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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 A S CARBON S O U R C E S FOR SULPHATE REDUCING BACTERIA IN PASSIVE TREATMENT OF HIGH SULPHATE WATER by AMBER BROWN B . S c , Queen's University, 2002  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE D E G R E E OF MASTER OF APPLIED SCIENCE in THE FACULTY OF GRADUATE STUDIES (Chemical and Biological Engineering)  THE UNIVERSITY OF BRITISH COLUMBIA April 2007 ©AmberBrown, 2007  Abstract Several natural and agricultural organic materials were a s s e s s e d a s 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 w a s that the system be low cost and simple to operate, since 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 ( S R B ) population was used in a m e s o c o s m experiment to determine its effectiveness at supporting sulphate reduction. Originally operated a s a continuous system, a flowrate of 6 mL min" flushed the nutrients out of the system. While operating in batch mode, 1  sulphate reduction was observed within the sediment, but not above in the water layer due to m a s s transfer limitations. Thus, recirculation of water through the sediment w a s required to increase the overall rate of sulphate-reduction.  Four different agricultural materials; barley, m o l a s s e s , hay, and silage, were supplied to a S R B inoculum and concentrated S 0 " water to test their effectiveness 2  4  as nutrient sources. A 1:1 (wt) mixture of hay and silage showed the highest drop in S 0 " and achieved a maximum S 0 " reduction rate of 14.4 mg S 0 ~ L: d" . A 2  2  4  2  4  1  1  4  second study, which tested the leachates of barley and orchard grass silages, found similar rates (19.4 ± 1.6 mg S 0 " L" d" ). 2  4  1  1  B a s e d on this work, a pair of m e s o c o s m s were built and supplied with a mixture of silage and hay. T h e s e systems achieved initial maximum rates of 33.8 mg S O 4 L" d" . After a second sulphate addition the maximum rate increased 1  2  1  to 132.4 mg S 0 " L" d" . The A S 0 " / A s C O D ratios were determined to be 1.10 2  4  1  1  2  4  and 0.91 g g" for m e s o c o s m s A and B. A halt in sulphate reduction w a s concomitant 1  with an increase in sulphide concentration to 323.5 mg L' . It is hypothesized that 1  sulphide inhibition of the S R B occurred. Recycling 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  T a b l e of C o n t e n t s  iv  List of T a b l e s  vi  List of F i g u r e s  vii  Glossary  x  List of Units Acknowledgements 1.0  Introduction  xii xiii 1  1.1 1.2  R a t i o n a l e for W o r k R e s e a r c h Objectives  1 3  1.3  T h e s i s Layout  4  2.0  B a c k g r o u n d Information and Literature R e v i e w 2.1  T h e Effect on Cattle of S a l i n e Drinking W a t e r  2.2 2.3 2.4  Sulphate Treatment T e c h n o l o g i e s for R e m o v i n g S u l p h a t e from W a t e r E x p e r i m e n t a l D e s i g n C o n s i d e r a t i o n s B a s e d U p o n the Literature R e v i e w 2.5 B a c k g r o u n d on the Field S t u d y S i t e s 3.0 Materials a n d M e t h o d s 3.1 3.2 3.3 3.4 3.5 3.6 4.0  L a c du B o i s G r a s s l a n d s P a r k L a k e s S a m p l i n g Laboratory S R B Inoculum C r e a t i o n a n d M a i n t e n a n c e L a k e 5 2 S a m p l i n g a n d In Situ S e t - U p B a t c h Bottle Experiments T e s t i n g Different Agricultural Materials M e s o c o s m Experiment S e t - u p a n d Operation Analytical M e t h o d s R e s u l t s and D i s c u s s i o n  4.1 4.2  5 5 6 10 35 43 45 ...45 .48 50 54 65 73 84  Characterization of High a n d L o w S u l p h a t e L a k e s 84 T h e Suitability of Agricultural Materials a s Substrates for Biological S u l p h a t e Reduction 100  iv  4.3  M e s o c o s m Experiments  137  4.4  In Situ Experiment  167  5.0  Conclusions and Recommendations for Future Work 5.1 5.2  Conclusions Future Work  180 180 183  Bibliography  185  Appendix 1 - R a w Data  195  Appendix 2 - Norwest Analysis of L a c du Bois and L a c du Bois Twin  281  V  List of Tables T a b l e 1 - A c t i v e s y s t e m operating parameters  22  T a b l e 2 - P a s s i v e s y s t e m operating parameters  28  T a b l e 3 - O r g a n i c substrates u s e d in batch reaction mixtures a s dry weight composition (wt %)  34  T a b l e 4 - In-situ treatment set-up  53  T a b l e 5 - Final v o l u m e s in leachate experiments  .....62  T a b l e 6 - S o d i u m sulphate a d d e d to a c h i e v e 1500 m g L" SO4 "  63  T a b l e 7 - C h a n g e s m a d e to the agricultural m e s o c o s m s  72  1  2  T a b l e 8 - A v e r a g e overall SO4 " c h a n g e a n d m a x i m u m sulphate reduction 2  rates ( S R R ) of n o n - p H adjusted agricultural bottle experiment  103  T a b l e 9 - C h a n g e a n d S R R s of p H adjusted agricultural bottle experiments.... 116 T a b l e 10 - Overall c h a n g e in SO4 " in leachate bottles o v e r 71 2  days and maximum S R R s  131  T a b l e 11 - p H of leachate  132  T a b l e 12 - A S 0  136  2 4  7 A s C O D ratio for l e a c h a t e experiment  T a b l e 13 - A m m o n i a - n i t r o g e n a n d o r t h o - P 0 " concentration in natural treatment s y s t e m T a b l e 14 - S u l p h a t e reduction w h e n a l g a e - s e d i m e n t w a s inoculated into P o s t g a t e B medium T a b l e 15 - C o m p a r i s o n of SO4 " levels in water c o l u m n a n d s e d i m e n t 3  4  145 147  2  of natural s y s t e m  148  T a b l e 16 - C h a n g e s m a d e to the agricultural m e s o c o s m s  161  T a b l e 17 - V i s u a l observations of in-situ buckets after 311 d a y s . . . .  169  T a b l e 18 - p H within L a k e 52  170  List of Figures Figure 1 - Simplified sulphur cycle  9  Figure 2 - U A S B reactor  19  Figure 3 - Sampling sites for L a c du Bois  46  Figure 4 - Sampling locations for Lake 52  50  Figure 5 - In-situ treatment set-up  52  Figure 6 - M e s o c o s m design  65  Figure 7 - Sampling sites for natural system materials  67  Figure 8 - Natural system sampling locations  70  Figure 9 - L a c du Bois Twin shoreline  85  Figure 10 - S 0 ~ concentration in L a c du Bois versus depth  88  Figure 11 - S 0 " concentration in L a c du Bois Twin versus depth  89  Figure 12 - Total sulphide concentration in L a c du Bois versus depth  90  Figure 13 - Total sulphide concentration in L a c du Bois Twin versus depth  90  2  4  2  4  Figure 14 - Nitrate and ammonia-nitrogen concentrations in L a c du B o i s and L a c du Bois Twin Figure 15 - O r t h o - P 0 " concentration in L a c du Bois and L a c du Bois Twin 3  4  91 92  Figure 16 - Soluble C O D concentration in L a c du Bois and L a c du Bois Twin... 92 Figure 17 - S 0 " concentration versus time for the non-pH-adjusted batch experiments 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  2  4  102  Figure 21 - O r t h o - P 0 ~ concentration in the non-pH-adjusted 3  4  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 ~ concentration v e r s u s time in the pH-adjusted batch experiments  114  Figure 25 - S 0 " concentration versus time in the pH-adjusted batch experiments containing mixtures of hay and silage  115  2  4  2  4  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 - O r t h o - P 0 ~ concentrations in the pH adjusted 3  4  batch experiments  120  Figure 29 - S 0 " 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 m e s o c o s m s  139  Figure 33 - pH in natural system m e s o c o s m s  141  2  4  Figure 34 - S 0 " concentration v e r s u s time in natural continuous s y s t e m ...... 143 2  4  Figure 35 - Total sulphide versus time in natural continuous system  143  Figure 36 - S 0 " concentration in the water column...  148  Figure 37 - S 0 " 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  2  4  2  4  Vlll  Figure 40 - Average agricultural m e s o c o s m pH versus time  154  Figure 41 - Average ammonium-nitrogen concentration versus time in m e s o c o s m s  155  Figure 42 - Average ortho-PGy " concentration in m e s o c o s m s versus time  155  Figure 43 - Average S 0 " and total sulphide concentrations in agricultural m e s o c o s m s versus time  156  Figure 44 - Average soluble C O D concentration in agricultural m e s o c o s m s versus time  158  3  2  4  Figure 4 5 - Average S 0 " and total sulphide concentrations versus time in 2  4  agricultural m e s o c o s m s  160  Figure 46 - Average soluble C O D versus time in agricultural m e s o c o s m s  160  Figure 47 - S R B presence in agricultural m e s o c o s m s  162  Figure 48 - S 0 " concentration in Lake 52  173  Figure 49 - Total sulphide concentration in Lake 52  173  Figure 50 - O r t h o - P 0 " 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  2  4  3  4  ix  Glossary 1+1  equal volumes a significance level silver sulphate Ag S0 acid mine drainage AMD barium ion Ba barium chloride BaCI barium sulphate (Barite) BaS0 British Columbia B.C. calcium sulphate (gypsum) CaS0 acetic acid CH3COOH C H C O O N a - 3 H 0 sodium acetate chlorine ion cr carbon dioxide C0 carbonate ion C0 " chemical oxygen demand COD constructed wetlands CW dissolved oxygen DO distilled water DW symbol for oxidation-reduction potential E environmental protection agency EPA Fe iron ion ferric chloride FeCI FeS iron sulphide FeS0 • 7H 0 iron sulphate heptahydrate FISH fluorescence in-situ hybridization FT proton bicarbonate ion HCO3water H 0 hydrogen sulphide H S sulphuric acid H S0 potassium dichromate K Cr 0 potassium phthalate KHP potassium phosphate KH P0 potassium nitrate KNO3 lactic acid bacteria LAB L a c du Bois LDB MgCI • 6 H 0 magnesium chloride hexahydrate Mn m a n g a n e s e ion MO microorganisms nitrogen g a s N sodium ion Na sodium hydroxide NaOH Na S • 9H 0 sodium sulphide nonahydrate 2  4  2 +  4  4  3  2  2  2  3  h  3 +  3  4  2  2  2  2  4  2  2  2  7  4  2  2  4 +  2  +  2  2  Na S0 NH NH -N 2  +  4  3  4  sodium sulphate ammonium ion ammonia nitrogen or total ammonia. The summed weight of nitrogen in both the ionized (ammonium, N H ) and molecular ( N H ) 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) +  4  3  N0 N0 N 0 O.D. ORC P 3  3  2  PCR PO  4  3  -  PFR PRB rDNA rRNA S°  s2  sCOD SMS S0 S0 " SOB SRB SRR T TAD TBD TEA TS UASB ZnS 2  2  4  +  XI  List of Units °C cm c m d" d g g L" g L" d" hr kg kg d" kg d" m" L L hr" m m ms" mg d" g" mg L" mg L ' V mg L" d~ g" mL min" mm um mol mmol L" mmol L" a" mV N nm ppm x g 1  1  1  1  1  1  3  1  2  1  1  1  1  1  1  1  1  1  1  1  degrees Celsius centimetres centimetres per day day grams grams per litre grams per litre per day hour kilograms kilograms per day kilograms per day per cubic metre litres litres per hour metres square metres metres per second milligram per day per gram milligram per litre milligram per litre per day milligram per litre per day per gram millilitre per minute millimetre micrometre mole millimoles per litre millimoles per litre per annum millivolt normal nanometre parts per million 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 B C 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.  Xlll  1.0  Introduction  1.1  Rationale for W o r k  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 c o m e s from natural water reservoirs, such a s lakes or ponds. However, natural water reservoirs can b e c o m e unsuitable a s livestock drinking water due to high salinity, which can be c a u s e d by the concentration of ions such a s chloride, carbonate and sulphate ( S 0 ) . While these substances could have their origins 2  4  from mankind's actions, these ions can also find their way into water reservoirs through the weathering and erosion of soil and rock. For SO4 ", bedrock deposits of 2  shale, coal and sandstone are the prime donators (Blinn 1993). Water reservoirs with salinity levels over 3 g L" (total salts) are considered saline and have low 1  biodiversity (Hammer 1986).  G o o d quality aquifers for livestock drinking water are becoming scarcer in arid regions of C a n a d a , such a s the a r e a s surrounding K a m l o o p s and Merrit, B . C . and in central S a s k a t c h e w a n . In the K a m l o o p s area the cattle herds graze in the hills surrounding the town, where the only drinking water available c o m e s from naturally occurring lakes and ponds. However, a good number of these lakes are highly saline due to S 0 " and the cattle either refuse to drink from them, or risk becoming sick if 2  4  they do. This appears to be a problem not only for this particular cattle station but with many farmers in the B . C . interior, a s well a s 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 " is typically the 2  4  dominant anion and the prime c a u s e 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 . C a n a d a Water Supply Expansion Program grant.  With that goal in mind, the focus of this study w a s on utilizing a passive system to treat the S 0 ~ in these lakes, which relied on the activity of a naturally occurring 2  4  group of microorganisms, specialized in reducing S 0 " , known a s sulphate reducing 2  4  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 p r o c e s s for sulphide removal is needed. However, this thesis focuses only on the initial step of sulphate reduction to sulphide.  1.2  Research Objectives  Overall objective To design a biological system to reduce levels of SO4 " to below 1000 mg L" 2  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 Agri-Food C a n a d a (Peterson 1999).  Sub-obiectives 1.  Conduct a field survey of high/low SO4 " lakes in the K a m l o o p s 2  region to s e e if there is anything obviously different about these lakes, which would account for their different salinities. 2.  P r o p o s e passive treatment configurations and test suitable nutrient sources for biological sulphate reduction.  3.  Determine the batch kinetics of sulphate reduction in open m e s o c o s m s with natural and agricultural substrates.  4.  Identify important parameters to control.  T h e initial intention was to run a continuous system but, due to time constraints, this w a s not done.  3  1.3  Thesis 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 subobjectives have been met. Conclusions and recommendations for future work are given in Chapter 5.  4  2.0  Background Information and Literature Review  2.1  T h e Effect o n Cattle of S a l i n e D r i n k i n g 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" , the fresh water intake was 2.3 kg (Dado and Allen 1994), 3 kg 1  (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" SO4 " 1  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" . In 1  addition, if water with lower SO4 " concentrations were available, the cattle would 2  reject the 2500 mg L" water. Smart, Cohen et al. (1986) found that cattle, which 1  were supplied water with a SO4 " concentration of 500 mg L" , had lower levels of 2  1  copper in their plasma and liver. While this did not have any health related effects to  5  the cattle, it w a s observed that their calves had lower weaning weights than those calves w h o s e parents were fed lower S 0 " concentration water. From a medical 2  4  standpoint, B e k e and Hironaka (1991) found that a saline well with a total dissolved solids concentration of 3875 mg L" (of which S 0 " and N a were the predominant 1  2  4  ions) w a s 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 polioencephalomalacia disorder. With this disorder, the S 0 " ingested by the cattle 2  4  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, C a n a d i a n water quality guidelines for livestock drinking water limit sulphate concentration to less than 1000 mg L* (Peterson 1999). 1  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. Consisting 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 a s salts or esters of sulphuric acid ( H S 0 ) and 2  4  is formed by replacing one or both of the hydrogen atoms with a metal or a radical, such a s sodium or ammonium. In the earth's crust, it is primarily found bound to metals to form mineral compounds such a s gypsum ( C a S 0 ) or barite ( B a S 0 ) . The 4  4  majority of sulphate compounds are water soluble, with the exception of lead, barium and strontium, and are easily eroded from the soil. O n c e it has entered the hydrosphere, it can remain a s 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 a s H S g a s or by the reduction of oxidized sulphur compounds by 2  S R B which are released a s H S g a s into the atmosphere. In aquatic environments, 2  sulphide can be oxidized to elemental sulphur aerobically by s u c h sulphur-oxidizing bacteria species a s 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 S0 ". 2  4  H S + / 0 1  2  2  2  ^ S° + H 0  (1)  2  S° + 1 / 0 + H 0 ^ S 0 " + 2 H 1  2  2  2  2  4  +  (2)  7  If both reactions are completed, then sulphuric acid is produced, which can then enter the local surface and groundwater and reduce the p H . T h e actions of these bacteria are the source of acid rock drainage, although in those c a s e s the H S is 2  generally replaced by a metal sulphide ore such a s iron sulphide (FeS).  Humans impact the sulphur cycle primarily in the production of sulphur dioxide (SO2) from industrial activities, s u c h as burning coal. The S 0 can precipitate and then be 2  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.  T h e majority of sulphur available to the biosphere exists in an oxidized form, either as sulphates in the lithosphere and hydrosphere or a s 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 w a y s (Figure 1). The first and most frequent method is known a s assimilatory sulphate reduction. Plants and most bacteria and archaea assimilate sulphate a s a source of sulphur for biosynthesis, where it is most often used to produce sulphur containing amino acids or co-factors (Postgate 1979).  8  SO  2-  SO4 reduction (assimilatory)  2 4  SO 4 reduction (dissimilatory)  Organic S  \.  I vl>  "yT / S  s  o  s Oxidation by sulfur oxidizing bacteria  Mineralization  Figure 1 - Simplified sulphur cycle  In the s e c o n d method, organisms utilize sulphate a s a terminal electron acceptor in the oxidation of organic matter. This process is known a s dissimilatory sulphate reduction and is accomplished by S R B and s o m e 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 C h i e n , Robertson et al. (1968) found that infants who were given formulas prepared with water containing 630 to 1150 mg L" S 0 " developed diarrhoea shortly after 1  2  4  ingestion. The C a n a d i a n guideline for the maximum acceptable concentration of  S 0 " in drinking water is 500 mg L" while in the U . S . the E P A has specified the 2  1  4  maximum contaminant level goal of drinking water to be 400 mg L"  1  (U.S.-EPA  1990). Sulphate is known to add an undesirable taste to water with a detectable taste threshold of 300-400 mg L~ (National-Academy-of-Sciences 1977). 1  Increasing levels of salinity have been shown to have a negative effect on plant-life and wildlife. Hammer (1986), Herbst and Blinn (1998) and Campbell and P r e p a s (1986) report that a s levels of salinity increase in aquatic ecosystems, the s p e c i e s diversity d e c r e a s e s . C a m p b e l l and P r e p a s (1986) also found with their findings that low levels of chlorophyll a also tend to be indicative of saline lakes in North A m e r i c a . 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 ~ in 2  4  groundwater and surface water. In addition, sulphate levels can also be increased from pyrite containing sediments undergoing desiccation, due to oxygen intrusion (Schuurkes, K e m p e r s et al. 1988, Lamers, van R o o z e n d a a l et al. 1998).  2.3  Treatment T e c h n o l o g i e s for R e m o v i n g S u l p h a t e from Water  There are two different branches of treatment systems that can be used to remove sulphate from water. T h e s e are chemical and biological treatments. S o m e of the  10  most popular physico-chemical treatments include reverse o s m o s i s , ion e x c h a n g e and distillation.  With reverse o s m o s i s , water-containing sulphate is forced through a s e m i permeable membrane. These units are used both for small-scale operations s u c h a s for home use, a s well as for industrial purposes. A household unit typically c a n 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 s e a water c a n have a capital cost anywhere between $1341 to 2379 m" day" per unit of daily capacity 3  1  and an operation and maintenance cost between $1.02 to 1.54 rrf per unit of 3  production (United-Nations-Environment-Programme 1997).  With an ion exchange unit, sulphate-containing water is p a s s e d through the unit where it c o m e s into contact with a resin. T h e sulphate ions in the water exchange places with other ions, usually chloride, which is on a resin. W h e n 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. A n ion exchange system designed to treat house water (max 12 gpm) can remove up to 2500 mg L" S 0 " and costs $2350 U . S . D ( R a i n D a n c e 1  2  4  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 ( S R B ) . 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 " and use it as a terminal electron acceptor (TEA) 2  4  in the consumption of carbonaceous materials. Although S R B are a morphologically and phylogenetically diverse group, they are viewed a s being physiologically unified b e c a u s e of this ability (Fauque 1995; Huycke and G a s k i n s 2004). Sulphate reducing  12  bacteria are generally considered strict anaerobes and the presence of oxygen inhibits their growth and SO4 " reduction activity. The presence of oxygen does not 2  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 " reducing activity w a s limited to the 2  anoxic z o n e (Okabe, Itoh et al. 1999).  Sulphate reducing bacteria, a s a group, are able to tolerate a range of environments with varying temperatures and p H . Psychrophilic s p e c i e s have been observed in environments with temperatures at -1.7 °C (Knoblauch, S a h m et al. 1999) a s well as thermophilic s p e c i e s in temperatures of 71 °C (Nakagawa, H a n a d a 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; I W A T a s k G r o u p 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" S 0 ~ . It is thought that the S R B are able to form biofilms around alkaline 1  2  4  substrates, which allow them to survive these otherwise harsh environments. A strong reducing environment is required for S R B to proliferate/with E values below h  -150 m V being ideal (Postgate 1979; F e n c h e l , King et al. 1998).  Several studies have indicated that S R B are typically found only within the top 10 c m of sediment in aquatic environments. Within rice paddy soil, Stubner (2002)  13  located S R B at depths of 5-10 c m using polymerase chain reaction ( P C R ) technology which targeted the 16s r D N A of Desulfotomaculum  lineage. Li, Purdy et  al. (1999) used oligonucleotide probes complementary to the 16S r R N A of the major phylogenetic groups of S R B to search for S R B within the sediments of a freshwater lake in J a p a n . There results showed the presence of 3 genera of S R B (Desulfobulbus,  Desulfobacterium  and Desulfovibrio)  within the first 6 c m of  sediment. Below this depth, the amount of r R N A recovered w a s lower by more than a factor of 4. Within marine arctic sediments, 7 3 % of all the S R B detected using F I S H and r R N A slot blot hybridization were located at a depth of 2.25 c m . Below a depth of 10 cm no S R B were detected (Ravenschlag, S a h m et al. 2000). This information is later used in the design of the m e s o c o s m 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 a s P 0 " . The limiting C : N ratio ranges between 45:1 - 1 2 0 : 1 (Okabe, Nielsen et al. 3  4  1992) while the C : P ratio is between 400:1 - 800:1 (Okabe and Characklis 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 ( H C O 3 ) and reduce the S 0 " to sulphide (S ~), which then combines with hydrogen 2  2  4  ions to form H2S g a s . 2CH3CHOHCOO" + 3 S 0 " + 2 H -> 6HCO3" + 3 H S 2  +  4  2  (3)  The S 0 " reduction reaction has a direct effect on the p H of its local environment. 2  4  Equation 3 indicates that the reaction removes protons to form H2S, which can then e s c a p e a s a gas, thus permanently removing the protons from the environment. This results in an increase in the p H . In addition, the bicarbonate formed can serve as a buffer to the system and c a n also bind with additional hydrogen ions to form H C 0 . 2  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 S g a s or remains dissolved in the local aquatic environment as 2  S " , HS" or H S species. This produces a distinct odour, which c a n act a s preliminary 2  2  indicator to the presence of S R B . O n c e released, the H S c a n either e s c a p e into the 2  atmosphere or undergo further reactions. If the local environment contains metal  15  cations, then the H S c a n react to form metal sulphides. This results in giving the 2  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 S g a s rises 2  through the sediment or aquatic environment, it enters oxic z o n e s . Within these z o n e s sulphur oxidizing bacteria can be found which c a n oxidize the H S to 2  elemental sulphur, or even further to S 0 ~ (Odom and Singleton 1993; Barton 2  4  1995).  2.3.1.3  S R B - Community Structure  In addition to requiring an anaerobic, reduced environment as well a s 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 reducers), F e  3 +  2  (aerobes), N 0 " (denitrifiers), M n 3  4 +  (manganese  (iron reducers) and C 0 (methanogens). Of these, only 2  methanogenesis is less thermodynamically favourable than sulphate reduction.  In the production of H S from S 0 2  2 4  \ S R B are able to create conditions that  encourage S R B proliferation, while discouraging the growth of other competing organisms. H S , in an environment with a pH of 7 and a temperature of 15°C, has an 2  equilibrium E of approximately -320 mV. This condition favours the activity of h  anaerobic organisms a s free oxygen is quickly depleted. In addition, H S is toxic to 2  16  many organisms and this also works to make conditions favourable for S R B . Although H S has been shown to have an inhibitory effect on S R B growth and 2  activity, noticeable at total sulphide (TS) levels of 200 mg L" , the effects are known 1  to be reversible if the sulphide is removed (Choi and Rim 1991; O k a b e , Nielsen et al. 1992; R e i s , 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, A l m e i d a et al. 1992) or by precipitating the sulphide out a s metal sulphides (Mitsch and W i s e 1998). While H S is the more toxic form of sulphide, this species is most prevalent at 2  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  S R B - Carbon sources  O n e 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 , H , lactate, ethanol, methanol, propionate, 2  2  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) a s well a s aliphatic and aromatic hydrocarbons like n-alkanes and alkylbenzenes (Rueter, R a b u s 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 w a s not until a paper w a s 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 " but primarily for their ability to produce 2  4  sulphide. The sulphide then reacts with metal ions in solution and precipitates them as well as increases the pH of the local environment. The d e c r e a s e of S 0 ~ is thus 2  4  s e e n 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 " reduction when heavy 2  4  metals are not present in the solution and in several studies the S 0 " reduction 2  4  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, a s well a s to monitor conditions within the system and make c h a n g e s as required. P a s s i v e 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. P a s s i v e 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 ( U A S B ) : U A S B reactors are a combination of both physical and biological processes. The physical component is the separation of solids and g a s e s from the liquid and the biological component is the degradation of d e c o m p o s a b l e organic matter under anaerobic conditions in the sludge blanket. The influent to be treated is pumped into the bottom of the reactor, where it c o m e s 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  7  t  1  Influent  Figure 2 - UASB reactor  19  P a c k e d bed reactor ( P B R ) : T h e s e reactors ideally follow a plug flow design and typically consist of a column containing immobilized microbial cells. T h e inlet c a n either be at the top or bottom of the column, depending on whether downwards or upwards flow is preferred. In c a s e s where S R B are utilized, H S g a s is one of the 2  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 u s e of a g a s 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 a s sediment, s e w a g e waste, or animal manure. S o m e 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, a s 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 " reduction ability. Active systems typically require a higher cost to 2  set-up and operate but in turn generally have faster sulphate reduction rates ( S R R ) . This higher cost is primarily due to the carbon source supplied to the bacteria,  20  usually a defined c o m p o u n d , a s well as the running cost of energy to keep the system active. Stoichiometrically, an S R B bioreactor that w a s 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 and H to support S R B gives 2  2  an estimated cost of $500 per tonne of sulphate reduced, a s s u m i n g the stoichiometric ratio of 4 moles of H are consumed per mole of sulphate reduced. 2  Several types of active bioreactors which bioremediate SO4 " are described below. 2  2.3.2.2  Active system applications in treating SO4  2  Several different types of active systems designed to treat S 0 ~ , while utilizing S R B , 2  4  have been developed over the years. While they may vary in design and nutrients supplied, they do share the s a m e overall purpose. All of the systems discussed here were developed for the removal of heavy metals from water. N o reports were found in the literature of active systems developed solely for the purpose of removing sulphate from high sulphate concentrated waters.  The P a q u e s C o m p a n y (T. de Boerstraat 24, 8561 A b Balk, T h e Netherlands ) creates the most well known commercial S R B bioreactors. T h e s e systems were successfully put into practice in 1992 at zinc refinery in B u d e l c o (The Netherlands), for the treatment of groundwater polluted with zinc ( S c h e e r e n , K o c h et al. 1993). B a s e d upon work completed by Barnes, J a n s s e n et al. (1991), these THIOPAQ™  21  reactors utilize an upflow anaerobic sludge blanket ( U A S B ) 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 b a s e of the tank, while the influent ( S 0 " contaminated ground water) flows upwards through the sludge. After 2  4  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" and is maintained at a pH of 7 and a temperature of 1  20°C. Further design details for this system are detailed in Table 1.  Table 1 - Active system operating parameters  Reference  Barnes, Janssen e t a l . 1991 Yamaguchi, Harada et al. 1999 Jong and Parry 2003 van Houten, Yun e t a l . 1997  Temperatur e and pH 20 pH 35 pH  °C =7 °C =7  25 °C pH = 4.57.5 55 °C ph = 7  Treatmen t System  Carbon Source  Retention Time  UASB  Ethanol  4 hours  UASB  Wastewater (2000 mg COD Lactate  9.9 days  14.4 hours  3.04 g, L" d"  80 % H and 20 % C0 3.3 L hf Molasses  4.5 hours  7.5 g L-'d"  0.019 hours  80% SO/removal  Packed Bed Reactor G a s lift reactor  2  Sulphate Reduction Rate 7.7 g L V  1  70.7 mg L" d  1  1  1  1  2  1  Annachhatre and Suktrakoolvait 2001  29.7 °C pH = 7.27.8  UASB  After their s u c c e s s with the U A S B reactor in Budelco, P a q u e s developed a g a s lift reactor which utilized a mixture C 0 and H a s an electron source. This system was 2  2  developed to treat acid rock drainage at Kennecitt's Bingham C a n y o n (Utah) copper mine, van Houten, Y u n et al. 's (1997) design consisted of an internal draft tube,  22  u s e d immobilized S R B in p u m i c e stones, which are maintained in s u s p e n s i o n in the reactor, to treat SO4 ". T o maintain the pumice s t o n e s in s u s p e n s i o n , a g a s recycle 2  flow rate of 350 L hr" w a s n e e d e d . T h e reactor w a s maintained at 55°C a n d a p H 1  7.0 a n d a S 0  2 4  " loading rate of 18 g L" d" w a s u s e d . 1  1  Y a m a g u c h i , H a r a d a et a l . (1999) utilized a 14.5 L U A S B to treat the high levels of C O D in sulphate c o n t a m i n a t e d wastewater (1000 m g S 0 " - S L" ). Instead of adding 2  1  4  a specific carbon s o u r c e to support S R B activity, the s y s t e m relied solely on the w a s t e w a t e r supplying all the n e c e s s a r y nutrients. T h e s y s t e m had lower S R R s than the other U A S B s m e n t i o n e d here. In order to k e e p s u l p h i d e levels within the c o l u m n low, a recycle s y s t e m w a s u s e d to p a s s the liquid in the reactor through a sulphide adsorption c o l u m n , w h i c h w a s p a c k e d with ferrous oxide pellets.  A n n a c h h a t r e a n d Suktrakoolvait (2001) operated a 5.7 L U A S B simply to o b s e r v e whether m o l a s s e s could support sulphate reduction by S R B , e v e n in the p r e s e n c e of m e t h a n e producing bacteria, which acts a s a competitor to S R B for nutrients. T h e p u r p o s e of this reactor w a s to treat wastewater from a tapioca/starch plant. T h e effluent w a s sent to a settling tank where the w a s h e d out b i o m a s s w a s r e m o v e d . A synthetic wastewater w a s provided to the s y s t e m , w h i c h included F e C b to convert the H2S to F e S . T h e influent had a very short r e s i d e n c e time within the s y s t e m (0.019 hrs), but low concentrations of S 0 per cent S 0  2 4  2 4  " were provided (450 m g L" S 0 " ) . Eighty 1  2  4  " removal w a s a c h i e v e d w h e n the C O D : S ratio w a s 10. S l u d g e from a n  a n a e r o b i c lagoon w a s u s e d to s e e d 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 c o a r s e pool filter sand and fed a solution with lactate, heavy metals and S 0 " (2500 mg L" ) at a rate of 2.61 mL min" . The 2  1  1  4  influent w a s initially pumped into a reservoir, which was continuously g a s s e d with N  2  in order to reduce the levels of oxygen. With a loading rate of 3.71 kg d" m" of 1  3  SO4 ", the reactor was able to remove more than 8 2 % of the SO4 ". 