. S0 4 2 _ (mg L"1) Average S0 4 (mg L"1) St. Dev. 24-Feb 0 33 0.308 1387.04 25 0.447 1440.65 20 0.564 1415.05 1414.25 26.81 10-Mar 14 25 0.423 1373.34 33 0.334 1483.30 20 0.595 1484.60 1447.08 63.87 24-Mar 28 33 0.249 1168.61 25 0.367 1216.27 20 0.463 1188.42 1191.10 23.94 7-Apr 42 25 0.363 1205.05 25 0.331 1115.30 1160.18 22-Apr 57 33 0.182 920.56 25 0.276 961.04 20 0.342 916.92 932.84 24.49 19-May 84 25 0.416 1128.09 20 0.596 1239.04 20 0.534 1123.11 1163.41 65.54 21-Jun 117 30 0.421 1305.86 25 0.519 1304.48 20 0.667 1304.87 1305.07 0.71 30-Jun 126 25 0.48 1218.42 30 0.393 1231.71 20 0.619 1220.13 1223.42 7.23 28-Jul 154 25 0.24 688.79 30 0.181 670.30 20 0.31 674.61 677.90 9.67 23-Aug 180 20 0.16 409.79 25 0.123 430.59 20 0.158 406.26 415.55 13.15 Barley 1 Date Time (Days) Dil. O.D. S0 4 2" (mg L-1) Dil. O.D. S0 4 2" (mg L"1) Dil. O.D. S0 4 2" (mg I."1) Average S0 4 2 ' (mg I-"1) St. Dev. 24-Feb 0 33 0.308 1387.04 25 0.447 1440.65 20 0.564 1415.05 1414.25 26.81 10-Mar 14 25 0.356 1185.42 33 0.312 1401.85 20 0.556 1397.10 1328.12 123.61 24-Mar 28 33 0.297 1346.32 25 0.331 1115.30 20 0.429 1112.13 1191.25 134.30 7-Apr 42 25 0.438 1415.41 25 0.446 1437.85 20 0.561 1408.32 1420.52 15.42 22-Apr 57 25 0.293 1008.72 33 0.2 987.20 20 0.378 997.70 997.87 10.76 19-May 84 25 0.517 1364.15 20 0.754 1534.48 20 0.662 1362.45 1420.36 98.83 21-Jun 117 25 0.575 1428.07 30 0.445 1369.42 20 0.714 1387.85 1395.11 29.99 30-Jun 126 25 0.552 1377.31 30 0.43 1329.69 20 0.707 1375.49 1360.83 26.98 28-Jul 154 25 0.409 1061.74 30 0.33 1064.88 20 0.534 1070.07 1065.56 4.21 23-Aug 180 20 0.474 964.14 25 0.363 960.22 962.18 2.77 Date Time (Days) Dil. O.D. S0 4 2" (mg L'1) Dil. O.D. S0 4 2" (mg L'1) Dil. O.D. S0 4 2" (mg L-1) Average S0 4 ^ (mg L-1) St. Dev. 24-Feb 0 33 0.308 1387.04 25 0.447 1440.65 20 0.564 1415.05 1414.25 26.81 10-Mar 14 25 0.375 1238.71 33 0.318 1424.07 20 0.559 1403.83 1355.53 101.68 24-Mar 28 33 0.221 1064.94 20 0.386 1015.65 25 0.304 1039.57 1040.05 24.65 7-Apr 42 25 0.349 1165.79 25 0.387 1272.37 20 0.449 1157.01 1198.39 64.22 22-Apr 57 25 0.29 1000.30 33 0.191 953.88 20 0.354 943.85 966.01 30.12 19-May 84 20 0.664 1366.19 25 0.483 1284.69 20 0.643 1326.92 1325.93 40.76 21-Jun 117 25 0.555 1383.93 30 0.416 1292.62 20 0.684 1334.88 1337.14 45.70 30-Jun 126 25 0.499 1260.35 30 0.414 1287.32 20 0.653 1280.16 1275.94 13.97 28-Jul 154 30 0.347 1109.90 25 0.427 1101.46 20 0.556 1108.91 1106.75 4.61 23-Aug 180 25 0.423 1092.63 20 0.564 1123.03 20 0.557 1110.67 1108.78 15.29 Silage 1 Date Time (Days) Dil. O.D. S0 4 2 ' (mg L'1) Dil. O.D. S0 4 2" (mg L-1) Dil. O.D. S0 4 2" (mg L"1) Average S0 4 2 ' (mg L-1) St. Dev. 24-Feb 0 33 0.308 1387.04 25 0.447 1440.65 20 0.564 1415.05 1414.25 26.81 10-Mar 14 33 0.301 1361.13 25 0.36 1196.64 25 0.379 1249.93 1269.23 83.93 24-Mar 28 20 0.35 934.87 25 0.29 1000.30 33 0.189 946.47 960.55 34.91 7-Apr 42 25 0.266 932.99 20 0.333 896.73 20 0.341 914.68 914.80 18.13 22-Apr 57 25 0.224 815.19 33 0.152 809.49 20 0.296 813.71 812.79 2.96 19-May 84 25 0.505 1336.11 20 0.593 1233.43 20 0.63 1302.62 1290.72 52.36 21-Jun 117 30 0.396 1239.66 25 0.493 1247.11 20 0.623 1227.19 1237.99 10.06 30-Jun 126 30 0.353 1125.79 25 0.457 1167.66 20 0.595 1177.76 1157.07 27.56 28-Jul 154 25 0.325 876.37 30 0.251 855.67 20 0.408 847.62 859.89 14.83 23-Aug 180 20 0.382 801.72 25 0.294 807.95 20 0.393 821.14 810.27 9.92 Date Time (Days) Dil. O.D. S0 4 2" (mg L-1) Dil. O.D. S0 4 2" (mg L-1) Dil. O.D. S0 4 2 ' (mg L"1) Average s o 4 2 -(mg L-1) St. Dev. 24-Feb 0 33 0.308 1387.04 25 0.447 1440.65 20 0.564 1415.05 1414.25 26.81 10-Mar 14 33 0.317 1420.36 25 0.417 1356.51 20 0.521 1318.56 1365.15 51.45 24-Mar 28 20 0.435 1125.60 25 0.384 1263.95 33 0.236 1120.48 1170.01 81.40 7-Apr 42 25 0.339 1137.74 20 0.501 1273.69 20 0.451 1161.50 1190.97 72.61 22-Apr 57 25 0.186 708.61 33 0.128 720.63 20 0.256 723.95 717.73 8.07 19-May 84 20 0.751 1528.87 25 0.493 1308.06 20 0.653 1345.62 1394.18 118.14 21-Jun 117 25 0.542 1355.24 30 0.428 1324.40 20 0.682 1331.35 1337.00 16.18 30-Jun 126 25 0.535 1339.79 30 0.453 1390.60 20 0.685 1336.65 1355.68 30.28 28-Jul 154 30 0.268 900.69 25 0.364 962.43 20 0.457 934.13 932.42 30.90 23-Aug 180 25 0.309 841.06 20 0.411 852.92 20 0.393 821.14 838.37 16.06 bo Molasses 1 Date Time (Days) Dil. O.D. S0 4 2" (mg L-1) Dil. O.D. SO42" (mg I-"1) Dil. O.D. S0 4 2" (mg L'1) Average S0 4 2 ' (mg L'1) St. Dev. 24-Feb 0 33 0.308 1387.04 25 0.447 1440.65 20 0.564 1415.05 1414.25 26.81 10-Mar 14 33 0.372 1623.99 25 0.494 1572.48 20 0.625 1551.92 1582.79 37.13 24-Mar 28 33 0.405 1746.16 25 0.494 1572.48 20 0.601 1498.07 1605.57 127.32 7-Apr 42 25 0.527 1665.03 20 0.705 1731.42 20 0.645 1596.80 1664.42 67.32 22-Apr 57 25 0.254 899.33 33 0.183 924.26 20 0.326 881.02 901.54 21.70 19-May 84 25 0.546 1431.94 20 0.674 1384.89 20 0.672 1381.15 1399.32 28.30 21-Jun 117 25 0.496 1253.73 30 0.377 1189.34 20 0.595 1177.76 1206.94 40.93 30-Jun 126 25 0.453 1158.84 30 0.352 1123.14 20 0.572 1137.16 1139.71 17.99 28-Jul 154 30 0.253 860.97 25 0.273 761.61 20 0.38 798.19 806.92 50.25 23-Aug 180 25 0.243 695.41 20 0.344 734.64 20 0.326 702.86 710.97 20.83 Molasses 2 Date Time (Days) Dil. O.D. SO42" (mg L"1) Dil. O.D. S0 4 2" (mg LZ1) Dil. O.D. SO4 2' (mg I-"1) Average S0 4 2 ' (mg L-1) St. Dev. 24-Feb 0 33 0.308 1387.04 25 0.447 1440.65 20 0.564 1415.05 1414.25 26.81 10-Mar 14 33 0.342 1512.92 25 0.456 1465.89 20 0.61 1518.26 1499.03 28.82 24-Mar 28 33 0.305 1375.94 25 0.479 1530.40 20 0.545 1372.41 1426.25 90.22 7-Apr 42 25 0.467 1496.75 20 0.535 1349.98 20 0.621 1542.94 1463.22 100.76 22-Apr 57 25 0.282 977.87 33 0.186 935.36 20 0.358 952.82 955.35 21.36 19-May 84 25 0.488 1296.37 20 0.556 1164.25 20 0.569 1188.55 1216.39 70.32 21-Jun 117 25 0.442 1134.56 30 0.337 1083.42 20 0.572 1137.16 1118.38 30.31 30-Jun 126 25 0.49 1240.49 30 0.402 1255.55 20 0.631 1241.32 1245.78 8.47 28-Jul 154 30 0.233 808.01 25 0.288 794.71 20 0.381 799.96 800.89 6.70 23-Aug 180 20 0.302 660.49 25 0.225 655.69 20 0.302 660.49 658.89 2.77 Silage:Hay 1:3 1 Date Time (Days) Dil. O.D. S0 42" (mg L-1) Dil. O.D. S0 4 2" (mg I/1) Dil. O.D. s o 4 2 -(mg L"1) Average S0 4 2" (mg L-1) St. Dev. 24-Feb 0 33 0.308 1387.04 25 0.447 1440.65 20 0.564 1415.05 1414.25 26.81 10-Mar 14 33 0.268 1238.95 25 0.371 1227.49 20 0.481 1228.81 1231.75 6.27 24-Mar 28 33 0.244 1150.10 20 0.451 1161.50 25 0.387 1272.37 1194.65 67.54 7-Apr 42 25 0.303 1036.77 25 0.316 1073.23 20 0.422 1096.43 1068.81 30.07 22-Apr 57 25 0.227 823.60 33 0.146 787.27 20 0.298 818.19 809.69 19.60 19-May 84 20 0.467 997.83 30 0.308 1050.79 25 0.361 999.54 1016.05 30.10 21-Jun 117 25 0.324 874.16 30 0.253 860.97 20 0.412 854.69 863.27 9.94 30-Jun 126 25 0.302 825.61 30 0.234 810.66 20 0.389 814.08 816.78 7.83 28-Jul 154 30 0.13 535.25 25 0.154 499.00 20 0.205 489.24 507.83 24.24 23-Aug 180 20 0.13 356.83 25 0.099 377.63 10 0.33 354.96 363.14 12.58 Date Time (Days) Dil. O.D. S0 4 2" (mg L-1) Dil. O.D. S0 4 2 ' (mg L'1) Dil. O.D. S0 4 2 ' (mg L-1) Average S0 4 2 ' (mg L-1) St. Dev. 24-Feb 0 33 0.308 1387.04 25 0.447 1440.65 20 0.564 1415.05 1414.25 26.81 10-Mar 14 25 0.39 1280.78 20 0.491 1251.25 1266.01 24-Mar 28 33 0.257 1198.23 25 0.366 1213.47 20 0.444 1145.79 1185.83 35.50 7-Apr 42 25 0.271 947.01 25 0.305 1042.38 20 0.439 1134.57 1041.32 93.78 22-Apr 57 33 0.131 731.74 25 0.194 731.05 20 0.257 726.20 729.66 3.02 19-May 84 30 0.281 975.06 25 0.361 999.54 20 0.479 1020.27 998.29 22.63 21-Jun 117 30 0.298 980.14 25 0.371 977.88 20 0.469 955.32 971.11 13.73 30-Jun 126 30 0.265 892.75 25 0.313 849.88 20 0.401 835.27 859.30 29.88 28-Jul 154 25 0.16 512.24 30 0.108 476.99 20 0.217 510.42 499.88 19.85 23-Aug 180 25 0.101 382.04 20 0.134 363.89 10 0.355 377.03 374.32 9.37 O S Silage:Hay 1:1 1 Date Time (Days) Dil. O.D. S0 4 2" (mg I-"1) Dil. O.D. SO42" (mg L-1) Dil. O.D. S0 4 2" (mg I-"1) Average S0 4 2" (mg I-"1) St. Dev. 24-Feb 0 33 0.308 1387.04 25 0.447 1440.65 20 0.564 1415.05 1414.25 26.81 10-Mar 14 25 0.414 1348.09 33 0.273 1257.46 20 0.492 1253.49 1286.35 53.51 24-Mar 28 20 0.445 1148.03 25 0.384 1263.95 33 0.244 1150.10 1187.36 66.34 7-Apr 42 25 0.32 1084.45 20 0.401 1049.31 1066.88 22-Apr 57 25 0.107 487.03 33 0.074 520.71 20 0.157 501.82 503.19 16.88 19-May 84 20 0.41 891.25 25 0.323 910.72 30 0.256 904.94 902.30 10.00 21-Jun 117 25 0.34 909.47 30 0.286 948.36 20 0.453 927.07 928:30 19.48 30-Jun 126 25 0.281 779.27 30 0.218 768.28 20 0.359 761.12 769.56 9.14 28-Jul 154 25 0.088 353.35 30 0.051 326.04 20 0.144 381.55 353.65 27.75 23-Aug 180 20 0.08 268.56 25 0.049 267.29 10 0.28 310.82 282.22 24.78 Date Time (Days) Dil. O.D. S0 4 2 ' (mg L-1) Dil. O.D. S0 4 2 ' (mg L'1) Dil. O.D. S0 4 2" (mg L-1) Average S 0 4 ^ (mg L'1) St. Dev. 24-Feb 0 33 0.308 1387.04 25 0.447 1440.65 20 0.564 1415.05 1414.25 26.81 10-Mar 14 33 0.298 1350.02 25 0.413 1345.29 25 0.401 1311.63 1335.65 20.93 24-Mar 28 20 0.401 1049.31 25 0.343 1148.96 33 0.223 1072.35 1090.20 52.17 7-Apr 42 25 0.254 899.33 25 0.259 913.36 20 0.356 948.33 920.34 25.24 22-Apr 57 33 0.098 609.56 25 0.147 599.22 20 0.202 602.79 603.86 5.25 19-May 84 20 0.408 887.51 30 0.254 899.33 25 0.319 901.37 896.07 7.48 21-Jun 117 30 0.207 739.16 25 0.28 777.06 20 0.347 739.93 752.05 21.66 30-Jun 126 30 0.194 704.73 25 0.269 752.78 20 0.337 722.28 726.60 24.32 28-Jul 154 30 0.09 429.32 25 0.104 388.66 20 0.155 400.97 406.32 20.85 23-Aug 180 20 0.091 287.98 14.28 0.166 300.16 10 0.272 303.76 297.30 8.27 Silage:Hay 3:1 1 Date Time (Days) Dil. O.D. S0 4 2" (mg L'1) Dil. O.D. S0 4 2" (mg L-1) Dil. O.D. S0 4 2 ' (mg L 1) Average S0 4 2" (mg L-1) St. Dev. 24-Feb 0 33 0.308 1387.04 25 0.447 1440.65 20 0.564 1415.05 1414.25 26.81 10-Mar 14 25 0.375 1238.71 33 0.263 1220.44 20 0.491 1251.25 1236.80 15.49 24-Mar 28 33 0.172 883.53 25 0.241 862.87 20 0.305 833.90 860.10 24.93 7-Apr 42 25 0.256 904.94 25 0.257 907.75 20 0.333 896.73 903.14 5.73 22-Apr 57 33 0.136 750.25 25 0.201 750.68 20 0.267 748.64 749.86 1.08 19-May 84 30 0.256 904.94 25 0.309 878.00 20 0.416 902.47 895.14 14.90 21-Jun 117 30 0.338 1086.06 25 0.402 1046.29 20 0.507 1022.40 1051.58 32.16 30-Jun 126 25 0.41 1063.94 30 0.327 1056.93 20 0.531 1064.77 1061.88 4.31 28-Jul 154 25 0.213 629.20 30 0.168 635.88 20 0.286 632.24 632.44 3.34 23-Aug 180 25 0.153 496.80 20 0.215 506.89 10 0.497 502.37 502.02 5.06 Date Time (Days) Dil. O.D. S0 42 ' (mg L-1) Dil. O.D. S0 4 2 ' (mg L-1) Dil. O.D. S0 4 2 ' (mg L-1) Average S0 4 2-(mg L'1) St. Dev. 24-Feb 0 33 0.308 1387.04 25 0.447 1440.65 . 20 0.564 1415.05 1414.25 26.81 10-Mar 14 25 0.415 1350.90 33 0.274 1261.17 20 0.503 1278.17 1296.75 47.66 24-Mar 28 33 0.182 920.56 25 0.244 871.29 20 0.347 928.14 906.66 30.87 7-Apr 42 25 0.247 879.70 20 0.337 905.70 25 0.243 868.48 884.63 19.09 22-Apr 57 25 0.168 658.12 33 0.119 687.31 20 0.224 652.15 665.86 18.81 19-May 84 20 0.403 878.16 30 0.256 904.94 25 0.315 892.02 891.71 13.39 21-Jun 117 30 0.292 964.25 25 0.39 1019.81 20 0.482 978.27 987.44 28.89 30-Jun 126 25 0.458 1169.87 30 0.36 1144.32 20 0.571 1135.39 1149.86 17.89 28-Jul 154 25 0.177 549.76 30 0.141 564.38 20 0.251 570.45 561.53 10.64 23-Aug 180 20 0.194 469.82 25 0.136 459.28 10 0.456 466.18 465.09 5.35 00 Positive Control Date Time (Days) Dil. O.D. S0 4 z" (mg L'1) Dil. O.D. 504'" (mg L-1) Dil. O.D. S0 4 " (mg I-"1) Average S0 4 2 ' (mg L"1) St. Dev. 24-Feb 0 33 0.308 1387.04 25 0.447 1440.65 20 0.564 1415.05 1414.25 26.81 14-Apr 50 10 0.003 65.11 20 -0.01 105.92 5 0 31.15 67.39 37.43 Abiotics Date Time (Days) Sample Dil. O.D. S 0 4 " (mg L'1) Dil. O.D. S O 4 " (mg L-1) Dil. O.D. S0 4 " (mg L"1) Average so/ (mg L-1) St. Dev. 24-Feb 0 initial 25 0.449 1446.26 33 0.317 1420.36 20 0.591 1475.63 1447.42 27.65 2-May 67 Hay 1 33 0.362 1586.97 25 0.435 1406.99 20 0.601 1498.07 1497.34 89.99 Hay 2 25 0.476 1521.99 33 0.319 1427.77 20 0.555 1394.85 1448.20 65.99 Barley 1 25 0.491 1564.06 33 0.354 1557.35 20 0.601 1498.07 1539.83 36.32 Barley 2 33 0.377 1642.50 25 0.399 1306.02 30 0.353 1412.41 1453.64 171.99 Silage 1 25 0.504 1600.52 33 0.366 1601.78 20 0.585 1462.17 1554.82 80.24 Silage 2 25 0.379 1249.93 33 0.371 1620.29 30 0.356 1422.50 1430.91 185.32 Molasses 1 25 0.465 1491.14 33 0.376 1638.80 20 0.602 1500.31 1543.42 82.73 Molasses 2 33 0.389 1686.93 20 0.603 1502.56 25 0.441 1423.82 1537.77 135.04 S:H (1 3)1 20 0.566 1419.53 30 0.412 1610.98 33 0.317 1420.36 1483.63 110.29 S:H (1 3) 2 33 0.335 1487.00 25 0.514 1628.57 20 0.561 1408.32 1507.96 111.61 S:H (1 1) 1 33 0.336 1490.71 25 0.501 1592.11 