2  2  While these active treatment systems s h o w high levels of SO4 " reduction and have 2  short residence times, these applications are not suitable for farmers for use in the field. T h e s e types of systems require a high capital cost to construct and operate, primarily due to the cost of the carbon s o u r c e s used, as well a s 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  T h e main advantages to using passive systems to treat S 0 " are their low cost to 2  4  set-up and operate a s 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, a s these passive treatments are generally  24  operated outside, they are affected by changing environmental conditions such a s temperature. T h e s e passive treatment systems are usually described a s either wetlands or treatment ponds. Most of these systems to date are designed almost solely treating wastewater or acid mine drainage. In the latter c a s e , they are utilized primarily for metal removal, and in s o m e c a s e s the sulphate levels are not even monitored. Several different types of passive systems are briefly d i s c u s s e d below followed by a literature review of how they have been applied to treat SO4 ". 2  Wetlands: Wetlands are complex biological systems that are home to many interconnected nutrient cycles and biotic associations. To be considered a wetland s y s t e m , the water table must be at or above ground level sufficiently during the year s o 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 a s 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 c a n be visually appealing and provide habitat for migratory waterfowl.  Natural wetlands are difficult to use a s treatment systems, primarily b e c a u s e 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 m e a n s 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 close to the water requiring treatment, rather than depending on a natural wetland being present, and they can receive more heavily polluted water, a s 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 a s cattails or bulrush. Constructed wetlands fall under two main categories, depending on the type of hydraulic flow in the s y s t e m . 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: T h e s e 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. T h e y 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 z o n e s of microbial activity. T h e upper z o n e is aerobic with oxygen being supplied by photosynthetic algae, while the lower zone is anaerobic. T h e decay of algae results in deposition and subsequently anaerobic decay. A n a e r o b i c ponds receive a heavier organic load than aerobic or facultative ponds, in order to ensure anaerobic conditions. They also p o s s e s s the deepest depths (between 2.5-5 m) (Reed, Crites e t a l . 1998; Shilton 2005).  In Situ Treatment S y s t e m s : A n in situ system involves the remediation of a site by using the natural p r o c e s s e s 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. T h e s e 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  2 4  Fortin, Goulet et al. (2000) observed the cycling of F e and S in a constructed wetland situated in Kanata, C a n a d a . 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. T h e system was colonized primarily by Typha latifolia and only surface flow w a s observed. The flow in the wetlands was less than 0.5 m s'\ After observing the S 0 ~ in the water column over the course of 9 months, 2  4  a total drop of 74 mg L" w a s observed from 132 mg L" S 0 " in D e c . 97 to 1  1  2  4  58 mg L  1  S 0 " in A u g , 98. Design features for this and the other passive systems 2  4  discussed further are listed in Table 2.  Table 2 - Passive system operating parameters  Reference  Mitsch and W i s e 1998 Fortin, Goulet et al. 2000 Mclntire, Edenborn et al. 1990 Benner, Blowes et al. 2002 Stark, Williams et al. 1995  Size  2 cells 927/868 m 6.37 x 1 0 m 27.4 m b  Total depth/depth of organic substrate (cm) 140/46  Carbon Source  Sulphate Reduction Rate (mg L d - ) 0 1  1  150/0  Animal waste/grain Nothing  75/46  Compost  0.19-57.6  360  Decaying leafs, wood chips SMS/whey  15.3-10.5  2  0.3/0.2  2  2  60  m  0.4  m  z  z  15/15  N/A  28  Mitsch and W i s e (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 " 2  4  reduction will occur. The system w a s 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 c m 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 w a s released into the sediment through subsurface infusion pipes. This w a s to increase the retention time in the anaerobic cells, a s well as ensure the contaminated water had a s much contact with the sediment as possible. With an average hydraulic conductivity of 4.6 c m d" the anaerobic cells received water with a pH of 2.82 and a 1  S 0 " concentration of 1216 mg L" . Unfortunately, the SO4 " concentration increased 2  1  2  4  within the anaerobic cells (1256 and 1286 mg L" S 0 ~ , cells 1 and 2 respectively), 1  2  4  rather than d e c r e a s e d . It w a s suspected that the sulphur compounds within the water were re-oxidized back into S 0 " and that the carbon source w a s not utilizable 2  4  by the S R B .  Mclntire, Edenborn et al. (2000) achieved s u c c e s s in using a smaller constructed wetland to treat acid mine drainage when they used compost (straw/manure/corncobs) as a substrate. W h e n the influent w a s directed to p a s s through the sediment by subsurface pipes, the pH of the effluent w a s 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 c a u s e of this variation.  T h e most recent treatment method developed to treat S 0 " contaminated water in 2  4  situ is the permeable reactive barrier ( P R B ) , which w a s first suggested by Blowes and Ptacek (1992). T h e s e 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. T h e s e treatments rely on the natural flow of contaminated groundwater through the P R B with S 0 " reduction occurring within the barrier. O n e 2  4  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 w a s 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 d a y s and an inlet concentration of 2592 mg L" S 0 1  2 4  \ the overall rate of S 0 " reduction w a s found to 2  4  decline from 15.3-10.5 mg L" d (Benner, Blowes et al. 2002). 1  1  Instead of using a large constructed wetlands or P R B to treat high sulphate water, Stark, Williams et al. (1995) developed a small scale m e s o c o s m to simulate a constructed wetlands treatment. P l a c e d in a greenhouse, e a c h m e s o c o s m (160x25x15 LxWxD) w a s filled with spent mushroom substrate a s a carbon source,  30  followed by a weekly injection of 600 mL of whey. T h e influent (simulated acid mine drainage water) into the m e s o c o s m w a s held constant at 30 mL min" with a 1  residence time of approximately 33 hours. E a c h m e s o c o s m had an inlet concentration of 50 mg L" F e for the first 60 days, and 150 mg L" F e for the next 1  1  60 d a y s . Unfortunately, the purpose of this system w a s to remove iron from the influent, rather than remove SO4 ", so no SO4 " concentrations were provided, except 2  2  to say that the simulated mine water contained ferrous sulphate and sulphuric acid. T h e m e s o c o s m w a s successful at removing 6 5 % of the iron, which Stark suggests w a s 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 W i s e 1998; Drury 1999; Edenborn and Brickett 2001). S o m e 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 l e s s a et al. 2004). M o l a s s e s is another substrate that has been used a s a carbon source in S R B remediation. Considered an inexpensive and easily obtained carbon substrate, its primary constituent is s u c r o s e . While it has been used successfully, particularly in upflow anaerobic sludge blanket reactors (described in detail above) it c a n 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 G a d d 1996; Utgikar, Harmon et al. 2002) or when enriching S R B cultures for later use (Hauser and Holder 1986; Lloyd, K l e s s a 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 " reduction is 2  obtained or even increases in the SO4 " levels. This w a s observed in the Mitsch and 2  W i s e (1998) study, previously mentioned above, where animal waste mixed with grain that had been fed to livestock and then recollected, w a s used a s the carbon substrate and no sulphate reduction w a s 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 s o m e c a s e s , the natural materials provided within lakes are more capable of supporting S R B activity than an added substrate. W e b b , M c G i n n e s s 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" sulphide) than the constructed wetland sediments 1  (4.5 mmol L" sulphide) after 7.5 days. 1  Hay and manure are c o m m o n substrates provided to p a s s i v e treatment systems. Barton and Karathanasis (1999) found that they could remediate a failed surface flow wetland, designed using spent mushroom compost a s 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. T h e s e alterations e n h a n c e d the m a s s S 0 ~ reduction by 5 5 % 2  4  (influent 3034 mg L" SO4 ", effluent 1352 mg L" S 0 ~ ) . 1  1  2  2  4  Dairy whey has also been used in several treatment s y s t e m s . 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 c o w manure w a s added to 19.6 L of acid mine water. After 203 days S R B were responsible for a S 0 " drop of 2  4  893 mg L" or a rate of 4.4 mg L" d" S 0 " (Christensen, L a a k e et al. 1996). Dairy 1  1  1  2  4  whey w a s also used a s a carbon amendment in the Stark, Williams 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 s o u r c e s under the s a m e operating conditions. In the majority of studies, experimenters simply use a material that has been used previously with s u c c e s s . However, since their experimental designs are usually different with varying loading rates and dissimilar inoculums, there is no consistency with the rates obtained. This m a k e s determining the effectiveness of a particular carbon material difficult.  One study which did test the effectiveness of a variety of different complex carbon sources w a s conducted by Waybrant, Blowes et al. (1998). In 1 L glass reaction flasks, 8 different mixtures of organic material were a d d e d , as described in Table 3 and the flasks were then filled with simulated mine drainage water with a S 0 " 2  4  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 Mixture 1 2 3 4 5 6 7 8  Sewage Sludge 100  Leaf Mulch  Wood Chips  Sheep Manure  Sawdust  Cejlulose  100  20 15  100 10 10 60 60  25 15  65 20  25 25 40 10 100  34  Over a period of 60 days mixture #5, containing 5 different organic s o u r c e s , had the highest sulphate reduction rate (4.23 mg L" d" g" ), while mixture #1, which 1  1  1  contained only s e w a g e sludge, had the lowest (0.14 mg L" d" g" ). Total combustible 1  1  1  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" d" g"\ mixtures 3, 5, 6, and 7, respectively). The overall conclusion of this 1  1  study w a s that a mixture of different organic materials w a s preferable to using a single carbon substrate in supporting S R B activity.  2.4 E x p e r i m e n t a l D e s i g n C o n s i d e r a t i o n s B a s e d U p o n the Literature R e v i e w  After completing this literature review of different passive systems and the carbon substrates provided, it w a s decided that a study, similar to that of Waybrant, B l o w e s et al. (1998) w a s 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 m o l a s s e s , commonly found on farms throughout C a n a d a 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 a s nutrient sources for growth of S R B and reduction of S 0  2 4  \ A s the overall goal of this  35  research w a s to try to build a passive treatment system that could be built and implemented by farmers, it w a s imperative to select materials that the average farmer would have on hand or could obtain with ease. O n e of the difficulties in designing passive treatment s y s t e m s is that a small fraction of the organic matter placed in passive treatment s y s t e m s are likely to be immediately available to S R B (Benner, Blowes et al. 1999) b e c a u s e S R B typically require simpler molecules (organic acids, alcohols, or H ) for energy (Widdel 1988). A n a e r o b i c degradation of 2  the complex material to simpler c o m p o u n d s by fermentative microbes is often required and may limit the rate at which utilizable substrates b e c o m e 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 w a s primarily to add structural support and surface area upon which the S R B c a n grow, which is also an important function. However, hay c a n 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 a s silage.  Silage: Silage is used as a feedstock primarily for the use of cattle, s h e e p and other cud-chewing animals. In the creation of silage there are 4 distinct stages. T h e s e 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. T h e 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. O n c e 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) b e c o m e 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 L A B produce a wide variety of materials such a s organic acids, ethanol, acetate mannitol and C 0 , in addition to 2  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 a s  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  T h e stable stage begins after the activity of the L A B has finished, either b e c a u s e 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 p H .  Feedout stage  At this point the silo is opened and the silage removed to feed to the animals. T h e 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, a s a cereal grain, contains high levels of starch which can be broken down into simple sugars, such a s sucrose, which are utilizable by the S R B (Lloyd, K l e s s a et al. 2004). It is also one of the highest grain stocks present in C a n a d a with 3489 x 1 0 tonnes in July 2005 ( C a n a d a 2005). 6  38  Molasses: Molasses has been used in many previous studies as a suitable carbon source for S R B (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 S R B .  After determining which of these organic substrates are effective at supporting S R B , 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 " concentrated water. Therefore, a simulated 2  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* ~ reduction by S R B . In addition to testing agricultural organic 2  4  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 S R B 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, a s this would introduce oxygen. In the m e s o c o s m which received natural materials removed from a lake, it w a s decided to s e e d the water's surface with biotic matter (algae/diatoms/microbes) which w a s observed floating on the surface of the lake, as this would supply the sediment with a continual carbon and electron source a s this matter died and d e c a y e d . 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 m e s o c o s m received a water cap of 18 c m . Dissolved oxygen measurements were taken to ensure that this water layer w a s 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 subsurface 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 w a s 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. S e c o n d , b e c a u s e 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 c m of sediment. For this reason, each m e s o c o s m received a 10 c m layer of sediment within which S R B were found to exist, a s an inoculum.  40  W h e n 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 a s s u m e d to be a black box and either one parameter ( M O 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. T h e s e 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 a s s e s s m e n t s of various pond d e s i g n s , but requires a complex development of nonsteady state equations for a multitude of variables (DO, algal cell m a s s , inorganic/organic carbon, p H , alkalinity, etc) which is too complex for the s c o p e 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 a s s u m e d 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. S e c o n d , the systems should be designed to be long and narrow, with length to width ratios greater than 4:1 (Shilton 2005). The m e s o c o s m s constructed in this study had length to width ratios of 7.88:1 and are similar to the m e s o c o s m s used by Stark, Williams 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 a s to dissipate inflow momentum in order to reduce short circuiting, which m e a n s that the influent p a s s e s 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 m e s o c o s m s enters the system vertically through the sub-surface pipes.  In addition to the two sets of m e s o c o s m s designed to simulate a passive treatment system, a second experiment w a s 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 m e s o c o s m systems.  42  2.5  B a c k g r o u n d o n the F i e l d S t u d y S i t e s  L a c du Bois, L a c du Bois Twin and L a k e 52 (which was the lake utilized for the insitu treatment, discussed further on in this thesis) are situated in the L a c Du Bois G r a s s l a n d s Provincial Park. This park is located approximately 6 km northwest of Kamloops, B C , near where the North T h o m p s o n and Thompson rivers converge. It is one of the largest publicly owned grasslands in British Columbia, and e n c o m p a s s e s 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. S o m e 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 c o m p o s e d of chernozem, or black earth. This blackcoloured 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. S o m e 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 a s C l a s s 3 under the C a n a d a 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. T h e s e 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 c a n be hindered during s o m e years due to droughts.  44  3.0  Materials and Methods  3.1  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 and the second on October 4 . Based upon measurements th  th  of a map provided in Ministry of Environment (2000) it is estimated that Lac du Bois has a surface area of 3.75 x 10 m while Lac du Bois Twin's area is 8 x 10 m . Two 5  2  4  2  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)  L a c 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 a s 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 L a c du Bois, s a m p l e s were removed from both the floating mat at L D B Algae and also from the rocks on the lake floor at L D B Rocky. From L a c du B o i s Twin, organic matter w a s removed from Site #1 both at the lake floor where it w a s attached to rocks and the middle of the water column where it were found s u s p e n d e d . 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 w a s added to preserve the organic matter, a s per instructions from Stockner (2004). Upon returning to V a n c o u v e r , 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 D O (dissolved oxygen), and a second to measure temperature and p H . The pH and temperature were measured using a  46  Mettler Toledo 1140 pH meter, while the D O was measured using a V W R Scientific Products S P 5 0 D Symphony probe. T h e s e 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 e a c h location, while a wet cloth w a s used to calibrate the D O probe before sampling at e a c h location. O n the July 6  th  trip, the pH and  temperature were measured 15 c m below the surface of the water, while on the s e c o n d trip these measurements were made at 5 cm intervals. Unfortunately the D O probe was inoperable during the July 6 during the Oct 4  t h  t h  trip, but measurements were obtained  sampling trip.  Water was removed from the water column in 5 cm intervals for S 0 " and T S tests 2  4  through the use of a syringe attached to a hose. At e a c h depth the syringe w a s 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 w a s performed to preserve any sulphide present in the water a s Z n S . After capping, the tubes were inverted several times to ensure mixing. T h e samples were stored in a cooler filled with ice, until returned to the V a n c o u v e r laboratory for analysis. In addition, at a depth of 20 c m , 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 ( s C O D ) .  47  3.2  Laboratory S R B Inoculum Creation and Maintenance  Sediment from L a c du Bois w a s removed from a location between L D B R o c k y and L D B A l g a e where there w a s a distinct odour of H S gas produced and g a s bubbles 2  released whenever the sediment w a s agitated. It is suspected that these g a s bubbles contained H S , or other g a s e o u s products of anaerobic degradation s u c h a s 2  methane, C 0 or H . T h e s e two factors strongly suggested the presence of S R B . A 2  2  500 m L plastic container w a s 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 w a s 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 a s follows. 1.  Create a 10X concentrated S R B stock solution for Postgate B media (Postgate 1979). P l a c e 750 mL of distilled water in a 1 L flask, then add the following salts. KH P0 NH CI CaS0 2  4  4  4  5g 10g 10g  M g S 0 •7H 0 F e S 0 •7H 0 4  4  2  2  20 g 5g  A d d 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.  A d d 1 g of yeast extract and fill the flask to the 880 mL mark with distilled water.  48  4.  Autoclave (Sanyo M L S - 3 7 8 0 ) the media containing flask, 200 mL of distilled water, a 160 m L bottle a n d cap and pipette tips for 10 m L a n d 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.  R e m o v e oxygen from the media by bubbling N through the solution for 2  8-10 min. 7.  A d d 2.5 mL of ethanol and 0.200 mL of methanol using a micropipette.  8.  A d d 10 ml of stock ascorbic acid solution (0.01 g mL" ) and 10 mL of stock 1  thioglycolic acid solution (0.01 g mL" ) to the media. Using the autoclaved 1  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 2 0 % of the total volume in the headspace.  10.  Using the autoclaved pipettes, add 1 0 % 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.  T h e 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 L a c du Bois.  49  3.3  Lake 52 S a m p l i n g a n d In Situ S e t - U p  L a k e 52 w a s the site selected for the in situ treatment experiment. This lake w a s selected b e c a u s e it w a s reported to have high levels of S 0 " . In addition, b e c a u s e it 2  4  w a s situated on privately owned land, and not the publicly owned park region, it w a s possible to get permission to conduct experiments within the lake.  O n the October 4, 2004 sampling expedition to K a m l o o p s , L a k e 52 w a s visited and sampled for the first time. The size of this lake is estimated a s 10 x 1 0 m b a s e d 4  2  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 a s sampling points (Figure 4). T h e s e sites were selected not only because of their accessibility, but also b e c a u s e 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 w a s removed and tested for S 0 " , T S , s C O D , P 0 ~ , N 0 " - N , N H - N , pH and D O . A s w a s the c a s e with the 2  3  4  4  3  3  sampling from L a c du Bois and L a c du Bois Twin, the water w a s removed using a syringe attached to a firm plastic tube to get water s a m p l e s at varying depths. Water was removed at 5 c m intervals for measurements of S 0 " and T S , and from a depth 2  4  of 20 cm for all other measurements. S a m p l e s for P 0 ~ , N 0 " - N , and N H - N tests 3  4  3  3  were analyzed at the site using a S M A R T portable colorimeter and LaMotte tests Phosphate - Low range (3653-SC), Nitrate-Nitrogen ( 3 6 4 9 - S C ) , and A m m o n i a Nitrogen (3642-SC). Soluble chemical oxygen demand s a m p l e s were taken back to laboratory in V a n c o u v e r 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 ~ and T S testing were immediately 2  4  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 V a n c o u v e r for analysis using the barium chloride ( S 0 ~ measurement) and methylene blue (TS measurement) 2  4  methods, respectively, which are described later in this section. T h e pH and temperature were measured using a Mettler Toledo 1140 pH meter, while the D O was measured using a V W R Scientific Products S P 5 0 D S y m p h o n y probe.  Site 2 w a s selected a s the location for the in-situ set-up, a s it w a s the more isolated area due to its position a s an inlet along the shore. There w a s also s o m e forest debris in the water, which helped to partially cordon off the area. This would protect the experiment more from outside interferences such a s snowmobiles.  51  Four feet from the shore, 6 plastic buckets, with a diameter of 35.5 c m and a total height of 57 c m , were placed into the sediment of Lake 52. The bottoms of the buckets were open to the lake floor. E a c h bucket w a s screwed into the sediment and then anchored by tying the buckets to metal fence posts, which were hammered into the lake floor. E a c h bucket w a s wired to two metal posts, on opposite s i d e s 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 Control  2 3 4 5 6 Inoculated Inoculated Amended Control Amended and and Amended 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  T h e buckets, specified as Control, received no additions through the course of the experiment. The bucket specified as A m e n d e d received 4 handfuls, approximately 1 kg, of a 1:1 hay:silage mixture. The hay w a s 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.  T h e buckets specified as Inoculated received exactly the s a m e materials and quantities a s the A m e n d e d buckets. However, they also received a 3 cm layer (2.96 L) of sediment removed from L D B . This was to act a s an inoculum source of SRB.  After making these additions, a metal mesh w a s placed over the top of each bucket and tied using plastic zips. No further additions or sampling w a s made to the in-situ treatment system until August 11, 2005 (311 days later) when the system w a s revisited to a s s e s s whether the amendments or inoculation had had any effect on reducing the SO4 " levels. It w a s a s s u m e d that the lake sampling of Site 2 outside 2  the buckets would serve a s a suitable time (t)=0 for S 0 " and T S . 2  4  53  During the August visit, all water removed from the lake for sampling purposes w a s immediately filtered using a hand pump and 11 urn pore sized filter paper. For the S 0 " tests, water was removed every 5 c m from all the buckets and from within the 2  4  lake at both Site 1 and 2. T h e s e s a m p l e s 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 T S and then brought back to Vancouver for analysis. Water w a s removed from the buckets and Sites l a n d 2 from three depths for D O , and nutrient tests. T h e depths tested were 0-5 c m , 10-11 c m , and the water/sediment interface. Dissolved oxygen samples were fixed at the lake to be analyzed using the L a Motte Winkler Colorimetric method ( 3 6 8 8 - S C ) , described later. All the other samples were preserved with 2 mL L" of concentrated sulphuric acid. The samples were then 1  placed in a cooler filled with ice and taken back to the laboratory in V a n c o u v e r for analysis of N H - N , N 0 " - N and P 0 " . 3  3  3  4  3.4 B a t c h Bottle E x p e r i m e n t s T e s t i n g Different A g r i c u l t u r a l Materials 3.4.1 Agricultural Experiment without pH adjustment set-up  Hay, silage, barley and m o l a s s e s were bagged from the Agricultural and Agri F o o d C a n a d a cattle research station in K a m l o o p s and brought to the laboratory in Vancouver. The hay w a s a mixed crop of alfalfa and orchard grass (65:35 mixture), the silage w a s fermented alfalfa crop and the molasses was Dried m o l a s s e s on S u g a r Beet Pulp Sweet 4 5 and was manufactured by Westway F e e d Products  54  Division. T h e materials were placed in a blender and cut up to reduce their size and increase homogeneity.  In duplicate, 3 grams of each material w a s individually placed in a 160 mL glass bottle. In addition 3 sets of duplicate bottles were made which contained 3 g of hay, silage or barley a s well as an addition of 0.3 g of molasses. A solution of 1750 mg L" SO4 " w a s created using N a S G v The SO4 " solution w a s de-oxygenated 1  2  2  for 10 minutes using N2 gas. E a c h bottle w a s filled % with the SO4 " solution and 2  then received 15 ml of inoculum of S R B , which w a s injected into the bottom of the s a m p l e bottles to reduce the S R B from encountering oxygen.  The S R B inoculum w a s created by placing sediment from L a c du Bois in modified Postgate B media (Postgate 1979) and transferring the bacteria into fresh media when the SO4 " levels had dropped. T h e protocol followed for creating this media 2  and performing S R B transfers w a s described previously.  The inoculum for this experiment w a s taken from the 7  th  transfer and the activity of  the S R B w a s ascertained by monitoring the SO4 " levels in the inoculum bottles. In 2  17 days the S R B of the inoculum had reduced 679.4 mg L" SO4 ", which indicates 1  2  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 " solution, and then capped with butyl rubber 2  4  inserts.  55  For a positive control, 1 0 % of the s a m e inoculum w a s placed in Postgate B media and the S 0 ~ concentration measured at both the start of this experiment and 2  4  12 days later. A d e c r e a s e in S 0 ~ would confirm that the S R B inoculum w a s active. 2  4  For negative controls, 3 g of e a c h agricultural material w a s blended and then placed in a 160 mL bottle. The bottles were placed in a S a n y o M L S - 3 7 8 0 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" S 0 " water. 1  2  4  All of the bottles were placed in an incubator set at 30°C. Tinfoil w a s 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  W h e n sampling for S 0 " concentrations over time, the bottle w a s inverted several 2  4  times to homogenize the liquid, and then 5 mL of liquid w a s removed and placed in 1 mL of 2 % (w/v) Zn-acetate to preserve the sulphide a s Zn-sulphide and prevent reoxygenation of sulphide into sulphate. The samples were stored in the fridge at 4°C and analyzed either that s a m e day or the next. If the s a m p l e s 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 w a s the time observed for the S R B  56  grown in Postgate B media in the laboratory transfers to undergo measurable SO4 " 2  reduction. S a m p l e s were taken at t=0, 16 and 33 d a y s . However, as little S 0 " 2  4  reduction w a s observed during this time, it w a s decided to increase the time between sampling and the next two samples were taken at t=75 and 170 days. T h e SO4 " analysis followed the method listed in the sulphate analysis protocol described 2  later.  After sampling, the amount removed w a s replenished with distilled, deoxygenated water in order to maintain an anaerobic environment. The dilution effect of the makeup water w a s taken into account when calculating the sulphate reduction due to S R B activity. T h e pH w a s measured both at t=0 d a y s 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 " , T S , s C O D , P O 4 " , and N H - N could be m e a s u r e d . E a c h analysis protocol is 2  3  4  3  described later in this section. The liquid in the bottles were filtered using 11 um pore filter paper to remove large precipitates. T o measure SO4 " and T S , 10 mL of 2  liquid w a s 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 " tests w a s removed and filtered through 0.45 urn pore filter 2  4  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 w a s b a s e d upon the previous experiment with several modifications and improvements.  Using N a 2 S 0 , 4500 mL of a 1500 mg L" S 0 " solution w a s created and d e g a s s e d 1  4  2  4  using N for 15 minutes. Barley, hay, silage and m o l a s s e s were again chopped in a 2  blender to reduce their size and increase homogeneity. Three grams of e a c h material w a s 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 m a s s ratios of 1:3, 1:1 and 3:1 all having a total m a s s of 3 g.  In this experiment, S R B cells were w a s h e d with distilled water to remove any vestiges of Postgate B medium. Into three 300 mL centrifuge tubes, 200 mL of distilled and deoxygenated water w a s added. Fifty millilitres of S R B inoculum w a s added to the 200 mL of distilled and deoxygenated water.  T h e 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 e a c h centrifuge bottle w a s removed and combined  58  with 140 mL of distilled deoxygenated water for a total volume of 200 mL, which w a s later used a s 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 .  E a c h experiment bottle containing agricultural material received 130 mL of deoxygenated 1500 mg L" S 0 ~ water. T h e bottles and inoculum were moved into 1  2  4  a C o y Laboratory Products Type A anaerobic chamber. The pH of e a c h bottle w a s measured using pH strips, and then the pH w a s adjusted to 7.5 through the use of a 10 N N a O H solution. The contents of e a c h bottle were well mixed and the pH w a s checked again after 10 minutes to ensure pH stability. Ten mL of the w a s h e d S R B cells inoculum w a s added to e a c h bottle and then the bottles were filled to the top with the 1500 mg L" S 0 " solution. T h e bottles were capped and inverted several 1  2  4  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 C o y Laboratory Products Type A Anaerobic C h a m b e r , and each received 1.5 mL of the s a m e inoculum a s above into 15 mL of Postgate B media and then filled to the top with media. S o m e of the media w a s s a v e d for S 0 " measurements. T h e vials were 2  4  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 M L S - 3 7 8 0 ) for 2 hours at 121°C to which 120 mL of a sterile 1500 mg L" S 0 " solution w a s a d d e d . The pHs were adjusted to 7.5 with the 1  2  4  10N N a O H . Negative controls were prepared in duplicate.  Sampling and Maintenance  Sampling occurred in the s a m e manner a s the previous experiment. However, b e c a u s e the experiment was isolated in the anaerobic chamber it w a s not necessary to replenish the liquid levels after every sampling to maintain an anaerobic system. T h e bottles had their levels replenished on t=29 days using distilled and deoxygenated water which had been spiked with N a 2 S 0 to the s a m e levels a s was 4  measured on the previous day. In addition, 0.08 g of K H P 0 2  4  w a s added to each  m o l a s s e s bottle on this day since a P 0 " measurement taken at t=28 days showed 3  4  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.  