20 0.565 1417.29 1500.04 87.78 S:H (1 1)2 30 0.367 1459.53 25 0.505 1603.33 20 0.541 1363.44 1475.43 120.73 S:H (3 1) 1 25 0.428 1387.36 33 0.365 1598.07 20 0.61 1518.26 1501.23 106.38 S:H (3 1)2 25 0.392 1286.39 33 0.204 1002.01 20 0.605 1507.04 1265.15 253.19 K3 A1-4b. Total sulphide concentrations in SRB growth with pH adjustment experiment Date Time (days) Sample Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg L-1) Average Sulphide (mg L-1) St. Dev. 22-Apr 57 Hay 1 5 0.2 2.11 5 0.191 2.01 4 0.311 2.67 2.26 0.36 Hay 2 4 0.211 1.78 5 0.172 1.80 4 0.221 1.87 1.82 0.05 Barley 1 5 0.186 1.95 4 0.22 1.86 5 0.189 1.98 1.93 0.06 Barley 2 4 0.17 1.42 5 0.137 1.41 5 0.136 1.40 1.41 0.01 Si lage 1 5 0.079 0.77 4 0.082 0.64 4 0.081 0.63 0.68 0.08 Si lage 2 4 0.154 1.28 5 0.121 1.23 4 0.151 1.25 1.25 0.02 Molasses 1 5 0.035 0.28 4 0.051 0.36 4 0.049 0.35 0.33 0.05 Molasses 2 4 0.017 0.06 5 0.001 0.00 2 0.044 0.15 0.07 0.08 Si lage:Hay (1:3)1 5 0.021 0.12 4 0.016 0.05 2 0.033 0.10 0.09 0.04 Si lage:Hay (1:3)2 5 0.108 1.09 4 0.12 0.98 2 0.26 1.11 1.06 0.07 Si lage:Hay (1:1) 1 5 0.069 0.66 4 0.077 0.59 2 0.151 0.63 0.63 0.03 S i lage:Hay (1:1)2 5 0.723 7.89 10 0.24 5.10 4 0.807 7.06 6.68 1.44 Si lage:Hay (3:1)1 5 1.115 Over 10 0.28 5.98 20 0.137 5.63 5.81 0.25 S i lage:Hay (3:1)2 5 0.354 3.81 4 0.615 5.36 10 0.234 4.96 4.71 0.80 21-Jun 117 Barley 2 10 over 100 0.162 41.26 50 0.322 40.22 40.74 Si lage 2 10 over 100 0.385 95.88 50 0.784 96.80 96.34 Si lage:Hay (1:1) 1 10 over 100 0.211 53.26 50 0.451 56.02 54.64 28-Jul 154 Si lage 2 100 0.097 25.34 100 0.082 21.67 23.50 S i lage:Hay (1:1) 1 100 0.176 44.69 200 0.091 47.74 46.22 O Date Time (days) Sample Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg L"1) Average Sulphide (mg L-1) St. Dev. 23-Aug 180 Hay 1 50 0.371 46.22 100 0.221 55.71 100 0.201 50.81 50.92 4.74 Hay 2 50 0.194 24.55 100 0.101 26.32 100 0.101 26.32 25.73 1.02 Barley 1 50 0.026 3.98 10 0.161 4.10 10 0.159 4.05 4.04 0.06 Barley 2 50 0.046 6.43 10 0.172 4.37 10 0.171 4.35 5.05 1.19 Silage 1 50 0.033 4.83 20 0.097 5.07 20 0.101 5.26 5.06 0.22 Silage 2 50 0.113 14.63 20 0.254 12.76 20 0.255 12.81 13.40 1.07 Molasses 1 50 0.042 5.94 10 0.166 4.22 10 0.173 4.40 4.85 0.94 Molasses 2 50 0.223 28.10 25 0.566 35.05 25 0.545 33.77 32.31 3.70 Silage:Hay (1:3)1 50 0.353 44.02 100 0.161 41.02 100 0.159 40.53 41.85 1.89 Silage:Hay (1:3)2 50 0.325 40.59 100 0.172 43.71 100 0.165 42.00 42.10 1.56 Silage:Hay (1:1) 1 50 0.172 21.86 100 0.088 23.14 100 0.071 18.97 21.32 2.13 Silage:Hay (1:1)2 50 0.425 52.84 100 0.221 55.71 100 0.207 52.28 53.61 1.84 Silage:Hay (3:1)1 50 0.21 26.51 100 0.091 23.87 100 0.103 26.81 25.73 1.62 Silage:Hay (3:1)2 50 0.353 44.02 100 0.176 44.69 100 0.172 43.71 44.14 0.50 Abiotic Time (days) Sample Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg I-"1) Dil. O.D. Sulphide (mg L-1) Average Sulphide (mg L"1) 2-May 67 Hay 1 2 0.025 0.07 2 0.021 0.05 0.06 Hay 2 2 0.028 0.08 2 0.025 0.07 0.07 Barley 1 2 0.028 0.08 2 0.025 0.07 0.07 Barley 2 2 -0.03 0.00 2 0 0.00 0.00 Silage 1 2 0.227 0.96 2 0.201 0.85 0.90 Silage 2 2 0.186 0.78 2 0.191 0.80 0.79 Molasses 1 2 0.021 0.05 2 0.005 -0.02 0.01 K> Abiotic Time (days) Sample Dil. O.D. Sulphide (mg L"1) Dil. O.D. Sulphide (mg L'1) Dil. O.D. Sulphide (mg I-"1) Average Sulphide (mg I."1) Molasses 2 2 0.018 0.04 2 0.01 0.00 0.02 Silage:Hay (1:3)1 2 0.035 0.11 2 0.041 0.14 0.12 Silage:Hay (1:3)2 2 0.01 0.00 2 0 -0.04 -0.02 Silage:Hay (1:1) 1 2 0.012 0.01 2 0.01 0.00 0.01 Silage:Hay (1:1)2 2 0.044 0.15 2 0.015 0.02 0.09 Silage:Hay (3:1)1 2 over over 10 0.313 6.71 6.71 Silage:Hay (3:1)2 2 over over 10 over over 20 0.911 39.90 39.90 A1-4c. pH in SRB growth with pH adjustment experiment Time (Days) Bottle 1 Bottle 2 Average PH Silage Time (Days) Bottle 1 Bottle 2 Average PH 0 5.0 5.0 5.0 0 7.0 7.0 7.0 0 7.5 7.5 7.5 0 7.5 7.5 7.5 14 5.3 5.5 5.4 14 6.0 6.0 6.0 14 7.8 7.5 7.7 14 7.5 7.5 7.5 29 5.8 6.7 6.3 29 7.0 6.8 6.9 29 7.5 7.5 7.5 29 7.5 7.5 7.5 57 7.5 7.2 7.4 57 7.8 7.5 7.7 57 7.5 7.2 7.4 57 7.8 7.5 7.7 84 7.0 7.5 7.3 84 7.5 7.6 7.6 119 7.1 7.5 7.3 119 7.8 7.8 7.8 126 7.5 7.6 7.6 126 7.6 7.6 7.6 154 7.4 7.5 7.5 154 7.6 7.6 7.6 180 7.7 7.5 7.6 180 7.7 7.6 7.7 Barley Silage:Hay 1:3 Time (Days) Bottle 1 Bottle 2 Average pH Molasses Time (Days) Bottle 1 Bottle 2 Average PH 0 4.0 4.0 4.0 0 4.0 4.0 4.0 0 7.5 7.5 7.5 0 7.5 7.5 7.5 14 4.5 4.0 4.3 14 4.3 4.5 4.4 14 7.5 7.5 7.5 14 7.5 7.5 7.5 29 4.0 4.5 4.3 29 4.5 4.5 4.5 29 7.5 7.5 7.5 29 7.5 7.5 7.5 57 4.5 5.0 4.8 57 6.0 5.8 5.9 57 7.5 7.5 7.5 57 7.5 7.5 7.5 84 6.8 7.0 6.9 84 7.1 7.1 7.1 119 7.0 7.2 7.1 119 6.8 6.9 6.9 126 7.3 7.6 7.5 126 7.2 7.2 7.2 154 7.1 7.3 7.2 154 7.0 . 7.1 7.1 180 7.2 7.3 7.3 180 7.0 7.2 7.1 Time (Days) Bottle 1 Bottle 2 Average pH Silage:Hay 1:1 Time (Days) Bottle 1 Bottle 2 Average PH 0 6.0 6.0 6.0 0 6.0 6.0 6.0 0 7.5 7.5 7.5 0 7.5 7.5 7.5 14' 5.0 5.5 5.3 14 6.0 6.0 6.0 14 7.5 7.8 7.7 14 7.5 7.5 7.5 29 6.2 6.5 6.4 29 7.0 5.5 6.3 29 7.5 7.5 7.5 29 7.5 7.5 7.5 57 6.5 6.8 6.7 57 6.8 7.0 6.9 57 7.5 7.5 7.5 57 7.5 7.5 7.5 84 7.6 7.5 7.6 84 7.6 7.1 7.4 119 7.4 7.6 7.5 119 7.8 7.5 7.7 126 7.6 7.7 7.7 126 7.5 7.5 7.5 154 7.6 7.6 7.6 154 7.5 7.7 7.6 180 7.7 7.6 7.7 180 7.6 7.6 7.6 Silage:Hay 3:1 Time (Days) Bottle 1 Bottle 2 Average PH 0 6.5 6.5 6.5 0 7.5 7.5 7.5 14 6.0 5.5 5.8 14 7.5 7.5 7.5 29 7.0 7.0 7.0 29 7.5 7.5 7.5 57 7.7 7.7 7.7 57 7.7 7.7 7.7 84 7.8 7.8 7.8 119 7.4 7.6 7.5 126 7.7 7.6 7.7 154 7.6 7.6 7.6 180 7.6 7.6 7.6 Before Adjustment Adjusted Date 24-Feb 24-Feb 2-May Time (Days) 0 0 67 Hay 1 5.0 7.5 4.5 Hay 2 5.0 7.5 4.5 Barley 1 5.0 7.5 4.2 Barley 2 5.0 7.5 4.2 Silage 1 7.0 7.5 6.0 Silage 2 7.0 7.5 6.2 Molasses 1 5.0 7.5 4.2 Molasses 2 5.0 7.5 4.3 Silage:Hay (1:3)1 6.0 7.5 6.5 4^ Before Adjustment Adjusted Date 24-Feb 24-Feb 2-May Silage:Hay (1:3)2 6.0 7.5 5.5 Silage:Hay (1:1) 1 6.0 7.5 5.5 Silage:Hay (1:1)2 6.0 7.5 5.5 Silage:Hay (3:1)1 6.0 7.5 6.0 Silage:Hay (3:1)2 6.0 7.5 7.0 A1-4d. Soluble COD in SRB growth with pH adjustment experiment Date Agricultural Material Dil. O.D. sCOD (mg L-1) Dil. O.D. sCOD (mg L"1) Dil. O.D. sCOD (mg I-"1) Average sCOD (mg L-1) St. Dev. 28-Feb Hay 1 2 0.421 over 10 0.216 5342.54 20 0.105 5194.14 5268.34 Hay 2 10 0.202 4996.27 20 0.099 4897.33 2 0.411 Over 4946.80 Barley 1 20 0.197 9745.20 10 0.356 8805.30 40 0.095 9398.92 9316.47 475.34 Barley 2 20 0.237 11723.92 40 0.116 11476.58 10 0.428 Over 11600.25 Silage 1 10 0.089 2201.33 10 0.098 2423.93 5 0.167 2065.29 2230.18 181.05 Silage 2 10 0.106 2621.80 10 0.106 2621.80 5 0.189 2337.36 2526.99 164.22 Molasses 1 10 0.422 over 20 0.244 12070.19 40 0.113 11179.77 11624.98 Molasses 2 10 0.432 over 20 0.235 11624.98 40 0.11 10882.96 11253.97 Silage:Hay (1:3)1 10 0.203 5021.00 20 0.099 4897.33 5 0.419 over 4959.17 Silage:Hay (1:3)2 10 0.206 5095.20 20 0.101 4996.27 40 0.046 4551.06 4880.84 289.86 Agricultural Material Dil. O.D. sCOD (mg L"1) Dil. O.D. sCOD (mg L-1) Dil. O.D. sCOD (mg L-1) Average sCOD (mg L-1) St. Dev. Silage:Hay (1:1) 1 10 0.156 3858.50 20 0.074 3660.63 5 0.31 3833.77 3784.30 107.81 Silage:Hay (1:1)2 20 0.071 3512.23 10 0.156 3858.50 5 0.301 3722.47 3697.73 174.46 Silage:Hay (3:1)1 10 0.133 3289.62 20 0.066 3264.89 5 0.259 3203.05 3252.52 44.59 Silage:Hay (3:1)2 10 0.125 3091.75 5 0T261 3227.79 20 0.059 2918.61 3079.38 154.96 23-Aug Hay 1 8 0.184 3640.84 10 0.155 3833.77 4 0.381 3769.46 3748.03 98.23 Hay 2 8 0.271 5362.33 4 0.397 3927.76 10 0.161 3982.17 4424.09 813.00 Barley 1 20 0.222 10981.90 40 0.121 11971.26 25 0.177 10944.80 11299.32 582.21 Barley 2 20 0.222 10981.90 25 0.174 10759.29 40 0.113 11179.77 10973.65 210.36 Silage 1 4 0.25 2473.40 8 0.129 2552.55 5 0.201 2485.77 2503.91 42.58 Silage 2 2 0.233 1152.60 4 0.122 1207.02 8 0.078 1543.40 1301.01 211.67 Molasses 1 20 0.227 11229.24 40 0.111 10981.90 25 0.172 10635.62 10948.92 298.18 Molasses 2 20 0.298 14741.46 40 0.119 11773.38 25 0.186 11501.31 12672.05 1797.32 Silage:Hay (1:3)1 4 0.327 3235.21 8 0.153 3027.44 10 0.121 2992.81 3085.15 131.10 Silage:Hay (1:3)2 4 0.302 2987.87 8 0.152 3007.65 10 0.124 3067.02 3020.85 41.19 Silage:Hay (1:1) 1 4 0.21 2077.66 8 0.103 2038.08 10 0.079 1953.99 2023.24 63.16 Silage:Hay (1:1)2 4 0.273 2700.95 8 0.13 2572.34 10 0.107 2646.54 2639.94 64.56 Silage:Hay (3:1)1 8 0.08 1582.98 4 0.162 1602.76 5 0.131 1620.08 1601.94 18.56 Silage:Hay (3:1)2 8 0.102 2018.29 4 0.198 1958.93 5 0.168 2077.66 2018.29 59.36 Abiotics Agricultural Material Dil. O.D. sCOD (mg L-1) Dil. O.D. sCOD (mg L-1) Dil. O.D. sCOD (mg L-1) Average sCOD (mg L-1) St. Dev. 12-May Hay 1 10 0.147 3635.90 5 0.328 4056.38 20 0.079 3907.97 3866.75 213.25 Hay 2 10 0.16 3957.44 5 0.321 3969.81 20 0.077 3809.04 3912.09 89.47 Barley 1 10 0.388 9596.79 20 0.213 10536.68 25 0.164 10140.94 10091.47 471.89 Barley 2 10 0.387 9572.06 20 0.198 9794.66 25 0.157 9708.10 9691.61 112.22 Silage 1 10 0.079 1953.99 5 0.163 2015.82 20 0.041 2028.19 1999.33 39.75 Silage 2 10 0.093 2300.26 5 0.166 2052.92 20 0.051 2522.87 2292.02 235.08 Molasses 1 10 0.39 9646.26 20 0.209 10338.81 25 0.155 9584.43 9856.50 418.84 Molasses 2 10 0.378 9349.45 20 0.201 9943.07 25 0.161 9955.44 9749.32 346.35 Silage:Hay (1:3)1 5 0.236 2918.61 10 0.119 2943.35 20 0.059 2918.61 2926.86 14.28 Silage.Hay (1:3)2 5 0.274 3388.56 10 0.137 3388.56 20 0.066 3264.89 3347.33 71.40 Silage:Hay (1:1) 1 5 0.203 2510.50 10 0.1 2473.40 20 0.045 2226.06 2403.32 154.63 Silage:Hay (1:1)2 5 0.206 2547.60 10 0.098 2423.93 20 0.048 2374.46 2448.67 89.18 Silage:Hay (3:1)1 5 0.176 2176.59 10 0.091 2250.79 20 0.043 2127.12 2184.84 62.25 Silage:Hay (3:1)2 5 0.194 2399.20 10 0.102 2522.87 20 0.05 2473.40 2465.16 62.25 A1-4e. NH3-N in SRB growth with pH adjustment experiment Agricultural Material Dil. O.D. NH3-N (mg L-1) Dil. O.D. NH3-N (mg L"1) Dil. O.D. NH3-N (mg L-1) Average NH3-N (mg L-1) Mar-24 Hay 1 10 over over 50 2.2 110 100 0.99 99 104.5 Hay 2 10 over over 50 1.7 85 100 0.75 75 80 Barley 1 10 1.18 11.8 20 0.45 9 10.4 Barley 2 10 over over 50 0.53 26.5 25 1.3 32.5 29.5 Silage 1 50 0.78 39 20 1.93 38.6 38.8 Agricultural Material Dil. O.D. NH3-N (mg L-1) Dil. O.D. NH3-N (mg I-"1) Dil. O.D. NH3-N (mg L-1) Average NH3 -N (mg L-1) Silage 2 20 1.89 37.8 50 0.76 38 37.9 Molasses 1 10 over over 50 over over 100 2.11 . 211 211 Molasses 2 10 over over 50 over over 100 2.03 203 203 Silage:Hay (1:3)1 20 over over 50 1.59 79.5 100 0.74 74 76.75 Silage:Hay (1:3)2 20 over over 50 1.96 98 50 1.86 93 95.5 Silage:Hay (1:1) 1 20 over over 50 1.66 83 100 0.84 84 83.5 Silage:Hay (1:1)2 20 over over 50 1.47 73.5 100 0.65 65 69.25 Silage:Hay (3:1)1 20 2.77 55.4 50 1.07 53.5 100 0.51 51 53.3 Silage:Hay (3:1)2 20 2.56 51.2 50 1.09 54.5 100 0.5 50 51.9 23-Aug Hay 1 50 1.75 87.5 100 0.96 96 91.75 Hay 2 100 1.09 109 50 2.3 115 112 Barley 1 100 1.83 183 100 1.9 190 186.5 Barley 2 100 2.34 234 100 1.84 184 209 Silage 1 100 0.88 88 50 1.8 90 89 Silage 2 100 0.82 82 50 1.91 95.5 88.75 Molasses 1 100 0.83 83 50 1.7 85 84 Molasses 2 100 0.99 99 50 1.85 92.5 95.75 Silage:Hay (1:3)1 100 1.2 120 50 2.59 129.5 124.75 Silage:Hay (1:3)2 100 0.98 98 50 2.07 103.5 100.75 Silage:Hay (1:1) 1 100 0.75 75 50 2.01 100.5 87.75 00 Agricultural Material Dil. O.D. NH3-N (mg L-1) Dil. O.D. NH3-N (mg L'1) Dil. O.D. NH3-N (mg L'1) Average NH3-N (mg L-1) Silage:Hay (1:1)2 100 1.05 105 50 2.06 103 104 Silage:Hay (3:1)1 100 0.96 96 50 2.13 106.5 101.25 Silage:Hay (3:1)2 100 0.93 93 50 1.74 87 90 Abiotic Agricultural Material Dil. O.D. NH3 -N (mg L"1) Dil. O.D. 1NH3-N (mg L"1) Average NH3 -N (mg L-1) May-02 Hay 1 50 0.68 34 20 1.97 39.4 36.7 Hay 2 50 0.59 29.5 20 1.6 32 30.75 Barley 1 50 over over 100 1.35 135 135 Barley 2 50 0.85 42.5 20 2.53 50.6 46.55 Silage 1 50 0.63 31.5 20 1.53 30.6 31.05 Silage 2 50 0.59 29.5 20 1.45 29 29.25 Molasses 1 50 over over 100 1.86 186 186 Molasses 2 50 over over 100 1.66 166 166 Silage:Hay (1:3)1 50 0.59 29.5 20 1.3 26 27.75 Silage:Hay (1:3)2 50 0.57 28.5 20 1.32 26.4 27.45 VO Agricultural Material Dil. O.D. NH3-N (mg L-1) Dil. O.D. NH3-N (mg I-"1) Average NH3-N (mg I."1) Silage:Hay (1:1) 1 50 0.45 22.5 20 0.84 16.8 19.65 Silage:Hay (1:1)2 50 0.37 18.5 20 0.87 17.4 17.95 Silage:Hay (3:1)1 50 0.96 48 20 1.4 28 38 Silage:Hay (3:1)2 50 0.77 38.5 20 1.55 31 34.75 A1-4f. PO4 3 " in SRB growth with pH adjustment experiment Agricultural Material Dil. O.D. P0 4J" (mg I-"1) Dil. O.D. P0 4 J" (mg L-1) Dil. O.D. P0 4 J ' (mg L-1) Average P0 4 3 ' (mg L-1) Mar-24 Hay 1 20 over over 50 0.61 30.5 100 0.28 28 29.25 Hay 2 20 over over 50 0.62 31 100 0.3 30 30.50 Barley 1 20 0.9 18 20 0.94 18.8 40 0.41 16.4 17.73 Barley 2 20 2.05 41 40 0.74 29.6 20 1.54 30.8 33.80 Silage 1 20 over over 50 1.71 85.5 100 0.91 91 88.25 Silage 2 20 over over 50 1.8 90 50 1.66 83 86.50 Molasses 1 10 0.07 0.7 20 0.03 0.6 0.65 Molasses 2 10 0.01 0.1 5 0 0 0.05 Silage:Hay (1:3)1 25 1.84 46 50 0.96 48 47.00 Silage:Hay (1:3)2 25 2.3 57.5 50 1.04 52 54.75 Silage:Hay (1:1) 1 25 2.57 64.25 50 1.22 61 62.63 Silage:Hay (1:1)2 25 2.81 70.25 50 1.28 64 67.13 O J O Agricultural Material Dil. O.D. P0 4 " (mg L-1) Dil. O.D. P0 4 J" (mg L-1) Dil. O.D. P0 4J" (mg L'1) Average P0 4 3" (mg L'1) Silage:Hay (3:1)1 25 2.64 66 50 1.4 70 68.00 Silage:Hay (3:1)2 25 2.85 71.25 50 1.39 69.5 70.38 23-Aug Hay 1 50 1.1 55 100 0.54 54 54.50 Hay'2 100 0.53 53 50 1.03 51.5 52.25 Barley 1 100 1.43 143 50 2.77 138.5 140.75 Barley 2 100 1.46 146 50 2.56 128 137.00 Silage 1 100 0.97 97 50 2.01 100.5 98.75 • Silage 2 100 0.98 98 50 2.12 106 102.00 Molasses 1 100 2.83 283 100 2.86 286 284.50 Molasses 2 100 2.91 291 100 2.88 288 289.50 Silage:Hay (1:3)1 100 0.63 63 50 1.31 65.5 64.25 Silage:Hay (1:3)2 100 0.63 63 50 1.41 70.5 66.75 Silage:Hay (1:1) 1 100 . 0.67 67 50 1.28 64 65.50 Silage:Hay (1:1)2 100 0.75 75 50 1.33 66.5 70.75 Silage:Hay (3:1)1 100 0.83 83 50 1.81 90.5 86.75 Silage:Hay (3:1)2 100 0.82 82 50 1.85 92.5 87.25 Abiotic Agricultural Material Dil. O.D. P0 4 3 ' (mg L-1) Dil. O.D. P0 4 3" (mg L-1) Average P0 4 3" (mg L-1) 2-May Hay 1 50 0.63 31.5 50 0.70 35.0 33.2 Hay 2 50 0.60 30.0 50 0.57 28.5 29.2 Barley 1 50 1.50 75.0 50 1.62 81.0 78.0 Barley 2 50 0.98 49.0 50 0.99 49.5 49.2 Silage 1 50 1.68 84.0 50 1.62 81.0 82.5 Silage 2 50 1.71 85.5 50 1.68 84.0 84.8 Molasses 1 10 1.15 11.5 5 2.55 12.8 12.1 Molasses 2 10 0.22 2.2 5 1.27 6.4 4.3 Silage:Hay (1:3)1 50 0.97 48.5 20 2.05 41.0 44.8 Silage:Hay (1:3)2 50 0.88 44.0 20 1.99 39.8 41.9 Silage:Hay (1:1) 1 50 1.19 59.5 20 2.89 57.8 58.6 Silage:Hay (1:1)2 50 1.17 58.5 20 2.84 56.8 57.6 Silage:Hay (3:1)1 50 1.59 79.5 20 2.91 58.2 68.8 Silage:Hay (3:1)2 50 1.51 75.5 20 over over 75.5 A1-5a. S 0 4 " concentrations in leachate experiment Barley 1 (TAD) Raw Date Time Dil. O.D. SO/" (mg L'1) Dil. O.D. S0 4 " (mg L-1) Dil. O.D. S0 4'" (mg L-1) Average S0 4 2 ' (mg L' 1) St. Dev 13-Jun 0 40 0.424 1751.74 30 0.586 1742.81 25 0.74 1792.19 1762.25 26.31 27-Jun 14 25 0.564 1403.79 30 0.471 1438.27 20 0.74 1433.75 1425.27 18.74 11-Jul 28 25 0.455 1163.25 30 0.374 1181.40 20 0.591 1170.70 1171.78 9.12 28-Jul 45 30 0.309 1009.27 40 0.178 883.14 25 0.388 1015.39 969.27 74.65 23-Aug 71 40 0.171 858.43 30 0.253 860.97 25 0.318 860.92 860.11 1.45 16-Sep 95 20 0.3 788.35 10 0.622 735.26 761.80 Barley 2 (TAD) Raw Date Time Dil. O.D. SO/" (mg L"1) Dil. O.D. S0 4 '" (mg I-"1) Dil. O.D. S0 4'" (mg L"1) Average S0 4 2" (mg L;1) St. Dev 13-Jun 0 30 0.592 1758.70 40 0.422 1744.68 25 0.72 1748.05 1750.48 7.32 27-Jun 14 30 0.512 1546.84 25 0.589 1458.96 20 0.713 1386.08 1463.96 80.50 11-Jul 28 30 0.355 1131.08 25 0.45 1152.22 25 0.444 1138.97 1140.76 10.68 28-Jul 45 30 0.319 1035.75 40 0.193 936.11 25 0.4 1041.88 1004.58 59.38 23-Aug 71 40 0.003 265.24 30 0.002 196.28 20 0.001 129.09 196.87 68.08 16-Sep 95 20 0.002 157.03 10 0.01 86.99 122.01 49.52 Barley 1 (TAD Flocced Date Time Dil. O.D. S0 4 z" (mg I/1) Dil. O.D. S0 4 " (mg L' 1) Dil. O.D. SO4'" (mg L-1) Average S0 4 2 ' (mg L"1) St. Dev 13-Jun 0 40 0.43 1772.93 30 0.6 1779.88 25 0.73 1770.12 1774.31 5.03 27-Jun 14 25 0.693 1688.47 30 0.572 1705.73 20 0.881 1682.68 1692.29 11.99 11-Jul 28 25 0.51 1284.62 30 0.406 1266.14 20 0.65 1274.86 1275.21 9.25 28-Jul 45 40 0.222 1038.50 30 0.314 1022.51 25 0.399 1039.67 1033.56 9.59 23-Aug 71 40 0.173 865.49 30 0.239 823.90 25 0.31 843.26 844.22 20.81 to Barley 2 (TAD) Flocced Date Time Dil. O.D. SO/ ' (mg L'1) Dil. O.D. SO/' (mg L'1) Dil. O.D. SO/' (mg L'1) Average SO/ ' (mg L'1) St. Dev 13-Jun 0 30 0.579 1724.27 40 0.42 1737.62 25 0.71 1725.98 1729.29 7.26 27-Jun 14 30 0.595 1766.64 25 0.696 1695.09 20 0.85 1627.95 1696.56 69.36 11-Jul 28 25 0.541 1353.03 30 0.432 1334.99 20 0.69 1345.48 1344.50 9.06 28-Jul 45 40 0.258 1165.62 30 0.355 1131.08 25 0.44 1130.15 1142.28 20.21 23-Aug 71 40 0.192 932.58 30 0.273 913.93 25 0.341 911.67 919.39 11.47 Bioethanol 1 (TBD) Raw Date Time Dil. O.D. SO/ ' (mg L'1) Dil. O.D. SO/ ' (mg L'1) Dil. O.D. SO/' (mg L'1) Average SO/" (mg L'1) St. Dev 13-Jun 0 40 0.341 1458.68 30 0.484 1472.70 25 0.58 1439.10 1456.82 16.87 27-Jun 14 30 0.492 1493.88 25 0.567 1410.41 20 0.721 1400.21 1434.83 51.39 11-Jul 28 25 0.509 1282.42 30 0.415 1289.97 20 0.659 1290.75 1287.71 4.60 28-Jul 45 40 0.263 1183.27 30 0.345 1104.60 25 0.423 1092.63 1126.83 49.24 23-Aug 71 20 0.616 1214.84 30 0.406 1266.14 25 0.477 1211.80 1230.92 30.53 Bioethanol 2 (TBD) Raw Date Time Dil. O.D. SO/ ' (mg L'1) Dil. O.D. SO/ ' (mg L'1) Dil. O.D. SO/' (mg L'1) Average SO/ ' (mg L'1) St. Dev 13-Jun 0 40 0.359 1522.23 30 0.497 1507.12 25 0.61 1505.30 1511.55 9.29 27-Jun 14 25 0.619 1525.16 30 0.467 1427.68 20 0.751 1453.17 1468.67 50.56 11-Jul 28 30 0.442 1361.47 25 0.491 1242.69 20 0.691 1347.24 1317.14 64.86 28-Jul 45 40 0.23 1066.75 30 0.323 1046.34 25 0.424 1094.84 1069.31 24.35 23-Aug 71 20 0.552 1101.85 30 0.344 1101.95 25 0.426 1099.25 1101.02 1.53 t o O J 4^ Bioethanol 1 (TBD) Flocced Date Time Dil. O.D. SO/ ' (mg L'1) Dil. O.D. SO/' (mg L'1) Dil. O.D. SO/' (mg L'1) Average SO/ ' (mg L-1) St. Dev 13-Jun 0 30 0.497 1507.12 40 0.364 1539.89 25 0.619 1525.16 1524.06 16.41 27-Jun 14 25 0.569 1414.82 30 0.465 1422.38 20 0.74 1433.75 1423.65 9.53 11-Jul 28 30 0.447 1374.71 25 0.563 1401.58 20 0.716 1391.38 1389.23 13.56 28-Jul 45 40 0.289 1275.07 30 0.387 1215.82 25 0.478 1214.01 1234.97 34.74 23-Aug 71 30 0.359 1141.68 20 0.543 1085.96 25 0.401 1044.08 1090.57 48.96 Bioethanol 2 (TBD) Flocced Date Time Dil. O.D. SO/ ' (mg L'1) Dil. O.D. SO/" (mg L"1) Dil. O.D. SO/" (mg L"1) Average SO4 (mg L"1L St. Dev 13-Jun 0 30 0.518 1562.73 40 0.373 1571.67 25 0.631 1551.65 1562.01 10.03 27-Jun 14 30 0.513 1549.49 25 0.632 1553.85 20 0.806 1550.27 1551.20 2.33 11-Jul 28 30 0.487 1480.64 25 0.602 1487.65 20 0.763 1474.35 1480.88 6.65 28-Jul 45 40 0.341 1458.68 30 0.488 1483.29 25 0.591 1463.37 1468.45 13.07 23-Aug 71 30 0.463 1417.08 20 0.729 1414.33 25 0.568 1412.62 1414.68 2.25 Orchard Grass 1 (PRC) Raw Date Time Dil. O.D. S04*" (mg L"1) Dil. O.D. S0 4 '" (mg L"1) Dil. O.D. SO4'" (mg L"1) Average SO4 (mg L"1) St. Dev 13-Jun 0 30 0.585 1740.16 40 0.408 1695.25 25 0.699 1701.71 1712.37 24.28 27-Jun 14 25 0.664 1624.47 30 0.579 1724.27 20 0.882 1684.44 1677.73 50.24 11-Jul 28 30 0.446 1372.07 25 0.535 1339.79 20 0.681 1329.59 1347.15 22.17 28-Jul 45 30 0.422 1308.51 40 0.285 1260.95 25 0.491 1242.69 1270.72 33.98 23-Aug 71 40 0.097 597.14 30 0.131 537.89 25 0.178 551.97 562.33 30.96 16-Sep 95 20 0.124 415.49 415.49 01 Orchard Grass 2 (ORC) Raw Date Time Dil. O.D. SO/ (mg L-1) Dil. O.D. SO/" (mg I-"1) Dil. O.D. SO/ -(mg L-1) Average so/" (mg L"1) St. Dev 13-Jun 0 40 0.426 1758.80 30 0.599 1777.23 25 0.722 1752.46 1762.83 12.87 27-Jun 14 25 0.678 1655.37 30 0.56 1673.96 20 0.866 1656.20 1661.84 10.50 11-Jul 28 30 0.422 1308.51 25 0.512 1289.04 20 0.661 1294.28 1297.28 10.08 28-Jul 45 40 0.254 1151.49 30 0.394 1234.36 25 0.485 1229.45 1205.10 46.49 23-Aug 71 40 0.166 840.77 30 0.255 866.27 25 0.323 871.95 859.66 16.60 16-Sep 95 20 0.283 752.33 752.33 Orchard 1 (ORC) Flocced Date Time Dil. O.D. SO/" (mg L"1) Dil. O.D. so/-(mg I."1) Dil. O.D. SO/" (mg L"1) Average SO4 (mg L"1) St. Dev 13-Jun 0 30 0.591 1756.05 40 0.417 1727.02 25 0.721 1750.26 1744.44 15.36 27-Jun 14 30 0.571 1703.09 25 0.694 1690.67 20 0.858 1642.07 1678.61 32.25 11-Jul 28 25 0.491 1242.69 30 0.406 1266.14 20 0.642 1260.74 1256.52 12.28 28-Jul 45 30 0.325 1051.64 40 0.203 971.42 25 0.382 1002.15 1008.40 40.47 23-Aug 71 40 0.236 1087.94 30 0.324 1048.99 25 0.38 997.74 1044.89 45.24 16-Sep 95 20 0.406 1012.91 1012.91 Orchard 2 (PRC) Flocced Date Time Dil. O.D. SO/" (mg L"1) Dil. O.D. SO/" (mg L"1) Dil. O.D. SO/' (mg L"1) Average S0 4 2" (mg L"1) St. Dev 13-Jun 0 40 0.432 1779.99 30 0.549 1644.83 25 0.701 1706.12 1710.31 67.68 27-Jun 14 25 0.673 1644.33 30 0.555 1660.71 20 0.868 1659.73 1654.92 9.19 11-Jul 28 25 0.563 1401.58 30 0.475 1448.86 20 0.722 1401.97 1417.47 27.18 28-Jul 45 40 0.252 1144.43 30 0.394 1234.36, 25 0.445 1141.18 1173.32 52.88 23-Aug 71 40 0.07 501.81 30 0.114 492.88 25 0.155 501.21 498.63 4.99 16-Sep 95 20 0.079 320.15 320.15 O S Ctrl #2 Date Time Dil. O.D. SO/" (mg L"1) Dil. O.D. SO/" (mg L"1) Dil. O.D. SO/" (mg L"1) Average SO/" (mg L"1) St. Dev 13-Jun 0 40 0.429 1769.40 30 0.573 1708.38 25 0.701 1706.12 1727.97 35.90 27-Jun 10 0.845 809.56 20 0.39 815.85 25 0.293 805.75 810.38 5.10 ±L A1-5b. Total sulphide concentrations in leachate experiment Date Sample Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg L'1) Average Sulphide (mg L-1)-St. Dev 28-Jul TAD 1 Raw 100 0.335 83.63 200 0.141 72.24 100 0.321 80.20 78.69 5.85 TAD 1 Floe 100 0.244 61.34 200 0.115 59.50 100 0.221 55.71 58.85 2.87 30-Aug TAD 1 Raw 50 0.084 11.08 10 0.422 10.49 10 0.412 10.25 10.61 0.43 TAD 2 Raw 50 0.115 14.87 10 0.623 15.42 10 0.601 14.88 15.06 0.31 TAD 1 Floe 50 0.199 25.16 10 1.15 over 20 0.568 28.14 26.65 TAD 2 Floe 50 0.159 20.26 10 1.21 over 20 0.429 21.33 20.80 TBD 1 Raw 50 0.293 36.67 10 1.22 over 20 0.799 39.46 38.06 TBD 2 Raw 50 0.033 4.83 10 0.157 4.00 10 0.141 3.61 4.15 0.62 TBD 1 Floe 50 0.057 7.77 10 0.459 11.40 10 0.462 11.47 10.22 2.12 TBD 2 Floe 50 0.473 58.72 10 1.66 over 100 0.227 57.18 57.95 Orch 1 Raw 50 0.035 5.08 10 0.234 5.89 10 0.241 6.06 5.68 0.53 Orch 2 Raw 50 0.034 4.96 10 0.187 4.74 10 0.191 4.84 4.84 0.11 Orch 1 Floe 50 0.015 2.63 10 0.113 2.93 10 0.101 2.63 2.73 0.17 Orch 2 Floe 50 0.019 3.12 10 0.258 6.48 10 0.203 5.13 4.91 1.69 A1-5c. pH in leachate experiment 13-Jun 27-Jun 11-Jul 28-Jul 23-Aug TAD 1 Raw 7.8 6.7 7.2 7.6 7.7 TAD 2 Raw 7.5 7.8 7.3 7.6 7.6 TAD 1 Flocced 7.4 6.5 7.3 7.5 7.6 TAD 2 Flocced 7.4 6.8 7.3 7.5 7.7 ORC 1 Raw 7.4 7.1 7.3 7.5 7.6 ORC 2 Raw 7.3 7.0 7.3 7.6 7.6 ORC 1 Flocced 7.1 7.1 7.3 7.5 7.6 OO 13-Jun 27-Jun 11-Jul 28-Jul 23-Aug ORC 2 Flocced 7.5 7.0 7.2 7.6 7.6 TBD 1 Raw 7.4 6.6 7.3 7.5 7.5 TBD 2 Raw 7.5 6.5 7.3 7.5 7.6 TBD 1 Flocced 7.5 6.7 7.2 7.5 7.5 TBD 2 Flocced 7.5 7.0 7.2 7.6 7.5 A1-5d. Soluble COD in leachate experiment Barley 1 (TAD) Raw Date Time Dil. O.D. sCOD (mg L-1) Dil. O.D. sCOD (mg I-"1) Dil. O.D. sCOD (mg I-"1) Average sCOD (mg L-1) St. Dev 13-Jun 0 20 0.228 11278.70 10 0.396 9794.66 10536.68 27-Jun 14 30 0.129 9572.06 20 0.196 9695.73 40 0.096 9497.86 9588.55 99.96 13-Jul 28 30 0.124 9201.05 20 0.184 9102.11 40 0.094 9299.98 9201.05 98.94 28-Jul 45 30 0.117 8681.63 20 0.178 8805.30 40 0.087 8607.43 8698.12 99.96 23-Aug 71 20 0.14 6925.52 30 0.1 7420.20 40 0.072 7123.39 7156.37 248.98 Barley 2 (TAD) Raw Date Time Dil. O.D. sCOD (mg I-"1) Dil. O.D. sCOD (mg L-1) Dil. O.D. sCOD (mg I-"1) Average sCOD (mg L"1) St. Dev 13-Jun 0 20 0.226 11179.77 10 0.399 9868.87 10524.32 27-Jun 14 30 0.134 9943.07 20 0.19 9398.92 40 0.095 9398.92 9580.30 314.16 13-Jul 28 30 0.124 9201.05 20 0.19 9398.92 40 0.093 9201.05 9267.01 114.24 28-Jul 45 30 0.121 8978.44 20 0.172 8508.50 40 0.083 8211.69 8566.21 386.62 23-Aug 71 20 0.127 6282.44 30 0.086 6381.37 40 0.064 6331.90 6331.90 49.47 Barley 1 (TAD) Flocced Date Time Dil. O.D. sCOD (mg L-1) Dil. O.D. sCOD (mg L-1) Dil. O.D. sCOD (mg L-1) Average sCOD (mg L-1) St. Dev 13-Jun 0 20 0.214 10586.15 10 0.391 9670.99 10128.57 27-Jun 14 30 0.14 10388.28 20 0.234 11575.51 40 0.103 10190.41 10718.07 749.13 13-Jul 28 30 0.121 8978.44 20 0.191 9448.39 40 0.095 9398.92 9275.25 258.23 28-Jul 45 30 0.117 8681.63 20 0.164 8112.75 40 0.084 8310.62 8368.34 288.80 23-Aug 71 20 0.152 7519.14 30 0.099 7346.00 40 0.075 7420.20 7428.44 86.86 Barley 2 (TAD) Flocced Date Time Dil. O.D. sCOD (mg L-1) Dil. O.D. sCOD (mg L-1) Dil. O.D. sCOD (mg L-1) Average sCOD (mg L-1) St. Dev 13-Jun 0 20 0.219 10833.49 10 0.392 9695.73 10264.61 27-Jun 14 30 0.122 9052.64 20 0.192 9497.86 40 0.091 9003.18 9184.56 272.45 13-Jul 28 30 0.119 8830.04 20 0.174 8607.43 40 0.089 8805.30 8747.59 122.01 28-Jul 45 30 0.108 8013.82 20 0.161 7964.35 40 0.08 7914.88 7964.35 49.47 23-Aug 71 20 0.14 6925.52 30 0.098 7271.80 40 0.069 6826.58 7007.97 233.78 Bioethanol 1 (TBD) Raw Date Time Dil. O.D. sCOD (mg L'1) Dil. O.D. sCOD (mg L-1) Dil. O.D. sCOD (mg L-1) Average sCOD (mg I-"1) St. Dev 13-Jun 0 5 0.146 1805.58 2 0.342 1691.81 10 0.085 2102.39 1866.59 211.98 27-Jun 14 4 0.172 1701.70 2 0.342 1691.81 8 0.086 1701.70 1698.40 5.71 13-Jul 28 4 0.143 1414.78 2 0.288 1424.68 8 0.072 1424.68 1421.38 5.71 28-Jul 45 8 0.055 1088.30 4 0.104 1028.93 8 0.052 1028.93 1048.72 34.27 23-Aug 71 2 0.199 984.41 4 0.099 979.47 8 0.048 949.79 971.22 18.73 ro O Bioethanol 2 (TBD) Raw Date Time Dil. O.D. sCOD (mg L-1) Dil. O.D. sCOD (mg L-1) Dil. O.D. sCOD (mg L-1) Average sCOD (mg L-1) St. Dev 13-Jun 0 5 0.155 1916.89 2 0.376 1860.00 10 0.073 1805.58 1860.82 55.66 27-Jun 14 4 0.198 1958.93 2 0.364 1800.64 8 0.088 1741.27 1833.61 112.51 13-Jul 28 2 0.311 1538.45 4 0.155 1533.51 8 0.077 1523.61 1531.86 7.56 28-Jul 45 8 0.052 . 