O n 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 " , 1.5 mL of sample was 2  4  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. T h e 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" solution of F e C I in 1  3  water was added to each bottle a s well a s 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  T h e previous experiments used silage that w a s produced through the fermentation of alfalfa crop. In this experiment, S R B would be inoculated in leachate obtained from two different types of silages, barley silage (TAD), and orchard grass silage ( O R C ) . The barley silage w a s obtained from Pound Maker Agventures (Moellenbeck 2006), while the orchard grass w a s taken from Pacific Agri-Food R e s e a r c h Centre (Veira 2004). In addition, leachate obtained from washing spent material obtained from a bio-ethanol plant that used corn (TBD), w a s also tested.  Thirty-five g of e a c h silage material w a s measured and placed into 273 mL of distilled water. The bottles were repeatedly shaken and allowed to soak over night. T h e leachate w a s filtered out using #1 Whatman 11 um pore filter paper and a water v a c u u m . The pH of the leachate w a s measured using a Mettler Toledo 1140 pH meter. Half of this liquid w a s s a v e d to be inoculated a s 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 w a s used. T h e s e bottles received 1 mL of a 100 g L" Z n S 0 solution per 100 mL of sample. 1  4  T h e bottles were then shaken vigorously for 1 minute and then had their pH adjusted  61  to 10.5 using 6 M 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. T h e s e 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 TAD 1 TAD 2 TBD 1 TBD2 ORC 1 0RC2  Raw Volume (mL) 120 120 100 78 100 96  Flocced V o l u m e (mL) 105 105 120 120 105 105  All of the raw and flocced leachate had their pHs adjusted to 7.5, using 6 M N a O H for the raw bottles and 1+1 HCI for the flocced bottles. E a c h bottle w a s g a s s e d with N  2  for 5 minutes to remove any oxygen and then w a s moved into the anaerobic chamber. E a c h bottle received N a S 0 to bring up their concentration to 2  4  1500 mg L" S 0 ~ (Table 6). The volumes used to calculate the amount of N a S 0 1  2  4  2  4  needed also included the volume of the 1 0 % inoculum.  62  Table 6 - Sodium sulphate added to achieve 1500 mg L"  1  SQ  4  Inoculum added (ml) 12  M a s s of Na S0  Raw TAD 1 TAD 2 TBD 1 TBD2 ORC 1 ORC2  Leachate Volume (ml) 120 120 100 78 100 96  12 10 7.8 10 9.6  0.293 0.244 0.190 0.244 0.234  Flocced TAD 1 TAD 2 TBD 1 TBD 2 ORC 1 ORC 2  105 105 120 120 105 105  10.5 10.5 12 12 10.5 10.5  0.256 0.256 0.293 0.293 0.256 0.256  2  4  (a) 0.293  E a c h bottle received an inoculation of S R B that w a s 1 0 % of their volume: The inoculum w a s obtained in the s a m e 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 w a s taken from the 2 1 transfer of S R B into new media derived from s t  the L D B sediment.  Three positive controls were created where 1.5 mL of the s a m e inoculum w a s placed in glass vials containing 13.5 mL of Postgate B media. T h e s e 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 w a s monitored for S 0 ~ and C O D . Before sampling, the liquid in 2  4  the bottles w a s homogenized by hand shaking for 2 min, followed by 1 hour of standing to settle the solids (bacteria). W h e n s a m p l e d , 2 mL w a s removed from the T A D and O R C bottles and placed into 18 mL of distilled water, while 3 mL w a s removed from the T B D bottles and placed in 3 mL of distilled water. T h e s e volumes were then analyzed for their S 0 " and C O D levels a s described by the protocols 2  4  listed later in this section.  O n t=17 days, all of the bottles received, 0.5 mL of a 17.5 g L" F e C b solution. This 1  was added to precipitate any sulphide produced and prevent it from becoming inhibitory to the activity of the S R B . This amount w a s determined using the Postgate B recipe and adding an adjusted amount to obtain the s a m e concentration. At this point, due to the presence of F e S precipitate, the s a m p l e s removed were filtered first using 11 pm filter paper. S a m p l e s were taken for S 0 " on t=28 days and C O D on 2  4  t=30 days  64  3.5  Mesocosm Experiment Set-up and Operation  Four m e s o c o s m boxes were constructed out of exterior grade plywood to the specifications provided in Figure 6.  T 29.2 cm  Inside dimensions 200 x 25.4 x 36 cm (LxWxD)  1  Top V i e w  - 203.8 cm r-A  Pump  -Q-  ISQ...  Side View  — A  |«—36.6 c m -  bulkhead  Crossbar to support equipment (loose)  T 28 cm  End View  Influent Reservoir 210L  Section A - A  Figure 6 - M e s o c o s m design  T h e m e s o c o s m design was based primarily on the design used by Stark, Williams et al. (1995), which had dimensions of 160x25x15 c m (LxWxD). This design w a s selected, with s o m e modifications. Justification for the dimensions and configuration chosen are given at the end of Chapter 2.  T h e m e s o c o s m s were placed in a greenhouse located on U B C c a m p u s . Two of the m e s o c o s m s used natural sediment and algae removed from L a c du Bois as a  65  carbon substrate, while the s e c o n d set utilized a 1:1 (wt) of silage and hay. E a c h box w a s lined with 2 layers of black polyethylene liner to prevent leaks. Most of the time, the room temperature w a s between 15-20°C without the need for cooling or heating, except from Nov. 04 - F e b . 05 during 6 pm - 8am when an electrical heater w a s required. The room temperature w a s monitored daily. In the natural treatment system, influent w a s introduced through a rigid polyethylene 0.5 c m diameter tube, perforated at 5 c m intervals, buried beneath the sediment and extending half the length of the m e s o c o s m . E a c h m e s o c o s m had a 3/8" bulkhead connectors attached at one end, 28 cm from the bottom of the m e s o c o s m , to allow effluent to leave the system. Underneath e a c h connector w a s 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  T h e treatment approach for the natural system w a s 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 m e s o c o s m s simulating the natural treatment system, water, sediment and algae were collected from the L a c du Bois (LDB) on July 6, 2004. The water w a s removed from the designated rocky site, while the algae/sediment w a s 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 w a s removed from the lake and placed into six 30 L barrels. T h e s e barrels were filled to the top with lake water and s e a l e d , in order to keep this material as anaerobic a s possible. Finally, yellow and green filamentous algae were removed from the lake's surface and placed into 30 L buckets containing lake water. T h e algae buckets were covered with translucent plastic bags in order to allow light to penetrate, so a s to keep the algae alive for the trip back to Vancouver. A botanist later identified which algae s p e c i e s were present (Stockner 2004). T h e s e two m e s o c o s m s are subsequently referred to a s natural treatment m e s o c o s m s .  T h e experiment began on July 7  t h  2004, when each of the natural treatment  m e s o c o s m s 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 c m of sediment. This layer was immediately covered, with a 15 cm layer of L D B water to act a s a water cap to maintain anaerobic conditions within the sediment. Finally, the  67  L D B algae were added to form a 0.6 c m thick layer on top of the water. In 11 days, the water level w a s observed to have dropped due to evaporation, thus 15 L distilled water (DW) w a s added to restore the s a m e water height. Subsequently, the water levels were restored to their original height at two-week intervals by adding D W . The m e s o c o s m s were left as a batch system to acclimate before introducing high sulphate concentration water continuously.  3.5.2 Natural Treatment System: Continuous Simulation  O n September 2 8  t h  (t=0), water containing 2500 mg L" S 0 ~ , a s N a S 0 , w a s 1  2  4  2  4  pumped at a rate of 6 mL min" into the two m e s o c o s m s through the subsurface 1  infusion pipe. This w a s the slowest possible pumping rate. At this rate, the residence time of the water flowing through the m e s o c o s m w a s estimated as 15.8 days. This residence time w a s tentatively selected since residence times vary greatly in passive treatment systems and the kinetics of this system w a s , a s yet, unknown. The Stark, Williams (1995) design, on which my system is based, had a residence time of 33 hours. However, studies in the laboratory with sediment removed from L a c 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 " by at least 800 mg L" (see 2  1  4  Appendix A-1). S i n c e it w a s a s s u m e d that the S R R would be slower in the m e s o c o s m , which would be open to the air and not using a growth medium to support S R B activity, a longer residence time w a s required.  68  Water samples were taken at 3 locations along the length of the m e s o c o s m (50, 100, 150 cm) and at two depths (10 and 20 centimetres below the water surface) for testing for S 0  2 4  \ In addition, 15 mL of water w a s taken at the 100 c m length and  15 cm depth for ammonia-nitrogen and phosphate analysis. Sampling w a s repeated on t=15 d a y s .  After this point, water samples were taken only of the influent and the outlet. O n t=43 days, the sulphate concentration in the feed reservoir w a s reduced from 2500 mg L" to 1500 mg L" . The inlet concentration w a s lowered since reduction of 1  1  2500 mg L" in one step is unlikely due to product inhibition from sulphide. For every 1  1000 mg L" sulphate reduced, 333 mg L" sulphide is produced. At concentrations 1  1  above this, sulphide b e c o m e s inhibitory to sulphate reduction.  The natural treatment m e s o c o s m s were run continuously from September 2 8 December 6  t h  t h  to  2004. Due to poor performance, the continuous system w a s stopped  and it w a s decided to run both the natural treatment m e s o c o s m s and the agricultural material m e s o c o s m s a s batch systems, rather than continuous, so as to determine the sulphate reduction kinetics. A n amendment of additional nutrients w a s made to the L D B m e s o c o s m as no ammonia could be measured in the system, and the levels of phosphate were low. It w a s believed that the continuous water flow w a s 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 m e s o c o s m s were  69  amended with 250 mL of lactic acid (8.65 mol C ) , 9.26 g of N H C I (0.17 mol N) and 4  3.36 g of K H P 0 2  4  in m e s o c o s m A and 3.69 g of K H P 0 2  4  in m e s o c o s m B which gives  a total phosphorus level of 0.03 mol. The amount of lactate added w a s 2.19 g L"  1  which is slightly less than the 2.78 g L~ suggested by (Postgate 1963) for the growth 1  of S R B from pure cultures and natural samples.  3.5.3 Natural Treatment System: Batch Simulation  The natural treatment system w a s 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. O n day 63, high S 0 " water was recycled from the water column into the sediment using a 2  4  peristaltic pump at 2 mL min" . This was done so a s to introduce high S 0 " into the 1  2  4  sediment layer. At t=91 days, sulphate concentrations were monitored weekly for an additional 84 days (this w a s considered t=0 for this new trial).  50 cm  Inlet  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 " concentration in the sediment and water column were compared. O represents the sampling location when the SO4 was measured in the sediment, after running the recycle system to increase the SO4 ' concentration in the sediment. 2  4  2  2  70  3.5.4 Agricultural Material Treatment System  A mixture of alfalfa and orchard grass hay and alfalfa-derived silage w a s removed from the Kamloops Agricultural and Agri Food C a n a d a R e s e a r c h station on October 4  t h  and placed in large black garbage bags for transportation back to K a m l o o p s . O n  October 2 0 , a 13 c m layer of hay and silage (1:1 ratio), weighing approximately 5 th  kg, w a s placed along the bottom of the second pair of m e s o c o s m s , henceforth referred to as the agricultural m e s o c o s m s . Tap water, previously allowed to sit for 2 days to remove any residual chlorine present, w a s used to fill the m e s o c o s m s to a height of 25.5 c m . The chlorine levels were measured using free and total chlorine test strips (2745050 H a c h C o m p a n y ) which have a range of 0-10 mg L~ and 1  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 m e s o c o s m s w a s adjusted from 5.5 to 7.5 using 10N N a O H . O n November 2 , 16 L of L D B sediment, as an n d  inoculum, w a s added along the length of each m e s o c o s m . In addition 4 L of Postgate B media w a s added to e a c h m e s o c o s m . T h e S R B were allowed to acclimatize to the agricultural m e s o c o s m environment for approximately 1 month prior to the initiation of the experiment. Two more pH adjustments were required on November 1 5 and D e c e m b e r 1 0 a s the pH had dropped to 6 in the m e s o c o s m s . th  th  71  T h e SO4 " concentrations in the agricultural m e s o c o s m s were increased to 1500 mg 2  L" by adding 1200 mL of water containing 304.21 g of N a S 0 to e a c h m e s o c o s m . 1  2  4  Sampling began on D e c e m b e r 1 0 (t=0 days) O n e sample w a s taken from e a c h th  m e s o c o s m weekly at 100 c m down the length of the m e s o c o s m and at a depth of 15 c m in the water column for S 0 ~ , T S , s C O D , N H - N and P 0 ~ . 2  3  4  3  4  Table 7 below is a record of all the changes made to the agricultural m e s o c o s m s over the course of this experiment.  Table 7 - Changes made to the agricultural mesocosms  Event # 1  Date of Event t=82d  2 3 4 5  t=109d t=159 t=174d t=222 d  Event A d d e d 1200 mL of water containing 304.21 mg of N a S 0 . A l s o a d d e d a 13 c m layer of pre-soaked silage and hay A d d e d 2 L of water containing 304.21 mg of N a S 0 A d d a 13 c m layer of silage and hay Placed a tarp over the system to prevent air from entering W a s h e d silage with tap water and added this wash-water to the system 2  2  4  4  3.5.5 S R B Activity  In order to determine if the S R B bacteria were still active, s a m p l e s of sediment were removed from the two m e s o c o s m s and placed in Postgate B media. T h e formation of black precipitate over time would indicate the presence of active S R B reducing S 0 " to sulphide, which would then form the black precipitate, F e S . At two different 2  4  locations within the m e s o c o s m s , sediment was removed at two depths. Five mL s a m p l e s were taken 50 c m away from the two m e s o c o s m ends within the bottom 0-3  72  cm of sediment and within the bottom 9-11 c m of sediment. T h e sediment w a s filtered through 0.22 um pore filter paper and the paper w a s placed within 50 mL tubes each filled with Postgate B media. T h e 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  Analytical Methods  3.6.1 Sulphate - Turbidimetric  Sulphate determination used the barium sulphate turbidimetric method (4500-SO ~ 2  4  E, American-Water-Works-Association 1975). Barium ions, added as barium chloride, react with sulphate ions to produce insoluble barium sulphate (Equation 4). Ba  2 +  + SO4 " -» B a S 0 2  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 c a n be measured using a spectrophotometer b e c a u s e the cloudiness reduces the amount of light that will pass through the sample. T h e minimum detection limit using the buffer A w a s 20 mg L" and the maximum detection limit w a s 100 mg L" . Dilutions 1  1  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 s a m e dilution a s that being a n a l y z e d . 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 s a m p l e s underwent filtration through 0.45 pm filter paper. If the sample w a s to be analyzed over the next two days, the filtered sample w a s stored in the fridge, otherwise it w a s frozen.  1. The spectrophotometer (Biochrom Ultrospec 1000) w a s turned on at least 0.5 hours before the analysis w a s to be undertaken. This time w a s necessary to allow the lamp time to warm up. The wavelength w a s set to 450 nm. 2. The s a m p l e s were allowed to warm up to room temperature and then diluted, if necessary, using distilled water. The final volume size w a s 10 mL and each sample w a s placed in identical 50 mL vials with magnetic stir bars, also of the s a m e s h a p e and size. 3. A standard S 0  2 4  solution of 100 mg L w a s created using N a S 0 and this 1  2  4  w a s 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 ( M g C I - 6 H 0 ) , 5 g of sodium acetate ( C H C O O N a - 3 H 0 ) , 1.0 g of potassium 2  2  3  2  nitrate ( K N 0 ) and 20 mL of acetic acid ( C H C O O H ) in 500 mL distilled water 3  3  and make up to 1000 mL. 5. E a c h sample of 10 mL received 2 mL of Buffer A .  74  This solution w a s temporarily poured into a cuvette in order to zero the spectrophotometer. The sample w a s placed on a magnetic stirrer and received 0.25 g of B a C b . T h e sample w a s stirred for 60 ± 2 s and then immediately removed and poured into a cuvette. After 5 ± 0.5 min, the sample w a s placed in the spectrophotometer and the optical density w a s recorded.  3.6.2 Sulphide - Methylene Blue  This determination of total sulphide is based on the standard methylene blue method (428C, American-Water-Works-Association 1975). This method is based on the ability of hydrogen sulphide and acid soluble sulphides to convert N,N-dimethyl-pphenylenediamine directly to methylene blue, a well known dye, in the presence of a mild oxidizing agent s u c h a s potassium dichromate. T h e accuracy of this test is reported to be about ± 1 0 % and have a minimum detection limit of 0.1 mg L"  1  Reagents 1. A m i n e sulphuric acid stock solution. Dissolve 27 g of N, N-dimethyl-pphenylenediamine oxalate in a cold mixture of 50 mL concentrated  H2SO4  and 20 mL of distilled water. Cool the mixture and dilute to 100 mL with distilled water. Store in a dark glass bottle. T h e amine oxalate must be fresh as an old supply c a n discolour to such a degree that the colour interferes with  75  the test. W h e n the stock is diluted and used in the procedure, it must yield a colourless solution. 2. A m i n e sulphuric acid reagent. Dilute 25 mL of the amine sulphuric acid stock solution in 975 mL 1+1 H S 0 and distilled water. 2  4  3. Ferric chloride solution. Dissolve 100g F e C I - 6 H 0 in 40 mL of water. 3  4.  2  Sulphuric acid solution. 1+1 H S 0 . A d d 1 mL of concentrated H S 0 to 1 2  4  2  4  mL of distilled water. 5. Diammonium hydrogen phosphate solution: Dissolve 400 g of ( N H ) H P 0 in 4  2  4  800 mL of distilled water. 6. N a S - 9 H 0 stock solution. Place 5 g of crushed N a S - 9 H 0 crystals in 2.5 2  2  2  2  mL of distilled water and maintain at 30°C overnight. This will create a saturated sulphide solution with a concentration of 9.2434 x 1 0 mg L" T S 4  1  (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. A n additional 2.5 mL is transferred to be a blanking solution.  76  5. T o the blanking solution add 0.167 mL of 1+1 H S 0 solution and to the test 2  4  samples add 0.167 mL of the amine sulphuric acid reagent. 6. Immediately add 1 drop of the F e C I solution to the s a m p l e s . C o v e r the 3  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 ) H P 0 solution. S h a k e 4  2  4  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  T h e total soluble C h e m i c a l O x y g e n D e m a n d ( s C O D ) is a measure of the oxygen required to oxidize all soluble c o m p o u n d s , both organic and inorganic, in water. This procedure is based upon the C O D Standard Methods 5 2 2 0 D , closed reflux, colorimetric method (American-Water-Works-Association 1975). T o 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 e x c e s s of potassium dichromate ( K C r 0 ) . After digestion, the remaining unreduced 2  K Cr 0 2  2  7  2  7  is titrated with ferrous ammonium sulfate to determine the amount of  K C r 0 consumed and the oxidizable matter is calculated in terms of the oxygen 2  2  7  equivalent. This test analyzes both biodegradable and non-biodegradable (refractory) organic matter. The results of this test are expressed a s mg L" 0 . 1  2  77  Procedure 1. S a m p l e s 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  H2SO4  and the sample is stored in the fridge at 4°C. S a m p l e s can be  stored for up to 28 d a y s under these conditions. 2. A d d 1 ml of a 100 g L" zinc sulphate solution to a 100 ml of sample and mix 1  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. T h e 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 H a c h company, at various dilutions. 6. Two reagents are prepared for the C O D test. a. Digestion Solution: A d d 10.216 g K C r 0 primary grade previously 2  2  7  dried at 103°C for 2hr, 167 mL concentrated H S 0 and 33.3 g H g S 0 2  4  4  to 500 mL of distilled water. Dissolve, cool to room temperature then dilute to 1000 mL. b. Sulphuric acid reagent: A d d silver sulphate ( A g S 0 ) , crystals or 2  4  powder, to concentrated H S 0 at a rate of 5.5 g A g S 0 2  4  2  per kg  4  H S 0 . Then let stand for 1 to 2 days to dissolve the A g S 0 . 2  4  2  4  78  7. T o 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 s a m e reagents to a vial containing D W to act as a blank to zero the spectrophotometer. 8. U s e an agitator (Thermolyne Maxi Mix II type 37600) to mix the vials for 15 s e c o n d s . 9. P l a c e sample vial into the heating block ( C O D 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 l a c e the C O D vial into a spectrophotometer ( D R / 2 0 0 0 , Hach Company) set at 600 nm and record the O . D . 11. A standard calibration curve is developed using potassium hydrogen phthalate. ( K H P ) 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  A m m o n i a nitrogen is the s u m m e d weight of nitrogen in both the ionized (ammonium, N H ) and un-ionized ( N H ) forms. In these aquatic s a m p l e s the ammonium s p e c i e s +  4  3  is generally the more prevalent. This protocol utilizes pre-calibrated reagents and a colourimeterforthe measurement (Nesslerization method, without preliminary distillation step) of ammonia-nitrogen a s modified from Standard Methods for the Examination of Water and Wastewater, method 4 5 0 0 - N H C (18 E d . , A P H A , th  3  79  A W W A , W E F , 1992). The reagents were purchased a s part of the L a Motte Smart A m m o n i a Nitrogen - High R a n g e kit code 3 6 4 2 - S C . In this method, Nessler's reagent, a solution of mercury (II) iodide (Hgl ) in potassium iodide and potassium 2  hydroxide, forms a brown colouration or precipitate in proportion to the amount of ammonia present in the sample. Rochelle 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 s a m p l e s 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 L a Motte). This sample is used to blank the colorimeter which has previously been set to S e q u e n c e #3 A m m o n i a - N High test. 3. E a c h sample vial receives 8 drops of A m m o n i a Nitrogen Reagent #1 (V4797) and then the vial is capped and mixed. 4. Using the 1.0 mL pipet (Code 0354), 1.0 mL of A m m o n i a 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 s c a n n e d .  80  3.6.5 Phosphate - Ascorbic A c i d  This protocol utilizes pre-calibrated reagents and a colorimeter for the measurement of ortho-phosphate in water samples a s outlined from (American-Water-WorksAssociation 1975) method 4 5 0 0 - 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 S0 2  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 L a Motte). This sample is used to blank the colorimeter which has previously been set to S e q u e n c e #33 Phosphate-Low. 3. E a c h 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 a d d e d . T h e vial is c a p p e d 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 s c a n n e d .  3.6.6 Dissolved Oxygen - Winkler Colorimetric Method  This method (LaMotte 3 6 8 8 - S C ) 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-Association 1975) (method 4 5 0 0 - O C ) . In this test, a manganese sulphate solution is a d d e d to the sample water and the m a n g a n e s e ions are liberated and loosely bound with e x c e s s hydroxide. In the presence of a strong base, the m a n g a n e s e 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 molecule for 2  each atom of oxygen. This results in a yellow/brown colour, w h o s e intensity can be measured using a colorimeter and which is proportional to the amount of D O 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. R e m o v e 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.  A d d two drops of m a n g a n e s e 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.  A d d two drops of sulphuric acid 1:1 (6141WT-G). C a p and gently s h a k e the vial until the precipitate has fully dissolved. N o w the s a m p l e is fixed.  6.  Set the colorimeter to test 27. Oxygen and place a clean colorimeter tube (LaMotte 0967) filled with sample water into the colorimeter c h a m b e r and scan blank.  7.  R e m o v e the blank and replace with the colorimeter tube containing the fixed water for analysis. S c a n the sample and record the result.  83  4.0  Results and Discussion  4.1  Characterization of H i g h a n d L o w S u l p h a t e L a k e s  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 L a c du Bois G r a s s l a n d s Park in Kamloops, were examined. This section describes site visits to L a c du Bois and L a c du Bois Twin and d i s c u s s e s the similarities and differences in the lakes' physical, chemical and biological makeup. T w o locations at each lake, R o c k y and A l g a e (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  L a c du Bois is one of the larger lakes in the L a c du Bois G r a s s l a n d s Park with a surface area estimated a s 3.75 x 1 0 m . 5  2  L D B R o c k y 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. S o m e 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 c m (6ft from shore). O n the surface of the water w a s a large yellow/green algae mat. Beneath this mat w a s a very thick layer of decaying algae matter which, at s o m e points, extended to the lake floor. W h e n e v e r the mat w a s disturbed, trapped g a s e s were able to e s c a p e a n d bubble up to the surface and the scent of sulphide w a s observed.  Lac du Bois Twin  L D B Twin w a s a considerably smaller lake, estimated at 8 x 1 0 m , when compared 4  2  to L a c du Bois. It w a s oval in shape and the entirety of the lake could be seen from any point around its surface, unlike L D B which w a s irregular in s h a p e . Around the shore of the lake, a thick white precipitate coated the ground a n d rocks (Figure 9).  Figure 9 - Lac du B o i s Twin shoreline. Oct 4, 2004  85  L D B Twin w a s shallow and the lake floor could be observed at all times. It is estimated that the maximum depth within the lake w a s 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 w a s 25 c m . The sediment layer on the floor of the lake w a s very thin, measuring 5-10 c m in places.  Physically, these two lakes were very different. Of the two, L a c du Bois w a s considerably larger in surface area. While it w a s not possible to determine the depth of L a c du Bois at distances further than 183 c m (6 ft) from shore, the lake floor could not be observed at greater distances from shore, unlike L a c du Bois Twin, which indicates that L a c du Bois w a s a deeper lake.  4.1.2 Chemical Characteristics Temperature/pH/DO  It w a s found that the two lakes differed greatly in their p H . L a c du Bois had the lower pH of 8.62 ± 0.04, (July 6, 2004) 8.63 ± 0.01 (Oct 4, 2004) while L a c 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 L a c du Bois w a s 22.95 ± 0.55°C (July 6, 2004) and 15.45 ± 0.22°C (Oct 4, 2004) while L a c du Bois Twin w a s 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 D O probe w a s only operable during the October visit to the lakes. L a c du Bois was found to have a D O level of 15-20% at the L D B R o c k y site, with higher readings near the water's surface. At L D B Algae readings of 12-15% saturated were observed in the first 5 cm of the algae mat at L D B A l g a e site. A s the probe w a s dropped lower, this level quickly dropped until it w a s 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  E v e n though the two lakes, L a c du Bois and L a c du Bois Twin are spatially very close to one another, the S 0 " concentrations in the two lakes vary greatly. L D B 2  4  w a s found to have low levels of S 0 " (Figure 10) with averages of 54.8 ± 2.0 mg L" 2  1  4  at the algae site and 60.1 ± 1.9 mg L" at the rocky site. The S 0 " concentration w a s 1  2  4  significantly lower at the L D B A l g a e site during the July 6  t h  visit w h e n 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 - (mg L" ) 2  1  4  40  45  H  0  50  .  .  55  .  60  65  .  ,  70  ,  Figure 10 - S0 " concentration (mg L' ) in Lac du Bois versus depth (cm). • represents S0 " 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 S 0 test. 2  1  2  4  4  2  4  In contrast, L D B Twin had exceptionally high levels of S 0 " averaging 2  4  6500 mg L" S 0 " (Figure 11). 1  2  4  88  S 0 - (mg L" ) 2  1  4  5800 0 -j  6000 ,  6200 ,  6400 .  6600 .  6800 ,  7000  7200  ,  ,  Figure 11 - S 0 ' concentration (mg L" ) in Lac du Bois Twin versus depth (cm). • represents S0 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. 2  1  4  2  4  Sulphide The presence of sulphide in natural water reservoirs indicates the presence of S R B . Sulphide w a s 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' 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. 1  TS (mg L" ) 1  0.00 0 H  0.50  1.00  1.50  2.00  '  '  '  •  Figure 13 - Total sulphide concentration (mg L" ) 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. 1  Nutrients T h e 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 w a s observed w a s that while there were low levels of nitrate in both lakes, there were higher levels of a m m o n i a , 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" ) 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. 1  Phosphate levels were higher in L D B Twin than in L D B (Figure 15). In addition, the levels of P 0 " were higher at L D B R o c k y than at L D B A l g a e . 3  4  91  6.00 -j 5.00 Li  4.00 3.00 -  CO  d CL  2.00 1.00 0.00 LDB Rocky  LDB Algae  LDB Twin Site 1 LDB Twin Site 2  Sample Location  Figure 15 - Ortho-P0 " concentration (mg L' ) 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. 3  1  4  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  400  TO 300  E, § 200 o 100 0  LDB Rocky  LDB Algae  LDB Twin Site 1 LDB Twin Site 2  Sample Location  Figure 1 6 - Soluble COD concentration (mg L" ) in Lac du Bois and Lac du Bois Twin. Sampled October 4, 2004. Error bars represent standard deviation of the test. 1  T h e higher levels of s C O D indicate higher levels of dissolved c a r b o n a c e o u s material present in L D B than in L D B Twin. A n assumption is being made that organic  92  material is the primary contributor to the chemical oxygen demand. A n earlier chemical analysis of these lakes, sampled by S u s a n Baldwin and analyzed by Norwest labs, found that the dissolved organic carbon ( D O C ) content of the water in the algae site to be 283 mg L" , while in L D B Twin the D O C w a s 113, 130 and 360 1  mg L" in 3 different locations. The complete Norwest chemical analysis of L D B and 1  L D B Twin can be found in Appendix A .  The chemical characteristics of the two lakes are very different, despite their close proximity. L a c du Bois Twin had higher levels of D O , pH and S 0 " when compared 2  4  to L D B . Nutrient levels were similar in the two lakes with L D B Twin having higher levels of P 0 " but L D B having higher levels of s C O D . 3  4  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. O n the second visit to the lake, a large group of cattle w a s 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 L a c du Bois Algae, a large population of cattails (Typha  93  latifolia) w a s observed growing around the shore. S e d g e (Carex) w a s also observed growing around the lake.  The plant-like matter of L a c du Bois w a s later identified by Dr. J o h n Stockner of E c o Logic Ltd. The samples removed from L D B were found to be primarily a green filamentous alga known a s Cladophora filaments of Ulothrix sp. and Mougeotia  cf. glomerata  sp. The Cladophora,  with s o m e scattered 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 L a c du Bois w a s completely coated with a wide variety of attached diatoms, the most dominant one being Achnanthes  minutissima.  diatom genera found on the filaments were: Navicula, Nitzschia, Cymbella,  Fragilaria,  Rhopalodia,  Amphora,  and Cocconeis.  Other  Frustrulia,  Dr. Stockner also  observed a wide variety of small bacteria and protozoan grazers within the samples.  The sediment of L D B w a s tested by J e n n y E n g , an undergraduate researcher at U B C , for the presence of S R B . Over a period of 51 days, the level of SO4 " w a s 2  monitored in 300 mL glass jars containing Postgate B media and 30 m L aliquots of L D B sediment. The results indicate that S R B were present in the L D B sediment a s S 0 " reduction w a s observed with an average drop of 1220.1 ± 49.5 mg L" S 0 " 2  1  4  2  4  from sediment sample #1 and 1071.4 ± 8.2 mg L" SO4 " from sediment sample #2. 1  2  94  This gives an overall average drop in S 0 " of 1145.8 ± 28.8 mg L" S 0 " for the 2  1  4  2  4  L D B sediment.  Lac du Bois Twin  In contrast to L a c du Bois, L D B Twin w a s not set in a lush environment. A s mentioned previously, instead of cattails and s e d g e surrounding the lakeshore, rock and soil covered in a thick crusty white precipitate, likely sulphate salt, w a s observed.  Within the lake itself, green organic matter w a s observed both on the sediment floor and suspended in the water. T h e organic matter did not look the s a m e as the algae found in L D B and instead had a granular appearance. By visual inspection, the amount of material present w a s also smaller. After being analyzed by Dr. John Stockner it w a s determined that no Cladophora  were present and the dark green  mat w a s primarily a filamentous blue-green Cyanobacteria known a s spumigena.  