1028.93 4 0.099 979.47 8 0.053 1048.72 1019.04 35.67 23-Aug 71 2 0.177 875.58 4 0.097 959.68 8 0.044 870.64 901.97 50.04 Bioethanol 1 (TBD) Flocced Date Time Dil. O.D. sCOD (mg I-"1) Dil. O.D. sCOD (mg L"1) Dil. O.D. sCOD (mg L-1) Average sCOD (mg L-1) St. Dev 13-Jun 0 5 0.122 1508.77 2 0.276 1365.32 10 0.069 1706.65 1526.91 171.39 27-Jun 14 4 0.157 1553.30 2 0.333 1647.28 8 0.079 1563.19 1587.92 51.65 13-Jul 28 4 0.124 1226.81 2 0.243 1202.07 8 0.062 1226.81 1218.56 14.28 28-Jul 45 8 0.066 1305.96 4 0.11 1088.30 8 0.053 1048.72 1147.66 138.51 23-Aug 71 2 0.142 702.45 4 0.08 831.06 8 0.036 712.34 748.62 71.57 Bioethanol 2 (TBD) Flocced Date Time Dil. O.D. sCOD (mg L-1) Dil. O.D. sCOD (mgl_-1) Dil. O.D. sCOD (mg L-1) Average sCOD (mg L'1) St. Dev 13-Jun 0 5 0.139 1719.01 2 0.317 1568.14 10 0.077 1904.52 1730.56 168.49 27-Jun 14 4 0.146 1444.47 2 0.29 1434.57 8 0.072 1424.68 1434.57 9.89 13-Jul 28 4 0.106 1048.72 2 0.208 1028.93 8 0.051 1009.15 1028.93 19.79 28-Jul 45 8 0.021 415.53 4 0.031 306.70 8 0.018 356.17 359.47 54.49 23-Aug 71 2 0.035 173.14 4 0.016 158.30 8 0.01 197.87 176.44 19.99 Orchard Grass 1 (ORC) Raw Date Time Dil. O.D. sCOD (mg L-1) Dil. O.D. sCOD (mg L-1) Dil. O.D. sCOD (mg I-"1) Average sCOD (mg L'1) St. Dev 13-Jun 0 10 0.335 8285.89 20 0.177 8755.84 8520.86 332.30 27-Jun 14 30 0.107 7939.61 20 0.154 7618.07 40 0.078 7717.01 7758.23 164.69 13-Jul 28 20 0.137 6777.12 30 0.089 6603.98 40 0.066 6529.78 6636.96 126.93 28-Jul 45 30 0.071 5268.34 20 0.104 5144.67 40 0.051 5045.74 5152.92 111.53 23-Aug 71 20 0.065 3215.42 30 0.049 3635.90 40 0.035 3462.76 3438.03 211.33 Orchard Grass 2 (ORC) Raw Date Time Dil. O.D. sCOD (mg L'1) Dil. O.D. sCOD (mg L'1) Dil. O.D. sCOD (mg L-1) Average sCOD (mg L"1) St. Dev 13-Jun 0 10 0.335 8285.89 20 0.179 8854.77 8570.33 402.26 27-Jun 14 30 0.113 8384.83 20 0.165 8162.22 40 0.083 8211.69 8252.91 116.89 13-Jul 28 20 0.13 6430.84 30 0.088 6529.78 40 0.063 6232.97 6397.86 151.13 28-Jul 45 30 0.077 5713.55 20 0.11 5441.48 40 0.054 5342.54 5499.19 192.12 23-Aug 71 20 0.077 3809.04 30 0.055 4081.11 40 0.039 3858.50 3916.22 144.93 Orchard 1 (ORC) Flocced Date Time Dil. O.D. sCOD (mg L-1) Dil. O.D. sCOD (mg L-1) Dil. O.D. sCOD (mg I/1) Average sCOD (mg L'1) St. Dev 13-Jun 0 10 0.327 8088.02 20 0.173 8557.96 8322.99 332.30 27-Jun 14 30 0.104 7717.01 20 0.156 7717.01 40 0.078 7717.01 7717.01 0.00 13-Jul 28 20 0.148 7321.26 30 0.096 7123.39 40 0.074 7321.26 7255.31 114.24 28-Jul 45 30 0.074 5490.95 20 0.113 5589.88 40 0.055 5441.48 5507.44 75.56 23-Aug 71 20 0.107 5293.08 30 0.075 5565.15 40 0.053 5243.61 5367.28 173.14 Orchard 2 (ORC) Flocced Date Time Dil. O.D. sCOD (mg L-1) Dil. O.D. sCOD (mg I/1) Dil. O.D. sCOD (mg L 1) Average sCOD (mg I-"1) St. Dev 13-Jun 0 20 0.173 8557.96 10 0.329 8137.49 8347.73 297.32 27-Jun 14 30 0.11 8162.22 20 0.156 7717.01 40 0.078 7717.01 7865.41 257.04 13-Jul 28 30 0.096 7123.39 20 0.148 7321.26 40 0.071 7024.46 7156.37 151.13 28-Jul 45 30 0.078 5787.76 20 0.112 5540.42 40 0.057 5639.35 5655.84 124.49 23-Aug 71 20 0.068 3363.82 30 0.049 3635.90 40 0.034 3363.82 3454.52 157.08 A1-5e. Ortho-phosphate in leachate experiment Sample Dil. O.D. P0 4J" (mg L-1) Dil. O.D. P0 4 3 -(mg L-1) Average P0 4 3 ' (mg L"1) 30-Aug TAD 1 Raw 50 1.89 94.5 100 0.96 96 95.3 TAD 2 Raw 50 1.38 69 100 0.75 75 72.0 TAD 1 Floe 50 1.54 77 100 0.55 55 66.0 TAD 2 Floe 50 1.45 72.5 100 0.61 61 66.8 TBD 1 Raw 50 1.99 99.5 100 1.04 104 101.8 TBD 2 Raw 50 1.83 91.5 100 1.01 101 96.3 TBD 1 Floe 50 1.75 87.5 100 0.81 81 84.3 TBD 2 Floe 50 1.31 65.5 100 0.73 73 69.3 Orch 1 Raw 50 1.71 85.5 100 0.91 91 88.3 Orch 2 Raw 50 1.58 79 100 0.73 73 76.0 Orch 1 Floe 50 1.3 65 100 0.71 71 . 68.0 Orch 2 Floe 50 1.22 61 100 0.55 55 58.0 A1-5f. Ammonia-nitrogen in leachate experiment Sample Dil. O.D. NH3-N (mg L"1) Dil. O.D. NH3-N (mg L"1) Dil. O.D. NH3-N (mg L-1) Average NH3-N (mg I-"1) 30-Aug TAD 1 Raw 50 over >150 100 over >300 250 1.43 357.5 TAD 2 Raw 250 1.44 360 200 1.86 372 366.0 TAD 1 Floe 250 1.49 372.5 200 1.88 376 374.3 TAD 2 Floe 250 1.46 365 200 1.71 342 353.5 TBD 1 Raw 50 1.6 80 100 0.77 77 78.5 TBD 2 Raw 50 1.91 95.5 100 0.92 92 93.8 TBD 1 Floe 50 1.55 77.5 100 0.82 82 79.8 TBD 2 Floe 50 1.04 52 100 0.49 49 50.5 Orch 1 Raw 100 over >300 250 1.35 337.5 200 1.61 322 329.8 Orch 2 Raw 250 1.45 362.5 200 1.77 354 358.3 Orch 1 Floe 250 1.47 367.5 200 1.82 364 365.75 Orch 2 Floe 250 1.42 355 200 1.83 366 360.5 A1-6a. SO/ - concentrations in natural treatment mesocosm Mesocosm A Date Length Position Height Position Dil. O.D. S0 4 2" (mg L-1) Dil. O.D. SO/" (mg L"1) Dil. O.D. SO/" (mg L"1) Averaae so/ (mg L"1) St. Dev. 28-Sep 50 10 5 0.175 135.55 5 0.157 125.45 3.33 0.285 131.50 130.84 5.08 28-Sep 100 10 5 0.179 137.80 3.33 0.294 134.87 5 0.175 135.55 136.07 1.53 28-Sep 150 10 3.33 0.252 119.16 5 0.167 131.06 5 0.147 119.84 123.36 6.68 28-Sep 50 20 5 -0.016 28.41 5 -0.005 34.58 3.33 -0.013 20.06 27.68 7.29 28-Sep 100 20 3.33 0.109 65.69 5 0.074 78.90 5 0.097 91.80 78.79 13.06 28-Sep 150 20 3.33 0.063 48.48 5 -0.03 20.56 5 -0.007 33.46 34.17 13.98 28-Sep Outlet 5 0.162 128.26 5 0.161 127.70 3.33 0.261 122.41 126.12 3.23 13-Oct Inlet 40 0.455 2340.94 30 0.65 2412.02 50 0.388 2550.34 2434.44 106.48 -fe. Date Length Position Height Position Dil. O.D. S0 4 2" (mg L'1) Dil. O.D. SO4 2 ' (mg L-1) Dil. O.D. S0 4 2" (mg L-1) Average S 0 4 ^ (mg L-1) St. Dev. 13-Oct 50 10 30 0.463 1782.63 40 0.324 1753.07 25 0.578 1808.08 1781.26 27.53 13-Oct 100 10 40 0.31 1690.24 30 0.441 1708.59 25 0.561 1760.39 1719.74 36.38 13-Oct 150 10 30 0.325 1318.17 20 0.528 1334.27 25 0.423 1373.34 1341.92 28.37 13-Oct 50 20 30 0.357 1425.87 40 0.259 1461.37 50 0.18 1383.56 1423.60 38.95 13-Oct 100 20 30 0.5 1907.16 40 0.348 1860.77 50 0.259 1826.71 1864.88 40.38 13-Oct 150 20 30 0.403 1580.69 50 0.206 1529.41 40 0.278 1546.64 1552.25 26.10 13-Oct Outlet 40 0.106 774.77 20 0.263 739.66 10 0.608 756.89 757.10 17.55 22-Oct Inlet 40 0.495 2520.45 40 0.476 2435.18 33 0.592 2438.49 2464.71 48.30 22-Oct Outlet 20 0.784 1908.68 40 0.375 1981.93 33 0.453 1923.87 1938.16 38.66 2-Nov Inlet 40 0.492 2506.99 33 0.589 2427.38 33 0.633 2590.28 2508.22 81.46 2-Nov Outlet 33 0.585 2412.58 33 0.58 2394.06 25 0.822 2492.44 2433.03 52.28 10-Nov Inlet 40 0.275 1533.17 25 0.526 1662.23 33 0.386 1675.82 1623.74 78.73 10-Nov Outlet 25 0.866 2615.85 40 0.542 2731.37 33 0.598 2460.71 2602.64 135.81 19-Nov Inlet 25 0.471 1507.97 20 0.603 1502.56 20 0.601 1498.07 1502.86 4.96 19-Nov Outlet 33 0.532 2216.35 40 0.431 2233.24 25 2224.80 11.94 30-Nov Inlet 25 0.481 1536.01 20 0.607 1511.53 1523.77 17.31 30-Nov Outlet 25 0.656 2026.85 20 0.837 2027.61 2027.23 0.54 Mesocosm B Date Length Position Height Position Dil. O.D. S0 4 2-(mg L-1) Dil. O.D. S0 4 2" (mg L'1) Dil. O.D. SO4 2 ' (mg L-1) Averaqe S 0 4 ^ (mg L-1) St. Dev. 28-Sep 50 10 5 0.109 98.53 3.33 0.187 94.86 5 0.125 107.50 100.30 6.51 28-Sep 100 10 5 0.124 106.94 3.33 0.22 107.20 5 0.134 112.55 108.90 3.17 28-Sep 150 10 3.33 0.223 108.32 5 0.12 104.70 5 0.124 106.94 106.65 1.83 28-Sep 50 20 5 0.077 80.58 3.33 0.087 57.46 5 0.059 70.48 69.51 11.59 28-Sep 100 20 5 0.001 37.95 3.33 0.002 25.67 5 0.001 37.95 33.85 7.09 28-Sep 150 20 3.33 0.093 59.70 5 0.018 47.48 5 0.003 39.07 48.75 10.38 13-Oct Inlet 40 0.455 2340.94 30 0.65 2412.02 50 0.388 2550.34 2434.44 106.48 13-Oct 50 10 40 0.293 1613.95 30 0.419 1634.54 25 0.517 1636.98 1628.49 12.65 13-Oct 100 10 40 0.306 1672.29 25 0.523 1653.81 30 0.395 1553.77 1626.62 63.77 13-Oct 150 10 25 0.531 1676.25 30 0.421 1641.27 40 0.319 1730.63 1682.72 45.03 Date Length Position Height Position Dil. O.D. S0 4 2 -(mg L"1) Dil. O.D. S0 4 2" (mg L-1) Dil. O.D. so 4 2 ' (mg L-1) Average S0 4 2 ' (mg L-1) St. Dev. 13-Oct 50 20 30 0.561 2112.47 50 0.297 2039.88 40 0.419 2179.39 2110.58 69.78 13-Oct 100 20 30 0.384 1516.74 50 0,192 1450.88 40 0.29 1600.49 1522.70 74.98 13-Oct 150 20 30 0.456 1759.07 50 0.225 1635.99 40 0.311 1694.73 1696.60 61.56 13-Oct Outlet 40 0.107 779.25 20 0.288 795.76 10 0.638 790.54 788.52 8.44 22-Oct Inlet 40 0.495 2520.45 40 0.476 2435.18 33 0.592 2438.49 2464.71 48.30 22-Oct Outlet 40 0.346 1851.79 33 0.407 1753.57 20 0.767 1870.54 1825.30 62.82 2-Nov Inlet 40 0.492 2506.99 33 0.589 2427.38 33 0.633 2590.28 2508.22 81.46 2-Nov Outlet 33 0.575 2375.55 33 0.56 2320.02 25 0.809 2455.97 2383.85 68.36 10-Nov Inlet 40 0.275 1533.172 25 0.526 1662.227 33 0.386 1675.82 1623.74 78.73 10-Nov Outlet 40 0.521 2637.126 25 0.859 2596.212 33 0.595 2449.60 2560.98 98.60 19-Nov Inlet 25 0.471 1507.965 20 0.603 1502.555 20 0.601 1498.07 1502.86 4.96 19-Nov Outlet 25 0.657 2029.651 40 0.394 2067.199 33 0.468 1979.41 2025.419 44.05 30-Nov Inlet 25 0.481 1536.013 20 0.607 1511.530 1523.772 30-Nov Outlet 25 0.662 2043.675 20 0.837 2027.606 2035.640 A1-6b. Total sulphide in natural treatment mesocosm Mesocosm A Date Time Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg L-1) Average Sulphide (mg L-1) St. Dev. 28-Sep 0 2 0.122 0.50 2 0.104 0.42 5 0.05 0.44 0.45 0.04 13-Oct 15 2 0.753 3.29 5 0.308 3.30 5 0.314 3.37 3.32 0.04 22-Oct 24 2 0.124 0.51 2 0.119 0.48 5 0.065 0.61 0.53 0.07 2-Nov 35 5 0.028 0.20 2 0.051 0.18 10 0.01 0.00 0.13 0.11 10-Nov 43 2 0.037 0.12 2 0.024 0.06 1 0.035 0.06 0.08 0.04 19-Nov 52 1 0.025 0.03 2 0.022 0.05 2 0.018 0.04 0.04 0.01 30-Nov 63 2 0.015 0.02 1 0.018 0.02 2 0.013 0.01 0.02 0.00 ON Mesocosm B Date Time Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg L-1) Average Sulphide (mg L-1) St. Dev. 28-Sep 0 20 0.012 0.10 20 0.015 0.23 40 0.05 3.56 1.30 1.96 13-Oct 15 20 0.644 28.08 40 0.343 29.50 40 0.363 31.27 29.62 1.60 22-Oct 24 40 0.103 8.25 20 0.217 9.17 20 0.186 7.80 8.41 0.70 2-Nov 35 40 0.025 1.34 20 0.034 1.07 40 0.021 0.99 1.14 0.19 10-Nov 43 2 0.029 0.08 2 0.03 0.09 1 0.03 0.04 0.07 0.02 19-Nov 52 2 0.018 0.04 2 0.021 0.05 1 0.021 0.02 0.04 0.01 30-Nov 63 1 0.018 0.02 2 0.022 0.05 2 0.015 0.02 0.03 0.02 A1-6c. pH in natural treatment mesocosm Mesocosm A Position along the length of the mesocosm 50 cm 100 cm 150 cm Depth (cm) Depth (cm) Depth (cm) Date 10 20 10 20 10 20 28-Sep 8.99 7.92 9.03 8.46 7.58 7.29 22-Oct 8.53 8.62 8.68 7.91 8.51 7.66 10-Nov 8.22 8 7.82 7.97 8.23 7.77 19-Nov 7.37 8.17 8.05 8.32 8.46 8.29 30-Nov 7.78 8.68 7.75 7.83 7.74 7.61 Mesocosm B 28-Sep 8.61 8.06 8.99 7.57 8.89 7.58 22-Oct 8.61 8.52 8.56 7.84 8.52 7.71 10-Nov 8.13 8.85 8.28 7.74 8.35 8.43 19-Nov 8.14 8.56 8.28 8.2 8.28 8.38 30-Nov 7.32 7.83 7.5 8.14 7.73 7.86 A1-6d. Dissolved oxygen (% saturation) in natural treatment mesocosm Mesocosm A Position along the length of the mesocosm 50 cm 100 cm 150 cm Depth (cm) Depth (cm) Depth (cm) Date 10 20 10 20 10 20 28-Sep 30.4 2.4 47.1 2.1 24.1 2.0 22-Oct 28.5 1.6 8.4 1.3 6.0 1.3 10-Nov 2.6 1.6 2.8 1.3 7.9 1.2 19-Nov 29.9 2.1 27.6 2.4 28.8 2.8 30-Nov 49.0 4.0 55.0 3.7 46.0 2.9 Mesocosm B 28-Sep 26.7 1.7 32.3 1.7 23.8 1.6 22-Oct 30.3 1.5 7.1 1.0 4.8 1.0 10-Nov 39.8 1.9 40.5 3.6 49.6 3.1 19-Nov 11.0 2.1 6.1 1.5 7.0 2.2 30-Nov 37.0 1.6 39.0 2.4 41.0 1.9 A1-6e. Ammonia-nitrogen in natural treatment mesocosm Mesocosm A Date Dil. O.D. NHj-N (mg L 1) Dil. O.D. NH3-N (mg I."1) Average NH3-N (mg L-1) 28-Sep 1 0.40 0.40 1 0.64 0.64 0.52 15-Nov 1 0.00 0.00 1 0.00 0.00 0.00 Mesocosm B Date Dil. O.D. NH3-N (mg L'1) Dil. O.D. NH3-N (mg L"1) Average NH3-N (mg L"1) 28-Sep 1 0.58 0.58 1 1.07 1.07 0.83 15-Nov 1 0.02 0.02 1 0.00 0.00 0.01 OO A1-6f. Ortho-phosphate in natural treatment mesocosm Mesocosm A Date Dil. O.D. P0 4 3 ' (mg L-1) Dil. O.D. P0 4 3 ' (mg L-1) Dil. O.D. P0 4 3" (mg L-1) Average P0 4 3 ' (mg L"1) 28-Sep 1 over >3 10 2.75 27.50 20 1.33 26.60 27.05 15-Nov '1 over >3 10 0.73 7.30 5 1.51 7.55 7.43 Mesocosm B Date Dil. O.D. P0 4 3" (mg L-1) Dil. O.D. P0 4 3" (mg L-1) Dil. O.D. P O 4 3 -(mg L'1) Average P0 4 3" (mg L'1) 28-Sep 2 over >6 10 2.49 24.90 20 1.13 22.60 23.75 15-Nov 10 0.48 4.80 5 1.01 5.05 4.93 A1-7a. S0 4 2 ' concentrations in mesocosms while operated as batch systems Natural Treatment Mesocosm A (water column) Date Time (Days) Dil. O.D. S0 42" (mg I/1) Dil. O.D. S0 4 2 ' (mg L 1) Dil. O.D. S0 4 2" (mg L-1) Average S0 4 ^ (mg L'1) St. Dev. 6-Dec 0 20 0.878 2119.60 20 0.840 2034.34 33 0.471 1990.52 2048.15 65.64 13-Dec 7 33 0.441 1879.45 33 0.442 1883.15 20 0.768 1872.78 1878.46 5.25 21-Dec 15 33 0.471 1990.52 33 0.482 2031.24 25 0.645 1995.99 2005.92 22.10 29-Dec 23 33 0.404 1742.46 33 0.397 1716.55 20 0.689 1695.52 1718.18 23.51 7-Jan 32 33 0.473 1997.92 33 0.488 2053.45 25 0.653 2018.43 2023.27 28.08 14-Jan 39 33 0.494 2075.67 33 0.498 2090.48 25 0.647 2001.60 2055.92 47.62 24-Jan 49 33 0.504 2112.69 33 0.501 2101.58 25 0.679 2091.36 2101.88 10.67 4-Feb 60 33 0.505 2116.39 33 0.497 2086.77 25 0.683 2102.58 2101.91 14.82 14-Feb 70 33 0.501 2101.58 33 0.485 2042.35 25 0.671 2068.92 2070.95 29.67 21-Feb 77 33 0.489 2057.16 33 0.477 2012.73 25 0.651 2012.82 2027.57 25.62 5-Jul 211 33 0.427 1827.61 25 0.601 1872.58 30 0.489 1870.14 1856.78 25.29 13-Sep 281 33 0.370 1616.58 30 0.397 1560.50 25 0.471 1507.97 1561.68 54.32 r-J 4^ v© Natural Treatment Mesocosm A Sediment (100 cm) Date Time (Days) Dil. O.D. S0 4 2" (mg I-"1) Dil. O.D. S0 4 2" (mg I-"1) Dil. O.D. so42-(mg L-1) Average S0 4 2 -(mg L-1) St. Dev. 7-Jan 32 33 0.12 691.01 20 0.236 679.08 25 0.168 658.12 676.07 16.65 14-Jan 39 20 0.21 620.74 33 0.121 694.72 25 0.162 641.30 652.25 38.19 14-Feb 70 33 0.195 968.68 25 0.264 927.38 20 0.345 923.65 939.91 24.99 21-Feb 77 33 0.234 1113.07 25 0.329 1109.69 20 0.387 1017.89 1080.22 54.00 28-Feb 84 20 0.102 378.41 10 0.259 365.34 33 0.032 365.21 369.65 7.58 7-Mar 91 33 0.08 542.92 25 0.101 470.20 20 0.152 490.60 501.24 37.51 6-Jun 182 33 0.276 1268.57 20 0.501 1273.69 1271.13 5-Jul 211 33 0.266 1181.92 25 0.368 1165.51 20 0.478 1165.45 1170.96 9.50 13-Sep 281 33 0.309 1332.23 20 0.545 1307.39 25 0.422 1308.51 1316.04 14.03 Natural Treatment Mesocosm A Sediment (50cm) Date Time (Days) Time (Days) (new exp.) Dil. O.D. S0 42 -(mg L-1) Dil. O.D. so42-(mg I."1) Dil. O.D. so42-(mg L'1) Average S0 4 2 -(mg L-1) St. Dev. 7-Mar 91 0 25 0.575 1799.66 33 0.374 1631.39 20 0.656 1621.48 1684.18 100.13 14-Mar 98 7 25 0.315 1070.42 33 0.209 1020.52 20 0.382 1006.67 1032.54 33.53 21-Mar 105 14 25 0.263 924.58 33 0.187 939.07 20 0.345 923.65 929.10 8.64 29-Mar 113 22 33 0.172 883.53 25 0.231 834.82 20 0.313 851.85 856.74 24.72 12-Apr 127 36 33 0.179 909.45 25 0.277 963.84 20 0.327 883.26 918.85 41.10 18-Apr 133 42 33 0.208 1016.81 25 0.319 1081.64 20 0.389 1022.38 1040.28 35.93 25-Apr 140 49 25 0.378 1247.12 33 0.246 1157.50 20 0.457 1174.96 1193.19 47.51 2-May 147 56 25 0.247 879.70 33 0.182 920.56 20 0.359 955.07 918.44 37.73 16-May 161 70 33 0.16 839.10 25 0.237 851.65 20 0.313 851.85 847.54 7.30 24-May 169 78 25 0.167 655.32 33 0.104 631.78 20 0.213 627.47 638.19 14.99 30-May 175 84 10 0.55 691.82 20 0.229 663.37 20 0.223 649.91 668.37 21.40 6-Jun 182 91 33 0.378 1646.20 20 0.684 1684.30 25 0.513 1625.77 1652.09 29.71 5-Jul 211 127 33 0.33 1405.64 25 0.425 1316.45 20 0.576 1373.06 1365.05 45.13 13-Sep 281 233 33 0.366 1531.48 20 0.601 1426.02 25 0.501 1517.71 1491.74 57.33 O Natural Treatment Mesocosm B (water column) Date Time (Days) Dil. O.D. S0 42 _ (mg L' 1) Dil. O.D. S0 42 -(mg L-1) Dil. O.D. S0 42 ' (mg L' 1) Average so42-(mg I-"1) St. Dev. 6-Dec 0 20 0.825 2000.68 20 0.828 2007 .'41 33 0.482 2031.24 2013.11 16.06 13-Dec 7 33 0.438 1868.34 33 0.441 1879.45 20 0.759 1852.59 1866.79 13.50 21-Dec 15 33 0.477 2012.73 33 0.478 2016.43 25 0.637 1973.56 2000.91 23.76 29-Dec 23 33 0.401 1731.36 33 0.406 1749.87 20 0.656 1621.48 1700.90 69.40 7-Jan 32 33 0.501 2101.58 33 0.492 2068.26 25 0.666 2054.89 2074.91 24.04 14-Jan 39 33 0.443 1886.85 33 0.451 1916.47 25 0.607 1889.41 1897.58 16.41 24-Jan 49 33 0.448 1905.36 33 0.448 1905.36 25 1905.36 4-Feb 60 33 0.455 1931.28 33 0.468 1979.41 25 0.627 1945.51 1952.07 24.73 14-Feb 70 33 0.465 1968.30 33 0.455 1931.28 25 0.613 1906.24 1935.27 31.22 21-Feb 77 33 0.447 1901.66 33 0.438 1868.34 25 0.599 1866.98 1878.99 19.64 5-Jul 211 25 0.581 1816.49 30 0.453 1748.98 33 0.429 1835.02 1800.16 45.29 13-Sep 281 33 0.356 1564.75 25 0.501 1592.11 30 0.374 1483.09 1546.65 56.72 Natural Treatment Mesocosm B Sediment (100 cm) Date Time (Days) Dil. O.D. S0 4 2" (mg L"1) Dil. O.D. S0 42 ' (mg L-1) Dil. O.D. S0 42 ' (mg I-"1) Average sof (mg I-"1) St. Dev. 7-Jan 32 33 0.061 472.58 20 0.090 351.48 25 0.055 341.19 388.42 73.07 14-Jan 39 20 0.438 1132.33 33 0.245 1153.80 1143.06 14-Feb 70 33 0.074 520.71 25 0.121 526.30 20 0.176 544.45 530.49 12.41 21-Feb 77 33 0.124 705.82 20 0.237 681.32 25 0.167 655.32 680.82 25.26 28-Feb 84 10 0.588 734.45 33 0.109 650.29 20 0.245 699.27 694.67 59.51 7-Mar 91 25 0.098 461.79 33 0.063 479.98 20 0.145 474.89 472.22 12.86 6-Jun 182 33 0.358 1572.16 20 0.645 1596.80 25 0.513 1625.77 1598.24 26.83 5-Jul 211 33 0.312 1342.72 25 0.431 1332.34 20 0.564 1347.64 1340.90 7.81 13-Sep 281 33 0.288 1258.83 20 0.533 1281.96 1270.40 to Natural Treatment Mesocosm B Sediment (50cm) Date Time (Days) Time (Days) (new exp.) Dil. O.D. S0 4 2 ' (mg L-1) Dil. O.D. S0 4 2 ' (mg L-1) Dil. O.D. S0 4 2" (mg L-1) Average S0 4 2 ' (mg L-1) St. Dev. 7-Mar 91 0 33 0.525 2190.44 25 0.672 2071.72 20 0.791 1924.39 2062.18 133.28 14-Mar 98 7 25 0.48 1533.21 33 0.345 1524.03 20 0.622 1545.19 1534.14 10.61 21-Mar 105 14 25 0.337 1132.13 33 0.267 1235.25 20 0.501 1273.69 1213.69 73.20 29-Mar 113 22 33 0.246 1157.50 25 0.376 1241.51 20 0.475 1215.35 1204.79 42.99 12-Apr 127 36 25 0.396 1297.61 33 0.275 1264.87 1281.24 18-Apr 133 42 25 0.339 1137.74 33 0.222 1068.65 20 0.402 1051.55 1085.98 45.63 25-Apr 140 49 33 0.144 779.87 25 0.224 815.19 25 0.240 860.07 818.38 40.19 2-May 147 56 25 0.268 938.60 33 0.197 976.09 20 0.345 923.65 946.11 27.01 16-May 161 70 25 0.087 430.94 33 0.066 491.09 10 0.326 440.5103 454.18 32.32 24-May 169 78 25 0.167 655.32 33 0.110 653.99 20 0.223 649.91 653.07 2.82 30-May 175 84 20 0.157 501.82 10 0.404 528.02 33 0.056 454.07 494.63 37.50 6-Jun 182 91 10 0.686 844.40 20 0.296 813.71 25 0.223 812.39 823.50 18.11 5-Jul 211 127 33 0.193 926.75 25 0.278 927.17 20 0.367 973.02 942.31 26.59 13-Sep 281 233 33 0.313 1346.22 20 0.571 1362.47 25 0.433 1337.64 1348.77 12.61 Agricultural Mesocosm A -100 cm down the length of the mesocosm Date Time (Days) Dil. O.D. SO4 2 ' (mg L-1) Dil. O.D. so42-(mg L'1) Dil. O.D. S0 4 2" (mg L-1) Average S0 4 ^ (mg L-1) St. Dev. 10-Dec 0 25 0.462 1482.72 20 0.566 1419.53 33 0.333 1479.60 1460.62 35.6 21-Dec 11 33 0.207 1013.11 33 0.214 1039.03 25 0.291 1003.11 1018.42 18.5 29-Dec 19 33 0.142 772.46 33 0.142 772.46 25 0.211 778.73 774.55 3.6 7-Jan 28 25 0.159 632.88 25 0.164 646.90 20 0.225 654.40 644.73 10.9 14-Jan 35 20 0.149 483.87 20 0.155 497.33 25 0.105 481.42 487.54 8.6 24-Jan 45 20 0.100 373.92 20 0.095 362.70 10 0.269 376.56 371.06 7.4 4-Feb 56 10 0.144 236.32 10 0.101 188.08 5 0.304 207.91 210.77 24.2 14-Feb 66 10 0.076 160.03 10 0.064 146.57 5 0.191 144.53 150.38 8.4 21-Feb 73 10 0.004 79.26 10 0.010 85.99 5 0.073 78.33 81.19 4.2 2-Mar 82 25 0.471 1507.97 33 0.343 1516.62 25 0.482 1538.82 1521.14 15.9 7-Mar 87 33 0.161 842.81 25 0.294 1011.52 20 0.335 901.21 918.52 85.7 Date Time (Days) Dil. O.D. SO42" (mg L-1) Dil. O.D. SO42" (mg L-1) Dil. O.D. S0 4 2" (mg L-1) Average so4^ (mg L-1) St. Dev. 9-Mar 89 33 0.065 487.39 20 0.152 490.60 10 0.385 506.70 494.90 10.3 14-Mar 94 25 0.007 206.56 5 0.493 313.93 10 0.222 323.83 281.44 65.0 21-Mar 101 10 0.063 145.45 5 . 0.157 125.45 10 0.059 140.96 137.29 10.5 29-Mar • 109 33 0.666 2712.46 25 0.920 2767.30 2739.88 30-Mar 110 33 0.341 1509.22 25 0.485 1547.23 20 0.613 1524.99 1527.15 19.1 12-Apr 123 20 0.614 1527.24 25 0.476 1521.99 33 0.343 1516.62 1521.95 5.3 18-Apr 129 25 0.453 1457.48 33 0.291 1324.10 20 0.589 1471.14 1417.58 81.2 25-Apr 136 20 0.883 2130.82 33 0.504 2112.69 25 0.703 2158.67 2134.06 23.2 2-May 143 33 0.447 1901.66 25 0.626 1942.70 20 0.801 1946.83 1930.40 25.0 16-May 157 33 0.519 2168.22 25 0.784 2385.86 20 0.887 2139.80 2231.29 134.6 24-May 165 33 0.432 1846.13 25 0.605 1883.80 20 0.763 1861.56 1863.83 18.9 30-May 171 33 0.247 1161.20 20 0.432 1118.86 25 0.331 1115.30 1131.79 25.5 6-Jun 178 25 0:259 913.36 33 0.169 872.43 20 0.341 914.68 900.15 24.0 20-Jun 192 20 0.840 1610.29 33 0.467 1570.44 25 0.639 1569.30 1583.35 23.3 5-Jul 207 20 0.371 938.76 33 0.206 972.19 30 0.242 998.21 969.72 29.8 19-Jul 221 25 0.651 1914.94 33 0.451 1828.60 30 0.507 1840.32 1861.29 46.8 26-Jul 228 25 0.787 2275.09 33 0.556 2195.64 30 0.628 2224.84 2231.85 40.2 2-Aug 235 33 0.431 1758.69 25 0.566 1689.84 30 0.461 1694.15 1714.23 38.6 9-Aug 242 33 0.473 1905.51 25 0.658 1933.48 30 0.531 1916.59 1918.52 14.1 17-Aug 250 25 0.640 1885.81 33 0.457 1849.58 30 0.511 1853.03 1862.81 20.0 13-Sep 277 33 0.339 1437.10 30 0.389 1465.34 25 0.467 1427.68 1443.37 19.60 Agricultural Mesocosm A - 50 cm down the length of the mesocosm Date Time (Days) Dil. O.D. S0 4 2 ' (mg L-1) Dil. O.D. S0 4 2 ' (mg I.'1) Dil. O.D. so42_ (mg I-"1) Average so4^ (mg I-"1) St. Dev. 6-Jun 178 20 0.331 892.24 33 0.181 916.85 25 0.25 874.09 894.39 21.5 20-Jun 192 20 0.713 1386.08 25 0.54 1359.1 30 0.44 1351.7 1365.63 18.1 5-Jul 207 20 0.398 995.96 33 0.217 1010.64 30 0.24 998.5 1001.70 7.8 19-Jul 221 33 0.454 1839.09 25 0.635 1872.57 30 0.51 1855.3 1855.65 16.7 26-Jul 228 33 0.563 2220.11 25 0.758 2198.29 30 0.62 2201.4 2206.60 11.8 2-Aug 235 33 0.41 1685.29 25 0.565 1687.20 30 0.46 1684.9 1685.79 1.2 9-Aug 242 25 0.633 1867.27 33 0.476 1915.99 30 0.52 1891.1 1891.45 24.4 17-Aug 250 33 0.451 1828.604 25 0.617 1824.900 30 0.50 1825.6 1826.368 2.0 Agricultural Mesocosm A -150 cm down the length of the mesocosm Date Time (Days) Dil. O.D. S0 4 2" (mg L-1) Dil. O.D. SO42" (mg L-1) Dil. O.D. SO42" (mg I-"1) Average S0 4 2 ' (mg L-1) St. Dev. 6-Jun 178 33 0.209 1020.52 25 0.306 1045.18 20 0.39 1031.36 1032.35 12.4 20-Jun 192 20 0.676 1320.76 33 0.37 1301.7 25 0.52 1306.4 1309.62 9.9 5-Jul 207 33 0.189 912.76 20 0.402 1004.44 30 0.23 944.7 953.97 46.5 19-Jul 221 33 0.442 1797.14 25 0.618 1827.55 30 0.49 1789 1804.56 20.3 26-Jul 228 25 0.759 2200.94 33 0.579 2276.04 30 0.62 2198.5 2225.16 44.1 2-Aug 235 33 0.424 1734.22 25 0.555 1660.71 30 0.46 1697.6 1697.51 36.8 9-Aug 242 33 0.465 1877.54 25 0.659 1936.12 30 0.54 1942.6 1918.76 35.8 17-Aug 250 25 0.633 1867.27 33 0.45 1825.11 30 0.52 1882.4 1858.26 29.7 Agricultural Mesocosm B -100 cm down the length of the mesocosm Date Time (Days) Dil. O.D. S0 4 2 ' (mg L-1) Dil. O.D. S0 4 2 _ (mg L-1) Dil. O.D. S0 4 2" (mg I/1) Average so 4 r (mg I-"1) St. Dev. 10-Dec 0 25 0.852 2576.58 20 1.002 2397.83 33 0.585 2412.58 2462.33 99.22 13-Dec 3 33 0.278 1275.97 33 0.287 1309.29 25 0.409 1334.07 1306.45 29.15 21-Dec 11 33 0.223 1072.35 33 0.228 1090.86 25 0.310 1056.40 1073.20 17.25 29-Dec 19 33 0.127 716.93 33 0.121 694.72 20 0.251 712.74 708.13 11.80 7-Jan 28 25 0.137 571.18 25 0.150 607.64 20 0.193 582.59 587.14 18.65 14-Jan 35 20 0.161 510.79 20 0.151 488.35 10 0.360 478.66 492.60 16.48 24-Jan 45 20 0.131 443.48 20 0.139 461.43 10 0.320 433.78 446.23 14.03 4-Feb 56 10 0.155 248.66 10 0.182 278.96 20 0.046 252.75 260.13 16.44 14-Feb 66 10 0.089 174.62 10 0.077 161.16 5 0.214 157.43 164.40 9.04 21-Feb 73 10 0.052 133.11 10 0.059 140.96 5 0.163 128.82 134.30 6.16 2-Mar 82 25 0.48 1522.40 33 0.336 1489.60 25 0.469 1502.36 1504.79 16.53 7-Mar 87 25 0.316 1073.23 33 0.204 1002.01 20 0.394 1033.60 1036.28 35.69 9-Mar 89 33 0.095 598.46 25 0.148 601.30 20 0.192 580.35 593.37 11.36 14-Mar 94 25 0.010 214.97 10 0.112 200.31 10 0.122 211.64 208.97 7.69 21-Mar 101 10 0.126 216.13 5 0.317 . 