Nodularia  A l s o present but not commonly found in the lake were the coccoid blue-  greens Chroococcus  sp. and Cyanothecae  sp. Unlike L a c 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, a m o e b a , Paramecium, and several species of rotifers).  95  The sediment of L D B Twin, a s analyzed by J e n n y E n g in the s a m e manner a s the sediment of L D B , found that S R B were present. A n average d e c r e a s e in S 0 ~ of 2  4  406.9 ± 266.8 mg L" S 0 " w a s obtained using sediment from location #1 and 1  2  4  1295.5 ± 555.0 mg L" S 0 " using sediment from location #2 giving an average 1  2  4  overall S 0 " drop of 851.2 ± 410.9 mg L" S 0 " . 2  1  4  2  4  Biologically, the two lakes are dissimilar. L a c du Bois has a healthy appearance with lush vegetation both surrounding and within the lake. In contrast, L a c 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 L D B had a greater potential for reducing S 0 " in Postgate B media. 2  4  4.1.4 Discussion and C o n c l u s i o n s  L a c du Bois and L a c du Bois Twin, although less than 500 m apart, had very different physical, chemical and biological attributes. Of the two, L D B w a s the larger lake, both in surface area, and in depth.  Dissolved oxygen levels within L a c du Bois were low and d e c r e a s e d 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, L D B Twin w a s 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 L D B Twin w a s saturated a s little plant life w a s observed either surrounding or within L D B 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 " within the lake. With the exception of the July 6 2  4  t h  LDB  R o c k y 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 s a m p l e s removed from these lakes and observed sulphate reduction in all s a m p l e s , confirms the presence of S R B in the sediment of both lakes. T h e medium containing L D B sediment had a higher rate of S 0 ~ reduction, which suggests that either there were 2  4  more S R B present in the L D B 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 a s 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 L D B , 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 L D B Twin waters. Cattails can survive in acidic, neutral and  97  basic p H , but Dyhr-Jensen and Brix (1996) found that growth is depressed w h e n the plants have prolonged exposure to pH 8.0 and completely suppressed at pH 3.5. L a c du Bois Twin, with a pH greater than 10, is likely too basic to allow their growth.  T h e cyanobacteria observed in L D B Twin, Nodularia  spumigena,  is well known for  its toxic blooms throughout the world in brackish lakes and waters (Runnegar, J a c k s o n et al. 1988; Sivonen, Kononen et al. 1989). It can live either as plankton, or attached to sediments, both of which were observed in L D B twin. The toxin produced is the cyclic pentapeptide nodularin which is known to be toxic to mammals, including humans. Karjalainen (2004) reported that nodularin w a s responsible for the deaths of 400 ducks in J a s m u n d e r B o d d e n , Germany, in 1963, and the death of 16 young cattle in Stelasund, G e r m a n y , in 1983. This toxin could be another deterrent to cattle drinking from the lake, in addition to the high levels of S 0 " . M a z u r - M a r z e c et al. (2005) found that the nodularin content of 2  4  spumigena  Nodularia  increases with salinity.  In contrast, the Cladophora  group of algae, observed in L D B , is common to nutrient-  rich lakes and ponds and has a very high tolerance for moderately saline lakes and even s o m e 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 m a s s e s which w a s h 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 decomposition they c a n be utilized by detrital feeders (Dodds and G u d d e r 1992).  L a c 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 E n g (2005) observed sulphate reduction in Postgate B medium inoculated with L a c du Bois Twin sediment, S R B may be present in this lake. In contrast, L D B had a lower pH which w a s more suitable for sustaining plant life and an anoxic z o n e w a s 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 " . 2  4  It w a s 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. A l g a e 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 m e s o c o s m - s c a l e laboratory model of such a treatment system w a s constructed and tested.  99  Although evidence for the presence of S R B was found in L D B Twin, little sulphate reduction was a s s u m e d to be occurring due to the lack of anaerobic conditions, high pH and a b s e n c e of utilizable electron donor carbon sources for S R B . T h e question arose that if an electron donor carbon source, proven to be suitable to support S R B , w a s supplied to L D B 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 w a s 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 w a s tested for their ability to support biological sulphate reduction. Initial batch test screenings of these materials is described next.  4.2 The Suitability of A g r i c u l t u r a l Materials a s S u b s t r a t e s for Biological Sulphate Reduction  Four different agricultural materials: hay, barley, silage and m o l a s s e s , were tested for their effectiveness a s nutrient s o u r c e s for supporting S 0 " reduction activity by 2  4  S R B . Initial experiments contained no additives other than the organic material and high sulphate water. However, it b e c a m e clear that pH adjustment w a s required a s well a s sulphide removal and/or nutrient (PIN) addition in s o m e c a s e s . Thus, subsequent experiments were performed to compare the organic materials under  100  m o r e ideal conditions for biological s u l p h a t e reduction.  4.2.1 Sulphate Reduction Without pH Adjustment  S i n c e the objective of my work w a s to d e v e l o p a low-cost p a s s i v e sulphate treatment, I d e c i d e d to s e e if S R B cultures could be maintained on t h e s e C - s o u r c e s without supplementation of additional c h e m i c a l s for nutrients or p H control. T h e materials w e r e tested both individually a n d with a s m a l l addition of m o l a s s e s to provide a n immediate s o u r c e of available c a r b o n . M o l a s s e s h a s b e e n u s e d to support S R B growth previously ( M a r e e a n d S t r y d o m 1987; T a r e a n d S a b u m o n 1995).  O f the four agricultural materials u s e d , silage p r o m o t e d the largest overall d e c r e a s e in S 0  2 4  " w h e n c o m p a r e d to the other materials (Figure 17, T a b l e 8).  101  T h e bottles containing H a y a n d B a r l e y did s h o w sulphate reduction o v e r the first 50 d a y s of the experiment, but after this time their rates s l o w e d drastically. O n l y in the bottles containing silage did the s u l p h a t e d e c r e a s e almost linearly for the entire experiment.  Figure 17 - (a)-(d) S0 " concentration (mg L" ) versus time for the non-pH-adjusted batch experiments. Points represent the average S 0 " concentration in duplicate bottles. Error bars represent the difference in S 0 between the two bottles in each pair. 2  1  4  2  4  2  4  102  Table 8 - Average overall S 0  4  " change and maximum sulphate reduction rates (SRR) of non-  Agricultural Material  Overall C h a n g e in S0 -(mgL- ) 2  Maximum S R R Obtained mg L" d" (mg d" g" substrate) 9.8 ± 0 . 9 R =0.98 (0.5 ± 0 . 0 ) 9.5 ± 2.1 R^=0.97 (0.5 ± 0 . 1 ) 8.9 ± 2 . 1 R^=0.99 (0.5 ± 0 . 1 ) N/A  1  4  1  1  Hay  523.5 ± 55.0  Barley  548.6 ± 13.7  Silage  1324.3 ±252.4  Molasses  286.2 ± 2 1 . 4  1  1  2  Significance w a s determined by the Tukey test which performed multiple pairwise comparisons of the mean change in S 0 " for all agricultural materials. A pair were 2  4  considered significantly different when P<0.05 and the level of significance w a s set to a=0.05. Except for the experiment with m o l a s s e s , sulphate concentrations decreased linearly with respect to time for the first 33 d a y s . 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 is provided. 2  The bottles containing an addition of m o l a s s e s s h o w e d 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 .  W h e n T S concentrations were measured at the end of the experiment, the concentration w a s highest in the bottle containing silage (Figure 18). Since 333 mg L~ sulphide is produced per 1000 mg L" S 0 " reduced, this result suggests 1  1  2  4  103  that the d e c r e a s e in S 0 ~ a s o b s e r v e d in the bottles containing s i l a g e w a s mainly 2  4  d u e to c h e m i c a l reduction of sulphate, likely carried out by S R B . H o w e v e r , the a m o u n t of sulphide m e a s u r e d at the e n d of the experiment in the bottle containing silage (172 mg L" ) only a c c o u n t s for 4 0 % of the sulphide p r o d u c e d from 1  1324 m g L" sulphate r e d u c e d . 1  200 -  1  160  o>  111  E  V  120  fw  80  3 o  40  Hay  Barley  Silage  Molasses  Agricultural Materials 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  T h e s C O D w a s o b s e r v e d to be high at the end of the experiment (Figure 19); h o w e v e r w e cannot c o n c l u d e from this data whether the s C O D that r e m a i n e d w a s d u e to organic materials that are not utilizable by the S R B . D e t e c t a b l e levels of N H - N w e r e o b s e r v e d in all of the bottles at the c o n c l u s i o n of the experiment (Figure 3  20). T h e silage h a d higher levels of a m m o n i a - nitrogen than the other materials but w a s still well under the levels obtained w h e n using P o s t g a t e B m e d i a w h e r e the levels of a m m o n i a present are 337 p p m . This is a s s u m i n g that the P o s t g a t e B m e d i u m contains non-inhibiting concentrations of a m m o n i u m for S R B growth.  104  12000 -  10000 -  8000 D)  Q O O  in  6000  HH IP  4000 2000  81111 Hay  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 D)  E 40  z  • CO  I  30  •• Sift  20  ijjj  10 0  Hay  Barley  Silage  Molasses  Agricultural Materials  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  E 1.50 A O  0. • o  1.00  A  0.50  A  o  0.00 Hay  Barley  Silage  Molasses  Agricultural Material  Figure 21 - Ortho-P0 " concentration in the non-pH-adjusted batch experiments. Error bars represent the difference between the concentrations in the replicates 3  4  T h e p H in each bottle remained largely unchanged over the course of the experiment, with slight increases in the hay and silage bottles, and d e c r e a s e s in the barley and molasses (Figure 22).  107  10  8  6  X  a  mm  4  V V  2  0  Hay  1^ Barley  pi!  .vs;  Silage  \YvMolasses  Agricultural materials  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  T h e largest reduction of sulphate occurred in the presence of silage. While silage did not have the highest maximum S R R of all the agricultural materials used (8.9 ± 2 . 1 mg L" d~ ), it w a s the only material that w a s able to sustain its rate 1  1  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 o n e s , 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 silage over time, which is not unusual since one of the products of S R B activity is carbonate which helps to increase the p H . For the other agricultural materials it appears the leachate w a s acidic from the beginning and little change was observed over time. In particular the pH of the barley and the m o l a s s e s w a s very low for supporting S R B activity. It w a s also observed that the agricultural materials, other than silage, 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 s o u r c e s required by S R B . T h e s e factors together likely contributed to the low sulphate reduction observed.  O n e other likely reason why silage outperformed the other agricultural materials is that in the production of silage, lactic acid bacteria ( L A B ) 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 w a s used is still much slower than S R R s measured with a defined carbon sources. A n identical amount of inoculum from the s a m e source grown in defined Postgate B medium was able to achieve S R R s of 40.0 mg L" d" . 1  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. O n e of the known c o m p o u n d s 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 5 0 % at a concentration of 54 mg L" acetic acid, by crossing the 1  membrane of the S R B and interfering with the cells proton motive forces. R z e c z y c k a and Blaszczykm (2005) found that S R B inhibition w a s observed when acetate concentrations were greater than 10 mg L" . 1  The sulphide measured in the bottles containing silage accounted for only 4 0 % of the total sulphide that would have been produced through the reduction of 1324 mg L" sulphate. It is hypothesized that the remaining 6 0 % e s c a p e d a s H S g a s during 1  2  sampling, when it w a s necessary to remove the cap to sample the fluid.  A second set of experiments w a s performed to s e e 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 s o m e concerns that the inoculum of S R B could have had nutrients present due to residual Postgate B media in which is w a s grown, which may have contributed to the initial S R R s . T o rectify this, the inoculum in the second experiment would be washed twice with distilled water before being used. Third, it w a s 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 s o m e of the negative controls did experience SO4 " d e c r e a s e . The negative controls were set-up to prove 2  110  that any drop in S 0 " w a s due solely to S R B activity and not by the absorption of 2  4  SO4 " on either the agricultural material or the interior of the bottle. 2  Ill  4.2.2 Sulphate Reduction With pH Adjustment  T h e p H in all the bottles of this new experiment dropped between sampling periods, particularly at the beginning of the experiment, a n d required further adjusting (Figure 23).  10 - Silage and Molasses 8 6 -j 4 * 2 0 50  100  150  200  I Q.  50  100  150  200  Time (days)  Time (days)  10  0  Silage & Hay mixtures  420 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, 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. A  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 w a s observed in the previous experiment where no pH adjustment w a s 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 silage, 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 Silage and Hay mixtures had the highest overall d e c r e a s e in S 0 " concentration. Statistically, the Hay and the Silage/Hay 2  4  mixtures 1:3 and 1:1, had significant overall d e c r e a s e s in their S O 4 concentration 2  (Table 9).  113  Replenish  Inoculum  H 0 lewis  & FeCI  2  (b) Barley  Replenish H 0 levels  3  2  1600  r_i  Inoculum & FeCI 3  1200  E CO  400  50  100 Time (days)  150  200  -I  50  100  150  200  Time (days)  Figure 24 - (a)-(d) S 0 ' concentration versus time in the pH-adjusted batch experiments. Lines represent the average S 0 concentration of each pair. Error bars represent the difference in S0 " between the two bottles in each pair. Stars indicate times when sulphide was measured in the bottles. 2  4  2  4  2  4  114  (b) Silage:Hay 1:1  (a) Silage: Hay 1:3 Replenish  Replenish  1600  H 0 levels Inoculum  H 0 levels |  2  & FeCI  2  |  & FeCI  1200  3  n o c u  u m  3  800  o  co 400  50  100  150  200  A  50  Time (days)  100  150  200  Time (days)  (c) Silage:Hay 3:1 1600  ^  1200  Inoculum & Replenish  FeCI  H 0 levels  3  |  2  O)  E  800  O co 400  50  100  150  200  Time (days)  Figure 25 - (a)-(c) S 0 " concentration versus time in the pH-adjusted batch experiments containing mixtures of hay and silage. Lines represent the average S 0 concentration of each pair. Error bars represent the difference in S0 " between the two bottles in each pair. Stars indicate times when sulphide was measured in the bottles. 4  2  4  2  4  115  Table 9 - Change and SRRs of pH adjusted agricultural bottle experiments. Results in brackets represent the mg d' g' substrate rates. Errors represent the difference between replicate bottles. 1  Agricultural Material  1  Overall C h a n g e in S0 " (mg L" ) after 180 days  Maximum S R R for first 50 days mg L ' d (mg d" g" substrate)  Barley  1099.6 ± 100.9 378.7 ± 73.3  8.3 ± 0.6 R^=0.91 (0.5 ± 0 . 1 ) N/A  Silage  589.9 ± 14.1  Molasses  729.3 ± 26.0  11.0 ± 2 . 5 R*=0.95 (0.6 ± 0 . 1 ) N/A  Silage/Hay 1:3 Silage/Hay 1:1  1045.5 ± 5 . 6  2  4  1  Hay  Silage/Hay 3:1  1124.517.5  930.7 ± 18.5  1  1  Maximum S R R after 126 days to day 150 mg L" d" (mg d" g substrate) 10.4 ± 4 . 7 R^=0.97 (0.5 ± 0 . 1 ) 5.3 ± 1 . 5 R^=0.90 (0.3 ± 0 . 1 ) 8.1 ± 1 . 4 R^=0.89 (0.4 ± 0 . 1 ) 9.5 ± 0 . 1 R^=0.93 (0.1 ± 0 . 1 ) 8.7 ± 0 . 7 R*=0.95 (0.4 ± 0.0) 8.5 ± 1 . 1 R =0.91 (0.5 ± 0 . 1 )  f  1  1  1  1  10.5 ± 1.0 R^=0.95 (0.6 ± 0 . 1 ) 14.4 ± 4 . 0 R^= 0.93 (0.8 ± 0.2) 12.6 ± 0 . 2 R =0.92 (0.7 ± 0.0)  1  2  11.6 ± 1.8 R =0.90 (0.6 ± 0 . 1 )  2  2  The drop in S 0 " w a s biphasic in most of the bottles, with an initial steep d e c r e a s e 2  4  in SO4 " followed by a period where the S 0 " levels began to plateau or increase. In 2  2  4  an attempt to remedy this, fresh inoculum w a s added on day 126. After this addition, all of the bottles again underwent an increased rate of S 0 " reduction. At the time of 2  4  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 w a s fit through the first 5 data points from day 0 to day 57. However, during this period the pH w a s not always in the optimum range for S R B . Another sulphate reduction rate w a s calculated after  116  fresh inoculum w a s added and when the pH w a s constant, between day 126 and day 150.  For the positive controls, black precipitate w a s observed throughout the vials, signifying that sulphide w a s being produced, which also suggests that the S R B in the original inoculum were active. After 50 d a y s , one positive control vial w a s sacrificed and the SO4 " concentration a n a l y z e d . All the readings were below the 2  minimum detection limit for S 0 " , signifying that S R B had utilized the S 0 " present 2  2  4  4  in the vial (Appendix A-4a).  The negative controls were analyzed after 67 days and the average SO4 " levels of 2  the bottles were found to be unchanged from the starting SO4 " concentrations. In 2  addition the sulphide levels were measured and found to be too low to be detected. The exception w a s 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" . That particular bottle 1  showed a d e c r e a s e of 182 ± 254.7 mg L" S 0 " . Statistically there w a s no significant 1  2  4  d e c r e a s e in SO4 ". The results from this experiment can be found in Appendix A - 4 a . 2  This signifies that without the addition of S R B to a SO4 " solution the SO4 " molecule 2  2  is stable. This also indicates that any loss of S 0 " in my experiments is due to the 2  4  presence of S R B .  Sulphide w a s 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 w a s observed for the bottles containing hay, silage, and the hay/silage mixtures, while the barley and molasses either increased or remained at the s a m e level (Figure 26).  12000  O)  9000  E, Q O  o  6000  •  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 pHadjusted batch experiments. Bars represent the average sCOD concentration in each pair of bottles. Error bars represent the difference concentration.  T h e 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 w a s found, after the first sampling, that the phosphate levels in the bottles containing m o l a s s e s were very low. It c a n be s e e n in the abiotic controls (Appendix A - 4 e ) that bottles containing molasses as a sole carbon source have much lower levels of phosphate than the other materials. After observing that the m o l a s s e s bottles had, up until this point, undergone no drop in SO4 ", it was concluded that the phosphate 2  118  w a s limiting and an addition of phosphate w a s made on t=29 d a y s . Shortly afterwards, SO4 " reduction w a s o b s e r v e d in the m o l a s s e s bottles. With the 2  exception of m o l a s s e s , all of the bottles had increased levels of N H - N over time. 3  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  —  1  250  Li  L O °-  1  150  NSS  50  -I  -50  .N  tf Agricultural Materials  Figure 28 - Ortho-P0 ' 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. 3  4  4.2.2.1  Discussion and Conclusions  T h e results from this experiment demonstrated that with closer monitoring and adjustment of p H , 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 p H , particularly at the beginning of the experiment. T h e s e 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 silage fodder (Barnett 1954). It is surprising that the silage a n d 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 a s a result of the fermentation. However, it has been reported that maceration of silage plant materials generally produce pHs of 6 or slightly higher (McDonald 1981).  In all of the bottles an increase in SO4 " was observed 84 days into the experiment. 2  It is possible that this increase w a s due to the oxidation of sulphide back into S 0 " 2  4  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 a s 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" (Reis, 1  Almeida et al. 1992), while others have found the S R B to have higher tolerances with only 5 0 % inhibition observed with a H S concentration of 1000 mg L" (Isa, 1  2  Grusenmeyer et al. 1986). Throughout the course of this experiment, the analyzed total sulphide concentrations never rose above 100 mg L" and at a pH of 7.4-7.5 the 1  H S species would only account for 1/3 of the total sulphide while HS", not 2  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 S0  2 4  " 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 d e c a y of the organic plant material by indigenous bacteria found on the plant material. Through the actions of d e c o m p o s e r bacteria, the organic plant material is converted into ammonium and phosphate, forms suitable to support S R B growth. M o l a s s e s , as it is not organic plant material but instead c o m p o s e d primarily of sugars containing little or no nitrogen or phosphorus compounds, would not undergo d e c a y and provide more N H - N or P 0 " to the 3  3  4  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 . A n addition of P 0 ~ w a s made to 3  4  the m o l a s s e s bottles at t=29 as measurements at the time indicated that PO4 " levels 3  were limited.  Barley w a s still unable to support sustained S R B activity (overall drop of 378 mg L"  1  S 0 " over 180 days), despite having the s e c o n d highest levels of s C O D after 2  4  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  M o l a s s e s did support higher S R B activity when the pH w a s 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 m o l a s s e s w a s able to achieve a maximum S R R of 9.5 ± 0.1 mg L' d" SO4 " this w a s not the highest rate obtained in 1  1  2  the experiment. Molasses has been used a s a successful carbon source in bioreactors treating high sulphate water, but  KH2PO4  w a s 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 m o l a s s e s bottles remained unchanged over the course of this experiment despite the activity of S R B . However, solid m o l a s s e s w a s 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 m o l a s s e s , 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 " by 2  4  S R B (1099.6 ± 100.9 mg L" S 0 " for hay and 1124.5 ± 7.5 mg L" S 0 ~ for 1:1 1  2  1  4  2  4  silage:hay) with high S R R s that were sustained for longer periods of time than in the bottles containing barley and m o l a s s e s . (8.3 ± 0.6 mg L" d" for hay and 1  1  123  14.4 ± 4.0 mg L" d" for 1:1 s i l a g e : h a y ) . S i n c e the 1:1 hay:silage mixture resulted in 1  1  the largest drop in S 0 ~ , this mixture w a s tested in a larger scale (137 L) o p e n 2  4  s y s t e m , typical of a configuration likely to be used in the field (Section 3.5).  Hay has not been used a s 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; H a m m a c k and Edenborn 1992). In the first study, no S R B activity w a s o b s e r v e d , while in the second an addition of lactate w a s required to promote S R B activity. In the third study, the rates of S 0 " reduction varied between 0.192 - 57.6 mg L" d" . 2  1  1  4  The hay and silage:hay S R R s compared well with those of other bench s c a l e experiments which supplied S R B with non-defined carbon sources. Christensen, L a a k e et al. (1996) achieved S 0 " rates ranging between 3-4.4 mg L" d" when 2  1  1  4  using 1250 mL of whey, which has high levels of lactose. Waybrant, B l o w e s et al. (1998) achieved rates of 0.14 mg L" d~ (g of organic substrate)" using s e w a g e 1  1  1  sludge and 0.76 mg L d" g" while using leaf mulch, manure and sawdust. T h e 1:1 1  1  1  silage:hay mixture performed better than these two substrates (0.8 ± 0.2 mg d" g" ) 1  1  however it did not achieve as high a S 0 " reduction rate a s the mixture of s e w a g e 2  4  sludge, leaf mulch, wood chips, sheep manure and sawdust (4.23 mg L" d" g" ). 1  1  1  Although following a second addition of S 0 " , this rate dropped to 1.70 mg L" d" g" . 2  1  1  1  4  124  While the bottles containing only silage did not have the s a m e degree of SO4 " 2  removal a s in the previous experiment (1324.3 ± 252.4 mg L" without pH adjustment 1  vs 589.9 ± 1 4 . 1 mg L" with pH adjustment), this could be due to the fact that the 1  silage material used was from the s a m e stock as the previous experiment, and w a s , therefore, not fresh. It is likely that, due to the time e l a p s e d , 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 a s 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 w a s 4814 ± 618 mg L" s C O D w h e r e a s the initial s C O D for 1  this experiment (pH adjustment) was 2378 ± 148 mg L" s C O D . This is a 4 9 % 1  decrease in s C O D . S i n c e the s a m e m a s s of silage from the s a m e stock was used in both trials, s o m e of the s C O D must have been degraded during the storage of the silage between experiments. T h e 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 e a c h of these agricultural materials could support S R B activity with a s little addition of other amendments as possible. T h e results revealed that silage and hay performed the best a s supporters of sulphate reduction and therefore these were used in a later mesocosm-scale experiment. B e c a u s e the product of silage fermentation is lactic acid, a known usable carbon source for S R B , one more experiment involving silage leachate was planned. T h e final batch experiment w a s designed using only silage leachate as a nutrient source. It w a s 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 w a s fresh and had not c o m e into much contact with oxygen. In this final batch experiment, two different types of silage, one derived from orchard grass and a s e c o n d 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 a s a substrate. In contrast to the two previous experiments, the agricultural materials were soaked with distilled water and filtered, and only the leachate w a s used as a nutrient source.  126  This w a s 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 c a n be removed from the material by washing. S i n c e 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 a s an effective nutrient source.  In this experiment the S 0 ~ and the s C O D were monitored at approximately 2 week 2  4  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 ( O R C ) based silages were able to support substantial S 0 ~ activity, although 2  4  there was clearly batch variability present in the T A D R a w and O R C flocced bottles. The rates of sulphate reduction are given in both milligrams of S 0 " per litre per 2  4  day, and milligrams of S 0 " per day per gram of substrate (the wet weight of solid 2  4  silage material from which leachate w a s obtained). Maximum sulphate reduction rates were calculated from the slope of a straight line fitted through the first 4 data points where there w a s no lag, or through the 2 , 3 n d  r d  and 4 , data points in c a s e s th  where there w a s a lag.  127  Figure 29 - (a)-(f) S0 " 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. 2  4  128  Table 10 - Overall change in SQ " in leachate bottles over 71 days and maximum SRRs 2  4  Barley  Orchard G r a s s Bioethanol Waste (mg L) A S0 " 1227.91325.7 1026.6 ± 123.4 318.2 ± 9 2 . 3 870.0 + 60.1 955.6 ± 2 5 6 . 1 290.4 ± 123.4 Maximum Sulphate Reduction R a t e s mg L V mg L" d" mg cTV mg L/'d" mg d ' g " mg d~V substrate substrate substrate 17.2 ± 13.6 ± 0.11 ± 0.14 ± 0.09 ± 11.4 ± 0.2 0.0 0.8 0.0 0.0 1.5 R =0.97 R =0.86 R =0.99 19.4 ± 0.15± 18.5 ± 0.14 ± 4.4 ± 1.8 0.03 ± 1.6 0.0 3.0 0.0 R =0.90 0.0 R =0.96 R =0.98 2  1  4  Raw Flocced  1  Raw  Flocced  1  2  2  2  2  1  1  1  2  2  In contrast to the silage experiments, the bioethanol waste (TBD) did not appear to strongly support S R B activity.  W h e n 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 silages.  131  12000 — 9000  10  20  30  40  50  60  70  80  Time (days)  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 w a s 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 c a u s e the pH to be low.  Table 11 - pH of leachate  Poundmaker 3.96  Orchard Grass 4.38  Bioethanol 3.99  After adjusting the pH to 7.5 and inoculating, the pH w a s 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 a b s e n c e of the solid agricultural material. Stirling a n d 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  80  60  80  Time (days)  (b) Barleysilage  8.0 6.0 X °- 4.0 2.0 0.0 20  40 Time (days)  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 Time (days)  60  80  4.2.3.1  Discussion and Conclusions  In this final experiment, it w a s demonstrated that silage leachate w a s 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" d" S 0 " 1  1  2  4  (flocced leachate), respectively. This is considerably higher than the rates obtained in the presence of the solid material. T h e maximum sulphate reduction rate measured when a 1:1 mixture of hay and silage w a s used w a s 14.4 mg S 0 ~ L" d" . 2  1  1  4  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" substrate, respectively) they had very different rates 1  of sulphate reduction. The solid barley grains, although briefly able to achieve a maximum S R R of 21.8 ± 6.3 mg L" d" S 0 ~ achieved a low overall change in the 1  1  2  4  S 0 " concentration (378.7 ± 73.3 mg L~ S 0 " ) . A s mentioned previously, the 2  4  1  2  4  carbohydrates in barley grains are primarily c o m p o s e d of starch, a non-utilizable carbon source for S R B . In contrast the barley silage gave w a s able to maintain a high S R R throughout the experiment, most likely due to the ensilage 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 7 A C O D ratio for S R B grown on lactic 2  4  acid, we s e e that 1 mol of lactate can convert 1.5 mol of sulphate (Eqn. 5) and 3 mol of C O D c a n oxidize 1 mol of lactate (Eqn. 6).  3S0 ~ + 2C H 0 2  3  CH0 3  6  3  6  -> 3HS~ + 6HCO~  3  + 30  2  + 3H  +  -> 3 C 0 + 3H 0  (6)  2  2  (5)  This means that if lactate is fully oxidized by the bacteria then the theoretical ratio of A S 0 7 A C O D is 1.5 g g" . However, the average A S 0 7 A C O D ratios found for this 2  1  2  4  4  experiment over 71 days are all well below this value (Table 12). Table 12 - A S 0 VAsCOD ratio for leachate ex periment 4  Tad Raw  T A D Floe  TBD Raw  T B D 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 w a s 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, especially if heterolactic bacteria instead of homolactic bacteria had fermented the silage. 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.  S i n c e S R B use only low molecular weight carbon compounds, we expect to find these in the soluble portion of the silage. Lactic acid, produced in the ensilage  136  process, is one of the carbon compounds found in silage w a s h 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 a s 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.  B a s e d on these results, a passive treatment system (mesocosm experiment) could be supplied with silage leachate as the carbon source, rather than the whole solid silage. Unfortunately the silage leachate experiment w a s undertaken after constructing and operating the m e s o c o s m s at the end of my studies, so this w a s not tested at the m e s o c o s m level.  4.3  M e s o c o s m Experiments  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 a s they are more space-efficient than larger-scale m e s o c o s m or field studies,  137  and are easier to maintain under uniform conditions. M e s o c o s m experiments, simulated field studies conducted in controlled environments s u c h a s artificial ponds, streams or large outdoor tanks, are designed to more closely approximate actual operating conditions, s u c h a s would exist in a fully functional passive treatment system. Unlike the previous microcosm bottle experiments, the m e s o c o s m experiments discussed here were operated under open air conditions, a s it may be impractical for farmers to place a cover on a treatment pond. This section outlines the two sets of m e s o c o s m experiments using different nutrient sources to stimulate S R B growth and activity. O n e pair of m e s o c o s m s used natural sediments and algae removed from L a c du Bois, and a second pair utilized a 1:1 silage:hay mixture.  4.3.1 Natural Treatment System - Continuous  A s mentioned previously in Section 4.1, L a c du Bois w a s 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 m e s o c o s m with high sulphate concentration water, and monitoring the S 0 ~ levels within the system. The rate at which the S 0 ~ is 2  4  2  4  reduced in the m e s o c o s m 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 " ponds. 2  4  138  Two m e s o c o s m s , containing high S 0 " water, decaying algae a n d sediment and 2  4  s e e d e d with live algae removed from L a c du Bois were initially operated as continuous systems. T h e m e s o c o s m s were fed a concentrated S 0 ~ (2500 mg L" ) 2  1  4  solution at a rate of 6 m L min" , giving an estimated residence time of 15.8 days 1  (residence time = V o l u m e / F l o w rate), assuming no short circuiting. T h e rational for this flowrate and retention time are outlined in Section 3.5. Both m e s o c o s m s had a thick layer of green algae from L D B 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 g a s e s from the sediment. This w a s visually ascertained whenever the sediment w a s moved in any way, s u c h as when the rigid tubing w a s first placed in the bottom of the m e s o c o s m .  