215.35 5 0.292 201.18 210.89 8.41 29-Mar 109 25 0.545 1715.52 33 0.377 1642.50 20 0.685 1686.55 1681.52 36.77 12-Apr 123 25 0.454 1460.29 33 0.301 1361.13 20 0.567 1421.78 1414.40 49.99 18-Apr 129 25 0.483 1541.62 33 0.346 1527.73 20 0.621 1542.94 1537.43 8.43 4^ Date Time (Days) Dil. O.D. S0 42 ' (mg L-1) Dil. O.D. S0 4 2" (mg L'1) Dil. O.D. S0 4 2 ' (mg L-1) Average SO*^ (mg I."1) St. Dev. 25-Apr 136 33 0.416 1786.89 25 0.636 1970.75 20 0.746 1823.42 1860.35 97.34 2-May 143 25 0.718 2200.74 33 0.511 2138.61 33 0.508 2127.50 2155.62 39.47 16-May 157 25 0.815 2472.80 33 0.593 2442.19 20 1.018 2433.74 2449.58 20.55 24-May 165 33 0.495 2079.37 25 0.725 2220.38 30 0.543 2051.89 2117.21 90.39 30-May 171 33 0.167 865.02 25 0.240 860.07 20 0.315 856.34 860.48 4.36 6-Jun 178 25 0.282 977.87 33 0.182 920.56 20 0.357 950.58 949.67 28.67 20-Jun 192 20 0.801 1541.44 33 0.459 1547.14 1544.29 5-Jul 207 20 0.797 1841.26 33 0.426 1741.21 25 0.601 1782.53 1788.33 50.27 19-Jul 221 25 0.676 1981.14 33 0.472 1902.01 20 0.790 1826.43 1903.19 77.36 26-Jul 228 33 0.581 2283.03 25 0.773 2238.01 30 0.596 2123.15 2214.73 82.44 2-Aug 235 33 0.445 1807.63 25 0.583 1734.86 30 0.446 1646.48 1729.66 80.70 9-Aug 242 25 , 0.699 2042.05 33 0.505 2017.36 30 0.536 1932.48 1997.30 57.48 17-Aug 250 33 0.459 1856.57 25 0.668 1959.96 30 0.510 1849.86 1888.79 61.72 13-Sep 277 33 0.399 1646.83 25 0.561 1676.60 30 0.451 1662.37 1661.94 14.89 Agricultural Mesocosm B - 50 cm down the length of the mesocosm Date Time (Days) Dil. O.D. S0 4 2" (mg L-1) Dil. O.D. S0 4 2" (mg L-1) Dil. O.D. SO42" (mg I/1) Average S 0 4 ^ (mg L-1) St. Dev. 6-Jun 178 25 0.373 1233.10 33 0.237 1124.18 20 0.494 1257.98 1205.09 71.16 20-Jun 192 33 0.457 1541.31 25 0.602 1487.65 20 0.789 1520.26 1516.41 27.04 5-Jul 207 33 0.496 1985.90 20 0.864 1983.20 25 0.653 1920.23 1963.11 37.16 19-Jul 221 33 0.549 2171.17 25 0.738 2145.33 30 0.591 2107.26 2141.25 32.15 26-Jul 228 33 0.595 2331.97 25 0.793 2290.98 30 0.647 2285.21 2302.72 25.49 2-Aug 235 33 0.452 1832.10 25 0.601 1782.53 30 0.492 1792.66 1802.43 26.19 9-Aug 242 33 0.491 1968.43 25 0.713 2079.12 20 0.905 2070.06 2039.20 61.46 17-Aug 250 25 0.660 1938.77 33 0.484 1943.96 30 0.527 1903.88 1928.87 21.80 to Oh Oh Agricultural Mesocosm B -150 cm down the length of the mesocosm Date Time (Days) Dil. O.D. S0 4 2 ' (mg L-1) Dil. O.D. SO42" (mg L-1) Dil. O.D. SO42" (mg L"1) Average S0 4 2-(mg L 1) St. Dev. 6-Jun 178 33 0.306 1379.64 25 0.435 1406.99 20 0.562 1410.51 1399.05 16.90 20-Jun 192 20 0.561 1117.74 33 0.315 1127.67 1122.70 5-Jul 207 33 0.416 1706.26 20 0.763 1769.23 25 0.559 1755.62 1743.70 33.13 19-Jul 221 33 0.456 1846.08 25 0.650 1912.29 30 0.521 1884.81 1881.06 33.26 26-Jul 228 25 0.807 2328.05 33 0.582 2286.52 30 0.606 2154.93 2256.50 90.38 2-Aug 235 33 0.471 1898.52 25 0.622 1838.14 30 0.503 1827.61 1854.76 38.26 9-Aug 242 33 0.502 2006.88 25 0.694 2028.81 20 0.901 2061.58 2032.42 27.53 17-Aug 250 33 0.474 1909.00 25 0.672 1970.55 30 0.533 1922.95 1934.17 32.27 A1-7b. Total sulphide in mesocosms while operated as batch systems Natural Treatment Mesocosm A (water column) Date Time (Days) Dil. O.D. Sulphide mg L'1 Dil. O.D. Sulphide mg L'1 Dil. O.D. Sulphide mg L'1 Average Sulphide (mg L'1) 6-Dec 0 1 0.131 0.27 1 0.135 0.28 1 0.130 0.22 0.26 13-Dec 7 1 0.031 0.05- 1 0.028 0.04 1 0.027 0.03 0.04 21-Dec 15 1 0.016 0.01 10 0.001 0.00 0.01 29-Dec 23 1 0.429 0.93 10 0.052 0.93 0.93 7-Jan 32 1 0.026 0.04 10 0.000 0.00 0.02 24-Jan 49 1 0.110 0.22 10 0.020 0.23 0.22 14-Feb 70 1 0.011 0.00 10 0.000 0.00 0.00 Natural Treatment Mesocosm A Sediment (100 cm) Date Time (Days) Dil. O.D. Sulphide mg L"1 Dil. O.D. Sulphide mg L"1 Dil. O.D. Sulphide mg L-1 7-Jan 32 20 over >27 80 over >108 320 0.358 246.64 Uh O S Natural Treatment Mesocosm A Sediment (50cm) Date Time (Days) Time (Days) (new exp.) Dil. O.D. Sulphide (mg L 1) Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg L-1) Average Sulphide (mg I-"1) St. dev 14-Mar 98 7 160 0.169 56.38 320 0.078 48.30 80 0.341 58.65 54.44 5.44 29-Mar 113 15 320 0.18 120.56 160 0.344 118.36 80 0.701 122.40 120.44 2.02 12-Apr 127 29 320 0.126 82.30 160 0.256 87.20 80 0.489 84.86 84.79 2.45 2-May 147 49 160 0.158 52.49 320 0.08 49.72 80 0.351 60.42 54.21 5.56 30-May 175 77 320 0.173 140.66 160 0.389 154.97 320 0.169 137.52 144.38 9.30 6-Jun 182 91 320 0.013 15.26 160 0.032 15.07 80 0.068 14.59 14.97 0.34 5-Jul 211 127 160 0.108 44.86 320 0.055 48.17 80 0.21 42.41 45.15 2.89 13-Sep 281 233 50 0.248 31.16 100 0.129 33.18 50 0.235 29.57 31.30 1.81 Natural Treatment Mesocosm B (water column) Date Time (Days) Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg L'1) Dil. O.D. Sulphide (mg L-1) Average Sulphide (mg L'1) 6-Dec 0 1 0.151 0.26 1 0.151 0.26 1 0.153 0.26 0.26 13-Dec 7 10 0.235 4.15 10 0.222 3.91 1 over 4.03 21-Dec 15 1 0.016 0.01 10 0.002 0.00 0.01 29-Dec 23 1 0.119 0.20 10 0.018 0.15 0.18 7-Jan 32 1 0.011 0.00 10 0.005 0.00 0.00 24-Jan 49 1 0.223 0.39 10 0.025 0.28 0.34 14-Feb 70 1 0.021 0.02 10 0 0.00 0.01 Natural Treatment Mesocosm B Sediment (100 cm) Date Time (Days) Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg L-1) 7-Jan 32 20 over >27 80 over >108 320 0.53 368.48 Natural Treatment Mesocosm B Sediment (50cm) Date Time (Days) Time (Days) (new exp.) Dil. O.D. Sulphide (mg L"1) Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg L-1) Average Sulphide (mg L-1) St. dev 14-Mar 98 7 160 0.094 29.82 320 0.038 16.64 80 0.162 22.46 22.97 6.60 29-Mar 113 15 320 0.165 109.93 80 0.622 108.41 160 0.312 107.03 108.46 1.45 12-Apr 127 29 320 0.134 87.97 160 0.241 81.88 80 0.412 71.22 80.36 8.48 2-May 147 49 160 0.243 82.59 320 0.125 68.00 80 0.396 56.99 69.17 12.87 30-May 175 77 320 0.278 222.95 160 0.491 194.94 208.95 14.00 6-Jun 182 91 320 0.150 122.63 80 0.621 122.94 160 0.304 121.66 122.41 0.67 5-Jul 211 127 160 0.126 51.91 320 0.068 58.36 55.14 13-Sep 281 183 50 0.222 27.98 100 0.118 30.48 50 0.227 28.59 29.02 1.31 Agricultural Mesocosm A -100 cm down the length of the mesocosm Date Time (Days) Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg I."1) Average Sulphide (mg L-1) 10-Dec 0 1 0.007 0.00 10 0 0.00 0.00 21-Dec 11 1 0.192 0.40 10 0 0.00 0.20 29-Dec 19 1 0.041 0.07 10 0.001 0.00 0.03 7-Jan 28 160 0.667 232.76 320 0.312 214.06 320 0.323 221.85 222.89 24-Jan 45 160 0.407 140.68 320 0.205 138.26 160 0.4 138.20 139.05 14-Feb 66 160 0.503 174.68 320 0.255 173.68 160 0.489 169.72 172.69 14-Mar 94 160 0.834 291.91 320 0.439 304.02 297.97 29-Mar 109 320 0.507 352.19 160 over 320 0.501 347.94 350.07 12-Apr 123 320 0.489 339.44 320 0.485 336.61 400 0.368 317.16 331.07 2-May 143 160 0.272 92.86 320 0.142 93.64 93.25 30-May 171 320 0.291 233.14 160 0.594 206.91 160 0.613 213.64 217.90 6-Jun 178 320 0.359 286.43 160 0.703 245.52 320 0.352 242.39 258.11 20-Jun 192 320 0.112 77.37 80 0.431 74.59 160 0.237 80.47 77.48 5-Jul 207 320 0.331 264.49 160 0.652 227.45 80 over 245.97 20-Jul 222 320 0.033 30.93 320 0.034 31.72 160 0.101 42.11 34.92 26-Jul 228 10 0.025 0.77 100 0.015 5.26 160 -0.005 0.58 2.20 2-Aug 235 20 0.014 1.00 10 0.028 0.84 10 0.91 22.45 8.10 OO Date Time (Days) Dil. O.D. Sulphide (mg L"1) Dil. O.D. Sulphide (mg I-"1) Dil. O.D. Sulphide (mg I-"1) Average Sulphide (mg I."1) 9-Aug 242 2 0.011 0.09 1 0.027 0.08 0.08 17-Aug 250 2 0.027 0.16 1 0.058 0.16 0.16 13-Sep 277 10 0.058 1.58 1 Over 5 0.112 1.45 1.51 Agricultural Mesocosm A - 50 cm down the length of the mesocosm Date Time (Days) Dil. O.D. Sulphide (mg L"1) Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg L'1) Average Sulphide (mg L-1) 6-Jun 182 320 0.39 310.73 160 0.741 292.91 80 over 301.82 5-Jul 211 160 .0.822 324.65 320 0.419 333.46 80 over 329.06 20-Jul 226 320 0.038 34.85 320 0.049 43.47 160 0.085 35.84 38.06 26-Jul 232 10 0.051 1.41 100 0.015 5.26 160 0 2.53 3.07 2-Aug 239 20 0.015 1.05 10 0.028 0.84 10 0.97 23.92 8.60 9-Aug 246 2 0.015 0.11 1 0.027 0.08 0.09 17-Aug 254 2 0.031 0.18 1 . 0.064 0.17 0.18 Agricultural Mesocosm A -150 cm down the length of the mesocosm Date Time (Days) Dil. O.D. Sulphide (mg I-"1) Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg L-1) Average Sulphide (mg L-1) 6-Jun 182 320 0.265 212.76 160 0.562 222.77 80 over 217.76 20-Jun 196 320 0.121 99.90 160 0.259 104.03 80 0.462 91.79 98.57 5-Jul 211 160 0.78 308.19 320 0.396 315.43 80 over 311.81 20-Jul 226 320 0.05 44.26 320 0.037 34.07 160 0.098 40.94 39.75 26-Jul 232 10 0.002 0.21 100 0 1.58 160 -0.001 2.14 1.31 2-Aug 239 20 0.01 0.81 10 0.022 0.70 10 0.018 0.60 0.70 9-Aug 246 2 0.014 0.10 1 0.03 0.09 0.09 17-Aug 254 2 0.027 0.16 1 0.04 0.11 0.14 Agricultural Mesocosm B -100 cm down the length of the mesocosm Date Time (Days) Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg L"1) Dil. O.D. Sulphide (mg L-1) Average Sulphide (mg L-1) 10-Dec 0 1 0.003 0.00 10 0.001 0.00 0.00 21-Dec 11 10 0.024 0.26 10 0.003 0.00 0.13 29-Dec 19 40 0.583 42.29 20 over 42.29 7-Jan 28 160 0.267 75.91 80 0.75 109.23 40 over 92.57 24-Jan 45 160 0.468 135.24 320 0.222 125.26 320 0.237 134.11 131.53 14-Feb 66 160 0.561 162.68 320 0.279 158.90 160.79 14-Mar 94 160 0.993 348.23 320 0.513 356.44 320 0.501 347.94 350.87 29-Mar 109 320 0.447 309.69 160 0.897 314.23 320 0.429 296.94 306.95 12-Apr 123 320 0.445 308.27 160 0.874 306.08 320 0.449 311.11 308.49 2-May 143 160 0.096 30.53 160 0.055 16.00 320 0.025 10.76 19.10 30-May 171 320 0.378 301.32 160 0.701 244.81 80 over 273.07 6-Jun 178 320 0.3 240.19 160 0.645 255.29 320 0.308 246.46 247.32 20-Jun 192 320 0.118 81.29 160 0.244 81.79 80 0.503 83.19 82.09 5-Jul 207 160 0.198 80.13 320 0.08 67.77 80 0.301 60.24 69.38 20-Jul 222 320 0.065 56.01 320 0.069 59.15 160 0.131 53.87 56.34 26-Jul 228 10 0.014 0.50 100 0.012 4.52 160 -0.002 1.75 2.26 2-Aug 235 20 0.01 0.81 10 0.021 0.67 10 0.018 0.60 0.69 9-Aug 242 2 0.013 0.10 1 0.032 0.09 0.09 17-Aug 250 2 0.015 0.11 1 0.022 0.07 0.09 13-Sep 277 1 over 10 0.026 0.80 5 0.056 0.76 0.78 O Agricultural Mesocosm B - 50 cm down the length of the mesocosm — 1 1 1 1 * ^ 1 r Date Time (Days) Dil. O.D. Sulphide (mg I/1) Dil. O.D. Sulphide (mg I-"1) Dil. O.D. Sulphide (mg L-1) Average Sulphide (mg L'1) 6-Jun 182 320 0.172 139.87 160 0.341 136.16 80 0.672 132.94 136.32 5-Jul 211 160 0.285 114.22 320 0.09 75.61 80 0.511 101.39 97.07 20-Jul 226 320 0.058 50.53 320 0.052 45.82 160 0.124 51.13 49.16 26-Jul 232 10 0.021 0.67 100 0.014 5.01 160 -0.005 0.58 2.09 2-Aug 239 20 0.009 0.76 10 0.024 0.75 10 0.027 0.82 0.77 9-Aug 246 2 0.021 0.13 1 0.039 0.11 0.12 17-Aug 254 2 0.012 0.09 1 0.031 0.09 0.09 Agricultural Mesocosm B -150 cm down the length of the mesocosm Date Time (Days) Dil. O.D. Sulphide (mg L 1) Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg L-1) Average Sulphide (mg L-1) 20-Jun 196 320 0.105 72.80 80 0.431 71.43 160 0.22 73.96 72.73 5-Jul 211 160 0.284 113.83 320 0.113 93.63 80 0.617 122.16 109.87 20-Jul 226 320 0.15 122.63 320 0.121 99.90 160 0.314 125.58 116.04 26-Jul 232 10 0.017 0.57 100 0 1.58 160 0.001 2.93 1.70 2-Aug 239 20 0.008 0.71 10 0.025 0.77 10 0.074 1.97 1.15 9-Aug 246 2 0.009 0.08 1 0.014 0.05 0.06 17-Aug 254 2 0.018 0.12 1 0.039 0.11 0.12 to ON A1-7c. pH in mesocosms while operated as batch systems Natural Treatment Mesocosm Water Column Mesocosm A Mesocosm B Date Time (Days) PH PH 6-Dec 0 7.41 7.43 7.32 7.27 13-Dec 7 7.37 7.45 7.45 7.41 21-Dec 15 7.24 7.19 7.58 7.59 7-Jan 32 7.24 7.22 7.32 7.33 14-Jan 39 7.31 7.45 7.28 7.12 24-Jan 49 7.55 7.58 7.77 7.56 14-Feb 70 7.40 7.31 7.62 7.65 21-Feb 77 7.57 7.62 7.10 7.12 28-Feb 84 8.43 8.32 8.94 8.68 12-Apr 127 8.50 8.43 8.48 8.52 20-Jul 226 9.10 9.01 9.30 9.35 Sediment 28-Feb 84 7.41 7.42 7.48 7.45 7-iViar 91 7.47 7.38 7.56 7.58 14-Mar 98 7.69 7.55 7.77 7.69 21-Mar 105 7.96 7.76 8.11 8.05 29-Mar 113 8.03 7.95 7.67 7.58 12-Apr 127 7.60 7.81 7.48 7.62 18-Apr 133 8.25 8.11 8.46 8.51 25-Apr 140 8.40 8.31 8.45 8.48 2-May 147 8.55 8.63 8.44 8.46 16-May 161 8.50 8.51 8.43 8.44 30-May 175 7.89 7.92 7.81 7.82 Agricultural Treatment Mesocosm Mesocosm A Mesocosm B Date Time (Days) PH pH 10-Dec 0 7.50 7.45 7.50 7.52 13-Dec 3 6.99 7.05 6.04 6.00 21-Dec 11 6.58 6.64 6.52 6.55 7-Jan 28 6.95 6.85 6.97 7.08 14-Jan 35 7.11 7.03 7.20 7.21 24-Jan 45 7.31 7.37 7.38 7.34 14-Feb 66 7.36 7.36 7.51 7.52 21-Feb 73 7.44 7.29 7.51 7.49 2-Mar 82 7.50 7.55 7.77 7.68 7-Mar 87 5.51 5.50 5.38 5.47 9-Mar 89 7.02 7.06 6.96 7.01 14-Mar 94 7.16 7.22 7.18 7.11 21-Mar 101 7.20 7.14 7.24 7.22 29-Mar 109 7.35 7.29 7.40 7.41 12-Apr 123 7.54 7.51 7.67 7.72 18-Apr 129 7.36 7.38 7.30 7.30 25-Apr 136 7.56 7.62 7.71 7.66 2-May 143 7.85 7.88 7.88 7.87 16-May 157 7.78 7.75 7.82 7.82 30-May 171 7.41 7.44 7.33 7.36 20-Jun 192 7.45 7.39 7.39 7.36 5-Jul 207 7.70 7.71 7.62 7.66 20-Jul 222 8.36 8.42 8.18 8.10 Mesocosm A Mesocosm B Date Time (Days) pH PH 20-Jun 196 7.80 7.67 7.82 7.88 5-Jul 211 7.40 7.51 7.60 7.64 20-Jul 226 8.30 8.42 8.38 8.21 26-Jul 232 8.00 8.05 8.00 7.98 2-Aug 239 8.40 8.42 8.40 8.03 9-Aug 246 8.00 8.00 8.20 8.22 17-Aug 254 8.00 8.03 8.00 8.05 13-Sep 281 7.80 7.77 7.80 7.69 Mesocosm A Mesocosm B Date Time (Days) PH pH 26-Jul 228 8.30 8.28 8.20 8.18 2-Aug 235 8.10 8.05 8.10 8.06 9-Aug 242 9.00 8.95 8.80 8.77 17-Aug 250 8.80 8.76 8.90 8.92 13-Sep 277 9.00 9.03 9.00 9.03 A1-7d. Soluble COD in mesocosms while operated as batch systems Natural Treatment Mesocosm