The pH in the m e s o c o s m s 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 w a s found on average to be 8.53 ± 0.8 and 7.89 ± 0.6 (st.dev. of 3 sampling points) in L D B A and 8.83 ± 0.2 and 7.74 ± 0.3 in L D B B. T h e s e measurements were made after the system had been left to equilibrate for a few months. It w a s 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 S e p t e m b e r 28  th  (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 w a s restricted only to the effluent end of the m e s o c o s m s and by day 24, the water's surface w a s 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 m e s o c o s m s 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). T h e inlet water had a pH of 7.1 ± 0.1.  140  Mesocosm B  £  8.00  -I-  10  20  30  40  50  60  Time (days) 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 D O probe was placed in the sediment layer itself, then the reading w a s too low to register, which indicates that the sediment w a s anaerobic.  If it is a s s u m e d that plug flow characteristics were achieved in the m e s o c o s m s , then S 0 " reduction w a s observed. After a residence time of 15.8 days, all of the water in 2  4  the m e s o c o s m s would have been replaced with the inlet concentrated S 0 ~ water, 2  4  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 " 2  4  141  concentration w a s 1677.3 ± 107.9 and 1645.9 ± 106.8 mg L" S 0 " for m e s o c o s m s 1  2  4  A and B, respectively. If ideal plug flow behaviour were a s s u m e d , then this difference would be due to sulphate reduction by S R B in the sediment.  However ideal plug flow behaviour in the m e s o c o s m , where the residence time is the s a m e for all fluid elements (Wilkes 2006), was unlikely due to the positioning of the inlet manifold, which extended half the length of the m e s o c o s m . In addition, s o m e axial mixing and short-circuiting might have been occurring. S i n c e no tracer studies were performed, the actual flow characteristics of the m e s o c o s m are unknown.  If ideal plug flow characteristics are not present in the m e s o c o s m s then the estimated residence time of 15.8 days is incorrect. While the flow characteristics in the natural treatment m e s o c o s m s are unknown, it is strongly suspected that the S R B were briefly active a s 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 s o m e S 0 " reduction did occur, particularly in 2  4  m e s o c o s m B (Figure 35), or this sulphide was present in the sediment pore water, prior to t=0, and the actions of the pump c a u s e d it to be pushed out. A n increase of 29.6 mg L" T S , a s s e e n in m e s o c o s m B, only corresponds with a d e c r e a s e of 88.9 1  mg L" S 0 " . 1  2  4  142  Mesocosm A  3000 o> E O cn  2000 1000  20  40  60  Time (days)  3000  Mesocosm B  2000  •  E  L  *  *\ ^  Z  ^  1000 \  O  CO  20  40  60  Time (days) Figure 34 - S 0 " 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. 4  40 30 cn 20 E £  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 o n d a y 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 w a s observed in L a c du Bois (0.67 and 0.39 mg L" N H - N , Rocky and A l g a e locations in July 04, 1  3  respectively). However, the P 0 ~ levels are high considering the levels in the lake 3  4  were 1.72 and 0.52 mg L" P 0 ~ (Rocky and Algae locations in July 04, 1  3  4  respectively). These high levels of PO4 " are likely due to the addition of plant growth 3  fertilizer, as mentioned previously in Section 3.5.1, which was added to encourage the growth of the algae. T h e s e levels of PO4 ", although higher than what w a s 3  observed in the lakes, are lower than the levels present in the Postgate B media (349 mg L" P 0 " ) . A s s u m i n g that the Postgate B media d o e s not have inhibitory 1  3  4  levels of P 0 " , it is a s s u m e d that these levels of P 0 " in the m e s o c o s m s are not 3  4  3  4  detrimental to S R B growth.  144  Table 13 - Ammonia-nitrogen and ortho-P0 concentration in natural treatment system. Errors are the difference between two test results. 3  4  M e s o c o s m A mgL" M e s o c o s m B mgL" M e s o c o s m A mgL" M e s o c o s m B mgL"  1  NH NH P0 P0  3  1  1  1  -N -N " "  3  3  4  3  4  Initial 0.52 ± 0 . 1 2 0.83 ± 0.25 27.05 ± 0.45 23.75 ± 1.15  After 48 days 0.00 ± 0.00 0.00 ± 0.01 7.43 ± 0 . 1 2 4.92 ± 0 . 1 3  It is a s s u m e d that the continuous process w a s h e d out the nutrients from the m e s o c o s m , faster than the degradation of algae could replenish them. In addition, as the algae growing on the water's surface w a s diminished, no fresh algae were provided to the sediment layer for decay. A s the fluid dynamics of the m e s o c o s m system were unknown, and the kinetics of the algae decomposition and sulphate reduction were not available, it w a s decided to terminate the continuous process. Instead, the system would be operated a s a batch reactor, so as to measure rates of sulphate reduction and nutrient depletion. This approach would allow us to determine s o m e 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 w a s h e d away the nutrients in the m e s o c o s m , the sulphate pump w a s shut down and the system w a s allowed to operate a s a batch instead. The m e s o c o s m s received an amendment of C : N : P in a 250:5:1 ratio, suggested a s an appropriate nutrient feed to anaerobic reactors (Metcalf and Eddy 1991), using lactic acid, ammonium chloride and potassium phosphate in order to replace s o m e of the nutrients lost and stimulate the re-growth  145  of the a l g a e , w h i c h had all but d i s a p p e a r e d w h e n the continuous s y s t e m w a s running, a n d S R B . T h e c a r b o n addition w a s intended to reach similar levels to what is s u p p l i e d in P o s t g a t e B m e d i a w h i c h g i v e s a concentration of 1.03 g C L" but there 1  w a s only sufficient quantities of lactic a c i d a v a i l a b l e to i n c r e a s e the concentration by 0.76 g C L" . 1  T h e s y s t e m w a s initially monitored, in o n e location, h a l f w a y d o w n the length of the m e s o c o s m (100 c m ) a n d at a depth of 15 c m from the surface, which w a s c l o s e to the water/sediment interface. F o u r d a y s after the addition of nutrients, a l g a e growth w a s o b s e r v e d o n the water's surface, which d e v e l o p e d into thick green a l g a e completely c o v e r i n g the surface in o n e month's time. This a l g a e w a s a g a i n sent out to a n limnologist specialist for identification, with the results s h o w i n g that what had initially b e e n Cladophora  glomerata  a l g a e (the s p e c i e s found in the healthy L a c du  Bois), had b e e n r e p l a c e d by Nodularia B o i s T w i n . W h i l e Cladophora  spumegena  glomerata  (the s p e c i e s found in L a c du  h a s b e e n o b s e r v e d to o c c u p y b r a c k i s h  environments with salinities of 17 g L" it is primarily a freshwater s p e c i e s (van den 1  H o e k 1963). It is s u s p e c t e d that the rate at w h i c h the salinity i n c r e a s e d in the m e s o c o s m tanks w a s either too fast to allow the freshwater s p e c i e s to adapt, or this strain of Cladophora  w a s not c a p a b l e of adapting to this salinity. T h u s , the  introduction of this Cladophora treatment s y s t e m . Nodularia,  sp. to saline waters is unlikely to s u c c e e d in a a s mentioned before, is a c y a n o b a c t e r i a that  d e m o n s t r a t e s preference for saline environments. H o r s t m a n n (1975) found that the growth of nodularia  strains in salinities ranging from 0-30 g,L" , exhibited the highest 1  146  growth rates between 5-15 g L" . Nordin and Stein 1980) found that the Uodularia 1  strains showed no preference for the different cations (CI", C 0 " , or SO4 "), and the 2  2  3  growth rate w a s highest at salinities between 5-10 g L" . Nodularia is an undesirable 1  algae due to its toxicity.  After monitoring the sulphate levels in the water column, it became apparent that SO4 " reduction w a s not occurring. It w a s known that viable S R B were present in the 2  sediment of the m e s o c o s m s a s of day 57. A small microcosm experiment which placed sediment taken from the m e s o c o s m s 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 " change (mg L" ) 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 1  2  However, it appeared that even though there were S R B present in the sediment, they were unable to significantly affect the S 0 " present at the water-sediment 2  4  interface (Figure 36).  147  2500  Z,  o> E.  1500 -  CN 6 1000 to 500 -  0  J  ,  ,  ,  ,  ,  ,  ,  ,  ,  0  10  20  30  40  50  60  70  80  90  Time (days)  Figure 36 - S0 " 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. 2  4  S i n c e 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 " s h o w e d that the concentration in the sediment w a s 2  4  considerably lower than what w a s present in the water column (Table .15).  Table 15 - Comparison of SQ " levels in water column and sediment of natural system 2  4  S a m p l e s taken J a n 7, 2005 t=101 days  Water C o l u m n (15 c m depth, 100 cm length) S 0 " mg L" 2023.27 ± 2 8 . 1 2074.91 ± 24.0 2  4  Mesocosm A Mesocosm B  1  Sediment (5 cm from sediment/water interface, 100 c m length) S 0 " mg L 676.07 ± 16.6 2  1  4  388.42 ± 7 3 . 1  148  Therefore there is a strong, vertical S 0 ~ concentration gradient within the 2  4  m e s o c o s m . 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" at the beginning of the batch experiment. The large S 0 ~ 1  2  4  concentration gradient implies that vertical S 0 " diffusion is slow. This is why 2  4  negligible sulphate reduction w a s 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. T o test the rate of sulphate reduction within the sediment, a recycle system w a s initiated which pumped water from the water column into the sediment through the rigid pipe system. This w a s continued until the S 0 " levels in the sediment had increased, at 2  4  which point the recycle system w a s turned off and the sediment layer w a s monitored over time for S 0 " reduction at the inlet location (50 cm down the length of the 2  4  m e s o c o s m , 5 cm into the sediment from the water/sediment interface).  Unlike the water column, which s h o w e d no decrease in S 0 " over time, there w a s 2  4  an observable decrease in S 0 " in both m e s o c o s m s within the sediment at this 2  4  location (Figure 37). M e s o c o s m A showed a decrease of 755.1 ± 100.5 mg L" S 0 " 1  2  4  over 14 days within the pore water, while m e s o c o s m B exhibited a drop of 848.5 ± 1 5 2 . 1 mg L" S 0 " . After this drop, there w a s a slow slight increase in the 1  2  4  sulphate concentration. Eventually, the sulphate reduction r e c o m m e n c e d , albeit at a much slower rate than that measured initially. The increase in S 0 " could be due to 2  4  several things. First, water from the water column could have accidentally mixed  149  with the sediment layer during sampling, thus increasing the S 0 " levels in the 2  4  sediment, since the water column did not show SO4 " reduction and maintained its 2  high concentration. Sampling the pore water required inserting a tube into the sediment, which always disturbed the sediment. C a r e w a s taken to insert the tube without an e x c e s s of axial movement in order to minimize this disturbance.  Figure 37 - S0 " concentration in natural system sediment under batch conditions. Solid line is mesocosm A, dotted line is mesocosm B 2  4  150  T h e total sulphide in the s y s t e m i n c r e a s e d over time a s the S 0  2 4  " d r o p p e d , which is  a s e x p e c t e d w h e n S R B are reducing SO4 " (Figure 38). 2  250 -  A  _l  150  TS (mg  200 •  100 -  50 00  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.  T h e drop in sulphide during the middle of the run c o i n c i d e s with a n i n c r e a s e in S 0 ~ 2  4  a n d could be due to a re-oxidation of sulphide to S 0 " . B e t w e e n d a y s 15-49 the 2  4  m e s o c o s m s e x p e r i e n c e d drops of 66.2 ± 5.9 a n d 39.2 ± 13.0 m g L" T S 1  ( m e s o c o s m s A a n d B, respectively). A s s u m i n g that the sulphide w a s re-oxidized completely back into S 0 " , a n i n c r e a s e of 198.7 a n d 117.8 m g L" 2  S0  1  4  2 4  "  ( m e s o c o s m s A a n d B, respectively) would be e x p e c t e d in the pore water of the m e s o c o s m s . During this s a m e time, m e s o c o s m A underwent a S 0  2 4  " i n c r e a s e of  264.1 ± 48.3 mg L" S 0 ~ , a n d m e s o c o s m B underwent a slight i n c r e a s e in S 0 1  2  4  67.5 mg L" S 0 1  2 4  2 4  " of  " b e t w e e n day 15-36 followed by a drop of  4 6 2 . 8 6 ± 83.5 mg L" S 0 " . Within the error range, the A S " c o r r e s p o n d s to A S 0 1  2  4  2  2 4  "  151  The pH in the m e s o c o s m s ' sediment (Figure 39), while it did fluctuate, always remained in the optimum range for S R B .  9.00 -,  7.00 -I 0  ,  r-  ,  ,  ,  ,  ,  ,  ,  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 s o m e conclusions can be made here. First, sulphate reduction w a s observed within the sediment, over the first 15 days. However, after this time the SO4 " slowly 2  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 m e s o c o s m were unable to survive at high salinities and were also insufficient for supplying nutrients to support continuous SO4 " reduction. 2  4.3.3 Agricultural Material Treatment System  In the previous experiment, natural sediment removed from L D B was used a s 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 i s c u s s e d in the previous section, A s mentioned in Section 2.3.2.5, many different agricultural materials have been used, with varying s u c c e s s , 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 w a s used in a scaled-up m e s o c o s m . T h e m e s o c o s m was operated a s a batch so a s to determine the sulphate reduction and C O D removal rates. Unfortunately, there w a s not enough time to run a continuous system.  Two m e s o c o s m s were set-up that received sediment from L a c du Bois a s an inoculum source. However, in contrast to the natural system which used algae, both alive and degrading, a s a nutrient source, the agricultural system used a 1:1 (13 c m layer) mixture of silage and hay. Initially, the hay/silage mixture floated on the water's surface but gradually b e c a m e water saturated over time. T h e system w a s run a s a batch operation with close monitoring of the nutrient levels within the mesocosm.  T h e pH within the m e s o c o s m s remained stable and well within the acceptable levels for S R B growth after being adjusted at the initiation of the experiment (Figure 40). T h e only time the pH dropped w a s after an addition of silage and hay w a s made to the m e s o c o s m s in order to increase the C O D levels. The pH in the m e s o c o s m  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 0  ,  20  :  r~  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 m e s o c o s m s (Figure 41 and Figure 42). While the levels reached in the agricultural m e s o c o s m s were high, they were less than what is provided to the S R B in Postgate B media (349 mg L" P 0 " and 337 mg 1  3  4  L" N H C I ) , a commonly used media for growing S R B , so it can be a s s u m e d that the 1  4  levels are non-inhibitory. Unlike the ortho-PGv " which appeared to generally follow a 3  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 w a s utilized by the S R B .  154  Time (Days)  Figure 41 - Average ammonium-nitrogen concentration versus time in mesocosms. is the agricultural system, • is the natural treatment system. Error bars represent the difference between the two replicate mesocosms. A  Figure 42 - Average ortho-P0 concentration in mesocosms versus time. is the agricultural mesocosm, • is the natural treatment mesocosm. . Error bars represent the difference between the two replicate mesocosms. 3  A  4  155  In contrast to the natural m e s o c o s m systems, where SO4 " reduction w a s observed 2  only in the sediment, in the agricultural treatment system SO4 " reduction w a s 2  observed in the water column. S a m p l e s taken halfway down the length of the m e s o c o s m and at a depth of 15 c m demonstrated that the system w a s capable of reducing S 0 " (Figure 43) 2  4  Time (days)  Figure 43 - Average S0 " and total sulphide concentrations in agricultural mesocosms versus time. • and • represent S 0 and TS concentrations, respectively, Error bars represent the difference between the two replicate mesocosms. 2  4  2  4  A s Figure 43 illustrates, SO4 " and T S concentrations in the two replicate 2  m e s o c o s m s followed the s a m e trends as evidenced by the relatively small error bars. The maximum S 0 " reduction rate after the first addition of S 0 " w a s 2  4  2  4  33.8 ± 2.3 mg L d " (R =0.99) calculated from the slope of a straight line fitted 1  1  2  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 a s 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" . O n day 82 a s e c o n d 1  addition of sulphate was made after which a much larger maximum S R R of 132.4 + 8.2 mg L" d" (R =0.97) w a s measured using data points for the first 7 days. 1  1  2  Another indicator that the S R B were successful in reducing the S 0 " present in the 2  4  system is the increasing concentration of sulphide in the m e s o c o s m . O v e r the course of the two additions of SO4 " mg L" total sulphide in the two m e s o c o s m s 2  1  followed an increasing trend to reach an average of 323.5 mg L" T S at the end of 1  the experiment (day 101).  The s C O D in the agricultural m e s o c o s m s followed a decreasing trend over time, which w a s expected, a s 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.  T h e second addition of silage and hay w a s soaked in H 0 prior to being added to 2  the m e s o c o s m s 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 w a s rapidly reduced. The average A S 0 7 A s C O D ratio was 1.005. 2  4  158  4.3.4 Agricultural Mesocosm System Failure  After the s e c o n d d e c r e a s e in S 0  2 4  ' a third addition of S 0 " was made. However, 2  4  instead of following the previous trend of decreasing o v e r t i m e , instead the S 0 " 2  4  levels began to increase. Simultaneously, sulphide levels d e c r e a s e d . From day 109/110 to day 157, sulphate increased by 704.1 ± 136.0 and 768.1 ± 42.1 for m e s o c o s m s A and B, respectively (Figure 45). During this time, s C O D w a s still being c o n s u m e d (Figure 46). Thus there was still bacterial activity in the system. Sulphide levels dropped 256.8 and 287.8 mg L" T S in m e s o c o s m s A and B, 2  respectively, which corresponds to an increase in S 0 " of 778.2 and 2  4  872.3 mg L" S 0 " for m e s o c o s m s A and B, respectively. It is clear that re-oxidation 1  2  4  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 m e s o c o s m (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 d e c r e a s e d and vice versa.  159  S0 ~ 2  4  100  150  200  250  300  Time (days)  Figure 45 - Average S 0 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. 2  4  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, a s stated in Table 16. Table 16 - Changes made to the agricultural mesocosms  Date of Event t= 159 days t=174 days  Event A d d a 13 cm layer of silage and hay (1:1 wt%) Placed a tarp over the system to restore anaerobic conditions and prevent S ' oxidation. A d d e d silage leachate to increase s C O D levels. 2  t=222 days  A tarp w a s placed over the system to exclude air and block out sunlight, which could c a u s e phototropic sulphur oxidizers to grow. The white slimy layer on the surface of the m e s o c o s m s visually disappeared over a 2-week period, but S 0 " concentrations 2  4  continued to increase. Since s C O D levels remained low (Figure 46), silage leachate w a s added on day 222.  After performing all these amendments, samples of sediment and water were removed from the m e s o c o s m , filtered, and then the filter paper w a s 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 m e s o c o s m 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 M e s o c o s m Experiments  Within the pore water of the natural treatment m e s o c o s m s , located a b o v e the buried inlet pipes, S R R s of 53.9 and 60.6 mg L" d" S 0 1  1  2 4  " were a c h i e v e d for the first 14  d a y s of operation ( m e s o c o s m s A & B, respectively). However, these rates of sulfate  162  reduction d e c r e a s e d after 14 d a y s , likely due to a lack of nutrients ( s C O D , N , P ) and eighty-four d a y s w e r e required to a c h i e v e a total S 0  2 4  " drop of at least 1000 m g L"  1  in both m e s o c o s m s in the batch s y s t e m . A s s u m i n g that a drop of 1000 m g L" SO4 " 1  2  is required in the final treatment s y s t e m , a n d a s s u m i n g that the initial high rates of SO4 " reduction could b e m a i n t a i n e d , a r e s i d e n c e time of 19 d a y s w o u l d be 2  sufficient. However, the natural treatment s y s t e m w a s u n a b l e to supply sufficient nutrients n e e d e d to continue the activity a n d the s y s t e m b e c a m e inactive.  A l t h o u g h sulfate reduction w a s occurring in the sediment, strong vertical SO4 " 2  concentration gradients indicated that diffusion from the water c o l u m n into the s e d i m e n t w a s very slow. T h u s in a treatment s y s t e m , high sulfate water would have to be directed through the s e d i m e n t layer.  It w a s o b s e r v e d in this experiment that the s u b s u r f a c e infusion pipes o c c a s i o n a l l y e x p e r i e n c e d clogging. A n alternative a p p r o a c h , utilized by Mclntire, E d e n b o r n et al. (1990) w h o a l s o e x p e r i e n c e d clogging with s u b s u r f a c e infusion pipes, involves placing a p i e c e of plexiglass in the m e s o c o s m , situated s o that the water is d a m m e d behind the plexiglass a n d is f o r c e d to flow d o w n w a r d s , b e n e a t h the water c o l u m n a n d through the sediment. A n o t h e r a d v a n t a g e to this s y s t e m is that the water would be introduced through the entire width of the sediment, rather than a l o n g o n e narrow region, a s with the s u b s u r f a c e infusion pipe. S i n c e the s e d i m e n t acts a s a barrier to diffusion, it is n e c e s s a r y 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 " reduction rate in the agricultural m e s o c o s m s after the 2  first addition of SO4 " w a s 33.8 mg L~ d"\ while after the s e c o n d sulphate addition 1  2  the rate increased to 132.4 mg L" d" . The rates obtained after the first SO4 " addition 1  1  2  are higher than what w a s obtained in the leachate experiments (19.4 mg L" d" raw 1  1  barley leachate), which w a s 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 s e c o n d 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 m e s o c o s m .  Sulphide is the product released by the dissimilatory sulphate reduction of sulphate by S R B . In the natural lake system of L a c du Bois, low levels of sulphide were observed in the water column. Sulphide in lakes is typically oxidized spontaneously by chemical oxidation in aerobic z o n e s or by the actions of chemolithotrophic bacteria or phototrophic bacteria, otherwise known a s sulphur oxidizing bacteria ( S O B ) into S° and S 0 " species (Thy, Fritz et al. 2000; Tonolla, Peduzzi et al. 2  4  2004). Sulphide c a n also be removed from the water column by mixing with a metal ion in solution, such a s iron, which will precipitate the sulphide a s a metal sulphide.  164  In the agricultural m e s o c o s m s , the sulphide levels increased rapidly until S 0 ~ 2  4  reduction stopped. The sulphide concentration increased faster than it could be removed through natural methods s u c h a s sulphur oxidizing bacteria or volatilization of H S gas. 2  Hydrogen sulphide is well recognized a s 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 m e s o c o s m s (297.97 and 350.87 mg L" T S ) are well above where inhibition has been observed 1  for S R B . T h e s e increased levels of sulphide are one obvious reason why the S 0 ~ 2  4  reduction stopped.  In addition to inhibiting S R B and slowing down the rate of S 0 ~ reduction, it is 2  4  believed that the high levels of sulphide also contributed to increasing the levels of S 0 " within the m e s o c o s m s . After 109 days, the S 0 " and the sulphide underwent 2  2  4  4  cycling, whereby the S 0 " and sulphide concentrations would alternate increasing 2  4  and decreasing. O n e obvious explanation for this is that S O B , demonstrated to be present in the m e s o c o s m by M c L e a n (2005), were oxidizing the sulphide in the system back into its S 0 " form. O n c e the sulphide levels had d e c r e a s e d , the S R B 2  4  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 " 2  4  165  inhibition and cycling from occurring is to remove sulphide from the m e s o c o s m a s quickly a s possible. In a continuous system, sulphide would not accumulate in the system. However, it would be n e c e s s a r y to remove sulphide from the effluent before the water could be released into the environment or fed to cattle.  O n e way to treat high sulphide concentrations in the effluent of a SO4 " reduction 2  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 c a n then be removed (Janssen, M a et al. 1997). O n e simple S O B reactor design w a s developed by Fox and Venkatasubbiah 1996) a n d 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 " to sulphide and then recycled sulphide treated water back to 2  the anaerobic system. By limiting the oxygen m a s s 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" sulphide. 1  T h e A S 0 4 7 A s C O D ratio for the m e s o c o s m s were 1.10 and 0.91 for m e s o c o s m s A 2  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 7 A C O D ratio of 1.5 gg" for 2  1  4  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 m e s o c o s m s 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 m e s o c o s m design is more appropriate for use in the field by farmers. T h e agricultural m e s o c o s m did not exhibit the s a m e m a s s transfer limitations a s the natural treatment system s o additional equipment is not needed to recycle water through the sediment. With the agricultural treatment system, the hay/silage w a s mixed with the sediment, thus increasing the porosity of the sediment and allowing water to have greater a c c e s s to the S R B . T h e agricultural material also provided more bioavailable nutrients. The agricultural m e s o c o s m s were also able to achieve the fastest rate of SO4 " reduction, measured after the 2  s e c o n d addition of SO4 ". However, if such a system were to be used successfully in 2  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 S i t u E x p e r i m e n t  Due to the s u c c e s s in using hay and silage as a nutrient source for S R B in both the agricultural bottle and m e s o c o s m experiments, one final experiment w a s 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 " concentrated lake, located in the L a c du Bois 2  G r a s s l a n d s Park. Unlike the bottle and m e s o c o s m 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 s e e n on the map presented in the materials and methods section, is in the northern region of the L a c du Bois G r a s s l a n d s Park. It is not easily accessible by road and is surrounded by sparse forests. Lake 52 is on private land and permission w a s obtained from the landowner to perform experiments within the lake.  The lake's surface area is estimated to be 2.4 x 1 0 m , which m a k e s it smaller than 5  2  L a c du Bois, but larger than L a c 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 w a s a 2.5 c m deep sediment layer followed by a 30.9 c m 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 L a c du Bois Twin. S e v e r a l 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 e a c h bucket (Table 17).  168  Table 17 - Visual observations of in-situ buckets after 311 days  #1 - Control  #2 - Inoculated Hay/silage/fertilizer and decaying algae/sediment from L a c du Bois #3 - Inoculated Hay/silage/fertilizer and decaying algae/sediment from L a c du Bois #4 - A m e n d e d Hay/silage/fertilizer  #5 - Control #6 - A m e n d e d Hay/silage/fertilizer  C o u l d s e e the sediment bottom. No signs of plant life, although a few small red insects were observed swimming. S a m e as w a s observed outside the buckets. This bucket had unfortunately tipped over and w a s no longer useable for the experiment.  All of the hay and silage had sunk and was well mixed with the added L D B sediment  Significant red tint to the water. Likely c a u s e d 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 w a s well mixed with the added L D B sediment C o u l d s e e the sediment bottom. N o signs of life. S a m e a s w a s observed outside the buckets. 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 w a s 11.9 and 13.9 °C at sites 1 and 2, respectively. T h e pH at each site w a s 9.67 and 9.32 at sites 1 and 2, respectively. After 311 days (August 11, 2005) the average temperature within the lakes w a s 23.6 and 21.2 °C.  169  The pH w a s 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  9.33  9.39  #1 Control 9.40  #3 Inoculated 9.40  #4 Amended 8.91  #5 Control 9.33  #6 Amended 9.34  Using A N O V A and Tukey tests to compare the p H s indicated that there were significant differences, not only between s o m e of the buckets containing different treatments, but also within the replicates (P<0.0001 a=0.05). What w a s particularly interesting w a s that, with the exception of the #4 A m e n d e d bucket, buckets situated next to e a c h other spatially were not significantly different. The pH of the lake water at site 2, the location for the in-situ experiment, w a s not significantly different from #1 Control, or #3 Inoculated. This suggests that the spatial location within the lake played an important role in the p H .  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 p H s between 6-9. Only recently have a few S R B species, Desulfonatronum the Desulfovibrio  alkalitolerans  cooperativum,  Desulfotomaculum  alkaliphilum  and  been discovered and identified as alkaliphilic (Pikuta,  Lysenko et al. 2000; Zhilina, Zavarzina et al. 2005; Abildgaard, Nielsen et al. 2006). Desulfonatronum  cooperativumare  greater than 10, while Desulfovibrio  is an obligate alkaliphile and requires a p H alkalitolerans  can tolerate lower p H s from 6-9.9,  with an ideal pH range from 9-9.4.  170  The dissolved oxygen (DO) w a s measured within the lake at the two location sites at the start of the experiment in October 2004. The D O , 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 w a s suspicious of the high D O readings found in Lake 52 and L a c 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, especially 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 d e c r e a s e the maximum amount of oxygen that can be dissolved in water, which m e a n s that the water can become saturated at lower concentrations of dissolved oxygen. However, during the sampling trip on day 311,1 w a s still under the impression that the D O probe w a s not functioning correctly in highly saline lakes, so I instead used the Winkler colorimetric method to test the D O in the lakes and the buckets. O n e of the steps in the test is to add a sulphuric acid reagent to the water sample, which c a u s e s 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 D O , this c a n take several minutes. W h e n this test w a s 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 D O concentration within the experiment, except to report that the D O w a s 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 S o u n d , North Carolina.  Sulphate and Sulphide  At the beginning of the experiment in October 2004, S 0 " levels were found to be 2  4  4188.7 ± 165.6 and 4156.0 ±179.5 mg L" and T S to be 0.14 ± 0.10 and 1  0.05 ± 0.02 mg L" at Sites 1 and 2, respectively. It w a s a s s u m e d that the levels of 1  S 0 ~ and T S in the buckets would be similar and there w a s no need to test these 2  4  levels individually in each bucket.  After 311 days, the buckets were sampled for SO4 " and T S (Figure 4 8 , Figure 49). 2  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 " levels. The results from an A N O V A test indicate that there w a s at 2  4  least one significantly different S 0 " m e a n (P=0.0001, a=0.05) while an additional 2  4  Tukey test found that #4 A m e n d e d and #5 Control had significantly higher S 0 " 2  4  m e a n s than the other buckets and the lake.  172  6000  6  — 4500  T  was*  li  ii If  O)  E. 3000  o  "mi  "PA  w 1500  M  "/''•>'  of  tr  tr  Figure 48 - S 0 " concentration in Lake 52. Taken on day 311. Average and st. dev. from 5 or more samples. 4  Sulphide levels in the in situ buckets, in contrast to the S 0 " levels, were all 2  4  significantly higher than in the lake ( A N O V A , p=0.0001 a=0.05, Tukey test). Amongst the buckets, #4 A m e n d e d w a s significantly different from all other buckets.  50 00 50 00  CD  E  50  CO  I-  1 0 0.  38.71  00 7.40  50 00 of  tr IT  9  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 s a m p l i n g of L a k e 52 found levels of N H - N (0.39 ± 0.03 m g L" ) similar to 1  3  t h o s e of L D B (0.44 ± 0.03 mg L" ) and L D B Twin (0.5 ± 0.02 m g L~ ), N 0 " - N levels 1  1  3  (0.05 ± 0.01 m g L" ) slightly higher than found in those lakes ( L a c du B o i s 1  0.02 ± 0.02 m g L" , L D B Twin 0.02 ± 0.0 m g L" ), a n d P 0 ~ levels (0.06 ± 0.03 1  1  3  4  m g L" ) which were c o n s i d e r a b l y lower ( L D B 0.56 ± 0.14 mg L" , L D B Twin 1  1  5.00 ± 0.70 m g L" ). All the m e a s u r e m e n t s w e r e taken on the O c t o b e r 4 , 2 0 0 4 1  expedition. C o n s i d e r i n g that p h o s p h a t e and a m m o n i a are both n e c e s s a r y nutrients for S R B growth, it w a s c o n s i d e r e d important that s o m e nutrient addition be m a d e to i n c r e a s e these levels in the in-situ treatment s y s t e m . For this r e a s o n , a slow r e l e a s e fertilizer w a s a d d e d to all buckets with the exception of the controls. During the A u g u s t 2 0 0 5 trip, the levels of P 0 ~ a n d N H - N w e r e tested a g a i n (Figure 5 0 , Figure 3  4  3  51). O n c e a g a i n it a p p e a r s that spatial location within the lake h a s had a n effect on nutrient levels. All 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 r i  0.00  do  Figure 50 - Ortho-P0 " concentration in Lake 52. Average and st. dev. of 3 depths. 