A Date Time (Days) Dil. O.D. sCOD (mg L-1) Dil. O.D. sCOD (mg L"1) Dil. O.D. sCOD (mg L-1) Average sCOD (mg L-1) St. Dev. 6-Dec 0 1 0.058 143.46 2 0.025 123.67 1 0.057 140.98 136.04 10.78 21-Dec 15 1 0.017 42.05 2 0.01 49.47 1 0.009 22.26 37.93 14.06 29-Dec 23 1 0.108 267.13 2 0.057 281.97 1 0.097 239.92 263.00 21.32 7-Jan 32 1 0.021 51.94 1 0.018 44.52 1 0.023 56.89 51.12 6.22 14-Jan 39 1 0.021 51.94 1 0.036 89.04 1 0.034 84.10 75.03 20.14 24-Jan 49 1 0.023 56.89 1 0.015 37.10 1 0.029 71.73 55.24 17.37 4-Feb 60 1 0.025 61.84 1 0.028 69.26 1 0.022 54.41 61.84 7.42 14-Feb 70 1 0.03 74.20 1 0.021 51.94 1 0.028 69.26 65.13 11.69 21-Feb 77 1 0.028 69.26 1 0.048 118.72 1 0.037 91.52 93.16 24.78 21-Mar 105 1 0.061 150.88 2 0.015 74.20 112.54 12-Apr 127 1 0.053 131.09 1 0.059 145.93 138.51 20-Jun 196 2 0.041 202.82 4 0.02 197.87 4 0.021 207.77 202.82 4.95 13-Sep 281 2 0.04 197.87 4 0.02 197.87 197.87 ON Natural Treatment Mesocosm B Date Time (Days) Dil. O.D. sCOD (mg L-1) Dil. O.D. sCOD (mg L'1) Dil. O.D. sCOD (mg L-1) Average sCOD (mg L-1) St. Dev. 6-Dec 0 1 0.008 19.79 1 0 0.00 1 0.015 37.10 18.96 18.56 21-Dec 15 1 0.012 29.68 1 0.014 34.63 1 0.008 19.79 28.03 7.56 29-Dec 23 1 0.057 140.98 0.025 123.67 0.029 143.46 136.04 10.78 7-Jan 32 1 0.024 59.36 1 0.033 81.62 1 0.018 44.52 61.84 18.67 14-Jan 39 1 0.041 101.41 1 0.035 86.57 1 0.042 103.88 97.29 9.36 24-Jan 49 1 0.036 89.04 1 0.021 51.94 1 0.048 118.72 86.57 33.46 4-Feb 60 1 0.034 84.10 1 0.027 66.78 1 0.021 51.94 67.61 16.09 14-Feb 70 1 0.021 51.94 1 0.023 56.89 1 0.023 56.89 55.24 2.86 21-Feb 77 1 0.054 133.56 1 0.033 81.62 1 0.049 121.20 112.13 27.13 21-Mar 105 1 0.032 79.15 2 0.012 59.36 69.26 13.99 12-Apr 127 1 0.047 116.25 1 0.054 133.56 124.91 12.24 20-Jun 196 2 0.041 202.82 4 0.021 207.77 4 0.022 217.66 209.41 7.56 13-Sep 281 2 0.038 187.98 4 0.031 306.70 10 0.012 296.81 263.83 65.87 Agricultural Treatment Mesocosm A (100 cm down the length of the mesocosm) Date Time (Days) Dil. O.D. sCOD (mg I-"1) Dil. O.D. sCOD (mg I-"1) Dil. O.D. sCOD (mg L-1) Average sCOD (mg L-1) St. Dev. 10-Dec 0 10 0.151 3734.83 20 0.078 3858.50 20 0.074 3660.63 3751.32 99.96 21-Dec 11 10 0.131 3240.15 20 0.075 3710.10 20 0.069 3413.29 3454.52 237.67 29-Dec 19 10 0.113 2794.94 20 0.071 3512.23 20 0.066 3264.89 3190.69 364.35 7-Jan 28 20 0.056 2770.21 20 0.059 2918.61 20 0.058 2869.14 2852.65 75.56 14-Jan 35 20 0.04 1978.72 40 0.019 1879.78 10 0.086 2127.12 1995.21 124.49 4-Feb 56 20 0.055 2720.74 40 0.021 2077.66 20 0.049 2423.93 2407.44 321.86 14-Feb 66 20 0.031 1533.51 40 0.016 1582.98 10 0.057 1409.84 1508.77 89.18 21-Feb 73 20 0.026 1286.17 40 0.023 2275.53 10 0.048 1187.23 1582.98 601.80 7-Mar 87 10 0.078 1929.25 20 0.042 2077.66 2003.45 14-Mar 94 2 0.279 1380.16 10 0.064 1582.98 20 0.037 1830.32 1597.82 225.45 21-Mar 101 2 0.289 1429.63 10 0.071 1756.11 5 0.144 1780.85 1655.53 196.03 29-Mar 109 2 0.279 1380.16 10 0.038 939.89 2 o;27o 1335.64 1218.56 242.36 ON 45* Date Time (Days) Dil. O.D. sCOD (mg L-1) Dil. O.D. sCOD (mg L'1) Dil. O.D. sCOD (mg L-1) Average sCOD (mg L"1) St. Dev. 12-Apr 123 2 0.111 549.09 2 0.168 831.06 10 0.031 766.75 715.64 147.77 2-May 143 2 0.113 558.99 5 0.046 568.88 563.94 24-May 165 2 0.275 1360.37 5 0.110 1360.37 10 0.057 1409.84 1376.86 28.56 6-Jun . 178 5 0.081 1001.73 2 0.188 930.00 965.86 20-Jun 192 2 0.167 826.12 4 0.072 712.34 4 0.082 811.28 783.24 61.85 5-Jul 207 2 0.166 821.17 4 0.089 880.53 4 0.084 831.06 844.25 31.80 20-Jul 222 4 0.122 1207.02 2 0.249 1231.75 4 0.118 1167.44 1202.07 32.44 13-Sep 277 2 0.126 623.2968 4 0.067 662.87 643.084 Agricultural Treatment Mesocosm A (50 cm down the length of the mesocosm) 20-Jun 192 2 0.158 781.59 4 0.076 751.91 4 0.067 662.87 732.13 61.79 5-Jul 207 2 0.184 910.21 4 0.084 831.06 4 870.64 20-Jul 222 4 0.167 1652.23 2 0.327 1617.60 4 0.159 1573.08 1614.31 39.68 Agricultural Treatment Mesocosm A (150 cm down the length of the mesocosm) 20-Jun 192 4 0.087 860.74 2 0.166 821.17 4 0.081 801.38 827.76 30.23 5-Jul 207 2 0.177 875.58 4 0.087 860.74 4 0.079 781.59 839.31 50.53 20-Jul 222 4 0.131 1296.06 2 0.254 1256.49 4 0.13 1286.17 1279.57 20.60 Agricultural Treatment Mesocosm B (100 cm down the length of the mesocosm) Date Time (Days) Dil. O.D. sCOD (mg L-1) Dil. O.D. sCOD (mg L"1) Dil. O.D. sCOD (mg L 1) Average sCOD (mg L"1) St Dev 10-Dec 0 10 0.149 3685.37 20 0.075 3710.10 20 0.074 3660.63 3685.37 24.73 21-Dec 11 10 0.19 4699.46 20 0.115 5688.82 20 0.105 5194.14 5194.14 494.68 29-Dec 19 10 0.155 3833.77 20 0.08 3957.44 20 0.075 3710.10 3833.77 123.67 7-Jan 28 20 0.053 2621.80 20 0.055 2720.74 20 0.056 2770.21 2704.25 75.56 14-Jan 35 20 0.038 1879.78 40 0.028 2770.21 10 0.103 2547.60 2399.20 463.39 4-Feb 56 20 0.033 1632.44 40 0.019 1879.78 10 0.077 1904.52 1805.58 150.45 14-Feb 66 20 0.026 1286.17 40 0.013 1286.17 10 0.054 1335.64 1302.66 28.56 21-Feb 73 20 0.034 1681.91 40 0.01 989.36 10 0.044 1088.30 1253.19 374.57 to ON Date Time (Days) Dil. O.D. sCOD (mg I-"1) Dil. O.D. sCOD (mg L-1) Dil. O.D. sCOD (mg L-1) Average sCOD (mg L-1) St Dev 7-Mar 87 10 0.079 1953.99 20 0.045 2226.06 40 0.019 1879.78 2019.94 182.32 14-Mar 94 2 0.286 1414.78 10 0.099 2448.67 20 0.046 2275.53 2046.33 553.74 21-Mar 101 2 0.323 1597.82 10 0.063 1558.24 1578.03 29-Mar 109 2 0.245 1211.97 2 0.228 1127.87 10 0.06 1484.04 1274.63 186.17 12-Apr 123 2 0.14 692.55 2 0.15 742.02 10 0.036 890.424 775.00 102.98 2-May 143 2 0.086 425.42 5 0.044 544.15 484.79 24-May 165 2 0.225 1113.03 5 0.11 1360.37 10 0.053 1310.902 1261.43 130.88 6-Jun 178 5 0.099 1224.33 2 0.207 1023.99 10 0.041 1014.094 1087.47 118.63 5-Jul 207 2 0.159 786.54 4 0.081 801.38 793.96 20-Jul 222 4 0.163 1612.66 2 0.348 1721.49 4 0.161 1592.869 1642.34 69.26 13-Sep 277 2 0.124 613.40 4 0.068 672.76 643.08 Agricultural Treatment Mesocosm B (50 cm down the length of the mesocosm) 20-Jun 192 2 0.146 722.23 4 0.068 672.76 4 0.071 702.45 699.15 24.90 5-Jul 207 2 0.153 756.86 4 0.079 781.59 4 0.079 781.59 773.35 14.28 20-Jul 222 4 0.102 1009.15 2 0.195 964.63 4 0.102 1009.15 994.31 25.70 Agricultural Treatment Mesocosm B (150 cm down the length of the mesocosm) 20-Jun 192 2 0.136 672.76 4 0.072 712.34 4 0.071 702.45 695.85 20.60 5-Jul 207 2 0.148 732.13 4 0.071 702.45 4 0.074 732.13 722.23 17.14 20-Jul 222 4 0.124 1226.81 2 0.228 1127.87 4 0.119 1177.34 1177.34 49.47 OS A1-7e. Ammonia-nitrogen in mesocosms while operated as batch systems Natural Treatment Mesocosm A Date Time (Days) Dil. O.D. NH3-N (mg LZ1) Dil. O.D. NH3-N (mg L"1) Average NH3-N (mg L/1) 6-Dec 0 10 2.00 20.0 10 1.32 13.2 16.6 13-Dec 7 11 0.57 6.3 11 0.56 6.2 6.2 21-Dec 15 11 0.20 2.2 11 0.21 2.3 2.3 29-Dec 23 11 2.45 27.0 11 2.38 26.2 26.6 7-Jan 32 . 11 0.35 3.9 11 0.37 4.1 4.0 14-Jan 39 5.5 0.20 1.1 5.5 0.25 1.4 1.2 24-Jan 49 5.5 0.36 2.0 5.5 0.26 1.4 1.7 4-Feb 60 2.04 0.56 1.1 2.04 0.46 0.9 1.0 14-Feb 70 2.04 0.18 0.4 2.04 0.18 0.4 0.4 21-Feb 77 1 0.44 0.4 1 0.46 0.5 0.5 21-Mar 105 10 1.67 16.7 20 0.87 17.4 17.1 2-May 147 10 0.46 4.6 20 0.25 5.0 4.8 5-Jul 211 10 0.45 4.5 20 0.24 4.8 4.7 13-Sep 281 20 0.21 4.2 ' 10 0.43 4.3 4.3 Natural Treatment Mesocosm B Date Time (Days) Dil. O.D. NH3-N (mg I-"1) Dil. O.D. NH3-N (mg L"1) Average NH3-N (mg L'1) 6-Dec 0 10 2.62 26.2 10 1.6 16.0 21.1 13-Dec 7 11 0.48 5.3 11 0.56 6.2 5.7 21-Dec 15 11 0.15 1.7 11 0.14 1.5 1.6 29-Dec 23 11 0.66 7.3 11 0.66 7.3 7.3 7-Jan 32 11 0.41 4.5 11 0.37 4.1 4.3 14-Jan 39 5.5 0.19 1.0 5.5 0.16 0.9 1.0 24-Jan 49 5.5 0.79 4.3 5.5 0.59 3.2 3.8 4-Feb 60 2.04 0.54 1.1 2.04 0.33 0.7 0.9 14-Feb 70 2.04 0.18 0.4 2.04 0.16 0.3 0.3 to ON ^0 Date Time (Days) Dil. O.D. NH3-N (mg I-"1) Dil. O.D. NH3-N (mg I-"1) Average NH3-N (mg L"1) 21-Feb 77 1 0.27 0.3 1 0.37 0.4 0.3 21-Mar 105 10 1.17 11.7 20 0.62 12.4 12.1 2-May 147 10 0.48 4.8 20 0.91 18.2 11.5 5-Jul 211 20 0.36 7.2 10 0.6 6.0 6.6 13-Sep 281 20 0.38 7.6 10 0.63 6.3 7.0 Agricultural Treatment Mesocosm A Date Time (Days) Dil. O.D. NH3-IM (mg I-"1) Dil. O.D. NH3-N (mg L"1) Average NH3-N (mg L-1) 10-Dec 0 41.6 2.9 120.6 41.6 2.81 116.9 118.8 21-Dec 11 41.6 2.9 120.6 41.6 2.89 120.2 120.4 29-Dec 19 41.6 3.1 129.0 41.6 3.1 129.0 129.0 7-Jan 28 55 2.17 119.4 55 2 110.0 114.7 14-Jan 35 55 2 111.7 55 2 107.3 109.5 24-Jan 45 55 2.22 122.1 55 2.01 110.6 116.3 4-Feb 56 51 2.45 125.0 51 2.51 128.0 126.5 14-Feb 66 51 2.8 142.8 51 2.8 142.8 142.8 21-Feb 73 50.1 2.6 130.3 50.1 2.66 133.3 131.8 7-Mar 87 100 1.85 185.0 100 1.8 180.0 182.5 21-Mar 101 100 1.85 185.0 100 1.9 190.0 187.5 12-Apr 123 100 1.31 131.0 100 1.42 142.0 136.5 2-May 143 100 0.94 94.0 50 1.89 94.5 94.3 24-May 165 50 2.61 130.5 100 1.24 124.0 127.3 6-Jun 178 50 2.33 116.5 100 0.89 89.0 102.8 20-Jun 192 50 over >150 100 1.4 140 140 13-Sep 277 100 0.31 31.0 50 0.59 29.5 30.3 as oo Agricultural Treatment Mesocosm B Date Time (Days) Dil. O.D. NH3-N (mg I-"1) Dil. O.D. NH3-N (mg L-1) Average NH3-N (mg I-"1) 10-Dec 0 41.6 2.70 112.3 41.6 2.30 95.7 104.0 21-Dec 11 41.6 3.00 124.8 41.6 3:00 124.8 124.8 29-Dec 19 41.6 3.10 129.0 41.6 3.10 129.0 129.0 7-Jan 28 55 2.10 115.5 55 2.15 118.3 116.9 14-Jan 35 55 2.14 117.7 55 2.30 126.5 122.1 24-Jan 45 55 2.34 128.7 55 2.19 120.5 124.6 4-Feb 56 51 2.55 130.1 51 2.35 119.9 125.0 14-Feb 66 51 2.81 143.3 51 2.79 142.3 142.8 21-Feb 73 50.1 2.67 133.8 50.1 2.66 133.3 133.5 7-Mar 87 100 1.83 183.0 100 1.84 184.0 183.5 21-Mar 101 100 1.71 171.0 100 1.84 184.0 177.5 12-Apr 123 100 1.34 134.0 100 1.37 137.0 135.5 2-May 143 100 1.00 100.0 50.0 2.03 101.5 100.8 24-May 165 100 1.41 141.0 50.0 2.66 133.0 137.0 6-Jun 178 50 2.66 133.0 100 1.05 105.0 119.0 20-Jun 192 50 over >150 100.0 1.32 132 132 13-Sep 277 100 0.3 30.0 50 0.53 26.5 28.3 A1-7f. Ortho-phosphate in mesocosms while operated as batch systems Natural Treatment Mesocosm A Date Time (Days) Dil. O.D. P0 4 3 ' (mg L-1) Dil. O.D. P O 4 3 ' (mg L'1) Average P0 4 3" (mg L'1) 6-Dec 0 10 1.29 12.9 10 1.35 13.5 13.2 13-Dec 7 11 1.68 18.5 11 1.61 17.7 18.1 21-Dec 15 11 0.13 1.4 11 0.15 1.7 1.5 29-Dec 23 11 2.14 23.5 11 2.2 24.2 23.9 7-Jan 32 11 0.68 7.5 11 0.64 7.0 7.3 14-Jan 39 11 0.77 8.5 11 0.8 8.8 8.6 OS VO Date Time (Days) Dil. O.D. P0 4 3 _ (mg L'1) Dil. O.D. P0 4 3" (mg L-1) Average P0 4 3" (mg I."1) 24-Jan 49 11 0.09 1.0 11 0.07 0.8 0.9 4-Feb 60 10 1.01 10.1 10 0.88 8.8 9.5 14-Feb 70 10 1.62 16.2 10 1.7 17.0 16.6 21-Feb 77 10 2.23 22.3 10 2.09 20.9 21.6 21-Mar 105 10 1.57 15.7 20 0.74 14.8 15.3 12-Apr 127 10 0.9 9.0 10 0.88 8.8 8.9 2-May 147 10 0.8 8.0 20 0.389 7.8 7.9 13-Sep 281 2 over >6 20 0.253 5.1 Natural Treatment Mesocosm B Date Time (Days) Dil. O.D. P0 4 3 ' (mg L-1) Dil. O.D. P O 4 3 -(mg L-1) Average P0 4 3 _ (mg I-"1) 6-Dec 0 10 1.21 12.1 10 1.18 11.8 12.0 13-Dec 7 11 1.59 17.5 11 1.62 17.8 17.7 21-Dec 15 11 0.05 0.6 11 0.1 1.1 0.8 29-Dec 23 11 0.12 1.3 11 0.15 1.7 1.5 7-Jan 32 11 0.56 6.2 11 0.5 5.5 5.8 14-Jan 39 11 - 0 0.0 11 0.01 0.1 0.1 24-Jan 49 11 0.07 0.8 11 0.06 0.7 0.7 4-Feb 60 10.2 0.58 5.9 10.2 0.45 4.6 5.3 14-Feb 70 10.2 1.78 18.2 10.2 1.7 17.3 17.7 21-Feb 77 10.02 1.63 16.3 10.02 1.54 15.4 15.9 21-Mar 105 10 1.04 10.4 10 1.14 11.4 10.9 12-Apr 127 10 0.81 8.1 10 0.8 8.0 8.1 2-May 147 10 0.73 7.3 20 0.34 6.8 7.1 13-Sep 281 2 over >6 20 0.3 6.0 t o o Agricultural Treatment Mesocosm A Date Time (Days) Dil. O.D. P0 4 3" (mg I-"1) Dil. O.D. P0 4 3" (mg L-1) Average P0 4 3" (mg L-1) 10-Dec 0 50 2.01 100.5 100 1.03 103.0 101.8 21-Dec 11 41.6 2.32 96.5 41.6 2.4 99.8 98.2 29-Dec 19 41.6 1.99 82.8 41.6 1.89 78.6 80.7 7-Jan 28 55 1.56 85.8 55 1.6 88.0 86.9 14-Jan 35 55 1.61 88.6 55 1.49 82.0 85.3 24-Jan 45 55 1.74 95.7 55 1.66 91.3 93.5 4-Feb 56 51 1.5 76.5 51 1.66 84.7 80.6 14-Feb 66 51 1.21 61.7 51 1.26 64.3 63.0 21-Feb 73 50.1 2.75 137.8 50.1 2.83 141.8 139.8 7-Mar 87 50 0.57 28.5 100 0.24 24.0 26.3 21-Mar 101 50 0.89 44.5 50 0.94 47.0 45.8 12-Apr 123 50 0.78 39.0 50 0.85 42.5 40.8 2-May 143 100 0.25 25.0 50 0.57 28.5 26.8 24-May 165 50 1.1 55.0 100 0.49 49.0 52.0 6-Jun 178 50 1.22 61.0 100 0.53 53.0 57.0 20-Jun 192 50 1.31 65.5 100 0.63 63.0 64.3 13-Sep 277 50 0.52 26.0 100 0.26 26.0 26.0 Agricultural Treatment Mesocosm B Date Time (Days) Dil. O.D. P0 4 3 ' (mg L-1) Dil. O.D. P0 4 3" (mg L-1) Average P0 4 3" (mg L-1) 10-Dec 0 50 1.97 98.5 100 0.96 96.0 97.3 21-Dec 11 41.6 1.99 82.8 41.6 1.89 78.6 80.7 29-Dec 19 41.6 1.7 70.7 41.6 1.8 74.9 72.8 7-Jan 28 55 3 165.0 55 2.56 140.8 152.9 14-Jan 35 55 1.26 69.3 55 1.3 71.5 70.4 24-Jan 45 55 1.14 62.7 55 1 55.0 58.9 4-Feb 56 51 2.01 102.5 51 1.75 89.3 95.9 14-Feb 66 51 2.54 129.5 51 2.68 136.7 133.1 t o Date Time (Days) Dil. O.D. P0 4 