4  1.50 _  1.20  Li  E. 2  CO  0.90 fe  0.60  I Z  0.30  M  ¥0,  Mr**!  0.00  of  #  -c>  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" ± 2.5 (difference between Sites 1 1  and 2) in October 2004. T h e s e levels are lower than what w a s observed in either L a c du Bois (350.0 ± 23.5 mg L" s C O D ) or L a c du Bois Twin (170.2 ± 22.7 mg L" 1  1  175  s C O D ) . It w a s 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" due to the silage material alone. The s C O D w a s 1  analyzed in each bucket after 311 days (Figure 52). O n c e again buckets to the left of the #4 A m e n d e d do not differ significantly, while the #4 A m e n d e d and #5 Control are significantly different from the other buckets. The #6 A m e n d e d , while significantly different from the #4 A m e n d e d , is not significantly different from buckets #1 or #3 or from the lake water outside the experiment.  250 200 en E.  150  g  100  isp  mm  O W  i  fry  |||  50  Rtf of  •hit:,  .0/  <f  4? 4>  Figure 52 - Soluble COD concentrations in Lake 52. Average and st dev. of 3 depths.  4.4.3 Discussion of In Situ Experiment  T h e results from these tests are surprising. There appears to be s o m e inconsistency within the results obtained from the replicate buckets. The #1 Control bucket had nutrient levels, S 0 * and T S levels which mimic those of the lake outside the 2  4  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 w a s observed in the lake, #4 and #6 A m e n d e d buckets had much higher levels of P 0 " 3  4  and N H . This is interesting considering that these buckets received exactly the 3  s a m e materials a s the inoculated bucket, with the exception that no sediment from L a c du Bois w a s added as a source of S R B inoculation. Overall, this experiment w a s 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.  T h e lake should have been visited and sampled prior to constructing any in situ treatment system. O n e of the major problems I encountered w a s that I had no physical or chemical information concerning L a k e 52, before I arrived to set up the system. I did not know how deep the lake w a s , which impacted the size of the buckets used, nor did I know how hard the lake floor would be, which affected how I w a s to anchor the buckets within the lake. Finally, I didn't know the lake p H , which could have prompted me to bring chemicals to adjust the pH to within the tolerance levels for the majority of S R B s p e c i e s (6-9). At the time, it w a s 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 a s 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.  T h e steel wire which I used to anchor #4 A m e n d e d bucket to its metal posts had its ends s u s p e n d e d over the bucket. After 311 days, this wire had corroded and iron oxides were released into the bucket, contaminating the experiment. T h e water in this bucket had a red tint, and a large aquatic plant w a s observed growing within. The plant w a s 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 s u s p e n d e d over the buckets that can c a u s e contamination.  4.  T h e biggest mistake I made in this experiment, was making the assumption that sampling the lake at two locations w a s sufficient for determining the t=0 conditions within the buckets. E a c h of the buckets should have been sampled at t=0 for S 0 " , T S , ammonia, phosphate, 2  4  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 c a u s e d by something happening during the 311 d a y s of the experiment. 5.  I should have brought along a D O meter during the experiment's final sampling, in addition to bringing the Winkler colourimetric test, even if I w a s suspicious of the results. S i n c e the high D O concentration within the  178  lake caused the Winkler test to fail, it would have been beneficial to have the D O 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  Conclusions  1.  L a c du Bois and L a c du Bois Twin had several differences which may have contributed to their different salinities a. L a c du Bois Twin w a s saturated with dissolved oxygen while L a c 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 L a c du B o i s had a greater potential for reducing SO4 ". 2  c.  L a c du Bois Twin had a high pH and contained toxin producing Nodularia  spumigena,  creating a more toxic environment for plants and  animals. L a c du B o i s Twin had lower levels of dissolved organic compounds and s C O D thus providing less matter to provide electron donor c o m p o u n d s to support S R B . 2. a. After testing several agricultural materials for their effectiveness a s a nutrient source for S R B , it w a s determined that silage, whether prepared from orchard grass (18.5 mg SO4 " L" d" ), alfalfa (14.4 mg 2  1  1  S 0 " L" d" ), or barley (19.4 mg S 0 " L" d~ ), w a s an effective 2  1  1  4  2  1  1  4  substrate. T h e s e materials are suitable for a treatment system with the following attributes  180  i. c h e a p 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 " concentration in water to below 2  4  1000 mg  I/ . 1  The leachate alone w a s sufficient for supporting S R B . The physical m a s s of silage w a s 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. M o l a s s e s requires an addition of phosphate to initiate sulphate reduction. ii. Barley in its grain form w a s 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 m e s o c o s m s , containing 1:1 hay:silage, demonstrated two successful batch d e c r e a s e s of SO4 " with maximum S R R s of 2  33.8 mg L" d" after the first addition of S 0 " and 132.4 mg L / V after 1  1  2  4  the s e c o n d . If the second rate could be maintained, then a residence time of 8 days would be sufficient for lowering SO4 " concentrations by 2  1000 mg L" . 1  b. In the natural treatment system, after raising the levels of S 0 ~ in the 2  4  sediment, S R R s of 53.9 and 63.6 mg L" d" SO4 " were achieved in the 1  1  2  first 14 days in m e s o c o s m s A and B, respectively. After this time, a brief period of sulphur oxidation w a s observed followed by sulphate reduction at a much slower rate than first observed. It w a s concluded that the natural treatment system w a s unable to supply sufficient nutrients to sustain active S R B activity.  a. In the natural treatment m e s o c o s m , operating a s a continuous s y s t e m , the rate at which new water flowed through the system (6 mL min' ) 1  w a s h e d 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 m e s o c o s m s inhibited the S R B and halted the reduction of S 0 " . 2  4  i. Sulphur oxidizing bacteria were observed within the m e s o c o s m s , and it is suspected that they are responsible for converting the sulphide within the m e s o c o s m back into SO4 ". 2  182  ii.  In a continuous s y s t e m , the sulphide could be prevented from reaching lethal toxic levels, by limiting the amount of sulphate reduced.  5.2  Future W o r k  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 c o m p o u n d s 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 w a s 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 " water throughout the 2  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 m a k e s sampling easier, as well a s 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 S0 " (mg L- )  Average S0 " (mg L" )  0.463 0.254 0.170 0.377 0.353 0.325  1701.75 1121.40 888.14 731.47 698.15 329.64  1705.91 1139.44 875.65 740.50 692.60 340.40  1568.46 1468.50 1074.19  0.421 0.362 0.233  1585.12 1421.29 1063.08  1576.79 1444.89 1068.64  0.414 0.002  1565.68 421.64  0.413 0.174  1562.91 449.6256  1564.29 435.63  0.458 0.939 0.577  1687.86 1511.75 1009.15  0.432 0.376 0.569  1615.67 1460.16 998.04  1651.76 1485.96 1003.60  5 0.53 5 0.103 5 0.203 6 data N/A 7 0.386 7 0.139  1887.79 702.10 489.89  0.528 0.287 0.216  1882.24 606.51 507.94  1885.02 654.31 498.91  1655.30 957.81  0.267 0.148  1649.08 983.22  1652.19 970.52  2  Time (days)  Transfer #  6-Mar 11-Mar 16-Mar 20-Mar 24-Mar 31-Mar  0 5 10 14 18 25  1 1 1 1 1 1  0.466 0.267 0.161 0.39 0.345 0.356  1710.08 1157.49 863.15 749.52 687.04 351.16  4-Apr 13-Apr 16-Apr  0 9 12  2 2 2  0.415 0.379 0.237  18-Apr 28-Apr  0 10  3 3  29-Apr 11-May 17-May  0 12 18  4 4 4  20-May 28-May 7-Jun  0 . 8 18 Transfer 0 17  Date  19-Jul 5-Aug  O.D.  SG-4 " 2  (mg L- ) 1  O.D.  4  1  z  4  1  A1-2a. S 0 ' concentrations in Lac du Bois and Lac du Bois Twin 2  4  6-Jul-04  Lac du Bois S0 ' (mg L- )  Dil.  62.29 61.73 59.75 61.16  1.2 1.2 1.2 1.2  2  Depth (cm)  Sample  Dil.  O.D.  4  1  S0 " (mg L- )  Dil.  0.235 0.253 0.245 0.220  61.44 63.99 62.86 59.33  1.2 1.2 1.2 1.2  S0 (mg L- )  Average S0 " (mg L- )  St. Dev.  0.234 0.227 0.222 0.256  61.30 60.32 59.61 64.41  61.68 62.01 60.74 61.63  0.53 1.85 1.84 2.57  2  O.D.  4  1  2_  O.D.  4  1  2  4  1  10 20 30 40  LDB LDB LDB LDB  Rocky Rocky Rocky Rocky  1.2  0.241 0.237 0.223 0.233  5 10  LDB Algae LDB Algae  1.2 1.2  0.201 0.198  56.64 56.22  1.2 1.2  0.181 0.187  53.82 54.67  1.2 1.2  0.172 0.192  52.55 55.37  54.34 55.42  2.10 0.78  10 20 30 40  LDB LDB LDB LDB  Rocky Rocky Rocky Rocky  1.2 1.2 1.2 1.2  0.471 0.462 0.441 0.44  60.75 59.78 57.51 57.40  2 2 2 2  0.189 0.181 0.174 0.161  60.49 58.76 57.24 54.43  1.2 1.2 1.2 1.2  0.475 0.465 0.452 0.449  61.19 60.10 58.70 58.37  60.81 59.55 57.82 56.73  0.35 0.70 0.78 2.05  5 10 20  LDB Algae LDB Algae LDB Algae  1.2 1.2  0.43 0.324 0.442  56.32 44.85 57.62  2 2 2  0.163 0.161 0.168  54.86 54.43 55.94  1.2 1.2 1.2  0.426 0.422 0.437  55.89 55.45 57.08  55.69 51.58 56.88  0.75 5.85 0.85  O.D.  S0 " (mg L- )  Dil.  O.D.  so -  Dil.  O.D.  S0 ' (mg L- )  Average S0 " (mg L- )  0.396  6675.92  95.17  0.388  6586.34  1.019 0.103 0.391 0.099  over 6789.64 6619.94 6700.06  95.17 95.17 190.33 95.17  0.413 0.381 0.103 0.365  6866.29 6507.96 6789.64 6328.80  6744.85 6731.91 6642.33 6698.32 6575.14  6669.04 6799.10 6646.64 6702.63 6534.67  1.2 1.2 1.2  4-Oct-04  6-Jul-04  Lac du Bois Twin  Depth (cm) 5 10 15 20 25  ON  1.2  2  2  Dil.  Sample LDB LDB LDB LDB LDB  Twin Twin Twin Twin Twin  Site Site Site Site Site  1 1 1 1 1  95.17 47.58 190.33 95.17 190.33  4  1  4  (mg L" ) 1  2  190.33 95.17 95.17 95.17 95.17  0.101 0.401 0.393 0.398 0.387  4  1  2  4  1  St. Dev. 79.48 140.89 84.93 188.91  Depth (cm) 5 10 15 20 25 4-Oct-04 5 10 15 20 25 5 10 15 20 25  95.17 95.17 95.17 190.33 190.33  0.386 0.381 0.403 0.101 0.095  6563.95 6507.96 6754.31 6744.85 6610.48  95.17 95.17 95.17 95.17 95.17  0.391 0.387 0.412 0.385 0.384  6619.94 6575.14 6855.09 6552.75 6541.55  190.33 190.33 95.17 95.17 95.17  0.092 0.098 0.428 0.37 0.386  6543.30 6677.67 7034.25 6384.78 6563.95  Average S0 " (mg L' ) 6575.73 6586.92 6881.22 6560.79 6571.99  1 1 1 1 1  80 80 80 80 80  0.635 0.597 0.641 0.666 0.672  6279.75 5950.85 6331.68 6548.06 6600.00  160 160 160 160 160  0.289 0.303 0.281 0.295 0.297  6570.06 6812.41 6431.57 6673.92 6708.54  80 80 80 80 80  0.635 0.661 0.653 0.671 0.662  6279.75 6504.79 6435.54 6591.34 6513.44  6376.52 6422.68 6399.60 6604.44 6607.33  167.61 436.61 58.85 63.94 97.76  Site 2 Site 2 Site 2 Site 2 Site 2  80 80 80 80 80  0.655 0.676 0.672 0.62 0.653  6452.86 6634.62 6600.00 6149.92 6435.54  160 160 160 160 160  0.287 0.292 0.284 0.285 0.289  6535.44 6621.99 6483.51 6500.82 6570.06  80 80 80 80 80  0.671 0.673 0.664 0.651 0.661  6591.34 6608.65 6530.75 6418.23 6504.79  6526.54 6621.75 6538.08 6356.32 6503.46  69.67 12.98 58.59 183.46 67.27  Sulphide (mg L- )  Dil.  O.D.  0.04 0.06 0.19 0.02  5 5 2 5  S0 " (mg L- )  Dil.  2  Dil.  Sample LDB LDB LDB LDB LDB  Twin Twin Twin Twin Twin  Site 2 Site 2 Site 2 Site 2 Site 2  LDB LDB LDB LDB LDB  Twin Twin Twin Twin Twin  Site Site Site Site Site  LDB LDB LDB LDB LDB  Twin Twin Twin Twin Twin  O.D.  4  1  S0 ' (mg L- )  Dil.  2  O.D.  4  1  O.D.  SO4 " 2  2  (mg L- ) 1  4  1  St. Dev. 39.65 85.47 141.79 180.17 35.16  A1-2b. Total sulphide concentrations in Lac du Bois and Lac du Bois Twin 6-Jul-04 Depth (cm) 10 20 30 40  SO  Lac du Bois Sample LDB LDB LDB LDB  Rocky Rocky Rocky Rocky  Dil. 1 1 1 1  O.D. 0.007 -0.004 0.014 0.009  Sulphide (mg L- )  Dil.  0.04 0.01 0.06 0.04  2 2 5 2  1  O.D. 0.001 0.004 0.007 -0.004  1  Average Sulphide Sulphide (mg L- ) (mg L- ) 1  1  -0.006 -0.001 0.014 -0.001  0.02 0.08 0.11 0.08  0.03 0.05 0.12 0.05  St. Dev. 0.01 0.04 0.07 0.03  Depth (cm)  Sample  5 10  LDB Algae LDB Algae  1 1  LDB LDB LDB LDB  Rocky Rocky Rocky Rocky  LDB Algae LDB Algae  4-Oct-04 10 20 30 40 5 10 6-Jul-04 Depth (cm)  vo OO  Sulphide (mg L- )  Dil.  0.074 0.049  0.22 0.15  2 2  1 1 1 1  0.016 0.013 0.175 0.238  0.01 0.01 0.37 0.51  1 1  0.096 0.101  0.19 0.20  Dil.  O.D.  Dil.  O.D.  Average Sulphide Sulphide (mg L' ) (mg L- ) 0.27 0.24 0.30 0.20  Sulphide (mg L- )  Dil.  0.037 0.024  0.24 0.17  5 5  0.013 0.015  1 1 1 1  0.017 0.011 0.166 0.225  0.02 0.00 0.35 0.48  2 2 2 2  0.02 0.01 0.089 0.111  0.05 0.00 0.35 0.45  0.02 0.00 0.35 0.48  0.02 0.00 0.01 0.03  1 1  0.121 0.096  0.25 0.19  2 2  0.062 0.037  0.23 0.12  0.22 0.17  0.03 0.04  Sulphide (mg L- )  Dil.  O.D.  Sulphide (mg L' )  Dil.  O.D.  1  O.D.  1  O.D.  1  1  St. Dev. 0.03 0.08  Lac du Bois Twin Sample  1  1  Average Sulphide Sulphide (mg L- ) (mg L- ) 1  1  St. Dev.  5 10 15 20  LDB LDB LDB LDB  Twin Twin Twin Twin  Site Site Site Site  1 1 1 1  1 1 1 1  0.094 0.06 0.762 0.41  0.27 0.18 2.05 1.11  2 2 2 2  0.07 0.029 0.442 0.248  0.41 0.19 2.40 1.36  1 1 5 5  0.112 0.065 0.171 0.112  0.32 0.19 2.38 1.59  0.33 0.19 2.28 1.36  0.07 0.01 0.19 0.24  5 10 15 20  LDB LDB LDB LDB  Twin Twin Twin Twin  Site 2 Site 2 Site 2 Site 2  1 1 1 1  0.077 0.057 0.036 0.264  0.22 0.17 0.12 0.72  2 2 2 2  0.097 0.037 0.018 0.109  0.56 0.24 0.13 0.62  1 5 5 5  0.123 0.019 0.01 0.048  0.35 0.35 0.23 0.74  0.38 0.25 0.16 0.69  0.17 0.09 0.06 0.06  4-Oct-04 Lac du Bois Twin Depth (cm) 5 10 15 20  Sample LDB LDB LDB LDB  Depth (cm) 5 10 15 20  Twin Twin Twin Twin  Site Site Site Site  1 1 1 1  Sample LDB LDB LDB LDB  Twin Twin Twin Twin  Site 2 Site 2 Site 2 Site 2  Dil.  O.D.  Sulphide (mg L" )  Dil.  O.D.  Sulphide (mg L" )  Dil.  O.D.  1 1 1 1  0.101 0.097 0.031 0.53  0.20 0.19 0.05 1.15  2 2 2 2  0.054 0.035 0.017 0.21  0.20 0.11 0.03 0.89  1 1 1 1  0.086 0.094 0.033 0.56  Dil.  O.D.  Sulphide (mg L" )  Dil.  O.D.  Sulphide (mg L' )  Dil.  O.D.  0.10 0.07 0.05 0.17  2 2 2 1  0.14 0.05 0.05 0.15  1 1 1 2  1 1 1 1  1  1  0.057 0.042 0.032 0.087  1  1  0.041 0.022 0.021 0.079  Average Sulphide Sulphide (mg L- ) (mg L- ) 0.17 0.19 0.19 0.16 0.04 0.05 1.22 1.09 1  1  Average Sulphide Sulphide (mg I.") (mg L- ) 0.11 0.12 0.06 0.06 0.04 0.05 0.17 0.18 1  1  0.061 0.037 0.028 0.051  A1-2c. Ammonia-nitrogen, nitrate-nitrogen, ortho-phosphate and sCOD concentrations in Lac du Bois and Lac du Bois Twin  6-Jul-04 LDB Rocky LDB Algae LDB Twin Site 1 LDB Twin Site 2  Dil.  O.D.  NH -N (mg L- )  Dil.  3  1  O.D.  NH -N (mg L' )  Dil.  3  1  O.D.  NH -N (mg L" )  Average NH -N (mg L- )  St. Dev.  3  1  3  1  1 1 1  0.72 0.39 0.55  0.72 0.39 0.55  2 2 2  0.31 0.19 0.3  0.62 0.38 0.6  1 1 1  0.68 0.4 0.54  0.68 0.4 0.54  0.67 0.39 0.56  0.05 0.01 0.03  1  0.49  0.49  2  0.24  0.48  1  0.51  0.51  0.49  0.02  St. Dev. 0.02 0.05 0.01 0.18 St. Dev. 0.02 0.01 0.01 0.01  LDB Rocky LDB Algae LDB Twin Site 1 LDB Twin Site 2  1 1 1  0.72 0.46 0.59  0.72 0.46 0.59  2 2 2  0.41 0.22 0.29  0.82 0.44 0.58  1 1 1  0.75 0.41 0.49  0.75 0.41 0.49  Average NH -N (mg L- ) 0.76 0.44 0.55  1  0.48  0.48  2  0.26  0.52  1  0.49  0.49  0.50  6-Jul-04  Dil.  O.D.  N0 "-N (mg L- )  Dil.  O.D.  N03-N (mg L- )  Dil.  O.D.  NO3-N (mg L- )  Average NO3-N (mg L" )  St. Dev.  4-Oct-04  LDB Rocky LDB Algae LDB Twin Site 1 LDB Twin Site 2  O O  Dil.  O.D.  NH -N (mg L- )  Dil.  3  1  3  1  O.D.  NH -N (mg L- )  Dil.  3  1  1  O.D.  NH -N (mg L- ) 3  1  1  3  St. Dev.  1  0.05 0.03 0.06 0.02  1  1 1 1  0.02 0.04 0.02  0.02 0.04 0.02  2 2 2  0 0 0  0 0 0  1 1 1  0.01 0.01 0  0.01 0.01 0  0.01 0.02 0.01  0.01 0.02 0.01  1  0.02  0.02  2  0.01  0.02  1  0.02  0.02  0.02  0.00  4-Oct-04  Dil.  O.D.  N0 '-N (mg L- )  Dil. 2 2 2 2  3  1  LDB Rocky LDB Algae LDB Twin Site 1 LDB Twin Site 2  1 1 1 1  0.03 0.03 0.01 0.02  0.03 0.03 0.01 0.02  6-Jul-04  Dil.  O.D.  P0 " (mg L- )  Dil.  0 0.01 0 0  0 0.02 0 0  1 1 1 1  0.02 0.04 0.01 0.01  0.02 0.04 0.01 0.01  O.D.  P0 ' (mg L- )  Dil.  O.D.  P0 ' (mg L' )  Average P0 ' (mg L" )  1  O.D.  3  4  1  1  3  4  1  St. Dev.  1  3  4  0.02 0.01 0.01 0.01  St. Dev.  1  LDB Rocky LDB Algae LDB Twin Site 1 LDB Twin Site 2  1 1 1 1  1.91 0.51 2.5 over  1.91 0.51 2.5 >3  10 10 10 10  0.18 0.06 0.31 0.41  1.8 0.6 3.1 4.1  2 2 2 5  0.72 0.22 1.4 0.88  1.44 0.44 2.8 4.4  1.72 0.52 2.80 4.25  0.25 0.08 0.30  4-Oct-04 LDB Rocky LDB Algae LDB Twin Site 1 LDB Twin Site 2  10 1 10 10  0.24 0.42 0.28 0.53  2.4 0.42 2.8 5.3  5 10 5 5  0.49 0.07 0.64 1.1  2.45 0.7 3.2 5.5  1 5 1 20  2.6 0.11 over 0.21  2.6 0.55 >3 4.2  2.48 0.56 3.00 5.00  0.10 0.14  4-Oct-04  Dil.  O.D.  sCOD (mg L- )  Dil.  O.D.  sCOD (mg L- )  Dil.  O.D.  sCOD (mg L' )  Average sCOD (mg L- )  St. Dev.  331.44 361.12 190.45 158.30  2 2 2 2  321.54 385.85 202.82 128.62  1 1 1 1  326.49 373.48 185.51 155.82  326.49 373.48 192.93 147.58  4.95 12.37 8.92 16.47  LDB Rocky LDB Algae LDB Twin Site 1 LDB Twin Site 2  O  1  Average NO3-N (mg L" ) 0.02 0.03 0.01 0.01  Dil.  3  4  NO3-N (mg L- )  N03-N (mg L' )  O.D.  1 1 1 1  0.134 0.146 0.077 0.064  1  0.065 0.078 0.041 0.026  1  0.132 0.151 0.075 0.063  1  0.70  1  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 " concentrations in SRB growth without pH adjustment experiment 2  4  Hay 1 Date 7-Aug 23-Aug 9-Sep 21-Oct 24-Jan  O  S0 " (mg L )  Dil.  1662.98 1433.62 1357.85 1235.54 1087.25  20 20 25 40 25  2  Time (Days)  Dil.  0 16 33 75 170  20 20 20 20 25  O.D. 0.678 0.572 0.538 0.484 0.321  4  1  O.D. 0.68 0.594 0.396 0.196 0.305  SO4 '  S0 " (mg L- )  Average S0 ^ (mg L- )  St. Dev.  1610.80 1475.56 1210.50 1259.39 1075.97  1647.03 1463.47 1291.59 1224.53 1068.53  31.45 26.01 74.79 41.48 23.34  2  2  (mg L- ) 1  1667.31 1481.22 1306.43 1178.65 1042.38  Dil. 25 25 25 33 20  O.D. 0.505 0.455 0.357 0.268 0.41  4  1  4  1  Hay 2  25 25 25 33 25  0.506 0.453 0.425 0.249 0.362  1613.51 1470.15 1386.26 1190.35 1212.84  25 25 25 40 25  0.554 0.486 0.413 0.216 0.345  1743.34 1559.41 1353.22 1268.40 1154.57  20 20 25 33 20  0.682 0.639 0.39 0.273 0.42  1671.64 1578.59 1289.91 1277.56 1097.99  1699.86 1561.38 1356.42 1309.12 1149.73  38.20 16.32 68.16 62.76 49.50  1686.78 1511.51 1439.33 1426.27 1334.07  20 25 25 40 25  0.706 0.484 0.441 0.22 0.395  1723.57 1554.00 1430.30 1286.35 1294.80  25 20 25 33 20  0.567 0.622 0.42 0.306 0.505  1778.50 1541.81 1372.49 1397.47 1285.18  1729.62 1535.77 1414.04 1370.03 1304.68  46.16 21.88 36.26 73.88 25.90  1610.80 1505.02 1410.70 1240.03 1115.30  20 20 25 40 25  0.673 0.609 0.425 0.18 0.332  1652.16 1513.68 1386.26 1106.85 1118.10  20 25 25 33 20  0.647 0.465 0.372 0.26 0.385  1595.90 1502.61 1240.36 1230.32 1020.92  1619.62 1507.10 1345.77 1192.40 1084.77  29.15 5.82 92.10 74.25 55.32  20 20 25 40 20  20 20 20 20 25  0.688 0.624 0.569 0.549 0.36  1684.62 1546.14 1426.11 1381.39 1196.64  20 20 20 20 25  0.689 0.608 0.575 0.569 0.409  25 20 20 20 25  0.505 0.605 0.562 0.486 0.331  Hay + Molasses 1 7-Aug 0 23-Aug 16 9-Sep 33 21-Oct 75 24-Jan 170 Hay + Molasses 2 0 7-Aug 23-Aug 16 9-Sep 33 21-Oct 75 24-Jan 170  O  1751.70 1489.88 1405.52 1268.40 1204.13  1684.62 1474.73 1377.67 1394.85 1227.49  20 20 20 20 25  0 16 33 75 170  0.719 0.598 0.432 0.216 0.47  Average S0 ' (mg I.") 1683.27 1478.25 1389.81 1284.54 1214.82  0.688 0.591 0.547 0.555 0.371  0 16 33 75 170  7-Aug 23-Aug 9-Sep 21-Oct 24-Jan  Dil.  Dil.  2  Dil.  7-Aug 23-Aug 9-Sep 21-Oct 24-Jan  S0 ' (mg L- )  S0 " (mg L- )  Time (Days)  Date  O.D.  4  1  2  O.D.  4  1  S0 " (mg L- ) 2  O.D.  4  1  2  4  St. Dev.  1  69.11 10.32 14.27 103.20 11.81  Barley 2  25 20 25 33 20  0.498 0.598 0.362 0.249 0.35  1591.87 1489.88 1212.84 1190.35 943.84  25 25 25 40 25  0.498 0.451 0.399 0.192 0.32  1591.87 1464.74 1314.69 1160.70 1084.45  20 20 25 33 20  0.653 0.613 0.366 0.251 0.36  1608.89 1522.33 1223.85 1197.62 965.86  1621.25 1464.19 1289.98 1193.47 1036.51  37.13 58.43 57.88 30.90 62.47  1643.51 1383.85 1485.57 1406.07 1196.64  20 20 25 40 25  0.691 0.553 0.415 0.238 0.371  1691.11 1392.50 1358.73 1367.13 1227.49  25 25 25 33 20  0.526 0.42 0.4 0.341 0.48  1667.60 1380.90 1317.44 1524.64 1230.12  1667.41 1385.75 1387.25 1432.61 1218.08  23.80 6.03 87.62 82.04 18.62  1927.26 1762.52 1622.10 1078.48 910.55  20 25 25 40 25  0.831 0.561 0.476 0.165 0.257  1994.05 1762.27 1526.64 1039.54 907.75  20 20 25 33 20  0.812 0.726 0.513 0.229 0.303  1952.93 1766.84 1628.49 1117.68 840.34  1958.08 1763.88 1592.41 1078.56 886.21  33.69 2.57 57.05 39.07 39.75  20 20 25 40 25  0.678 0.559 0.526 0.478 0.311  1662.98 1405.49 1331.42 1222.08 1059.20  Barley + Molasses 1 7-Aug 0. 16 23-Aug 9-Sep 33 75 21-Oct 24-Jan 170  20 20 20 20 25  Barley + Molasses 2 7-Aug 0 23-Aug 16 9-Sep 33 21-Oct' 75 24-Jan 170  20 20 20 20 25  0.669 0.549 0.596 0.56 0.36  25 20 20 20 25  0.622 0.724 0.658 0.414 0.258  O  1587.25 1528.83 1207.33 1088.90 1081.64  1626.20 1502.61 1329.22 1262.47 1092.86  20 25 20 20 25  0 16 33 75 170  0.643 0.616 0.36 0.176 0.319  Average S0 " (mg L' ) 1601.77 1507.10 1249.80 1180.57 1039.45  0.661 0.465 0.525 0.496 0.323  0 16 33 75 170  7-Aug 23-Aug 9-Sep 21-Oct 24-Jan  Dil.  Dil.  2  Dil.  7-Aug 23-Aug 9-Sep 21-Oct 24-Jan  S0 " (mg L- )  S0 " (mg L" )  Time (Days)  Date  O.D.  4  1  4  1  so 2  2  O.D.  O.D.  4  (mg L" ) 1  2  4  St. Dev.  1  21.28 19.86 68.84 87.20 82.99  Silage 2 S0 " (mg L- )  Dil.  0.777 0.72 0.507 0.217 0.15  1877.20 1753.86 1611.97 1272.89 243.06  25 20 25 33 4  0.552 0.738 0.473 0.28 0.605  1737.93 1792.81 1518.38 1302.99 301.08  Average S0 " (mg I.") 1830.77 1770.55 1607.64 1300.39 254.05  25 20 25 40 25  0.604 0.572 0.418 0.165 0.202  1878.57 1433.62 1366.99 1039.54 753.49  20 20 25 33 20  0.781 0.603 0.43 0.248 0.31  1885.85 1500.70 1400.02 1186.72 855.76  1887.03 1469.96 1440.28 1105.32 783.84  9.11 33.89 99.72 74.83 62.54  1939.95 1606.72 1401.89 878.78 1076.03  25 20 25 40 25  0.633 0.645 0.44 0.142 0.305  1957.01 1591.58 1427.55 936.32 1042.38  20 25 25 33 20  0.83 0.495 0.382 0.199 0.38  1991.88 1583.75 1267.89 1008.68 1009.91  1962.95 1594.02 1365.77 941.26 1042.77  26.47 11.68 85.74 65.09 33.06  1890.18 1898.84 1879.75 1803.23 1536.01  20 25 25 40 25  0.773 0.614 0.508 0.271 0.5  1868.54 1905.62 1614.73 1515.22 1589.30  25 20 25 33 20  0.569 0.786 0.492 0.354 0.6  1783.91 1896.67 1570.68 1571.88 1494.38  1847.54 1900.38 1688.39 1630.11 1539.90  56.16 4.67 167.18 152.58 47.58  S0 ' (mg I-")  Dil.  0.777 0.562 0.69 0.524 0.322  1877.20 1764.97 1692.57 1325.29 218.01  20 20 25 40 10  20 25 20 20 25  0786 0.455 0.627 0.419 0.198  1896.67 1475.56 1553.84 1089.69 742.27  Silage + Molasses 2 0 7-Aug 23-Aug 16 9-Sep 33 21-Oct 75 24-Jan 170  20 20 20 20 25  0.806 0.652 0.558 0.325 0.317  Molasses 1 7-Aug 23-Aug 9-Sep 21-Oct 24-Jan  20 20 20 20 25  0.783 0.787 0.775 0.737 0.481  2  Time (Days)  Dil.  7-Aug 23-Aug 9-Sep 21-Oct 24-Jan  0 16 33 75 170  20 25 20 20 5  7-Aug 23-Aug 9-Sep 21-Oct 24-Jan  0 16 33 75 170  Date  K> O  0 16 33 75 170  O.D.  4  1  2  O.D.  4  1  S0 ' (mg L- ) 2  O.D.  4  1  2  4  St. Dev.  1  80.41 20.06 87.17 26.30 42.61  Molasses 2 Date 7-Aug 23-Aug 9-Sep 21-Oct 24-Jan  Time (Days)  Dil.  0 16 33 75 170  20 20 20 20 25  20 25 25  S0 " (mg I.")  Dil.  0.739 0.833 0.735 0.653 0.485  1794.97 1998.37 1791.67 1614.75 1547.23  20 20 25 40 25  0.644 0.407 0.418  1591.27 1328.46 1359.31  0.622 0.417 0.341  (mg L- )  Average S0 " (mg I.")  St. Dev.  0.575 0.611 0.489 0.367 0.612  1800.14 1897.51 1562.43 1619.11 1520.80  1787.32 1930.85 1656.27 1524.69 1522.53  17.92 58.48 120.14 159.78 23.89  25 33 20  0.451 0.326 0.485  1457.82 1470.14 1241.13  1542.68 1381.10 1290.94  73.75 77.54 61.24  1529.39 1192.11 1168.59  25 33 20  0.429 0.285 0.443  1397.27 1321.16 1148.64  1489.83 1289.93 1153.53  80.44 86.53 13.31  0.477 0.253 0.48  1529.39 1434.45 1533.21  25 33 20  0.454 0.336 0.58  1466.08 1506.47 1450.34  1536.26 1512.88 1494.37  73.85 81.82 41.68  0.47 0.264 0.465  1510.13 1483.81 1491.14  25 33 20  0.459 0.329 0.574  1479.85 1481.04 1437.12  1519.01 1528.33 1476.87  44.28 79.53 34.88  S0 ' (mg L' )  Dil.  0.726 0.786 0.508 0.232 0.468  1766.84 1896.67 1614.73 1340.21 1499.55  25 25 25 33 20  25 40 25  0.495 0.233 0.387  1578.94 1344.69 1272.37  1542.83 1356.51 1143.35  25 40 25  0.477 0.199 0.35  0.654 0.503 0.468  1613.29 1597.72 1499.55  25 40 25  0.633 0.511 0.469  1567.05 1620.16 1502.36  25 40 25  O.D.  2  4  1  O.D.  2  4  1  O.D.  so  2 4  -  2  1  4  1  Negative Control Hay 9-Sep 21-Oct 24-Jan  0 42 137  Negative Control Barley 9-Sep 21-Oct 24-Jan  0 42 137  20 25 25  Negative Control Silage 9-Sep 21-Oct 24-Jan  0 42 137  20 25 25  Negative Control Molasses 9-Sep 21-Oct 24-Jan  O  0 42 137  20 25 25  A1-3b. Total sulphide concentrations in SRB growth without pH adjustment experiment at T=170 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 Silage + Molasses 1 Silage + Molasses 2 Molasses 1 Molasses 2 Control Hay Control Barley Control Silage Control Molasses  O  2.4 1.2  0.016 0.036  0.03 0.06  1.2 1.2  0.048 0.044  0.08 0.08  1.2 1.2  0.046 0.037  0.08 0.06  Average Sulphide (mg L- ) 0.06 0.06  1.2  0.088  0.17  1.2  0.078  0.15  1.2  0.077  0.15  0.16  0.01  1.2  0.077  0.15  1.2  0.064  0.12  1.2  0.058  0.11  0.13  0.02  9.6 2.4  0.113 0.025  1.83 0.07  2.4 1.2  0.434 0.052  1.88 0.09  2.4 1.2  0.437 0.066  1.89 0.12  1.87 0.10  0.03 0.03  2.4  0.023  0.06  1.2  0.05  0.09  1.2  0.049  0.09  0.08  0.02  2.4  0.104  0.42  1.2  0.223  0.47  1.2  0.201  0.42  0.44  0.03  192 192  0.525 0.484  182.47 167.95  192 192  0.53 0.477  184.24 165.47  192 192  0.501 0.469  173.97 162.64  180.23 165.35  5.49 2.66  192  0.087  27.34  96  0.193  32.44  96  0.201  33.86  31.21  3.43  192  0.057  16.71  96  0.121  19.69  96  0.122  19.87  18.76  1.77  2.4 2.4 1.2 1.2 1.2 1.2  0.03 0.021 0.036 0.009 0.01 0.005  0.09 0.05 0.06 -0.00 0.00 -0.01  1.2 1.2 1.2 1.2 1.2 1.2  0.054 0.044 0.022 0.011 0.008 0.014  0.10 0.08 0.03 0.00 -0.00 0.01  1.2 1.2 1.2 1.2 1.2 1.2  0.055 0.039 0.03 0.015 0.013 0.013  0.10 0.06 0.04 0.01 0.01 0.01  0.10 0.06 0.04 0.00 0.00 0.00  0.01 0.01 0.02 0.01 0.01 0.01  Dil.  O.D.  Sulphide (mg L- )  Dil.  1  O.D.  Sulphide (mg L- )  Dil.  1  O.D.  Sulphide (mg I-") 1  St. Dev.  1  0.03 0.01  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 Hay 2 Hay 1 Material 5 5 PH  Barley 1  Barley 2  Silage 1  Silage 2  4  4  7  7  Barley 1  Barley 2  3.5 Control Hay 5.2  3.5 Control Barley 3.5  Final pH Agricultural Hay + Hay + Hay 1 Hay 2 Material Molasses 1 Molasses 2 5.3 5.4 5.2 5.2 PH Agricultural Silage + Silage + Molasses 1 Molasses 2 Material Molasses 1 Molasses 2 7.3 7.8 3.5 3.5 PH  Molasses 1 Molasses 2 4  4  Barley + Barley + Molasses 1 Molasses 2 3.6 3.6 Control Control Silage Molasses 7.3 3.4  Silage 1  Silage 2  8  7.9  A1-3d. Ammonia-nitrogen in SRB growth without pH adjustment experiment at T=170.  Agricultural Material Hay 1 Hay 2 Hay + Molasses 1 Hay + Molasses 2 Barley 1 Barley 2 Barley + Molasses 1  O OO  11 5.5  0.18 0.34  1.98 1.87  11 2.2  0.16 0.83  1.76 1.83  5.5 2.2  0.32 0.84  1.76 1.85  Average NH -N (mg L- ) 1.83 1.85  4.4  0.54  2.38  5.5  0.45  2.48  4.4  0.59  2.60  2.48  0.11  4.4  0.66  2.90  4.4  0.59  2.60  5.5  0.47  2.59  2.70  0.18  5.5 2.2  0.85 2.48  4.68 5.46  2.2 4.4  2.33 1.2  5.13 5.28  2.2 2.2  2.21 2.4  4.86 5.28  4.89 5.34  0.23 0.10  11  0.49  5.39  11  0.45  4.95  11  0.47  5.17  5.17  0.22  Dil.  O.D.  NH -N (mg L- )  Dil.  3  1  O.D.  NH -N (mg L" )  Dil.  3  1  O.D.  NH -N (mg I-") 3  1  3  St. Dev.  1  0.13 0.02  Barley + Molasses 2  2.2  2.08  4.58  11  0.4  4.40  2.2  2.06  4.53  Average NH -N (mg L" ) 4.50  Silage 1 Silage 2 Silage + Molasses 1  55 55 55  1.34 1.37 1.32  73.70 75.35 72.60  55 55 55  1.32 1.37 1.33  72.60 75.35 73.15  55 55 55  1.28 1.35 1.33  70.40 74.25 73.15  72.23 74.98 72.97  1.68 0.64 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 Molasses 2 Control Hay Control Barley Control Silage Control Molasses  5.5 4.4 2.2 4.4 55 4.4  0.51 0.61 0.58 1.2 0.72 0.2  2.81 2.68 1.28 5.28 39.60 0.88  4.4 4.4 2.2 5.5 55 5.5  0.57 0.48 0.49 0.95 0.77 0.15  2.51 2.11 1.08 5.23 42.35 0.83  4.4 5.5 2.2 4.4 55 4.4  0.59 0.45 0.55 1.2 0.76 0.2  2.60 2.48 1.21 5.28 41.80 0.88  2.64 2.42 1.19 5.26 41.25 0.86  0.15 0.29 0.10 0.03 1.46 0.03  Agricultural Material  O VO  Dil.  O.D.  NH -N (mg L- )  Dil.  3  1  O.D.  NH -N (mg L' )  Dil.  3  1  O.D.  NH -N (mg L' ) 3  1  3  St. Dev.  1  0.09  A1-3e. Ortho-phosphate in SRB growth without pH adjustment experiment at T=170. 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 Silage + Molasses 1 Silage + Molasses 2 Molasses 1 Molasses 2 Control Hay Control Barley Control Silage Control Molasses  O  P0 ' (mg I.")  Dil.  3  Dil.  O.D.  4  1  P0 " (mg L- )  Average P0 " (mg I-")  3  O.D.  4  1  3  4  1  5.50 2.75  0 0  0.00 0.00  1.1 1.1  0.01 0  0.01 0.00  0.01 0.00  1.38  0  0.00  1.1  0.05  0.06  0.03  1.38  0.04  0.06  1.1  0.08  0.09  0.07  2.75 2.75  0 0  0.00 0.00  1.1 1.1  0.02 0  0.02 0.00  0.01 0.00  2.75  0  0.00  1.1  0  0.00  0.00  2.75  0  0.00  1.1  0  0.00  0.00  11.00 1.83  0.22 1.14  2.42 2.09  1.1  0.21 1.8  1.16 1.98  1.79 2.04  1.83  0  0.00  1.1  0  0.00  0.00  1.83  0  0.00  1.1  0  0.00  0.00  1.38 1.38 1.38 1.38 1.38  0 0 0 0 0  0.00 0.00 0.00 0.00 0.00  1.1 1.1 1.1 1.1 1.1  0 0.06 0 0 0  0.00 0.07 0.00 0.00 0.00  0.00 0.03 0.00 0.00 0.00  1.38  0  0.00  1.1  0  0.00  0.00  A1-3f. Soluble COD in SRB growth without pH adjustment experiment at T=170. 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 Silage + Molasses 1 Silage + Molasses 2 Molasses 1 Molasses 2 Control Hay Control Barley Control Silage Control Molasses  Dil.  O.D.  sCOD (mg L" )  Dil.  1  O.D.  sCOD (mg L- )  Dil.  1  O.D.  sCOD (mg I-")  Average sCOD (mg L- )  St. Dev.  1  1  20 10  0.095 0.16  4699.46 3957.44  10 20  0.195 0.079  4823.13 3907.97  10 10  0.196 0.177  4847.86 4377.92  4790.15 4081.11  79.51 258.23  10  0.23  5688.82  10  0.233  5763.02  20  0.111  5490.95  5647.60  140.64  20  0.108  5342.54  10  0.221  5466.21  10  0.231  5713.55  5507.44  188.91  20 10  0.121 0.204  5985.63 5045.74  10 10  0.222 0.198  5490.95 4897.33  10 20  0.221 0.101  5466.21 4996.27  5647.60 4979.78  293.00 75.56  10  0.345  8533.23  20  0.151  7469.67  10  0.311  7692.27  7898.39  560.94  20  0.222  10981.90  20  0.215  10635.62  20 10  0.09 0.222  4452.12 5490.95  10 10  0.163 0.212  4031.64 5243.61  10 10  0.166 0.225  4105.84 5565.15  4196.54 5433.24  224.43 168.36  10  0.123  3042.28  10  0.143  3536.96  10  0.156  3858.50  3479.25  411.16  10  0.149  3685.37  10  0.114  2819.68  10  0.151  3734.83  3413.29  514.68  10 10 10 20 5  0.323 0.315 0.318 0.195 0.119  7989.08 7791.21 7865.41 9646.26 1471.67  10 10 10 20 10  0.333 0.32 0.334 0.2 0.096  8236.42 7914.88 8261.16 9893.60 2374.46  10 10 10 20 10  0.298 0.341 0.317 0.205 0.057  7370.73 8434.29 7840.68 10140.94 1409.84  7865.41 8046.79 7989.08 9893.60 1751.99  445.90 341.23 235.95 247.34 539.96  20  0.197  9745.20  20  0.205  10140.94  20  0.186  9201.05  9695.73  471.89  10808.76  A1-4a. S 0 ' concentrations in SRB growth with pH adjustment experiment 2  4  Hay 1 Dil.  24-Feb 10-Mar 24-Mar 7-Apr 22-Apr 19-May 21-Jun 30-Jun 28-Jul 23-Aug  0 14 28 42 57 84 117 126 154 180  33 33 33 25 33 20 25 25 30 20  Date  Time (Days)  Dil.  0 14 28 42 57 84 117 126 154 180  33 25 33 25 33 25 30 25 25 20  24-Feb 10-Mar 24-Mar 7-Apr 22-Apr 19-May 21-Jun 30-Jun 28-Jul 23-Aug  S0 " (mg L- )  Dil.  0.308 0.311 0.241 0.375 0.185 0.404 0.211 0.174 0.055 0.034  1387.04 1398.15 1138.99 1238.71 931.66 880.03 624.79 543.14 336.63 187.35  25 20 25 25 25 25 30 30 25 25  O.D.  S0 (mg L- )  Dil.  1387.04 1373.34 1168.61 1205.05 920.56 1128.09 1305.86 1218.42 688.79 409.79  25 33 25 25 25 20 25 30 30 25  2  Time (Days)  Date  O.D.  4  1  S0 " (mg L- )  Dil.  0.447 0.601 0.351 0.354 0.297 0.295 0.159 0.12 0.07 0.039  1440.65 1498.07 1171.39 1179.81 1019.94 845.27 612.04 508.77 313.63 245.22  20 25 20 20 20 20 20 20 10 10  0.564 0.453 0.451 0.465 0.379 0.393 0.273 0.217 0.272 0.164  O.D.  S0 ' (mg L' )  Dil.  O.D.  2  O.D.  2_  0.308 0.423 0.249 0.363 0.182 0.416 0.421 0.48 0.24 0.16  4  1  4  1  O.D.  2  0.447 0.334 0.367 0.331 0.276 0.596 0.519 0.393 0.181 0.123  4  1  1440.65 1483.30 1216.27 1115.30 961.04 1239.04 1304.48 1231.71 670.30 430.59  Average S0 (mg L- )  St. Dev.  1415.05 1457.48 1161.50 1192.91 999.94 859.46 609.29 510.42 303.76 208.43  1414.25 1451.23 1157.29 1203.81 983.85 861.59 615.37 520.78 318.01 213.67  26.81 50.25 16.61 30.93 46.29 17.48 8.27 19.38 16.87 29.29  S0 (mg L" )  Average S0 (mg L" )  St. Dev.  so  2 4  -  (mg L" ) 1  2_  4  1  20 20 20  0.564 0.595 0.463  1415.05 1484.60 1188.42  20 20 20 20 20 20  0.342 0.534 0.667 0.619 0.31 0.158  916.92 1123.11 1304.87 1220.13 674.61 406.26  4  1  4  1  1414.25 1447.08 1191.10 1160.18 932.84 1163.41 1305.07 1223.42 677.90 415.55  26.81 63.87 23.94 24.49 65.54 0.71 7.23 9.67 13.15  Barley 1 Dil.  24-Feb 10-Mar 24-Mar 7-Apr 22-Apr 19-May 21-Jun 30-Jun 28-Jul 23-Aug  0 14 28 42 57 84 117 126 154 180  33 25 33 25 25 25 25 25 25 20  Date  Time (Days)  Dil.  0 14 28 42 57 84 117 126 154 180  33 25 33 25 25 20 25 25 30 25  24-Feb 10-Mar 24-Mar 7-Apr 22-Apr 19-May 21-Jun 30-Jun 28-Jul 23-Aug  S0 " (mg L- )  Dil.  0.308 0.356 0.297 0.438 0.293 0.517 0.575 0.552 0.409 0.474  1387.04 1185.42 1346.32 1415.41 1008.72 1364.15 1428.07 1377.31 1061.74 964.14  25 33 25 25 33 20 30 30 30 25  0.447 0.312 0.331 0.446 0.2 0.754 0.445 0.43 0.33 0.363  1440.65 1401.85 1115.30 1437.85 987.20 1534.48 1369.42 1329.69 1064.88 960.22  O.D.  S0 " (mg L' )  Dil.  O.D.  1387.04 1238.71 1064.94 1165.79 1000.30 1366.19 1383.93 1260.35 1109.90 1092.63  25 33 20 25 33 25 30 30 25 20  2  Time (Days)  Date  O.D.  4  1  2  0.308 0.375 0.221 0.349 0.29 0.664 0.555 0.499 0.347 0.423  4  1  S0 " (mg L" ) 2  O.D.  4  1  O.D.  Average S0 ' (mg I-")  St. Dev. 26.81 123.61 134.30 15.42 10.76 98.83 29.99 26.98 4.21 2.77  4  1  2  4  1  20 20 20 20 20 20 20 20 20  0.564 0.556 0.429 0.561 0.378 0.662 0.714 0.707 0.534  1415.05 1397.10 1112.13 1408.32 997.70 1362.45 1387.85 1375.49 1070.07  1414.25 1328.12 1191.25 1420.52 997.87 1420.36 1395.11 1360.83 1065.56 962.18  S0 " (mg L' )  Dil.  O.D.  S0 " (mg L- )  1440.65 1424.07 1015.65 1272.37 953.88 1284.69 1292.62 1287.32 1101.46 1123.03  20 20 25 20 20 20 20 20 20 20  Average S0 ^ (mg L- ) 1414.25 1355.53 1040.05 1198.39 966.01 1325.93 1337.14 1275.94 1106.75 1108.78  2  0.447 0.318 0.386 0.387 0.191 0.483 0.416 0.414 0.427 0.564  S0 " (mg I.") 2  Dil.  4  1  2  0.564 0.559 0.304 0.449 0.354 0.643 0.684 0.653 0.556 0.557  4  1  1415.05 1403.83 1039.57 1157.01 943.85 1326.92 1334.88 1280.16 1108.91 1110.67  4  St. Dev.  1  26.81 101.68 24.65 64.22 30.12 40.76 45.70 13.97 4.61 15.29  Silage 1 Dil.  24-Feb 10-Mar 24-Mar 7-Apr 22-Apr 19-May 21-Jun 30-Jun 28-Jul 23-Aug  0 14 28 42 57 84 117 126 154 180  33 33 20 25 25 25 30 30 25 20  Date  Time (Days)  Dil.  0 14 28 42 57 84 117 126 154 180  33 33 20 25 25 20 25 25 30 25  24-Feb 10-Mar 24-Mar 7-Apr 22-Apr 19-May 21-Jun 30-Jun 28-Jul 23-Aug  bo  S0 ' (mg L' )  Dil.  0.308 0.301 0.35 0.266 0.224 0.505 0.396 0.353 0.325 0.382  1387.04 1361.13 934.87 932.99 815.19 1336.11 1239.66 1125.79 876.37 801.72  25 25 25 20 33 20 25 25 30 25  O.D.  S0 " (mg L- )  Dil.  1387.04 1420.36 1125.60 1137.74 708.61 1528.87 1355.24 1339.79 900.69 841.06  25 25 25 20 33 25 30 30 25 20  2  Time (Days)  Date  O.D.  4  1  Dil.  0.447 0.36 0.29 0.333 0.152 0.593 0.493 0.457 0.251 0.294  1440.65 1196.64 1000.30 896.73 809.49 1233.43 1247.11 1167.66 855.67 807.95  20 25 33 20 20 20 20 20 20 20  O.D.  S0 " (mg L- )  Dil.  1440.65 1356.51 1263.95 1273.69 720.63 1308.06 1324.40 1390.60 962.43 852.92  20 20 33 20 20 20 20 20 20 20  2  0.308 0.317 0.435 0.339 0.186 0.751 0.542 0.535 0.268 0.309  4  1  2  4  1  S0 " (mg L" )  Average S0 ' (mg L- )  St. Dev.  0.564 0.379 0.189 0.341 0.296 0.63 0.623 0.595 0.408 0.393  1415.05 1249.93 946.47 914.68 813.71 1302.62 1227.19 1177.76 847.62 821.14  1414.25 1269.23 960.55 914.80 812.79 1290.72 1237.99 1157.07 859.89 810.27  26.81 83.93 34.91 18.13 2.96 52.36 10.06 27.56 14.83 9.92  S0 ' (mg L" )  Average  O.D.  1415.05 1318.56 1120.48 1161.50 723.95 1345.62 1331.35 1336.65 934.13 821.14  1414.25 1365.15 1170.01 1190.97 717.73 1394.18 1337.00 1355.68 932.42 838.37  2  S0 " (mg L- )  O.D.  O.D.  2  0.447 0.417 0.384 0.501 0.128 0.493 0.428 0.453 0.364 0.411  4  1  4  1  2  0.564 0.521 0.236 0.451 0.256 0.653 0.682 0.685 0.457 0.393  4  1  2  4  1  so  2 4  St. Dev.  -  (mg L- ) 1  26.81 51.45 81.40 72.61 8.07 118.14 16.18 30.28 30.90 16.06  Molasses 1 Date  Dil.  0 14 28 42 57 84 117 126 154 180  33 33 33 25 25 25 25 25 30 25  Time (Days)  Dil.  0 14 28 42 57 84 117 126 154 180  33 33 33 25 25 25 25 25 30 20  24-Feb 10-Mar 24-Mar 7-Apr 22-Apr 19-May 21-Jun 30-Jun 28-Jul 23-Aug  S0 " (mg L- )  Dil.  0.308 0.372 0.405 0.527 0.254 0.546 0.496 0.453 0.253 0.243  1387.04 1623.99 1746.16 1665.03 899.33 1431.94 1253.73 1158.84 860.97 695.41  25 25 25 20 33 20 30 30 25 20  0.447 0.494 0.494 0.705 0.183 0.674 0.377 0.352 0.273 0.344  1440.65 1572.48 1572.48 1731.42 924.26 1384.89 1189.34 1123.14 761.61 734.64  20 20 20 20 20 20 20 20 20 20  0.564 0.625 0.601 0.645 0.326 0.672 0.595 0.572 0.38 0.326  1415.05 1551.92 1498.07 1596.80 881.02 1381.15 1177.76 1137.16 798.19 702.86  O.D.  SO4 "  Dil.  O.D.  S0 " (mg LZ )  Dil.  O.D.  SO4 '  1440.65 1465.89 1530.40 1349.98 935.36 1164.25 1083.42 1255.55 794.71 655.69  20 20 20 20 20 20 20 20 20 20  2  Time (Days)  O.D.  4  1  O.D.  S0 " (mg L' ) 2  SO4 " 2  (mg I-") 1  Dil.  O.D.  4  1  Average S0 ' (mg L' ) 1414.25 1582.79 1605.57 1664.42 901.54 1399.32 1206.94 1139.71 806.92 710.97 2  4  St. Dev.  1  26.81 37.13 127.32 67.32 21.70 28.30 40.93 17.99 50.25 20.83  Molasses 2 Date 24-Feb 10-Mar 24-Mar 7-Apr 22-Apr 19-May 21-Jun 30-Jun 28-Jul 23-Aug  0.308 0.342 0.305 0.467 0.282 0.488 0.442 0.49 0.233 0.302  2  2  (mg L" ) 1  1387.04 1512.92 1375.94 1496.75 977.87 1296.37 1134.56 1240.49 808.01 660.49  25 25 25 20 33 20 30 30 25 25  0.447 0.456 0.479 0.535 0.186 0.556 0.337 0.402 0.288 0.225  4  1  0.564 0.61 0.545 0.621 0.358 0.569 0.572 0.631 0.381 0.302  2  Average S0 ' (mg L- ) 1414.25 1499.03 1426.25 1463.22 955.35 1216.39 1118.38 1245.78 800.89 658.89 2  (mg I-") 1  1415.05 1518.26 1372.41 1542.94 952.82 1188.55 1137.16 1241.32 799.96 660.49  4  St. Dev.  1  26.81 28.82 90.22 100.76 21.36 70.32 30.31 8.47 6.70 2.77  Silage:Hay 1:3 1 Time (Days)  Dil.  24-Feb 10-Mar 24-Mar 7-Apr 22-Apr 19-May 21-Jun 30-Jun 28-Jul 23-Aug  0 14 28 42 57 84 117 126 154 180  33 33 33 25 25 20 25 25 30 20  Date  Time (Days)  Dil.  0 14 28 42 57 84 117 126 154 180  33 25 33 25 33 30 30 30 25 25  Date  24-Feb 10-Mar 24-Mar 7-Apr 22-Apr 19-May 21-Jun 30-Jun 28-Jul 23-Aug  OS  Dil.  0.308 0.268 0.244 0.303 0.227 0.467 0.324 0.302 0.13 0.13  1387.04 1238.95 1150.10 1036.77 823.60 997.83 874.16 825.61 535.25 356.83  25 25 20 25 33 30 30 30 25 25  O.D.  S0 " (mg L- )  Dil.  1387.04 1280.78 1198.23 947.01 731.74 975.06 980.14 892.75 512.24 382.04  25 20 25 25 25 25 25 25 30 20  2  4  1  S0 " (mg I/ )  Dil.  0.447 0.371 0.451 0.316 0.146 0.308 0.253 0.234 0.154 0.099  1440.65 1227.49 1161.50 1073.23 787.27 1050.79 860.97 810.66 499.00 377.63  20 20 25 20 20 25 20 20 20 10  0.564 0.481 0.387 0.422 0.298 0.361 0.412 0.389 0.205 0.33  O.D.  S0 ' (mg L' )  Dil.  O.D.  2  S0 " (mg L- )  O.D.  O.D.  2  0.308 0.39 0.257 0.271 0.131 0.281 0.298 0.265 0.16 0.101  4  1  4  1  O.D.  2  0.447 0.491 0.366 0.305 0.194 0.361 0.371 0.313 0.108 0.134  4  1  1440.65 1251.25 1213.47 1042.38 731.05 999.54 977.88 849.88 476.99 363.89  Average S0 " (mg L- )  St. Dev.  1415.05 1228.81 1272.37 1096.43 818.19 999.54 854.69 814.08 489.24 354.96  1414.25 1231.75 1194.65 1068.81 809.69 1016.05 863.27 816.78 507.83 363.14  26.81 6.27 67.54 30.07 19.60 30.10 9.94 7.83 24.24 12.58  S0 ' (mg L- )  Average S0 ' (mg L- )  St. Dev.  1414.25 1266.01 1185.83 1041.32 729.66 998.29 971.11 859.30 499.88 374.32  so  2 4  -  2  (mg L" ) 1  2  4  1  20  0.564  1415.05  20 20 20 20 20 20 20 10  0.444 0.439 0.257 0.479 0.469 0.401 0.217 0.355  1145.79 1134.57 726.20 1020.27 955.32 835.27 510.42 377.03  4  1  2  4  1  26.81 35.50 93.78 3.02 22.63 13.73 29.88 19.85 9.37  Silage:Hay 1:1 1 Dil.  24-Feb 10-Mar 24-Mar 7-Apr 22-Apr 19-May 21-Jun 30-Jun 28-Jul 23-Aug  0 14 28 42 57 84 117 126 154 180  33 25 20 25 25 20 25 25 25 20  Date  Time (Days)  Dil.  0 14 28 42 57 84 117 126 154 180  33 33 20 25 33 20 30 30 30 20  24-Feb 10-Mar 24-Mar 7-Apr 22-Apr 19-May 21-Jun 30-Jun 28-Jul 23-Aug  S0 " (mg I-")  Dil.  0.308 0.414 0.445 0.32 0.107 0.41 0.34 0.281 0.088 0.08  1387.04 1348.09 1148.03 1084.45 487.03 891.25 909.47 779.27 353.35 268.56  25 33 25 20 33 25 30 30 30 25  0.447 0.273 0.384 0.401 0.074 0.323 0.286 0.218 0.051 0.049  1440.65 1257.46 1263.95 1049.31 520.71 910.72 948.36 768.28 326.04 267.29  O.D.  S0 ' (mg L- )  Dil.  O.D.  1387.04 1350.02 1049.31 899.33 609.56 887.51 739.16 704.73 429.32 287.98  25 25 25 25 25 30 25 25 25 14.28  2  Time (Days)  Date  O.D.  4  1  O.D.  2  0.308 0.298 0.401 0.254 0.098 0.408 0.207 0.194 0.09 0.091  4  1  SO4 " 2  (mg L- ) 1  O.D.  4  1  20 20 33  0.564 0.492 0.244  1415.05 1253.49 1150.10  20 30 20 20 20 10  0.157 0.256 0.453 0.359 0.144 0.28  501.82 904.94 927.07 761.12 381.55 310.82  S0 ' (mg L' )  Dil.  O.D.  S0 " (mg L- )  1440.65 1345.29 1148.96 913.36 599.22 899.33 777.06 752.78 388.66 300.16  20 25 33 20 20 25 20 20 20 10  2  0.447 0.413 0.343 0.259 0.147 0.254 0.28 0.269 0.104 0.166  S0 " (mg I-") 2  Dil.  4  1  2  0.564 0.401 0.223 0.356 0.202 0.319 0.347 0.337 0.155 0.272  4  1  1415.05 1311.63 1072.35 948.33 602.79 901.37 739.93 722.28 400.97 303.76  Average S0 " (mg I-") 1414.25 1286.35 1187.36 1066.88 503.19 902.30 928:30 769.56 353.65 282.22 2  4  St. Dev.  1  Average S0 ^ (mg L' ) 1414.25 1335.65 1090.20 920.34 603.86 896.07 752.05 726.60 406.32 297.30 4  26.81 53.51 66.34 16.88 10.00 19.48 9.14 27.75 24.78  St. Dev.  1  26.81 20.93 52.17 25.24 5.25 7.48 21.66 24.32 20.85 8.27  Silage:Hay 3:1 1 Dil.  24-Feb 10-Mar 24-Mar 7-Apr 22-Apr 19-May 21-Jun 30-Jun 28-Jul 23-Aug  0 14 28 42 57 84 117 126 154 180  33 25 33 25 33 30 30 25 25 25  Date  Time (Days)  Dil.  0 14 28 42 57 84 117 126 154 180  33 25 33 25 25 20 30 25 25 20  24-Feb 10-Mar 24-Mar 7-Apr 22-Apr 19-May 21-Jun 30-Jun 28-Jul 23-Aug  00  S0 " (mg L' )  Dil.  0.308 0.375 0.172 0.256 0.136 0.256 0.338 0.41 0.213 0.153  1387.04 1238.71 883.53 904.94 750.25 904.94 1086.06 1063.94 629.20 496.80  25 33 25 25 25 25 25 30 30 20  O.D.  S0 ' (mg L- )  Dil.  1387.04 1350.90 920.56 879.70 658.12 878.16 964.25 1169.87 549.76 469.82  25 33 25 20 33 30 25 30 30 25  S0 " (mg L- )  Dil.  0.447 0.263 0.241 0.257 0.201 0.309 0.402 0.327 0.168 0.215  1440.65 1220.44 862.87 907.75 750.68 878.00 1046.29 1056.93 635.88 506.89  20 20 20 20 20 20 20 20 20 10  0.564 0.491 0.305 0.333 0.267 0.416 0.507 0.531 0.286 0.497  1415.05 1251.25 833.90 896.73 748.64 902.47 1022.40 1064.77 632.24 502.37  O.D.  S0 ' (mg L- )  Dil.  O.D.  S0 ' (mg L- )  2  Time (Days)  Date  O.D.  4  1  2  O.D.  0.308 0.415 0.182 0.247 0.168 0.403 0.292 0.458 0.177 0.194  1  1  O.D.  2  2  4  0.447 0.274 0.244 0.337 0.119 0.256 0.39 0.36 0.141 0.136  4  1  2  4  1  1440.65 1261.17 871.29 905.70 687.31 904.94 1019.81 1144.32 564.38 459.28  S0 ' (mg L ) 2  4  .  20 20 20 25 20 25 20 20 20 10  0.564 0.503 0.347 0.243 0.224 0.315 0.482 0.571 0.251 0.456  4  1  1415.05 1278.17 928.14 868.48 652.15 892.02 978.27 1135.39 570.45 466.18  Average S0 " (mg L- ) 1414.25 1236.80 860.10 903.14 749.86 895.14 1051.58 1061.88 632.44 502.02 2  4  St. Dev.  1  Average S0 (mg L' ) 1414.25 1296.75 906.66 884.63 665.86 891.71 987.44 1149.86 561.53 465.09 2  4  26.81 15.49 24.93 5.73 1.08 14.90 32.16 4.31 3.34 5.06  St. Dev.  1  26.81 47.66 30.87 19.09 18.81 13.39 28.89 17.89 10.64 5.35  Positive Control Date Time (Days)  Dil.  O.D.  S0 " (mg L' )  Dil.  z  4  O.D.  1  504'" (mg L- )  Dil.  O.D.  1  S0 " (mg I-") 4  1  Average S0 ' (mg L" )  St. Dev.  1414.25 67.39  26.81 37.43  2  4  1  24-Feb 14-Apr  Abiotics Date  0 50  33 10  Time (Days)  Sample  0.308 0.003  Dil.  1387.04 65.11  O.D.  25 20  S0 " (mg L' ) 4  0.447 -0.01  Dil.  O.D.  1440.65 105.92  20 5  SO4"  Dil.  0.564 0  O.D.  (mg L- )  1  1  1415.05 31.15  S0 " (mg L" ) 4  1  Average  so/  St. Dev.  (mg L- ) 1  24-Feb 2-May  K3  0 67  initial Hay 1 Hay 2 Barley 1 Barley 2 Silage 1 Silage 2 Molasses 1 Molasses 2 S:H (1 3)1 S:H (1 3) 2 S:H (1 1) 1 S:H (1 1)2 S:H (3 1) 1 S:H (3 1)2  25 33 25 25 33 25 25 25 33 20 33 33 30 25 25  0.449 0.362 0.476 0.491 0.377 0.504 0.379 0.465 0.389 0.566 0.335 0.336 0.367 0.428 0.392  1446.26 1586.97 1521.99 1564.06 1642.50 1600.52 1249.93 1491.14 1686.93 1419.53 1487.00 1490.71 1459.53 1387.36 1286.39  33 25 33 33 25 33 33 33 20 30 25 25 25 33 33  0.317 0.435 0.319 0.354 0.399 0.366 0.371 0.376 0.603 0.412 0.514 0.501 0.505 0.365 0.204  1420.36 1406.99 1427.77 1557.35 1306.02 1601.78 1620.29 1638.80  20 20 20 20 30 20 30 20  1502.56 1610.98 1628.57 1592.11 1603.33 1598.07 1002.01  25 33 20 20 20 20 20  0.591 0.601 0.555 0.601 0.353 0.585 0.356 0.602 0.441 0.317 0.561 0.565 0.541 0.61 0.605  1475.63 1498.07 1394.85 1498.07 1412.41 1462.17 1422.50 1500.31 1423.82 1420.36 1408.32 1417.29 1363.44 1518.26 1507.04  1447.42 1497.34 1448.20 1539.83 1453.64 1554.82 1430.91 1543.42 1537.77 1483.63 1507.96 1500.04 1475.43 1501.23 1265.15  27.65 89.99 65.99 36.32 171.99 80.24 185.32 82.73 135.04 110.29 111.61 87.78 120.73 106.38 253.19  A1-4b. Total sulphide concentrations in SRB growth with pH adjustment experiment  Date 22-Apr  Time (days) 57  Sample  Dil.  O.D.  Dil.  1  O.D.  Sulphide (mg L- )  Dil.  1  O.D.  Average Sulphide Sulphide (mg L- ) (mg L- ) 1  St. Dev.  1  Hay 1 Hay 2 Barley 1 Barley 2 Silage 1 Silage 2 Molasses 1 Molasses 2 Silage:Hay (1:3)1  5 4 5 4 5 4 5 4 5  0.2 0.211 0.186 0.17 0.079 0.154 0.035 0.017 0.021  2.11 1.78 1.95 1.42 0.77 1.28 0.28 0.06 0.12  5 5 4 5 4 5 4 5 4  0.191 0.172 0.22 0.137 0.082 0.121 0.051 0.001 0.016  2.01 1.80 1.86 1.41 0.64 1.23 0.36 0.00 0.05  4 4 5 5 4 4 4 2 2  0.311 0.221 0.189 0.136 0.081 0.151 0.049 0.044 0.033  2.67 1.87 1.98 1.40 0.63 1.25 0.35 0.15 0.10  2.26 1.82 1.93 1.41 0.68 1.25 0.33 0.07 0.09  0.36 0.05 0.06 0.01 0.08 0.02 0.05 0.08 0.04  Silage:Hay (1:3)2  5  0.108  1.09  4  0.12  0.98  2  0.26  1.11  1.06  0.07  Silage:Hay (1:1) 1  5  0.069  0.66  4  0.077  0.59  2  0.151  0.63  0.63  0.03  Silage:Hay (1:1)2  5  0.723  7.89  10  0.24  5.10  4  0.807  7.06  6.68  1.44  Silage:Hay (3:1)1  5  1.115  Over  10  0.28  5.98  20  0.137  5.63  5.81  0.25  Silage:Hay (3:1)2  5  0.354  3.81  4  0.615  5.36  10  0.234  4.96  4.71  0.80  100 100 100  0.162 0.385 0.211  41.26 95.88 53.26  50 50 50  0.322 0.784 0.451  40.22 96.80 56.02  40.74 96.34 54.64  100 200  0.082 0.091  21.67 47.74  21-Jun  117  Barley 2 Silage 2 Silage:Hay (1:1) 1  10 10 10  over over over  28-Jul  154  Silage 2 Silage:Hay (1:1) 1  100 100  0.097 0.176  O  Sulphide (mg L- )  25.34 44.69  23.50 46.22  Date 23-Aug  Abiotic 2-May  K>  Time (days) 180  Time (days) 67  Sample  Dil.  O.D.  Sulphide (mg L- )  Dil.  1  O.D.  Sulphide (mg L- )  Dil.  1  O.D.  Average Sulphide Sulphide (mg L" ) (mg L- ) 50.92 50.81 26.32 25.73 4.04 4.05 4.35 5.05 5.26 5.06 12.81 13.40 4.40 4.85 33.77 32.31 40.53 41.85 1  St. Dev.  1  Hay 1 Hay 2 Barley 1 Barley 2 Silage 1 Silage 2 Molasses 1 Molasses 2 Silage:Hay (1:3)1  50 50 50 50 50 50 50 50 50  0.371 0.194 0.026 0.046 0.033 0.113 0.042 0.223 0.353  46.22 24.55 3.98 6.43 4.83 14.63 5.94 28.10 44.02  100 100 10 10 20 20 10 25 100  0.221 0.101 0.161 0.172 0.097 0.254 0.166 0.566 0.161  55.71 26.32 4.10 4.37 5.07 12.76 4.22 35.05 41.02  100 100 10 10 20 20 10 25 100  0.201 0.101 0.159 0.171 0.101 0.255 0.173 0.545 0.159  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  Sample  Dil.  O.D.  Sulphide (mg L- )  Dil.  O.D.  Sulphide (mg I-")  Dil.  O.D.  0.07 0.08 0.08 0.00 0.96 0.78 0.05  2 2 2 2 2 2 2  Hay 1 Hay 2 Barley 1 Barley 2 Silage 1 Silage 2 Molasses 1  2 2 2 2 2 2 2  0.025 0.028 0.028 -0.03 0.227 0.186 0.021  1  0.021 0.025 0.025 0 0.201 0.191 0.005  1  0.05 0.07 0.07 0.00 0.85 0.80 -0.02  Average Sulphide Sulphide (mg L- ) (mg L" ) 0.06 0.07 0.07 0.00 0.90 0.79 0.01 1  1  4.74 1.02 0.06 1.19 0.22 1.07 0.94 3.70 1.89  Abiotic  Molasses 2 Silage:Hay (1:3)1  2 2  0.018 0.035  0.04 0.11  2 2  0.01 0.041  0.00 0.14  Average Sulphide (mg I.") 0.02 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  Time (days)  Dil.  Sample  O.D.  Sulphide (mg L" )  Dil.  1  O.D.  Sulphide (mg L' ) 1  Dil.  20  O.D.  0.911  Sulphide (mg I-") 1  1  39.90  39.90  Average PH 7.0 7.5 6.0 7.5 6.9 7.5 7.7 7.7 7.6 7.8 7.6 7.6 7.7  A1-4c. pH in SRB growth with pH adjustment experiment Time (Days)  Bottle 1  Bottle 2  0 0 14 14 29 29 57 57 84 119 126 154 180  5.0 7.5 5.3 7.8 5.8 7.5 7.5 7.5 7.0 7.1 7.5 7.4 7.7  5.0 7.5 5.5 7.5 6.7 7.5 7.2 7.2 7.5 7.5 7.6 7.5 7.5  Average PH 5.0 7.5 5.4 7.7 6.3 7.5 7.4 7.4 7.3 7.3 7.6 7.5 7.6  Silage  Time (Days)  Bottle 1  Bottle 2  0 0 14 14 29 29 57 57 84 119 126 154 180  7.0 7.5 6.0 7.5 7.0 7.5 7.8 7.8 7.5 7.8 7.6 7.6 7.7  7.0 7.5 6.0 7.5 6.8 7.5 7.5 7.5 7.6 7.8 7.6 7.6 7.6  Barley  Silage:Hay 1:3  Time (Days)  Bottle 1  Bottle 2  Average pH 4.0 7.5 4.3 7.5 4.3 7.5 4.8 7.5 6.9 7.1 7.5 7.2 7.3  Molasses  0 0 14 14 29 29 57 57 84 119 126 154 180  4.0 7.5 4.5 7.5 4.0 7.5 4.5 7.5 6.8 7.0 7.3 7.1 7.2  4.0 7.5 4.0 7.5 4.5 7.5 5.0 7.5 7.0 7.2 7.6 7.3 7.3  Time (Days) 0 0 14' 14 29 29 57 57 84 119 126 154 180  Bottle 1  Bottle 2  Average pH  Silage:Hay 1:1  6.0 7.5 5.0 7.5 6.2 7.5 6.5 7.5 7.6 7.4 7.6 7.6 7.7  6.0 7.5 5.5 7.8 6.5 7.5 6.8 7.5 7.5 7.6 7.7 7.6 7.6  6.0 7.5 5.3 7.7 6.4 7.5 6.7 7.5 7.6 7.5 7.7 7.6 7.7  Time (Days) 0 0 14 14 29 29 57 57 84 119 126 154 180  Bottle 1 4.0 7.5 4.3 7.5 4.5 7.5 6.0 7.5 7.1 6.8 7.2 7.0 . 7.0  Bottle 2 4.0 7.5 4.5 7.5 4.5 7.5 5.8 7.5 7.1 6.9 7.2 7.1 7.2  Time (Days)  Bottle 1  Bottle 2  0 0 14 14 29 29 57 57 84 119 126 154 180  6.0 7.5 6.0 7.5 7.0 7.5 6.8 7.5 7.6 7.8 7.5 7.5 7.6  6.0 7.5 6.0 7.5 5.5 7.5 7.0 7.5 7.1 7.5 7.5 7.7 7.6  Average PH 4.0 7.5 4.4 7.5 4.5 7.5 5.9 7.5 7.1 6.9 7.2 7.1 7.1  Average PH 6.0 7.5 6.0 7.5 6.3 7.5 6.9 7.5 7.4 7.7 7.5 7.6 7.6  Silage:Hay 3:1  Time (Days)  Bottle 1  Bottle 2  0 0 14 14 29 29 57 57 84 119 126 154 180  6.5 7.5 6.0 7.5 7.0 7.5 7.7 7.7 7.8 7.4 7.7 7.6 7.6  6.5 7.5 5.5 7.5 7.0 7.5 7.7 7.7 7.8 7.6 7.6 7.6 7.6  Average PH 6.5 7.5 5.8 7.5 7.0 7.5 7.7 7.7 7.8 7.5 7.7 7.6 7.6  Adjusted Before Adjustment Date Time (Days) Hay 1 Hay 2 Barley 1 Barley 2 Silage 1 Silage 2 Molasses 1 Molasses 2 Silage:Hay (1:3)1  4^  24-Feb 0  24-Feb 0  2-May 67  5.0 5.0 5.0 5.0 7.0 7.0 5.0 5.0 6.0  7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5 7.5  4.5 4.5 4.2 4.2 6.0 6.2 4.2 4.3 6.5  Before Adjusted Adjustment 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- )  Dil.  O.D.  1  sCOD (mg L" )  Dil.  O.D.  1  sCOD (mg I-") 1  Average sCOD (mg L- )  St. Dev.  1  28-Feb  Hay 1 Hay 2 Barley 1 Barley 2 Silage 1 Silage 2 Molasses 1 Molasses 2 Silage:Hay (1:3)1  2 10 20 20 10 10 10 10 10  0.421 0.202 0.197 0.237 0.089 0.106 0.422 0.432 0.203  over 4996.27 9745.20 11723.92 2201.33 2621.80 over over 5021.00  10 20 10 40 10 10 20 20 20  0.216 0.099 0.356 0.116 0.098 0.106 0.244 0.235 0.099  5342.54 4897.33 8805.30 11476.58 2423.93 2621.80 12070.19 11624.98 4897.33  20 2 40 10 5 5 40 40 5  0.105 0.411 0.095 0.428 0.167 0.189 0.113 0.11 0.419  5194.14 5268.34 Over 4946.80 9398.92 9316.47 Over 11600.25 2065.29 2230.18 2337.36 2526.99 11179.77 11624.98 10882.96 11253.97 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  475.34 181.05 164.22  289.86  Agricultural Material  Dil.  O.D.  sCOD (mg L" )  Dil.  O.D.  sCOD (mg L- )  Dil.  O.D.  sCOD (mg L- )  St. Dev.  Silage:Hay (1:1) 1  10  0.156  3858.50  20  0.074  3660.63  5  0.31  3833.77  Average sCOD (mg L- ) 3784.30  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  0 261  3227.79  20  0.059  2918.61  3079.38  154.96  Hay 1 Hay 2 Barley 1 Barley 2 Silage 1 Silage 2 Molasses 1 Molasses 2 Silage:Hay (1:3)1  8 8 20 20 4 2 20 20 4  0.184 0.271 0.222 0.222 0.25 0.233 0.227 0.298 0.327  3640.84 5362.33 10981.90 10981.90 2473.40 1152.60 11229.24 14741.46 3235.21  10 4 40 25 8 4 40 40 8  0.155 0.397 0.121 0.174 0.129 0.122 0.111 0.119 0.153  3833.77 3927.76 11971.26 10759.29 2552.55 1207.02 10981.90 11773.38 3027.44  4 10 25 40 5 8 25 25 10  0.381 0.161 0.177 0.113 0.201 0.078 0.172 0.186 0.121  3769.46 3982.17 10944.80 11179.77 2485.77 1543.40 10635.62 11501.31 2992.81  3748.03 4424.09 11299.32 10973.65 2503.91 1301.01 10948.92 12672.05 3085.15  98.23 813.00 582.21 210.36 42.58 211.67 298.18 1797.32 131.10  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  1  1  1  1  23-Aug  T  107.81  Abiotics  Agricultural Material  Dil.  O.D.  sCOD (mg L-1)  Dil.  O.D.  sCOD (mg L-1)  Dil.  O.D.  sCOD Average (mg L-1) sCOD (mg L-1)  St. Dev.  12-May  Hay 1 Hay 2 Barley 1 Barley 2 Silage 1 Silage 2 Molasses 1 Molasses 2 Silage:Hay (1:3)1  10 10 10 10 10 10 10 10 5  0.147 0.16 0.388 0.387 0.079 0.093 0.39 0.378 0.236  3635.90 3957.44 9596.79 9572.06 1953.99 2300.26 9646.26 9349.45 2918.61  5 5 20 20 5 5 20 20 10  0.328 0.321 0.213 0.198 0.163 0.166 0.209 0.201 0.119  4056.38 3969.81 10536.68 9794.66 2015.82 2052.92 10338.81 9943.07 2943.35  20 20 25 25 20 20 25 25 20  0.079 0.077 0.164 0.157 0.041 0.051 0.155 0.161 0.059  3907.97 3866.75 3809.04 3912.09 10140.94 10091.47 9708.10 9691.61 2028.19 1999.33 2522.87 2292.02 9584.43 9856.50 9955.44 9749.32 2918.61 2926.86  213.25 89.47 471.89 112.22 39.75 235.08 418.84 346.35 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  O.D.  NH -N (mg L" )  Dil.  O.D.  NH -N (mg L- )  Average NH -N (mg L- )  A1-4e. NH -N in SRB growth with pH adjustment experiment Agricultural Dil. O.D. NH -N Dil. Material (mg L- ) 3  3  1  3  1  3  1  3  1  Mar-24  Hay 1 Hay 2 Barley 1 Barley 2 Silage 1  10 10 10 10 50  over over 1.18 over 0.78  over over 11.8 over 39  50 50 20 50 20  2.2 1.7 0.45 0.53 1.93  110 85 9 26.5 38.6  100 100  0.99 0.75  99 75  25  1.3  32.5  104.5 80 10.4 29.5 38.8  Agricultural Material  Dil.  O.D.  NH -N (mg L- ) 3  Dil.  O.D.  1  NH -N (mg I-") 3  Dil.  O.D.  1  NH -N (mg L- ) 3  1  Average NH3-N  Silage 2 Molasses 1 Molasses 2 Silage:Hay (1:3)1  20 10 10 20  1.89 over over over  37.8 over over over  50 50 50 50  0.76 over over 1.59  38 over over 79.5  100 100 100  2.11 . 2.03 0.74  211 203 74  (mg L- ) 37.9 211 203 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  Hay 1 Hay 2 Barley 1 Barley 2 Silage 1 Silage 2 Molasses 1 Molasses 2 Silage:Hay (1:3)1  50 100 100 100 100 100 100 100 100  1.75 1.09 1.83 2.34 0.88 0.82 0.83 0.99 1.2  87.5 109 183 234 88 82 83 99 120  100 50 100 100 50 50 50 50 50  0.96 2.3 1.9 1.84 1.8 1.91 1.7 1.85 2.59  96 115 190 184 90 95.5 85 92.5 129.5  91.75 112 186.5 209 89 88.75 84 95.75 124.75  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  1  23-Aug  00  Dil.  O.D.  NH3-N (mg L- )  Dil.  O.D.  Dil.  O.D.  NH -N (mg L' )  Silage:Hay (1:1)2  100  1.05  105  50  2.06  103  Average NH -N (mg L- ) 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  Dil.  O.D.  Dil.  O.D.  Agricultural Material  NH3-N  (mg L' )  1  1  3  1  3  1  Abiotic Agricultural Material  NH3-N  (mg L" )  1NH3-N  (mg L" )  1  1  Average NH3-N  (mg L- ) 1  May-02  VO  Hay 1 Hay 2 Barley 1 Barley 2 Silage 1 Silage 2 Molasses 1 Molasses 2 Silage:Hay (1:3)1  50 50 50 50 50 50 50 50 50  0.68 0.59 over 0.85 0.63 0.59 over over 0.59  34 29.5 over 42.5 31.5 29.5 over over 29.5  20 20 100 20 20 20 100 100 20  1.97 1.6 1.35 2.53 1.53 1.45 1.86 1.66 1.3  39.4 32 135 50.6 30.6 29 186 166 26  36.7 30.75 135 46.55 31.05 29.25 186 166 27.75  Silage:Hay (1:3)2  50  0.57  28.5  20  1.32  26.4  27.45  Silage:Hay (1:1) 1  50  0.45  22.5  20  0.84  16.8  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  O.D.  P0 " (mg L- )  Dil.  O.D.  NH -N (mg L- ) 3  Dil.  O.D.  NH -N (mg I-")  Average NH -N (mg I.") 19.65  Agricultural Material  3  1  1  3  1  A1-4f.  PO4 " 3  in SRB growth with pH adjustment experiment  Agricultural Material  Dil.  O.D.  P0 " (mg I-") J  4  Dil.  1  J  4  Dil.  O.D.  1  P0 ' (mg L- ) J  4  1  Average P0 ' (mg L- ) 3  4  1  Mar-24  OJ  O  Hay 1 Hay 2 Barley 1 Barley 2 Silage 1 Silage 2 Molasses 1 Molasses 2 Silage:Hay (1:3)1  20 20 20 20 20 20 10 10 25  over over 0.9 2.05 over over 0.07 0.01 1.84  over over 18 41 over over 0.7 0.1 46  50 50 20 40 50 50 20 5 50  0.61 0.62 0.94 0.74 1.71 1.8 0.03 0 0.96  30.5 31 18.8 29.6 85.5 90 0.6 0 48  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  100 100 40 20 100 50  0.28 0.3 0.41 1.54 0.91 1.66  28 30 16.4 30.8 91 83  29.25 30.50 17.73 33.80 88.25 86.50 0.65 0.05 47.00  Agricultural Material  Dil.  Silage:Hay (3:1)1  25  Silage:Hay (3:1)2  O.D.  J  J  2.64  66  50  1.4  70  Average P0 " (mg L' ) 68.00  25  2.85  71.25  50  1.39  69.5  70.38  Hay 1 Hay'2 Barley 1 Barley 2 Silage 1 • Silage 2 Molasses 1 Molasses 2 Silage:Hay (1:3)1  50 100 100 100 100 100 100 100 100  1.1 0.53 1.43 1.46 0.97 0.98 2.83 2.91 0.63  55 53 143 146 97 98 283 291 63  100 50 50 50 50 50 100 100 50  0.54 1.03 2.77 2.56 2.01 2.12 2.86 2.88 1.31  54 51.5 138.5 128 100.5 106 286 288 65.5  54.50 52.25 140.75 137.00 98.75 102.00 284.50 289.50 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  4  1  1  Dil.  P0 " (mg L' )  Dil.  4  O.D.  P0 " (mg L- )  P0 " (mg L- )  O.D.  4  1  3  4  1  23-Aug  Abiotic Agricultural Material  Dil.  O.D.  P0 ' (mg L- ) 3  4  Dil.  O.D.  P0 " (mg L- ) 3  4  1  1  Average P0 " (mg L- ) 3  4  1  2-May  Hay 1 Hay 2 Barley 1 Barley 2 Silage 1 Silage 2 Molasses 1 Molasses 2 Silage:Hay (1:3)1  50 50 50 50 50 50 10 10 50  0.63 0.60 1.50 0.98 1.68 1.71 1.15 0.22 0.97  31.5 30.0 75.0 49.0 84.0 85.5 11.5 2.2 48.5  50 50 50 50 50 50 5 5 20  0.70 0.57 1.62 0.99 1.62 1.68 2.55 1.27 2.05  35.0 28.5 81.0 49.5 81.0 84.0 12.8 6.4 41.0  33.2 29.2 78.0 49.2 82.5 84.8 12.1 4.3 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 " concentrations in leachate experiment 4  Date  Time  Dil.  O.D.  SO/" (mg L' ) 1  Barley 1 (TAD) Raw Dil. O.D. S0 " (mg L- ) 4  Dil.  O.D.  S0 '" (mg L- ) 4  1  1  Average S0 ' (mg L' )  St. Dev  2  4  1  13-Jun 27-Jun 11-Jul 28-Jul 23-Aug 16-Sep  0 14 28 45 71 95  40 25 25 30 40 20  0.424 0.564 0.455 0.309 0.171 0.3  1751.74 1403.79 1163.25 1009.27 858.43 788.35  30 30 30 40 30 10  Date  Time  Dil.  O.D.  SO/" (mg L" )  Barley 2 (TAD) Raw S0 '" Dil. O.D. (mg I-")  1  0.586 0.471 0.374 0.178 0.253 0.622  1742.81 1438.27 1181.40 883.14 860.97 735.26  4  25 20 20 25 25  0.74 0.74 0.591 0.388 0.318  1792.19 1433.75 1170.70 1015.39 860.92  1762.25 1425.27 1171.78 969.27 860.11 761.80  26.31 18.74 9.12 74.65 1.45  Dil.  O.D.  S0 '" (mg L" )  Average S0 " (mg L; )  St. Dev  1748.05 1386.08 1138.97 1041.88 129.09  1750.48 1463.96 1140.76 1004.58 196.87 122.01  7.32 80.50 10.68 59.38 68.08 49.52  SO4'"  Average S0 ' (mg L" )  St. Dev  1774.31 1692.29 1275.21 1033.56 844.22  5.03 11.99 9.25 9.59 20.81  1  4  1  2  4  1  13-Jun 27-Jun 11-Jul 28-Jul 23-Aug 16-Sep  0 14 28 45 71 95  30 30 30 30 40 20  0.592 0.512 0.355 0.319 0.003 0.002  1758.70 1546.84 1131.08 1035.75 265.24 157.03  40 25 25 40 30 10  Date  Time  Dil.  O.D.  S0 " (mg I/ )  Barley 1 (TADFlocced Dil. O.D. S0 " (mg L' )  z  4  1  0.422 0.589 0.45 0.193 0.002 0.01  1744.68 1458.96 1152.22 936.11 196.28 86.99  4  25 20 25 25 20  0.72 0.713 0.444 0.4 0.001  Dil.  O.D.  (mg L- ) 1  1  2  4  1  13-Jun 27-Jun 11-Jul 28-Jul 23-Aug  to  0 14 28 45 71  40 25 25 40 40  0.43 0.693 0.51 0.222 0.173  1772.93 1688.47 1284.62 1038.50 865.49  30 30 30 30 30  0.6 0.572 0.406 0.314 0.239  1779.88 1705.73 1266.14 1022.51 823.90  25 20 20 25 25  0.73 0.881 0.65 0.399 0.31  1770.12 1682.68 1274.86 1039.67 843.26  Date  Time  Dil.  O.D.  SO/' (mg L' ) 1  Barley 2 (TAD) Flocced SO/' Dil. O.D. (mg L' )  Dil.  O.D.  1  SO/' (mg L' ) 1  Average SO/' (mg L' )  St. Dev  1  13-Jun 27-Jun 11-Jul 28-Jul 23-Aug  0 14 28 45 71  30 30 25 40 40  0.579 0.595 0.541 0.258 0.192  1724.27 1766.64 1353.03 1165.62 932.58  40 25 30 30 30  1737.62 1695.09 1334.99 1131.08 913.93  25 20 20 25 25  0.71 0.85 0.69 0.44 0.341  1725.98 1627.95 1345.48 1130.15 911.67  1729.29 1696.56 1344.50 1142.28 919.39  7.26 69.36 9.06 20.21 11.47  Date  Time  Dil.  O.D.  SO/' (mg L' )  Bioethanol 1 (TBD) Raw SO/' Dil. O.D. (mg L' )  Dil.  O.D.  SO/' (mg L' )  Average SO/" (mg L' )  St. Dev  1  0.42 0.696 0.432 0.355 0.273  1  1  1  13-Jun 27-Jun 11-Jul 28-Jul 23-Aug  0 14 28 45 71  40 30 25 40 20  0.341 0.492 0.509 0.263 0.616  1458.68 1493.88 1282.42 1183.27 1214.84  30 25 30 30 30  1472.70 1410.41 1289.97 1104.60 1266.14  25 20 20 25 25  0.58 0.721 0.659 0.423 0.477  1439.10 1400.21 1290.75 1092.63 1211.80  1456.82 1434.83 1287.71 1126.83 1230.92  16.87 51.39 4.60 49.24 30.53  Date  Time  Dil.  O.D.  SO/' (mg L' )  Bioethanol 2 (TBD) Raw Dil. O.D. SO/' (mg L' )  Dil.  O.D.  SO/' (mg L' )  Average SO/' (mg L' )  St. Dev  1511.55 1468.67 1317.14 1069.31 1101.02  9.29 50.56 64.86 24.35 1.53  1  0.484 0.567 0.415 0.345 0.406  1  1  1  13-Jun 27-Jun 11-Jul 28-Jul 23-Aug  to OJ  4^  0 14 28 45 71  40 25 30 40 20  0.359 0.619 0.442 0.23 0.552  1522.23 1525.16 1361.47 1066.75 1101.85  30 30 25 30 30  0.497 0.467 0.491 0.323 0.344  1507.12 1427.68 1242.69 1046.34 1101.95  25 20 20 25 25  0.61 0.751 0.691 0.424 0.426  1505.30 1453.17 1347.24 1094.84 1099.25  Date  Time  Dil.  O.D.  SO/' (mg L' ) 1  Bioethanol 1 (TBD) Flocced Dil. O.D. SO/' (mg L' )  Dil.  O.D.  1  SO/' (mg L' ) 1  Average SO/' (mg L- )  St. Dev  1  13-Jun 27-Jun 11-Jul 28-Jul 23-Aug  0 14 28 45 71  30 25 30 40 30  0.497 0.569 0.447 0.289 0.359  1507.12 1414.82 1374.71 1275.07 1141.68  Date  Time  Dil.  O.D.  SO/' (mg L' ) 1  40 30 25 30 20  0.364 0.465 0.563 0.387 0.543  1539.89 1422.38 1401.58 1215.82 1085.96  Bioethanol 2 (TBD) Flocced SO/" Dil. O.D. (mg L" )  25 20 20 25 25  0.619 0.74 0.716 0.478 0.401  1525.16 1433.75 1391.38 1214.01 1044.08  1524.06 1423.65 1389.23 1234.97 1090.57  16.41 9.53 13.56 34.74 48.96  Dil.  O.D.  SO/" (mg L" )  Average  St. Dev  1  1  SO4  (mg L" L 1  13-Jun 27-Jun 11-Jul 28-Jul 23-Aug  0 14 28 45 71  30 30 30 40 30  0.518 0.513 0.487 0.341 0.463  1562.73 1549.49 1480.64 1458.68 1417.08  Date  Time  Dil.  O.D.  S04*" (mg L" ) 1  40 25 25 30 20  0.373 0.632 0.602 0.488 0.729  1571.67 1553.85 1487.65 1483.29 1414.33  Orchard Grass 1 (PRC) Raw S0 '" Dil. O.D. (mg L" ) 4  25 20 20 25 25  0.631 0.806 0.763 0.591 0.568  Dil.  O.D.  1  1551.65 1550.27 1474.35 1463.37 1412.62  1562.01 1551.20 1480.88 1468.45 1414.68  10.03 2.33 6.65 13.07 2.25  SO4'"  Average  St. Dev  (mg L" )  SO4  1  (mg L" ) 1  13-Jun 27-Jun 11-Jul 28-Jul 23-Aug 16-Sep  01  0 14 28 45 71 95  30 25 30 30 40 20  0.585 0.664 0.446 0.422 0.097 0.124  1740.16 1624.47 1372.07 1308.51 597.14 415.49  40 30 25 40 30  0.408 0.579 0.535 0.285 0.131  1695.25 1724.27 1339.79 1260.95 537.89  25 20 20 25 25  0.699 0.882 0.681 0.491 0.178  1701.71 1684.44 1329.59 1242.69 551.97  1712.37 1677.73 1347.15 1270.72 562.33 415.49  24.28 50.24 22.17 33.98 30.96  Date  Time  Dil.  O.D.  SO/ (mg L- ) 1  Orchard Grass 2 (ORC) Raw Dil. O.D. SO/" (mg I-")  Dil.  O.D.  1  SO/ (mg L- ) -  1  Average  so/"  St. Dev  (mg L" ) 1  13-Jun 27-Jun 11-Jul 28-Jul 23-Aug 16-Sep  0 14 28 45 71 95  40 25 30 40 40 20  0.426 0.678 0.422 0.254 0.166 0.283  1758.80 1655.37 1308.51 1151.49 840.77 752.33  Date  Time  Dil.  O.D.  SO/" (mg L" ) 1  30 30 25 30 30  0.599 0.56 0.512 0.394 0.255  1777.23 1673.96 1289.04 1234.36 866.27  25 20 20 25 25  0.722 0.866 0.661 0.485 0.323  1752.46 1656.20 1294.28 1229.45 871.95  1762.83 1661.84 1297.28 1205.10 859.66 752.33  12.87 10.50 10.08 46.49 16.60  Orchard 1 (ORC) Flocced Dil. O.D. so/(mg I.")  Dil.  O.D.  SO/" (mg L" )  Average  St. Dev  1  1  SO4  (mg L" ) 1  13-Jun 27-Jun 11-Jul 28-Jul 23-Aug 16-Sep  0 14 28 45 71 95  30 30 25 30 40 20  0.591 0.571 0.491 0.325 0.236 0.406  1756.05 1703.09 1242.69 1051.64 1087.94 1012.91  Date  Time  Dil.  O.D.  SO/" (mg L" ) 1  1727.02 1690.67 1266.14 971.42 1048.99  25 20 20 25 25  0.721 0.858 0.642 0.382 0.38  1750.26 1642.07 1260.74 1002.15 997.74  1744.44 1678.61 1256.52 1008.40 1044.89 1012.91  15.36 32.25 12.28 40.47 45.24  Orchard 2 (PRC) Flocced SO/" Dil. O.D. (mg L" )  Dil.  O.D.  SO/' (mg L" )  Average S0 " (mg L" )  St. Dev  1710.31 1654.92 1417.47 1173.32 498.63 320.15  67.68 9.19 27.18 52.88 4.99  40 25 30 40 30  0.417 0.694 0.406 0.203 0.324  1  1  2  4  1  13-Jun 27-Jun 11-Jul 28-Jul 23-Aug 16-Sep  OS  0 14 28 45 71 95  40 25 25 40 40 20  0.432 0.673 0.563 0.252 0.07 0.079  1779.99 1644.33 1401.58 1144.43 501.81 320.15  30 30 30 30 30  0.549 0.555 0.475 0.394 0.114  1644.83 1660.71 1448.86 1234.36, 492.88  25 20 20 25 25  0.701 0.868 0.722 0.445 0.155  1706.12 1659.73 1401.97 1141.18 501.21  Date  Time  Dil.  O.D.  SO/" (mg L" )  Dil.  Ctrl #2 O.D.  1  ±L  SO/" (mg L" )  Dil.  O.D.  1  SO/" (mg L" ) 1  Average SO/" (mg L" )  St. Dev  1727.97 810.38  35.90 5.10  1  13-Jun 27-Jun  0  40 10  0.429 0.845  1769.40 809.56  30 20  0.573 0.39  1708.38 815.85  25 25  0.701 0.293  1706.12 805.75  A1-5b. Total sulphide concentrations in leachate experiment  Date  Sample  Dil.  O.D.  Sulphide (mg L- )  Dil.  O.D.  1  Sulphide (mg L- )  Dil.  O.D.  1  Sulphide (mg L' ) 1  Average Sulphide (mg L- )-  St. Dev  78.69 58.85 10.61 15.06 26.65 20.80 38.06 4.15 10.22 57.95 5.68 4.84 2.73 4.91  5.85 2.87 0.43 0.31  1  28-Jul  TAD 1 Raw TAD 1 Floe 30-Aug TAD 1 Raw TAD 2 Raw TAD 1 Floe TAD 2 Floe TBD 1 Raw TBD 2 Raw TBD 1 Floe TBD 2 Floe Orch 1 Raw Orch 2 Raw Orch 1 Floe Orch 2 Floe  100 100 50 50 50 50 50 50 50 50 50 50 50 50  0.335 0.244 0.084 0.115 0.199 0.159 0.293 0.033 0.057 0.473 0.035 0.034 0.015 0.019  83.63 61.34 11.08 14.87 25.16 20.26 36.67 4.83 7.77 58.72 5.08 4.96 2.63 3.12  200 200 10 10 10 10 10 10 10 10 10 10 10 10  A1-5c. pH in leachate experiment  TAD 1 Raw TAD 2 Raw TAD 1 Flocced TAD 2 Flocced ORC 1 Raw ORC 2 Raw ORC 1 Flocced  OO  13-Jun 7.8 7.5 7.4  27-Jun 6.7 7.8 6.5  11-Jul 7.2 7.3 7.3  28-Jul 7.6 7.6 7.5  23-Aug 7.7 7.6 7.6  7.4  6.8  7.3  7.5  7.7  7.4 7.3 7.1  7.1 7.0 7.1  7.3 7.3 7.3  7.5 7.6 7.5  7.6 7.6 7.6  0.141 0.115 0.422 0.623 1.15 1.21 1.22 0.157 0.459 1.66 0.234 0.187 0.113 0.258  72.24 59.50 10.49 15.42 over over over 4.00 11.40 over 5.89 4.74 2.93 6.48  100 100 10 10 20 20 20 10 10 100 10 10 10 10  0.321 0.221 0.412 0.601 0.568 0.429 0.799 0.141 0.462 0.227 0.241 0.191 0.101 0.203  80.20 55.71 10.25 14.88 28.14 21.33 39.46 3.61 11.47 57.18 6.06 4.84 2.63 5.13  0.62 2.12 0.53 0.11 0.17 1.69  ORC 2 Flocced TBD 1 Raw TBD 2 Raw TBD 1 Flocced TBD 2 Flocced  13-Jun 7.5  27-Jun 7.0  11-Jul 7.2  28-Jul 7.6  23-Aug 7.6  7.4 7.5 7.5  6.6 6.5 6.7  7.3 7.3 7.2  7.5 7.5 7.5  7.5 7.6 7.5  7.5  7.0  7.2  7.6  7.5  A1-5d. Soluble COD in leachate experiment  Date  Time  Dil.  O.D.  sCOD (mg L- ) 1  Barley 1 (TAD) Raw Dil. O.D. sCOD (mg I-")  Dil.  O.D.  1  sCOD (mg I-") 1  Average sCOD (mg L- )  St. Dev  1  13-Jun 27-Jun 13-Jul 28-Jul 23-Aug  0 14 28 45 71  30 30 30 20  0.129 0.124 0.117 0.14  9572.06 9201.05 8681.63 6925.52  20 20 20 20 30  Date  Time  Dil.  O.D.  sCOD (mg I-")  Barley 2 (TAD) Raw Dil. O.D. sCOD (mg L- )  1  0.228 0.196 0.184 0.178 0.1  11278.70 9695.73 9102.11 8805.30 7420.20  10 40 40 40 40  0.396 0.096 0.094 0.087 0.072  9794.66 9497.86 9299.98 8607.43 7123.39  10536.68 9588.55 9201.05 8698.12 7156.37  Dil.  O.D.  sCOD (mg I-")  Average sCOD (mg L" )  1  1  99.96 98.94 99.96 248.98  St. Dev  1  13-Jun 27-Jun 13-Jul 28-Jul 23-Aug  0 14 28 45 71  20 30 30 30 20  0.226 0.134 0.124 0.121 0.127  11179.77 9943.07 9201.05 8978.44 6282.44  10 20 20 20 30  0.399 0.19 0.19 0.172 0.086  9868.87 9398.92 9398.92 8508.50 6381.37  40 40 40 40  0.095 0.093 0.083 0.064  9398.92 9201.05 8211.69 6331.90  10524.32 9580.30 9267.01 8566.21 6331.90  314.16 114.24 386.62 49.47  Date  Time  Dil.  O.D.  sCOD (mg L- ) 1  Barley 1 (TAD) Flocced Dil. O.D. sCOD (mg L- )  Dil.  O.D.  1  sCOD (mg L- ) 1  Average sCOD (mg L- )  St. Dev  1  13-Jun 27-Jun 13-Jul 28-Jul 23-Aug  0 14 28 45 71  20 30 30 30 20  0.214 0.14 0.121 0.117 0.152  10586.15 10388.28 8978.44 8681.63 7519.14  Date  Time  Dil.  O.D.  sCOD (mg L- ) 1  10 20 20 20 30  0.391 0.234 0.191 0.164 0.099  9670.99 11575.51 9448.39 8112.75 7346.00  Barley 2 (TAD) Flocced Dil. O.D. sCOD (mg L- )  40 40 40 40  0.103 0.095 0.084 0.075  Dil.  O.D.  10128.57 10190.41 10718.07 9398.92 9275.25 8310.62 8368.34 7420.20 7428.44  sCOD (mg L- ) 1  1  Average sCOD (mg L- )  749.13 258.23 288.80 86.86  St. Dev  1  13-Jun 27-Jun 13-Jul 28-Jul 23-Aug  0 14 28 45 71  20 30 30 30 20  0.219 0.122 0.119 0.108 0.14  10833.49 9052.64 8830.04 8013.82 6925.52  Date  Time  Dil.  O.D.  sCOD (mg L' ) 1  10 20 20 20 30  0.392 0.192 0.174 0.161 0.098  9695.73 9497.86 8607.43 7964.35 7271.80  Bioethanol 1 (TBD) Raw Dil. O.D. sCOD (mg L- )  40 40 40 40  0.091 0.089 0.08 0.069  9003.18 8805.30 7914.88 6826.58  Dil.  