3" (mg L"1) Dil. O.D. P0 4 3" (mg L'1) Average P0 4 3" (mg L'1) 21-Feb 73 50.1 0.83 41.6 50.1 0.86 43.1 42.3 7-Mar 87 50 0.75 37.5 100 0.34 34.0 35.8 21-Mar 101 50 0.81 40.5 50 0.86 43.0 41.8 12-Apr 123 50 0.69 34.5 50 0.7 35.0 34.8 2-May 143 100 0.27 27.0 50 0.56 28.0 27.5 24-May 165 100 0.51 51.0 50 1.02 51.0 51.0 6-Jun 178 50 1.26 63.0 100 0.57 57.0 60.0 20-Jun 192 50 1.27 63.5 100 0.64 64.0 63.8 13-Sep 277 50 0.59 29.5 100 0.29 29.0 29.3 A1-8a. S0 4 2" concentration in Lake 52 in situ experiment Location Depth (cm) Dil. O.D. SO4 2 ' (mg L-1) Dil. O.D. so42-(mg L-1) Dil. O.D. S0 4 2" (mg L1) Average S0 4 2 ' (mg L'1) St. Dev. 4-Oct-04 Site 1 5 40 0.794 3827.97 80 0.375 4029.38 50 0.645 3978.94 3945.43 104.80 Site 1 10 80 0.434 4540.04 40 0.898 4278.04 50 0.659 4054.67 4290.92 242.94 Site 1 15 50 0.727 4422.52 80 0.377 4046.69 40 0.794 3827.97 4099.06 300.72 Site 1 20 80 0.419 4410.21 50 0.686 4200.73 40 0.862 4122.25 4244.40 148.87 Site 1 25 80 0.393 4185.18 40 0.813 3910.19 50 0.705 4303.51 4132.96 201.79 Site 1 30 50 0.731 4444.16 40 0.903 4299.68 80 0.431 4514.08 4419.31 109.34 Site 2 5 40 0.795 3832.30 80 0.375 4029.38 50 0.664 4081.72 3981.13 131.53 Site 2 10 40 0.833 3996.75 80 0.354 3847.62 50 0.626 3876.16 3906.84 79.16 Site 2 15 80 0.422 4436.18 50 0.684 4189.91 40 0.882 4208.80 4278.30 137.06 Site 2 20 50 0.705 4303.51 40 0.891 4247.75 80 0.419 4410.21 4320.49 82.55 Site 2 25 80 0.389 4150.56 40 0.882 4208.80 50 0.656 4038.44 4132.60 86.59 Site 2 30 80 0.411 4340.97 50 0.711 4335.97 40 0.897 4273.71 4316.88 37.47 rO ro Location Depth (cm) Dil. O.D. S0 4 2 ' (mg L-1) Dil. O.D. S0 4 2 ' (mg L-1) Dil. O.D. so42-(mg L-1) Average SO/ (mg L-1) St. Dev. 11-Aug-05 Site 1 5 40 0.983 4470.60 80 0.532 5119.38 50 0.881 5048.03 4879.34 355.77 Site 1 10 40 0.890 4076.56 80 0.460 4509.25 50 0.742 4311.84 4299.22 216.62 Site 1 15 80 0.471 4602.46 40 0.914 4178.25 50 0.744 4322.43 4367.71 215.70 Site 1 20 40 0.969 4411.28 80 0.516 4983.80 50 0.773 4476.03 4623.70 313.53 Site 1 25 50 0.770 4460.14 80 0.497 4822.79 40 0.999 4538.40 4607.11 190.84 Site 1 30 80 0.466 4560.09 50 0.746 4333.03 40 1.000 4542.63 4478.58 126.36 Site 2 5 80 0.508 4916.00 40 0.960 4373.15 50 0.746 4333.03 4540.73 325.62 Site 2 10 50 0.720 4195.32 80 0.433 4280.45 40 0.931 4250.28 4242.01 43.16 Site 2 15 80 0.390 4242.40 50 0.640 4085.92 40 0.810 4049.06 4125.79 102.65 Site 2 20 80 0.499 4436.43 50 0.797 4219.54 40 1.050 4358.28 4338.08 109.84 Site 2 25 50 0.727 4232.40 80 0.445 4382.14 40 0.928 4237.57 4284.03 85.00 Site 2 30 80 0.491 4771.94 50 0.776 4491.92 40 0.988 4491.79 4585.22 161.71 #1 Control 5 50 0.804 4640.21 80 0.486 4729.57 40 1.030 4669.75 4679.84 45.53 #1 Control 10 50 0.787 4550.18 80 0.514 4966.85 40 1.050 4754.49 4757.17 208.35 #1 Control 15 80 0.469 4585.51 50 0.751 4359.51 40 0.989 4496.03 4480.35 113.82 #1 Control 20 50 0.807 4656.10 80 0.516 4983.80 40 1.020 4627.37 4755.76 198.01 #1 Control 25 80 0.513 4958.37 50 0.754 4375.40 40 1.030 4669.75 4667.84 291.49 #1 Control 30 50 0.787 4550.18 80 0.489 4755.00 40 1.030 4669.75 4658.31 102.89 #3 Inoculated 5 80 0.518 5000.75 50 0.802 4629.62 60 0.669 4710.25 4780.21 195.20 #3 Inoculated 10 80 0.448 4407.56 50 0.727 4232.40 60 0.600 4271.72 4303.89 91.91 #3 Inoculated 15 50 0.815 4698.47 80 0.496 4814.31 60 0.675 4748.39 4753.72 58.11 #3 Inoculated 20 50 0.786 4544.88 80 0.461 4517.72 60 0.641 4532.30 4531.63 13.59 #3 Inoculated 25 80 0.473 4619.41 50 0.782 4523.69 60 0.651 4595.85 4579.65 49.87 #4 Amended 5 80 0.619 5856.63 50 0.920 5254.59 60 0.780 5415.72 5508.98 311.67 #4 Amended 10 50 0.907 5185.73 80 0.597 5670.20 60 0.770 5352.17 5402.70 246.15 #4 Amended 15 50 0.993 5641.22 80 0.668 6271.86 60 0.810 5606.39 5839.82 374.56 Location Depth (cm) Dil. O.D. S0 4 2 ' (mg L-1) Dil. O.D. S0 4 2 ' (mg L"1) Dil. O.D. S0 4 2" (mg L-1) Average so/ (mg L-1) St. Dev. #4 Amended 20 80 0.590 5610.88 50 0.918 5243.99 60 0.741 5167.85 5340.91 236.88 #4 Amended 25 50 0.918 5243.99 80 0.583 5551.56 60 0.782 5428.43 5408.00 154.80 #5 Control 5 50 0.843 4846.77 80 0.554 5305.81 60 0.741 5167.85 5106.81 235.53 #5 Control 10 80 0.557 5331.24 50 0.829 4772.62 60 0.733 5117.01 5073.62 281.82 #5 Control 15 80 0.547 5246.49 50 0.840 4830.88 60 0.735 5129.72 5069.03 214.35 #5 Control 20 50 0.898 5138.07 80 0.603 5721.04 60 0.734 5123.37 5327.49 340.91 #5 Control 25 80 0.610 5780.36 50 0.863 4952.70 60 0.755 5256.83 5329.96 418.65 #5 Control 30 50 0.918 5243.99 80 0.619 5856.63 60 0.771 5358.52 5486.38 325.72 #5 Control 35 80 0.513 4958.37 50 0.816 4703.77 60 0.662 4665.76 4775.97 159.11 #6 Amended 5 50 0.834 4799.10 80 0.528 5085.49 60 0.711 4977.19 4953.93 144.60 #6 Amended 10 80 0.442 4356.71 50 0.717 4179.43 60 0.631 4468.74 4334.96 145.87 #6 Amended 15 80 0.426 4221.13 50 0.688 4025.84 60 0.601 4278.07 4175.01 132.29 #6 Amended 20 50 0.814 4693.18 80 0.516 4983.80 60 0.650 4589.50 4755.49 204.40 #6 Amended 25 50 0.768 4449.55 80 0.466 4560.09 60 0.633 4481.45 4497.03 56.90 #6 Amended 30 50 0.733 4264.17 80 0.453 4449.93 60 0.617 4379.76 4364.62 93.80 A1-8b. Total sulphide in Lake 52 in situ experiment Location Depth (cm) Dil. O.D. Sulphide (mg L 1) Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg L'1) Average Sulphide (mg L"1) St. Dev. 4-Oct-04 Site 1 5 1 0.139 0.29 1 0.144 0.30 2 0.068 0.26 0.28 0.02 Site 1 10 1 0.068 0.13 2 0.031 0.09 1 0.061 0.11 0.11 0.02 Site 1 15 1 0.122 0.25 1 0.115 0.23 2 0.072 0.28 0.25 0.02 Site 1 20 1 0.064 0.12 2 0.021 0.05 1 0.058 0.11 0.09 0.04 Site 1 25 0.018 0.04 1 0.040 0.07 1 0.033 0.05 0.05 0.02 Site 1 30 1 0.023 0.03 2 0.020 0.05 1 0.040 0.07 0.05 0.02 Site 2 5 1 0.028 0.04 1 0.034 0.05 2 0.024 0.06 0.05 0.01 Site 2 10 0.021 0.05 1 0.050 0.09 1 0.045 0.08 0.07 0.02 Site 2 15 1 0.022 0.03 1 0.026 0.04 2 0.018 0.04 0.03 0.01 Site 2 20 1 0.029 0.04 2 0.017 0.03 1 0.025 0.03 0.04 0.01 4^ Location Depth (cm) Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg L-1) Average Sulphide (mg L-1) St. Dev. 11-Aug-05 Site 1 5 1 0.020 0.06 2 0.000 0.03 1 0.030 0.09 0.06 0.03 Site 1 10 1 0.050 0.14 2 0.010 0.08 1 0.041 0.12 0.11 0.03 Site 1 15 1 0.043 0.12 2 0.019 0.12 1 0.022 0.07 0.11 0.03 Site 1 20 1 0.039 0.11 2 0.015 0.11 1 0.028 0.08 0.10 0.01 Site 1 25 1 0.083 0.22 2 0.030 0.18 1 0.077 0.20 0.20 0.02 Site 1 30 1 0.038 0.11 2 0.015 0.11 1 0.041 0.12 0.11 0.01 Site 2 5 1 0.057 0.16 2 0.020 0.13 1 0.042 0.12 0.13 0.02 Site 2 10 1 0.041 0.12 2 0.022 0.14 1 0.037 0.11 0.12 0.02 Site 2 15 1 0.030 0.10 2 0.000 0.03 1 0.038 0.12 0.08 0.04 Site 2 20 1 0.045 0.12 2 0.018 0.11 1 0.061 0.15 0.13 0.02 Site 2 25 1 0.037 0.11 2 0.015 0.11 1 0.037 0.11 0.11 0.00 Site 2 30 1 0.045 0.13 2 0.015 0.11 1 0.046 0.13 0.12 0.01 #1 Control 5 1 0.437 1.09 2 0.167 0.85 5 0.068 0.91 0.95 0.12 #1 Control 10 1 0.419 1.04 2 0.170 0.86 5 0.055 0.75 0.89 0.15 #1 Control 15 1 0.318 0.79 2 0.176 0.89 5 0.055 0.75 0.81 0.07 #1 Control 20 1 0.343 0.86 2 0.135 0.69 5 0.057 0.78 0.78 0.08 #1 Control 25 1 0.190 0.48 2 0.071 0.38 5 0.041 0.58 0.48 0.10 #1 Control 30 1 0.113 0.29 2 0.055 0.30 5 0.015 0.26 0.29 0.02 #3 Inoculated 5 1 0.435 1.08 2 0.208 1.05 5 0.079 1.05 1.06 0.02 #3 Inoculated 10 1 0.415 1.03 2 0.234 1.18 5 0.081 1.07 1.09 0.08 #3 Inoculated 15 1 0.343 0.86 2 0.175 0.89 5 0.061 0.83 0.86 0.03 #3 Inoculated 20 1 0.106 0.28 2 0.062 0.34 5 0.017 0.29 0.30 0.03 #3 Inoculated 25 1 0.092 0.24 2 0.010 0.08 5 0.000 0.08 0.13 0.09 #4 Amended 5 1 0.923 2.28 2 0.683 3.38 5 0.200 2.53 2.73 0.58 #4 Amended 10 1 1.007 2.48 2 0.674 3.33 5 0.210 2.65 2.82 0.45 t o - J U l Location Depth (cm) Dil. O.D. Sulphide (mg L 1) Dil. O.D. Sulphide (mg L-1) Dil. O.D. Sulphide (mg L-1) Average Sulphide (mg L-1) St. Dev. #4 Amended 15 1 0.911 2.25 2 0.631 3.12 5 0.252 3.17 2.84 0.52 #4 Amended 20 1 1.252 3.08 2 0.764 3.77 5 0.241 3.03 3.30 0.42 #4 Amended 25 1 0.747 1.85 2 0.487 2.42 5 0.151 1.93 2.06 0.31 #5 Control 5 1 0.526 1.30 2 0.339 1.69 5 0.111 1.44 1.48 0.20 #5 Control 10 1 0.333 0.83 2 0.247 1.24 5 0.071 0.95 1.01 0.21 #5 Control 15 1 0.324 0.81 2 0.222 1.12 5 0.065 0.88 0.93 0.16 #5 Control 20 1 0.314 0.78 2 0.253 1.27 5 0.072 0.96 1.01 0.25 #5 Control 25 1 0.316 0.79 2 0.238 1.20 5 0.071 0.95 0.98 0.21 #5 Control 30 1 0.163 0.42 2 0.099 0.52 5 0.031 0.46 0.46 0.05 #5 Control 35 10 1.530 37.63 50 0.308 38.51 20 0.810 39.99 38.71 1.19 #6 Amended 5 1 0.131 0.34 2 0.084 0.44 5 0.025 0.39 0.39 0.05 #6 Amended 10 1 0.105 0.27 2 0.029 0.17 5 0.015 0.26 0.24 0.05 #6 Amended 15 1 0.043 0.12 2 0.018 0.12 5 0.000 0.08 0.11 0.02 #6 Amended 20 1 0.016 0.06 2 0.004 0.05 5 0.000 0.08 0.06 0.02 #6 Amended 25 1 0.075 0.20 2 0.022 0.14 5 0.010 0.20 0.18 0.04 #6 Amended 30 5 0.644 7.97 10 0.26 6.53 20 0.151 7.71 7.40 0.77 A1-8c. Temperature, pH, DO in Lake 52 in situ experiment 4-Oct-04 Depth (cm) Temperature (°C) PH DO % Saturation Site 1 0-5 11.9 9.65 95.6 5-10 11.9 9.67 94 10-15 11.9 9.62 89.9 15-20 11.9 9.68 91 20-25 11.9 9.69 25-30 11.9 9.72 94.8 Site 2 0-5 14.5 9.36 94.8 5-10 14.2 9.32 94.3 10-15 13.7 9.2 92.3 15-20 13.7 9.3 91.7 20-25 13.7 9.38 93.6 25-30 13.7 9.37 93.9 11-Aug-05 Depth (cm) Temperature (°C) PH Site 1 5 23.7 9.34 12.5 23.6 9.32 20 23.4 9.33 Site 2 5 21.2 9.38 12.5 21.2 9.4 20 21.2 9.39 #1 Control 5 20.9 9.40 12.5 21.0 9.40 20 21.0 9.40 #3 Inoculated 5 21.0 9.40 12.5 21.0 9.40 20 21.0 9.40 —J 11-Aug-05 Depth (cm) Temperature (°C) PH #4 Amended 5 20.6 8.92 12.5 20.5 8.91 20 20.5 8.90 #5 Control 5 20.1 9.34 12.5 20.3 9.32 20 20.2 9.32 #6 Amended 5 20.1 9.34 12.5 20.0 9.34 20 20.0 9.33 A1-8d. Soluble COD in Lake 52 in situ experiment Sample Depth (cm) Dil. O.D. sCOD (mg L-1) Dil. O.D. sCOD (mg L-1) Dil. O.D. sCOD (mg L"1) Average sCOD (mg L 1) 4-Oct-04 Site 1 15 1 0.058 143.46 1 0.056 138.51 1 0.063 155.82 145.93 Site 2 15 1 0.057 140.98 1 0.06 148.40 1 0.054 133.56 140.98 11-Aug-05 Site 1 5 1 0.059 145.93 1 0.061 150.88 1 0.061 150.88 149.23 10 1 0.058 143.46 1 0.055 136.04 1 0.056 138.51 139.33 20 1 0.055 136.04 1 0.054 133.56 1 0.054 133.56 134.39 Site 2 5 1 0.054 133.56 1 0.048 118.72 1 0.051 126.14 126.14 10 1 0.058 143.46 1 0.053 131.09 1 0.057 140.98 138.51 20 1 0.058 143.46 1 0.051 126.14 1 0.05 123.67 131.09 #1 Control 5 1 0.054 133.56 1 0.058 143.46 1 0.055 136.04 137.69 10 1 0.055 136.04 1 0.06 148.40 1 0.056 138.51 140.98 20 1 0.051 126.14 1 0.056 138.51 1 0.051 126.14 130.27 #3 Inoculated 5 1 0.057 140.98 1 0.055 136.04 1 0.057 140.98 139.33 10 1 0.071 175.61 1 0.057 140.98 1 0.06 148.40 155.00 20 1 0.057 140.98 1 0.074 183.03 1 0.061 150.88 158.30 #4 Amended 5 1 0.087 215.19 1 0.087 215.19 1 0.079 195.40 208.59 10 1 0.079 195.40 1 0.079 195.40 1 0.079 195.40 195.40 20 1 0.078 192.93 1 0.095 234.97 1 0.081 200.35 209.41 #5 Control 5 1 0.086 212.71 1 0.088 217.66 1 0.086 212.71 214.36 10 1 0.08 197.87 1 0.077 190.45 1 0.08 197.87 195.40 20 1 0.08 197.87 1 0.081 200.35 1 0.08 197.87 198.70 #6 Amended 5 0.066 1.63.24 1 0.061 150.88 1 0.061 150.88 155.00 10 1 0.063 155.82 1 0.066 163.24 1 0.065 160.77 159.95 20 1 0.064 158.30 1 0.055 136.04 1 0.059 145.93 146.76 A1-8e. Ortho-phosphate and ammonia-nitrogen in Lake 52 in situ experiment 4-Oct-04 1 2 3 Average PCV" (mg L"1) 0.04 0.04 0.10 0.06 N03"-N (mg L"1) 0.06 0.04 0.05 0.05 NH3-N (mg L-1) 0.38 0.42 0.37 0.39 11-Aug-05 Depth (cm) P0 4 3" (mg L-1) P0 4 3" (mg L-1) Average P0 4 3 ' (mg L"1) Site 1 5 0.00 0.00 0.00 12.5 0.00 0.01 0.01 20 0.01 0.00 0.01 Site 2 5 0.04 0.01 0.03 12.5 0.00 0.00 0.00 20 0.00 0.00 0.00 #1 Control 5 0.01 0.00 0.01 12.5 0.02 0.01 0.02 20 0.02 0.01 0.02 #3 Inoculated 5 0.01 0.00 0.01 12.5 0.01 0.00 0.01 20 0.06 0.04 0.05 #4 Amended 5 0.30 0.21 0.26 12.5 0.34 0:54 0.44 20 0.26 0.27 0.27 11-Aug-05 Depth (cm) NH3-N (mg L-1) NH3-N (mgL 1) Average NH3-N (mg L"1) Site 1 5 0.39 0.35 0.37 12.5 0.34 0.36 0.35 20 0.46 0.38 0.42 Site 2 5 0.54 0.50 0.52 12.5 0.43 0.45 0.44 20 0.43 0.43 0.43 #1 Control 5 0.45 0.46 0.46 12.5 0.45 0.44 0.45 20 0.44 0.46 0.45 #3 Inoculated 5 0.45 0.38 0.42 12.5 0.39 0.42 0.41 20 0.46 0.44 0.45 #4 Amended 5 1.20 1.19 1.20 12.5 1.18 1.17 1.18 20 1.19 1.21 1.20 SO 11-Aug-05 Depth (cm) P0 4 3 ' (mg L-1) P0 4 3" (mg L'1) Average P0 4 3 ' (mg L-1) 11-Aug-05 Depth (cm) NH3-N (mg L 1) NH3-N (mg L-1) Average NH3 -N (mg L-1) #5 Control 5 0.00 0.04 0.02 #5 Control 5 0.82 0.81 0.82 12.5 0.09 0.05 0.07 12.5 0.80 0.80 0.80 20 0.09 0.04 0.07 20 0.84 0.82 0.83 #6 Amended 5 0.23 0.23 0.23 #6 Amended 5 0.76 0.72 0.74 12.5 0.23 0.24 0.24 12.5 0.72 0.71 0.72 20 0.21 0.19 0.20 20 0.76 0.75 0.76 ro 00 O 

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