O.D.  sCOD (mg L- )  1  1  10264.61 9184.56 8747.59 7964.35 7007.97  272.45 122.01 49.47 233.78  Average sCOD (mg I-")  St. Dev  1866.59 1698.40 1421.38 1048.72 971.22  211.98 5.71 5.71 34.27 18.73  1  13-Jun 27-Jun 13-Jul 28-Jul 23-Aug  ro O  0 14 28 45 71  5 4 4 8 2  0.146 0.172 0.143 0.055 0.199  1805.58 1701.70 1414.78 1088.30 984.41  2 2 2 4 4  0.342 0.342 0.288 0.104 0.099  1691.81 1691.81 1424.68 1028.93 979.47  10 8 8 8 8  0.085 0.086 0.072 0.052 0.048  2102.39 1701.70 1424.68 1028.93 949.79  Date  Time  Dil.  O.D.  sCOD (mg L- ) 1  Bioethanol 2 (TBD) Raw Dil. O.D. sCOD (mg L- )  Dil.  O.D.  1  sCOD (mg L- ) 1  Average sCOD (mg L- )  St. Dev  1  13-Jun 27-Jun 13-Jul 28-Jul 23-Aug  0 14 28 45 71  5 4 2 8 2  Date  Time  Dil.  0.155 0.198 0.311 0.052 . 0.177  1916.89 1958.93 1538.45 1028.93 875.58  O.D.  sCOD (mg I-") 1  2 2 4 4 4  0.376 0.364 0.155 0.099 0.097  1860.00 1800.64 1533.51 979.47 959.68  10 8 8 8 8  0.073 0.088 0.077 0.053 0.044  1805.58 1741.27 1523.61 1048.72 870.64  1860.82 1833.61 1531.86 1019.04 901.97  55.66 112.51 7.56 35.67 50.04  Bioethanol 1 (TBD) Flocced Dil. O.D. sCOD (mg L" )  Dil.  O.D.  sCOD (mg L- )  Average sCOD (mg L- )  St. Dev  1  1  1  13-Jun 27-Jun 13-Jul 28-Jul 23-Aug  0 14 28 45 71  5 4 4 8 2  0.122 0.157 0.124 0.066 0.142  1508.77 1553.30 1226.81 1305.96 702.45  Date  Time  Dil.  O.D.  sCOD (mg L- ) 1  2 2 2 4 4  0.276 0.333 0.243 0.11 0.08  1365.32 1647.28 1202.07 1088.30 831.06  10 8 8 8 8  0.069 0.079 0.062 0.053 0.036  1706.65 1563.19 1226.81 1048.72 712.34  1526.91 1587.92 1218.56 1147.66 748.62  171.39 51.65 14.28 138.51 71.57  Bioethanol 2 (TBD) Flocced Dil. O.D. sCOD (mgl_- )  Dil.  O.D.  sCOD (mg L- )  Average sCOD (mg L' )  St. Dev  1730.56 1434.57 1028.93 359.47 176.44  168.49 9.89 19.79 54.49 19.99  1  1  1  13-Jun 27-Jun 13-Jul 28-Jul 23-Aug  0 14 28 45 71  5 4 4 8 2  0.139 0.146 0.106 0.021 0.035  1719.01 1444.47 1048.72 415.53 173.14  2 2 2 4 4  0.317 0.29 0.208 0.031 0.016  1568.14 1434.57 1028.93 306.70 158.30  10 8 8 8 8  0.077 0.072 0.051 0.018 0.01  1904.52 1424.68 1009.15 356.17 197.87  Date  Time  Dil.  O.D.  sCOD (mg L- ) 1  Orchard Grass 1 (ORC) Raw Dil. O.D. sCOD (mg L- )  Dil.  O.D.  1  sCOD (mg I-") 1  Average sCOD (mg L' )  St. Dev  8520.86 7758.23 6636.96 5152.92 3438.03  332.30 164.69 126.93 111.53 211.33  Average sCOD (mg L" )  St. Dev  8570.33 8252.91 6397.86 5499.19 3916.22  402.26 116.89 151.13 192.12 144.93  Average sCOD (mg L' )  St. Dev  8322.99 7717.01 7255.31 5507.44 5367.28  332.30 0.00 114.24 75.56 173.14  1  13-Jun 27-Jun 13-Jul 28-Jul 23-Aug  0 14 28 45 71  10 30 20 30 20  0.335 0.107 0.137 0.071 0.065  8285.89 7939.61 6777.12 5268.34 3215.42  Date  Time  Dil.  O.D.  sCOD (mg L' ) 1  20 20 30 20 30  0.177 0.154 0.089 0.104 0.049  8755.84 7618.07 6603.98 5144.67 3635.90  Orchard Grass 2 (ORC) Raw Dil. O.D. sCOD (mg L' )  40 40 40 40  0.078 0.066 0.051 0.035  7717.01 6529.78 5045.74 3462.76  Dil.  O.D.  sCOD (mg L- )  1  1  1  13-Jun 27-Jun 13-Jul 28-Jul 23-Aug  0 14 28 45 71  10 30 20 30 20  0.335 0.113 0.13 0.077 0.077  8285.89 8384.83 6430.84 5713.55 3809.04  Date  Time  Dil.  O.D.  sCOD (mg L- ) 1  20 20 30 20 30  0.179 0.165 0.088 0.11 0.055  8854.77 8162.22 6529.78 5441.48 4081.11  Orchard 1 (ORC) Flocced Dil. O.D. sCOD (mg L- )  40 40 40 40  0.083 0.063 0.054 0.039  8211.69 6232.97 5342.54 3858.50  Dil.  O.D.  sCOD (mg I/ )  1  1  1  13-Jun 27-Jun 13-Jul 28-Jul 23-Aug  0 14 28 45 71  10 30 20 30 20  0.327 0.104 0.148 0.074 0.107  8088.02 7717.01 7321.26 5490.95 5293.08  20 20 30 20 30  0.173 0.156 0.096 0.113 0.075  8557.96 7717.01 7123.39 5589.88 5565.15  40 40 40 40  0.078 0.074 0.055 0.053  7717.01 7321.26 5441.48 5243.61  Date  Time  Dil.  O.D.  sCOD (mg L- ) 1  Orchard 2 (ORC) Flocced Dil. O.D. sCOD (mg I/ )  Dil.  O.D.  1  sCOD (mg L ) 1  Average sCOD (mg I-")  St. Dev  8347.73 7865.41 7156.37 5655.84 3454.52  297.32 257.04 151.13 124.49 157.08  1  13-Jun 27-Jun 13-Jul 28-Jul 23-Aug  0 14 28 45 71  20 30 30 30 20  0.173 0.11 0.096 0.078 0.068  8557.96 8162.22 7123.39 5787.76 3363.82  10 20 20 20 30  0.329 0.156 0.148 0.112 0.049  8137.49 7717.01 7321.26 5540.42 3635.90  40 40 40 40  0.078 0.071 0.057 0.034  A1-5e. Ortho-phosphate in leachate experiment Sample  Dil.  O.D.  P0 " (mg L- ) J  4  Dil.  O.D.  1  P0 (mg L- ) 3-  4  1  Average P0 ' (mg L" ) 3  4  1  30-Aug  TAD 1 Raw TAD 2 Raw TAD 1 Floe TAD 2 Floe TBD 1 Raw TBD 2 Raw TBD 1 Floe TBD 2 Floe Orch 1 Raw Orch 2 Raw Orch 1 Floe Orch 2 Floe  50 50 50 50 50 50 50 50 50 50 50 50  1.89 1.38 1.54 1.45 1.99 1.83 1.75 1.31 1.71 1.58 1.3 1.22  94.5 69 77 72.5 99.5 91.5 87.5 65.5 85.5 79 65 61  100 100 100 100 100 100 100 100 100 100 100 100  0.96 0.75 0.55 0.61 1.04 1.01 0.81 0.73 0.91 0.73 0.71 0.55  96 75 55 61 104 101 81 73 91 73 71 55  95.3 72.0 66.0 66.8 101.8 96.3 84.3 69.3 88.3 76.0 . 68.0 58.0  7717.01 7024.46 5639.35 3363.82  A1-5f. Ammonia-nitrogen in leachate experiment Dil.  Sample  O.D.  NH -N (mg L" )  Dil.  3  O.D.  1  NH -N (mg L" )  Dil.  3  O.D.  1  NH -N (mg L- ) 3  1  Average NH -N (mg I-") 3  1  30-Aug TAD 1 Raw TAD 2 Raw TAD 1 Floe TAD 2 Floe TBD 1 Raw TBD 2 Raw TBD 1 Floe TBD 2 Floe Orch 1 Raw Orch 2 Raw Orch 1 Floe Orch 2 Floe  50 250 250 250 50 50 50 50 100 250 250 250  over 1.44 1.49 1.46 1.6 1.91 1.55 1.04 over 1.45 1.47 1.42  >150 360 372.5 365 80 95.5 77.5 52 >300 362.5 367.5 355  100 200 200 200 100 100 100 100 250 200 200 200  over 1.86 1.88 1.71 0.77 0.92 0.82 0.49 1.35 1.77 1.82 1.83  >300 372 376 342 77 92 82 49 337.5 354 364 366  250  200  1.43  1.61  357.5  322  366.0 374.3 353.5 78.5 93.8 79.8 50.5 329.8 358.3 365.75 360.5  A1-6a. S O / concentrations in natural treatment mesocosm -  Mesocosm A Date 28-Sep 28-Sep 28-Sep 28-Sep 28-Sep 28-Sep 28-Sep 13-Oct  -fe.  Length Position 50 100 150 50 100 150 Outlet Inlet  S0 " (mg L- )  Dil.  135.55 137.80 119.16 28.41 65.69 48.48 128.26 2340.94  5 3.33 5 5 5 5 5 30  2  Height Position 10 10 10 20 20 20  Dil. 5 5 3.33 5 3.33 3.33 5 40  O.D. 0.175 0.179 0.252 -0.016 0.109 0.063 0.162 0.455  4  1  O.D. 0.157 0.294 0.167 -0.005 0.074 -0.03 0.161 0.65  SO/" (mg L" )  Dil.  125.45 134.87 131.06 34.58 78.90 20.56 127.70 2412.02  3.33 5 5 3.33 5 5 3.33 50  1  O.D.  SO/" (mg L" ) 1  0.285 131.50 0.175 135.55 0.147 119.84 -0.013 20.06 0.097 91.80 -0.007 33.46 122.41 0.261 0.388 2550.34  Averaae  so/  (mg L" ) 130.84 136.07 123.36 27.68 78.79 34.17 126.12 2434.44  St. Dev.  1  5.08 1.53 6.68 7.29 13.06 13.98 3.23 106.48  Date 13-Oct 13-Oct 13-Oct 13-Oct 13-Oct 13-Oct 13-Oct 22-Oct 22-Oct 2-Nov 2-Nov 10-Nov 10-Nov 19-Nov 19-Nov 30-Nov 30-Nov  Length Position 50 100 150 50 100 150 Outlet Inlet Outlet Inlet Outlet Inlet Outlet Inlet Outlet Inlet Outlet  S0 " (mg L' )  Dil.  0.463 0.31 0.325 0.357 0.5 0.403 0.106 0.495 0.784 0.492 0.585 0.275 0.866 0.471 0.532 0.481 0.656  1782.63 1690.24 1318.17 1425.87 1907.16 1580.69 774.77 2520.45 1908.68 2506.99 2412.58 1533.17 2615.85 1507.97 2216.35 1536.01 2026.85  40 30 20 40 40 50 20 40 40 33 33 25 40 20 40 20 20  0.324 0.441 0.528 0.259 0.348 0.206 0.263 0.476 0.375 0.589 0.58 0.526 0.542 0.603 0.431 0.607 0.837  1753.07 1708.59 1334.27 1461.37 1860.77 1529.41 739.66 2435.18 1981.93 2427.38 2394.06 1662.23 2731.37 1502.56 2233.24 1511.53 2027.61  O.D.  S0 (mg L- )  Dil.  O.D.  98.53 106.94 108.32 80.58 37.95 59.70 2340.94 1613.95 1672.29 1676.25  3.33 3.33 5 3.33 3.33 5 30 30 25 30  2  Height Position 10 10 10 20 20 20  Dil. 30 40 30 30 30 30 40 40 20 40 33 40 25 25 33 25 25  Height Position 10 10 10 20 20 20  Dil.  O.D.  4  1  O.D.  Average S0 ^ (mg L- ) 1808.08 1781.26 1760.39 1719.74 1373.34 1341.92 1383.56 1423.60 1826.71 1864.88 1546.64 1552.25 756.89 757.10 2438.49 2464.71 1923.87 1938.16 2590.28 2508.22 2492.44 2433.03 1675.82 1623.74 2460.71 2602.64 1498.07 1502.86 2224.80 1523.77 2027.23 S0 " (mg L- ) 2  SO4 ' 2  (mg L- ) 1  Dil.  O.D.  25 25 25 50 50 40 10 33 33 33 25 33 33 20 25  0.578 0.561 0.423 0.18 0.259 0.278 0.608 0.592 0.453 0.633 0.822 0.386 0.598 0.601  S0 " (mg L' )  Dil.  O.D.  94.86 107.20 104.70 57.46 25.67 47.48 2412.02 1634.54 1653.81 1641.27  5 5 5 5 5 5 50 25 30 40  4  1  4  St. Dev.  1  27.53 36.38 28.37 38.95 40.38 26.10 17.55 48.30 38.66 81.46 52.28 78.73 135.81 4.96 11.94 17.31 0.54  Mesocosm B Date 28-Sep 28-Sep 28-Sep 28-Sep 28-Sep 28-Sep 13-Oct 13-Oct 13-Oct 13-Oct  Length Position 50 100 150 50 100 150 Inlet 50 100 150  2  10 10 10  5 5 3.33 5 5 3.33 40 40 40 25  0.109 0.124 0.223 0.077 0.001 0.093 0.455 0.293 0.306 0.531  4  1  2  0.187 0.22 0.12 0.087 0.002 0.018 0.65 0.419 0.523 0.421  4  1  Averaqe S0 ^ (mg L- ) (mg L- ) 107.50 100.30 112.55 108.90 106.94 106.65 70.48 69.51 37.95 33.85 39.07 48.75 2550.34 2434.44 1636.98 1628.49 1553.77 1626.62 1730.63 1682.72 SO4 ' 2  1  4  St. Dev.  1  0.125 0.134 0.124 0.059 0.001 0.003 0.388 0.517 0.395 0.319  6.51 3.17 1.83 11.59 7.09 10.38 106.48 12.65 63.77 45.03  Date 13-Oct 13-Oct 13-Oct 13-Oct 22-Oct 22-Oct 2-Nov 2-Nov 10-Nov 10-Nov 19-Nov 19-Nov 30-Nov 30-Nov  Length Position 50 100 150 Outlet Inlet Outlet Inlet Outlet Inlet Outlet Inlet Outlet Inlet Outlet  Dil.  Dil.  30 30 30 40 40 40 40 33 40 40 25 25 25 25  2112.47 1516.74 1759.07 779.25 2520.45 1851.79 2506.99 2375.55 1533.172 2637.126 1507.965 2029.651 1536.013 2043.675  50 50 50 20 40 33 33 33 25 25 20 40 20 20  O.D.  4  1  0.561 0.384 0.456 0.107 0.495 0.346 0.492 0.575 0.275 0.521 0.471 0.657 0.481 0.662  S0 " (mg L- )  O.D. 0.297 0,192 0.225 0.288 0.476 0.407 0.589 0.56 0.526 0.859 0.603 0.394 0.607 0.837  so ' 2  2  S0 (mg L" ) 2 -  Height Position 20 20 20  4  Dil.  1  2039.88 1450.88 1635.99 795.76 2435.18 1753.57 2427.38 2320.02 1662.227 2596.212 1502.555 2067.199 1511.530 2027.606  40 40 40 10 33 20 33 25 33 33 20 33  O.D. 0.419 0.29 0.311 0.638 0.592 0.767 0.633 0.809 0.386 0.595 0.601 0.468  4  (mg L- ) 1  2179.39 1600.49 1694.73 790.54 2438.49 1870.54 2590.28 2455.97 1675.82 2449.60 1498.07 1979.41  Average S0 ' (mg L- ) 2110.58 1522.70 1696.60 788.52 2464.71 1825.30 2508.22 2383.85 1623.74 2560.98 1502.86 2025.419 1523.772 2035.640 2  4  St. Dev.  1  69.78 74.98 61.56 8.44 48.30 62.82 81.46 68.36 78.73 98.60 4.96 44.05  A1-6b. Total sulphide in natural treatment mesocosm Mesocosm A Date 28-Sep 13-Oct 22-Oct 2-Nov 10-Nov 19-Nov 30-Nov  ON  Time 0 15 24 35 43 52 63  Dil. 2 2 2 5 2 1 2  O.D. 0.122 0.753 0.124 0.028 0.037 0.025 0.015  Sulphide (mg L- )  Dil.  0.50 3.29 0.51 0.20 0.12 0.03 0.02  2 5 2 2 2 2 1  1  O.D. 0.104 0.308 0.119 0.051 0.024 0.022 0.018  Sulphide (mg L- )  Dil.  0.42 3.30 0.48 0.18 0.06 0.05 0.02  5 5 5 10 1 2 2  1  O.D.  Average Sulphide Sulphide (mg L- ) (mg L- ) 0.44 0.45 3.37 3.32 0.61 0.53 0.00 0.13 0.06 0.08 0.04 0.04 0.01 0.02 1  St. Dev.  1  0.05 0.314 0.065 0.01 0.035 0.018 0.013  0.04 0.04 0.07 0.11 0.04 0.01 0.00  Mesocosm B Date 28-Sep 13-Oct 22-Oct 2-Nov 10-Nov 19-Nov 30-Nov  Time 0 15 24 35 43 52 63  Dil. 20 20 40 40 2 2 1  O.D.  Sulphide (mg L- )  Dil.  0.10 28.08 8.25 1.34 0.08 0.04 0.02  20 40 20 20 2 2 2  0.012 0.644 0.103 0.025 0.029 0.018 0.018  1  O.D. 0.015 0.343 0.217 0.034 0.03 0.021 0.022  A1-6c. pH in natural treatment mesocosm Mesocosm A Position along the length of the mesocosm 100 cm 50 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 8.62 22-Oct 8.53 8.68 7.91 8.51 7.66 7.77 10-Nov 8.22 8 7.82 7.97 8.23 19-Nov 8.32 7.37 8.17 8.05 8.46 8.29 30-Nov 7.74 7.78 8.68 7.75 7.83 7.61 Mesocosm B 28-Sep 8.61 22-Oct 8.61 10-Nov 8.13 19-Nov 8.14 30-Nov 7.32  8.06 8.52 8.85 8.56 7.83  8.99 8.56 8.28 8.28 7.5  7.57 7.84 7.74 8.2 8.14  8.89 8.52 8.35 8.28 7.73  7.58 7.71 8.43 8.38 7.86  Sulphide (mg L- )  Dil.  0.23 29.50 9.17 1.07 0.09 0.05 0.05  40 40 20 40 1 1 2  1  O.D.  Average Sulphide Sulphide (mg L- ) (mg L- ) 3.56 1.30 31.27 29.62 8.41 7.80 0.99 1.14 0.04 0.07 0.02 0.04 0.02 0.03 1  St. Dev.  1  0.05 0.363 0.186 0.021 0.03 0.021 0.015  1.96 1.60 0.70 0.19 0.02 0.01 0.02  A1-6d. Dissolved oxygen (% saturation) in natural treatment mesocosm Mesocosm A Position along the length of the mesocosm 150 cm 50 cm 100 cm Depth (cm) Depth (cm) Depth (cm) 10 20 Date 10 20 10 20 30.4 2.4 47.1 2.1 24.1 2.0 28-Sep 8.4 6.0 1.3 22-Oct 28.5 1.6 1.3 7.9 1.2 10-Nov 2.6 1.6 2.8 1.3 2.4 28.8 2.8 19-Nov 2.1 27.6 29.9 3.7 46.0 2.9 30-Nov 49.0 4.0 55.0 Mesocosm B 26.7 28-Sep 22-Oct 30.3 10-Nov 39.8 19-Nov 11.0 30-Nov 37.0  1.7 1.5 1.9 2.1 1.6  1.7 1.0 3.6 1.5 2.4  32.3 7.1 40.5 6.1 39.0  23.8 4.8 49.6 7.0 41.0  1.6 1.0 3.1 2.2 1.9  A1-6e. Ammonia-nitrogen in natural treatment mesocosm Mesocosm A Date 28-Sep 15-Nov  Dil. 1 1  NHj-N (mg L )  Dil.  0.40 0.00  0.40 0.00  1 1  0.64 0.00  0.64 0.00  O.D.  NH -N (mg L' )  Dil.  O.D.  NH -N (mg L")  0.58 0.02  1 1  O.D.  1  O.D.  NH -N (mg I.") 3  1  Average NH -N (mg L- ) 0.52 0.00 3  1  Mesocosm B Date 28-Sep 15-Nov  OO  Dil. 1 1  0.58 0.02  3  1  1.07 0.00  3  1  1.07 0.00  Average NH -N (mg L" ) 0.83 0.01 3  1  A1-6f. Ortho-phosphate in natural treatment mesocosm Mesocosm A P0 ' (mg L- )  Dil.  over over  >3 >3  10 10  O.D.  P0 " (mg L- )  Dil.  >6 4.80  10 5  P0 ' (mg L- )  Dil.  2.75 0.73  27.50 7.30  20 5  1.33 1.51  O.D.  P0 " (mg L- )  Dil.  O.D.  3  Date  Dil.  1 28-Sep 15-Nov '1 Mesocosm B  O.D.  4  1  3  O.D.  3  Date  Dil.  28-Sep 15-Nov  2 10  over 0.48  4  1  4  1  3  2.49 1.01  4  1  24.90 5.05  P0 " (mg L- ) 3  O.D.  20  4  1  26.60 7.55 PO 3  1.13  Average P0 ' (mg L" ) 27.05 7.43 3  4  1  Average P0 " (mg L' ) 3  4  (mg L' ) 1  22.60  4  1  23.75 4.93  A1-7a. S 0 ' concentrations in mesocosms while operated as batch systems 2  4  Natural Treatment Mesocosm A (water column) Date 6-Dec 13-Dec 21-Dec 29-Dec 7-Jan 14-Jan 24-Jan 4-Feb 14-Feb 21-Feb 5-Jul 13-Sep  r-J  4^  v©  Time (Days)  Dil.  0 7 15 23 32 39 49 60 70 77 211 281  20 33 33 33 33 33 33 33 33 33 33 33  O.D. 0.878 0.441 0.471 0.404 0.473 0.494 0.504 0.505 0.501 0.489 0.427 0.370  Dil.  2119.60 1879.45 1990.52 1742.46 1997.92 2075.67 2112.69 2116.39 2101.58 2057.16 1827.61 1616.58  20 33 33 33 33 33 33 33 33 33 25 30  2  4  1  S0 ' (mg L )  Dil.  2034.34 1883.15 2031.24 1716.55 2053.45 2090.48 2101.58 2086.77 2042.35 2012.73 1872.58 1560.50  33 20 25 20 25 25 25 25 25 25 30 25  2  S0 " (mg I/ )  O.D. 0.840 0.442 0.482 0.397 0.488 0.498 0.501 0.497 0.485 0.477 0.601 0.397  4  1  S0 " (mg L- ) 2  O.D. 0.471 0.768 0.645 0.689 0.653 0.647 0.679 0.683 0.671 0.651 0.489 0.471  4  1  1990.52 1872.78 1995.99 1695.52 2018.43 2001.60 2091.36 2102.58 2068.92 2012.82 1870.14 1507.97  Average S0 ^ (mg L' ) 2048.15 1878.46 2005.92 1718.18 2023.27 2055.92 2101.88 2101.91 2070.95 2027.57 1856.78 1561.68 4  St. Dev.  1  65.64 5.25 22.10 23.51 28.08 47.62 10.67 14.82 29.67 25.62 25.29 54.32  Natural Treatment Mesocosm A Sediment (100 cm) Date 7-Jan 14-Jan 14-Feb 21-Feb 28-Feb 7-Mar 6-Jun 5-Jul 13-Sep  S0 " (mg I-")  Dil.  691.01 620.74 968.68 1113.07 378.41 542.92 1268.57 1181.92 1332.23  20 33 25 25 10 25 20 25 20  2  Time (Days)  Dil.  32 39 70 77 84 91 182 211 281  33 20 33 33 20 33 33 33 33  O.D. 0.12 0.21 0.195 0.234 0.102 0.08 0.276 0.266 0.309  4  1  Natural Treatment Mesocosm A Sediment (50cm) Time Time (Days) Dil. O.D. Date (Days) (new exp.) 7-Mar 91 0 25 0.575 14-Mar 98 7 0.315 25 21-Mar 105 14 25 0.263 29-Mar 113 22 0.172 33 12-Apr 127 36 33 0.179 18-Apr 133 42 33 0.208 25-Apr 140 49 25 0.378 2-May 147 0.247 56 25 16-May 161 0.16 70 33 0.167 24-May 169 78 25 30-May 175 84 0.55 10 6-Jun 182 91 33 0.378 211 5-Jul 127 33 0.33 13-Sep 281 233 33 0.366  O  S0 " (mg I-") 4  1  Dil.  O.D.  679.08 694.72 927.38 1109.69 365.34 470.20 1273.69 1165.51 1307.39  25 25 20 20 33 20  0.168 0.162 0.345 0.387 0.032 0.152  20 25  0.478 0.422  S0 (mg L- )  Dil.  O.D.  so -  1  1799.66 1070.42 924.58 883.53 909.45 1016.81 1247.12 879.70 839.10 655.32 691.82 1646.20 1405.64 1531.48  33 33 33 25 25 25 33 33 25 33 20 20 25 20  2  2 -  1  4  0.374 0.209 0.187 0.231 0.277 0.319 0.246 0.182 0.237 0.104 0.229 0.684 0.425 0.601  2-  4  St. Dev.  1  0.236 0.121 0.264 0.329 0.259 0.101 0.501 0.368 0.545  4  Average S0 (mg L- ) (mg L- ) 658.12 676.07 641.30 652.25 923.65 939.91 1017.89 1080.22 365.21 369.65 501.24 490.60 1271.13 1165.45 1170.96 1308.51 1316.04  so 2  2  O.D.  4  (mg I.") 1  1631.39 1020.52 939.07 834.82 963.84 1081.64 1157.50 920.56 851.65 631.78 663.37 1684.30 1316.45 1426.02  so 2  Dil. 20 20 20 20 20 20 20 20 20 20 20 25 20 25  O.D. 0.656 0.382 0.345 0.313 0.327 0.389 0.457 0.359 0.313 0.213 0.223 0.513 0.576 0.501  4  (mg L' ) 1  1621.48 1006.67 923.65 851.85 883.26 1022.38 1174.96 955.07 851.85 627.47 649.91 1625.77 1373.06 1517.71  16.65 38.19 24.99 54.00 7.58 37.51 9.50 14.03  Average S0 (mg L- ) 1684.18 1032.54 929.10 856.74 918.85 1040.28 1193.19 918.44 847.54 638.19 668.37 1652.09 1365.05 1491.74 2-  4  St. Dev.  1  100.13 33.53 8.64 24.72 41.10 35.93 47.51 37.73 7.30 14.99 21.40 29.71 45.13 57.33  Natural Treatment Mesocosm B (water column) Date 6-Dec 13-Dec 21-Dec 29-Dec 7-Jan 14-Jan 24-Jan 4-Feb 14-Feb 21-Feb 5-Jul 13-Sep  Time (Days)  Dil.  0 7 15 23 32 39 49 60 70 77 211 281  20 33 33 33 33 33 33 33 33 33 25 33  S0 (mg L' )  Dil.  2000.68 1868.34 2012.73 1731.36 2101.58 1886.85 1905.36 1931.28 1968.30 1901.66 1816.49 1564.75  20 33 33 33 33 33 33 33 33 33 30 25  S0 " (mg L" )  Dil.  472.58 1132.33 520.71 705.82 734.45 461.79 1572.16 1342.72 1258.83  20 33 25 20 33 33 20 25 20  S0 (mg L- )  Dil.  0.828 0.441 0.478 0.406 0.492 0.451 0.448 0.468 0.455 0.438 0.453 0.501  2007 .'41 1879.45 2016.43 1749.87 2068.26 1916.47 1905.36 1979.41 1931.28 1868.34 1748.98 1592.11  33 20 25 20 25 25 25 25 25 25 33 30  0.482 0.759 0.637 0.656 0.666 0.607  2031.24 1852.59 1973.56 1621.48 2054.89 1889.41  0.627 0.613 0.599 0.429 0.374  1945.51 1906.24 1866.98 1835.02 1483.09  O.D.  S0 ' (mg L- )  Dil.  O.D.  S0 ' (mg I-")  2_  O.D. 0.825 0.438 0.477 0.401 0.501 0.443 0.448 0.455 0.465 0.447 0.581 0.356  4  1  2-  O.D.  4  1  S0 ' (mg L' ) 2  O.D.  4  1  Average  so 2  St. Dev.  4  (mg I-") 2013.11 1866.79 2000.91 1700.90 2074.91 1897.58 1905.36 1952.07 1935.27 1878.99 1800.16 1546.65 1  16.06 13.50 23.76 69.40 24.04 16.41 24.73 31.22 19.64 45.29 56.72  Natural Treatment Mesocosm B Sediment (100 cm) Date 7-Jan 14-Jan 14-Feb 21-Feb 28-Feb 7-Mar 6-Jun 5-Jul 13-Sep  to  Time (Days)  Dil.  32 39 70 77 84 91 182 211 281  33 20 33 33 10 25 33 33 33  2  O.D. 0.061 0.438 0.074 0.124 0.588 0.098 0.358 0.312 0.288  4  1  0.090 0.245 0.121 0.237 0.109 0.063 0.645 0.431 0.533  2  4  1  351.48 1153.80 526.30 681.32 650.29 479.98 1596.80 1332.34 1281.96  2  4  1  25  0.055  341.19  20 25 20 20 25 20  0.176 0.167 0.245 0.145 0.513 0.564  544.45 655.32 699.27 474.89 1625.77 1347.64  Average  sof  St. Dev.  (mg I-") 388.42 1143.06 530.49 680.82 694.67 472.22 1598.24 1340.90 1270.40 1  73.07 12.41 25.26 59.51 12.86 26.83 7.81  Natural Treatment Mesocosm B Sediment (50cm) Time Time (Days) Dil. O.D. Date (Days) (new exp.) 7-Mar 91 0 33 0.525 14-Mar 7 0.48 98 25 21-Mar 14 0.337 105 25 29-Mar 22 33 0.246 113 12-Apr 127 36 25 0.396 18-Apr 133 42 25 0.339 25-Apr 49 0.144 140 33 2-May 147 56 25 0.268 0.087 16-May 161 70 25 0.167 24-May 169 78 25 84 0.157 30-May 175 20 6-Jun 182 91 10 0.686 5-Jul 211 127 0.193 33 13-Sep 281 233 33 0.313  S0 ' (mg L- )  Dil.  2190.44 1533.21 1132.13 1157.50 1297.61 1137.74 779.87 938.60 430.94 655.32 501.82 844.40 926.75 1346.22  25 33 33 25 33 33 25 33 33 33 10 20 25 20  S0 ' (mg L- )  2  4  1  2  4  O.D.  1  S0 " (mg L- )  Average S0 ' (mg L- ) 2062.18 1534.14 1213.69 1204.79 1281.24 1085.98 818.38 946.11 454.18 653.07 494.63 823.50 942.31 1348.77  2  Dil.  4  O.D.  1  0.672 0.345 0.267 0.376 0.275 0.222 0.224 0.197 0.066 0.110 0.404 0.296 0.278 0.571  2071.72 1524.03 1235.25 1241.51 1264.87 1068.65 815.19 976.09 491.09 653.99 528.02 813.71 927.17 1362.47  20 20 20 20  0.791 0.622 0.501 0.475  1924.39 1545.19 1273.69 1215.35  20 25 20 10 20 33 25 20 25  0.402 0.240 0.345 0.326 0.223 0.056 0.223 0.367 0.433  1051.55 860.07 923.65 440.5103 649.91 454.07 812.39 973.02 1337.64  so -  Dil.  O.D.  2  4  Agricultural Mesocosm A -100 cm down the length of the mesocosm Date 10-Dec 21-Dec 29-Dec 7-Jan 14-Jan 24-Jan 4-Feb 14-Feb 21-Feb 2-Mar 7-Mar  Time (Days)  Dil.  0 11 19 28 35 45 56 66 73 82 87  25 33 33 25 20 20 10 10 10 25 33  2  SO4 ' 2  O.D. 0.462 0.207 0.142 0.159 0.149 0.100 0.144 0.076 0.004 0.471 0.161  (mg L- )  Dil.  1482.72 1013.11 772.46 632.88 483.87 373.92 236.32 160.03 79.26 1507.97 842.81  20 33 33 25 20 20 10 10 10 33 25  1  O.D. 0.566 0.214 0.142 0.164 0.155 0.095 0.101 0.064 0.010 0.343 0.294  4  (mg L' ) 1  1419.53 1039.03 772.46 646.90 497.33 362.70 188.08 146.57 85.99 1516.62 1011.52  Average S0 " S0 ^ (mg L- ) (mg L- ) 1479.60 1460.62 1003.11 1018.42 778.73 774.55 654.40 644.73 481.42 487.54 376.56 371.06 207.91 210.77 150.38 144.53 78.33 81.19 1538.82 1521.14 901.21 918.52 2  4  1  4  St. Dev.  1  33 25 25 20 25 10 5 5 5 25 20  0.333 0.291 0.211 0.225 0.105 0.269 0.304 0.191 0.073 0.482 0.335  St. Dev.  1  35.6 18.5 3.6 10.9 8.6 7.4 24.2 8.4 4.2 15.9 85.7  133.28 10.61 73.20 42.99 45.63 40.19 27.01 32.32 2.82 37.50 18.11 26.59 12.61  Date  Time (Days)  9-Mar 89 14-Mar 94 21-Mar 101 29-Mar • 109 30-Mar 110 12-Apr 123 18-Apr 129 25-Apr 136 2-May 143 16-May 157 24-May 165 30-May 171 6-Jun 178 20-Jun 192 5-Jul 207 221 19-Jul 26-Jul 228 2-Aug 235 9-Aug 242 17-Aug 250 13-Sep 277  Dil. 33 25 10 33 33 20 25 20 33 33 33 33 25 20 20 25 25 33 33 25 33  O.D. 0.065 0.007 0.063 0.666 0.341 0.614 0.453 0.883 0.447 0.519 0.432 0.247 0:259 0.840 0.371 0.651 0.787 0.431 0.473 0.640 0.339  SO4 " 2  (mg L- ) 1  487.39 206.56 145.45 2712.46 1509.22 1527.24 1457.48 2130.82 1901.66 2168.22 1846.13 1161.20 913.36 1610.29 938.76 1914.94 2275.09 1758.69 1905.51 1885.81 1437.10  Dil. 20 5 5 . 25 25 25 33 33 25 25 25 20 33 33 33 33 33 25 25 33 30  O.D. 0.152 0.493 0.157 0.920 0.485 0.476 0.291 0.504 0.626 0.784 0.605 0.432 0.169 0.467 0.206 0.451 0.556 0.566 0.658 0.457 0.389  Average S0 " so4^ (mg L- ) (mg L- ) 506.70 494.90 281.44 323.83 140.96 137.29 2739.88 1524.99 1527.15 1516.62 1521.95 1471.14 1417.58 2158.67 2134.06 1946.83 1930.40 2139.80 2231.29 1861.56 1863.83 1115.30 1131.79 914.68 900.15 1569.30 1583.35 998.21 969.72 1840.32 1861.29 2224.84 2231.85 1694.15 1714.23 1916.59 1918.52 1853.03 1862.81 1427.68 1443.37 2  SO4 " 2  (mg L- ) 1  Dil.  O.D.  4  1  St. Dev.  1  490.60 313.93 125.45 2767.30 1547.23 1521.99 1324.10 2112.69 1942.70 2385.86 1883.80 1118.86 872.43 1570.44 972.19 1828.60 2195.64 1689.84 1933.48 1849.58 1465.34  10 10 10  0.385 0.222 0.059  20 33 20 25 20 20 20 25 20 25 30 30 30 30 30 30 25  0.613 0.343 0.589 0.703 0.801 0.887 0.763 0.331 0.341 0.639 0.242 0.507 0.628 0.461 0.531 0.511 0.467  S0 ' (mg I.')  Dil.  O.D.  10.3 65.0 10.5 19.1 5.3 81.2 23.2 25.0 134.6 18.9 25.5 24.0 23.3 29.8 46.8 40.2 38.6 14.1 20.0 19.60  Agricultural Mesocosm A - 50 cm down the length of the mesocosm Date 6-Jun 20-Jun 5-Jul 19-Jul 26-Jul 2-Aug 9-Aug 17-Aug  S0 ' (mg L- ) 2  Time (Days)  Dil.  178 192 207 221 228 235 242 250  20 20 20 33 33 33 25 33  O.D. 0.331 0.713 0.398 0.454 0.563 0.41 0.633 0.451  4  1  892.24 1386.08 995.96 1839.09 2220.11 1685.29 1867.27 1828.604  2  Dil. 33 25 33 25 25 25 33 25  O.D. 0.181 0.54 0.217 0.635 0.758 0.565 0.476 0.617  4  1  916.85 1359.1 1010.64 1872.57 2198.29 1687.20 1915.99 1824.900  25 30 30 30 30 30 30 30  0.25 0.44 0.24 0.51 0.62 0.46 0.52 0.50  so  Average  2_ 4  (mg I-") 1  874.09 1351.7 998.5 1855.3 2201.4 1684.9 1891.1 1825.6  so ^ 4  (mg I-") 894.39 1365.63 1001.70 1855.65 2206.60 1685.79 1891.45 1826.368  St. Dev.  1  21.5 18.1 7.8 16.7 11.8 1.2 24.4 2.0  Agricultural Mesocosm A -150 cm down the length of the mesocosm Date 6-Jun 20-Jun 5-Jul 19-Jul 26-Jul 2-Aug 9-Aug 17-Aug  S0 " (mg L- )  Dil.  1020.52 1320.76 912.76 1797.14 2200.94 1734.22 1877.54 1867.27  25 33 20 25 33 25 25 33  2  Time (Days)  Dil.  178 192 207 221 228 235 242 250  33 20 33 33 25 33 33 25  O.D. 0.209 0.676 0.189 0.442 0.759 0.424 0.465 0.633  4  1  O.D. 0.306 0.37 0.402 0.618 0.579 0.555 0.659 0.45  SO4 " 2  (mg L- ) 1  Dil.  O.D.  Average S0 ' (mg I-") (mg L- ) 1031.36 1032.35 1306.4 1309.62 944.7 953.97 1789 1804.56 2198.5 2225.16 1697.6 1697.51 1942.6 1918.76 1882.4 1858.26 SO4 " 2  2  1  4  St. Dev.  1  1045.18 1301.7 1004.44 1827.55 2276.04 1660.71 1936.12 1825.11  20 25 30 30 30 30 30 30  0.39 0.52 0.23 0.49 0.62 0.46 0.54 0.52  S0 (mg L- )  Dil.  O.D.  2397.83 1309.29 1090.86 694.72 607.64 488.35 461.43 278.96 161.16 140.96 1489.60 1002.01 601.30 200.31 . 215.35 1642.50 1361.13 1527.73  33 25 25 20 20 10 10 20 5 5 25 20 20 10 5 20 20 20  12.4 9.9 46.5 20.3 44.1 36.8 35.8 29.7  Agricultural Mesocosm B -100 cm down the length of the mesocosm Date 10-Dec 13-Dec 21-Dec 29-Dec 7-Jan 14-Jan 24-Jan 4-Feb 14-Feb 21-Feb 2-Mar 7-Mar 9-Mar 14-Mar 21-Mar 29-Mar 12-Apr 18-Apr  4^  S0 ' (mg L- )  Dil.  2576.58 1275.97 1072.35 716.93 571.18 510.79 443.48 248.66 174.62 133.11 1522.40 1073.23 598.46 214.97 216.13 1715.52 1460.29 1541.62  20 33 33 33 25 20 20 10 10 10 33 33 25 10 5 33 33 33  2  Time (Days)  Dil.  0 3 11 19 28 35 45 56 66 73 82 87 89 94 101 109 123 129  25 33 33 33 25 20 20 10 10 10 25 25 33 25 10 25 25 25  O.D. 0.852 0.278 0.223 0.127 0.137 0.161 0.131 0.155 0.089 0.052 0.48 0.316 0.095 0.010 0.126 0.545 0.454 0.483  4  1  1.002 0.287 0.228 0.121 0.150 0.151 0.139 0.182 0.077 0.059 0.336 0.204 0.148 0.112 0.317 0.377 0.301 0.346  4  1  Average S0 " so 4 r (mg I/ ) (mg I-") 2412.58 2462.33 1334.07 1306.45 1056.40 1073.20 712.74 708.13 582.59 587.14 478.66 492.60 433.78 446.23 252.75 260.13 164.40 157.43 128.82 134.30 1502.36 1504.79 1033.60 1036.28 580.35 593.37 211.64 208.97 201.18 210.89 1686.55 1681.52 1421.78 1414.40 1542.94 1537.43 2  2_  O.D.  4  1  St. Dev.  1  0.585 0.409 0.310 0.251 0.193 0.360 0.320 0.046 0.214 0.163 0.469 0.394 0.192 0.122 0.292 0.685 0.567 0.621  99.22 29.15 17.25 11.80 18.65 16.48 14.03 16.44 9.04 6.16 16.53 35.69 11.36 7.69 8.41 36.77 49.99 8.43  Date 25-Apr 2-May 16-May 24-May 30-May 6-Jun 20-Jun 5-Jul 19-Jul 26-Jul 2-Aug 9-Aug 17-Aug 13-Sep  Time (Days) 136 143 157 165 171 178 192 207 221 228 235 242 250 277  Dil. 33 25 25 33 33 25 20 20 25 33 33 25 , 33 33  O.D. 0.416 0.718 0.815 0.495 0.167 0.282 0.801 0.797 0.676 0.581 0.445 0.699 0.459 0.399  Dil.  1786.89 2200.74 2472.80 2079.37 865.02 977.87 1541.44 1841.26 1981.14 2283.03 1807.63 2042.05 1856.57 1646.83  25 33 33 25 25 33 33 33 33 25 25 33 25 25  2  4  1  S0 " (mg L' ) 2  S0 ' (mg L- )  O.D. 0.636 0.511 0.593 0.725 0.240 0.182 0.459 0.426 0.472 0.773 0.583 0.505 0.668 0.561  4  1  1970.75 2138.61 2442.19 2220.38 860.07 920.56 1547.14 1741.21 1902.01 2238.01 1734.86 2017.36 1959.96 1676.60  S0 ' (mg L- ) 2  Dil.  O.D.  4  1  20 33 20 30 20 20  0.746 0.508 1.018 0.543 0.315 0.357  1823.42 2127.50 2433.74 2051.89 856.34 950.58  25 20 30 30 30 30 30  0.601 0.790 0.596 0.446 0.536 0.510 0.451  1782.53 1826.43 2123.15 1646.48 1932.48 1849.86 1662.37  S0 " (mg L- )  Dil.  O.D.  1124.18 1487.65 1983.20 2145.33 2290.98 1782.53 2079.12 1943.96  20 20 25 30 30 30 20 30  Average SO*^ (mg I.") 1860.35 2155.62 2449.58 2117.21 860.48 949.67 1544.29 1788.33 1903.19 2214.73 1729.66 1997.30 1888.79 1661.94  St. Dev.  1  97.34 39.47 20.55 90.39 4.36 28.67 50.27 77.36 82.44 80.70 57.48 61.72 14.89  Agricultural Mesocosm B - 50 cm down the length of the mesocosm Date 6-Jun 20-Jun 5-Jul 19-Jul 26-Jul 2-Aug 9-Aug 17-Aug  to  Oh Oh  S0 " (mg L- )  Dil.  1233.10 1541.31 1985.90 2171.17 2331.97 1832.10 1968.43 1938.77  33 25 20 25 25 25 25 33  2  Time (Days)  Dil.  178 192 207 221 228 235 242 250  25 33 33 33 33 33 33 25  O.D. 0.373 0.457 0.496 0.549 0.595 0.452 0.491 0.660  4  1  2  O.D. 0.237 0.602 0.864 0.738 0.793 0.601 0.713 0.484  4  1  Average S0 ^ (mg I/ ) (mg L- ) 1257.98 1205.09 1520.26 1516.41 1920.23 1963.11 2107.26 2141.25 2285.21 2302.72 1792.66 1802.43 2070.06 2039.20 1903.88 1928.87 SO4 " 2  1  4  St. Dev.  1  0.494 0.789 0.653 0.591 0.647 0.492 0.905 0.527  71.16 27.04 37.16 32.15 25.49 26.19 61.46 21.80  Agricultural Mesocosm B -150 cm down the length of the mesocosm S0 ' (mg L- )  Dil.  0.306 0.561 0.416 0.456 0.807 0.471 0.502  1379.64 1117.74 1706.26 1846.08 2328.05 1898.52 2006.88  25 33 20 25 33 25 25  0.474  1909.00  25  (mg L- )  Dil.  0.435 0.315 0.763 0.650 0.582 0.622 0.694  1406.99 1127.67 1769.23 1912.29 2286.52 1838.14 2028.81  20  0.562  25 30 30 30 20  0.559 0.521 0.606 0.503 0.901  Average S0 (mg L" ) (mg L ) 1410.51 1399.05 1122.70 1755.62 1743.70 1884.81 1881.06 2154.93 2256.50 1827.61 1854.76 2061.58 2032.42  0.672  1970.55  30  0.533  1922.95  SO4 "  2  Time (Days)  Dil.  6-Jun 20-Jun 5-Jul 19-Jul 26-Jul 2-Aug 9-Aug  178 192 207 221 228 235 242  33 20 33 33 25 33 33  17-Aug  250  33  Date  O.D.  4  1  SO4 "  2  O.D.  1  2  O.D.  1  2  4  1934.17  A1-7b. Total sulphide in mesocosms while operated as batch systems Natural Treatment Mesocosm A (water column) Date 6-Dec 13-Dec 21-Dec 29-Dec 7-Jan 24-Jan 14-Feb  Time (Days)  Dil.  0 7 15 23 32 49 70  1 1 1 1 1 1 1  O.D. 0.131 0.031 0.016 0.429 0.026 0.110 0.011  Sulphide mg L'  Dil.  0.27 0.050.01 0.93 0.04 0.22 0.00  1 1 10 10 10 10 10  0.135 0.028 0.001 0.052 0.000 0.020 0.000  0.28 0.04 0.00 0.93 0.00 0.23 0.00  Dil.  O.D.  1  Natural Treatment Mesocosm A Sediment (100 cm) Time Dil. O.D. Sulphide Date (Days) mg L"  O.D.  1  7-Jan  Uh OS  32  20  over  >27  Sulphide mg L' 1  Dil.  O.D.  over  Average Sulphide Sulphide mg L' (mg L' ) 0.22 0.26 0.04 0.03 0.01 0.93 0.02 0.22 0.00 1  1  1 1  0.130 0.027  Sulphide mg L"  Dil.  O.D.  >108  320  1  80  St. Dev.  1  Sulphide mg L1  0.358  246.64  16.90 33.13 33.26 90.38 38.26 27.53 32.27  Natural Treatment Mesocosm A Sediment (50cm) Time Time Sulphide (Days) Date Dil. O.D. (Days) (mg L ) (new exp.) 14-Mar 7 160 98 0.169 56.38 29-Mar 15 320 0.18 120.56 113 12-Apr 127 29 320 0.126 82.30 147 2-May 49 160 0.158 52.49 30-May 77 320 140.66 175 0.173 6-Jun 182 91 320 0.013 15.26 127 5-Jul 211 160 0.108 44.86 13-Sep 281 233 50 0.248 31.16 1  Dil.  O.D.  Sulphide (mg L- )  Dil.  1  O.D.  Average Sulphide Sulphide (mg L- ) (mg I-") 54.44 58.65 122.40 120.44 84.86 84.79 60.42 54.21 137.52 144.38 14.97 14.59 42.41 45.15 29.57 31.30 1  320 160 160 320 160 160 320 100  0.078 0.344 0.256 0.08 0.389 0.032 0.055 0.129  48.30 118.36 87.20 49.72 154.97 15.07 48.17 33.18  80 80 80 80 320 80 80 50  O.D.  Sulphide (mg L' )  Dil.  O.D.  0.341 0.701 0.489 0.351 0.169 0.068 0.21 0.235  Natural Treatment Mesocosm B (water column) Date 6-Dec 13-Dec 21-Dec 29-Dec 7-Jan 24-Jan 14-Feb  Time (Days)  Dil.  0 7 15 23 32 49 70  1 10 1 1 1 1 1  O.D. 0.151 0.235 0.016 0.119 0.011 0.223 0.021  Sulphide (mg L- )  Dil.  0.26 4.15 0.01 0.20 0.00 0.39 0.02  1 10 10 10 10 10 10  0.151 0.222 0.002 0.018 0.005 0.025 0  0.26 3.91 0.00 0.15 0.00 0.28 0.00  Dil.  O.D.  1  Natural Treatment Mesocosm B Sediment (100 cm) Dil. O.D. Sulphide Date Time (Days) (mg L- ) 7-Jan 32 20 over >27 1  1  over  Average Sulphide Sulphide (mg L- ) (mg L' ) 0.26 0.26 4.03 0.01 0.18 0.00 0.34 0.01 1  1  1 1  0.153 over  Sulphide (mg L- )  Dil.  O.D.  >108  320  1  80  St. dev  1  Sulphide (mg L- ) 1  0.53  368.48  5.44 2.02 2.45 5.56 9.30 0.34 2.89 1.81  Natural Treatment Mesocosm B Sediment (50cm) Time Sulphide Time (Days) Dil. O.D. Date (Days) (mg L" ) (new exp.) 14-Mar 7 0.094 29.82 98 160 29-Mar 113 15 0.165 109.93 320 12-Apr 127 29 0.134 87.97 320 2-May 147 49 160 0.243 82.59 77 30-May 175 320 0.278 222.95 6-Jun 182 91 0.150 122.63 320 211 127 5-Jul 160 0.126 51.91 0.222 13-Sep 281 183 50 27.98 1  Dil.  Sulphide (mg L- )  O.D.  320 80 160 320 160 80 320 100  1  0.038 0.622 0.241 0.125 0.491 0.621 0.068 0.118  16.64 108.41 81.88 68.00 194.94 122.94 58.36 30.48  Sulphide (mg L- )  Dil.  Dil.  O.D.  Average Sulphide Sulphide (mg L- ) (mg L- ) 22.97 22.46 107.03 108.46 71.22 80.36 56.99 69.17 208.95 121.66 122.41 55.14 28.59 29.02 1  80 160 80 80  0.162 0.312 0.412 0.396  160  0.304  50  0.227  Agricultural Mesocosm A -100 cm down the length of the mesocosm Date 10-Dec 21-Dec 29-Dec 7-Jan 24-Jan 14-Feb 14-Mar 29-Mar 12-Apr 2-May 30-May 6-Jun 20-Jun 5-Jul 20-Jul 26-Jul 2-Aug  OO  Time (Days)  Dil.  0 11 19 28 45 66 94 109 123 143 171 178 192 207 222 228 235  1 1 1 160 160 160 160 320 320 160 320 320 320 320 320 10 20  O.D. 0.007 0.192 0.041 0.667 0.407 0.503 0.834 0.507 0.489 0.272 0.291 0.359 0.112 0.331 0.033 0.025 0.014  Sulphide (mg L- )  Dil.  0.00 0.40 0.07 232.76 140.68 174.68 291.91 352.19 339.44 92.86 233.14 286.43 77.37 264.49 30.93 0.77 1.00  10 10 10 320 320 320 320 160 320 320 160 160 80 160 320 100 10  1  O.D. 0 0 0.001 0.312 0.205 0.255 0.439 over 0.485 0.142 0.594 0.703 0.431 0.652 0.034 0.015 0.028  1  0.00 0.00 0.00 214.06 138.26 173.68 304.02 336.61 93.64 206.91 245.52 74.59 227.45 31.72 5.26 0.84  Average Sulphide Sulphide (mg I.") (mg L- ) 0.00 0.20 0.03 0.323 221.85 222.89 0.4 138.20 139.05 0.489 169.72 172.69 297.97 0.501 347.94 350.07 0.368 317.16 331.07 93.25 0.613 213.64 217.90 0.352 242.39 258.11 0.237 80.47 77.48 over 245.97 42.11 0.101 34.92 -0.005 0.58 2.20 0.91 22.45 8.10 O.D.  1  1  320 160 160 320 400 160 320 160 80 160 160 10  St. dev  1  6.60 1.45 8.48 12.87 14.00 0.67 1.31  Date 9-Aug 17-Aug 13-Sep  Time (Days)  Dil.  242 250 277  2 2 10  O.D. 0.011 0.027 0.058  Sulphide (mg L" )  Dil.  0.09 0.16 1.58  1 1 1  1  O.D.  Sulphide (mg I-") 1  0.027 0.058 Over  Dil.  O.D.  Average Sulphide Sulphide (mg I-") (mg I.") 0.08 0.16 1.45 1.51 1  1  0.08 0.16 5  0.112  Agricultural Mesocosm A - 50 cm down the length of the mesocosm Date 6-Jun 5-Jul 20-Jul 26-Jul 2-Aug 9-Aug 17-Aug  Time (Days)  Dil.  182 211 226 232 239 246 254  320 160 320 10 20 2 2  O.D. 0.39 .0.822 0.038 0.051 0.015 0.015 0.031  Sulphide (mg L" )  Dil.  310.73 324.65 34.85 1.41 1.05 0.11 0.18  160 320 320 100 10 1 1.  1  O.D.  Sulphide (mg L- ) 1  0.741 0.419 0.049 0.015 0.028 0.027 0.064  292.91 333.46 43.47 5.26 0.84 0.08 0.17  Dil.  Average Sulphide Sulphide (mg L' ) (mg L- ) over 301.82 over 329.06 35.84 0.085 38.06 3.07 0 2.53 0.97 23.92 8.60 0.09 0.18 O.D.  1  1  80 80 160 160 10  Agricultural Mesocosm A -150 cm down the length of the mesocosm Date 6-Jun 20-Jun 5-Jul 20-Jul 26-Jul 2-Aug 9-Aug 17-Aug  Time (Days)  Dil.  182 196 211 226 232 239 246 254  320 320 160 320 10 20 2 2  O.D. 0.265 0.121 0.78 0.05 0.002 0.01 0.014 0.027  Sulphide (mg I-")  Dil.  212.76 99.90 308.19 44.26 0.21 0.81 0.10 0.16  160 160 320 320 100 10 1 1  1  O.D. 0.562 0.259 0.396 0.037 0 0.022 0.03 0.04  Sulphide (mg L- ) 1  222.77 104.03 315.43 34.07 1.58 0.70 0.09 0.11  Dil.  Average Sulphide Sulphide (mg L- ) (mg L- ) over 217.76 0.462 98.57 91.79 over 311.81 0.098 40.94 39.75 2.14 -0.001 1.31 0.018 0.60 0.70 0.09 0.14 O.D.  1  1  80 80 80 160 160 10  Agricultural Mesocosm B -100 cm down the length of the mesocosm Date 10-Dec 21-Dec 29-Dec 7-Jan 24-Jan 14-Feb 14-Mar 29-Mar 12-Apr 2-May 30-May 6-Jun 20-Jun 5-Jul 20-Jul 26-Jul 2-Aug 9-Aug 17-Aug 13-Sep  O  Time (Days)  Dil.  0 11 19 28 45 66 94 109 123 143 171 178 192 207 222 228 235 242 250 277  1 10 40 160 160 160 160 320 320 160 320 320 320 160 320 10 20 2 2 1  O.D. 0.003 0.024 0.583 0.267 0.468 0.561 0.993 0.447 0.445 0.096 0.378 0.3 0.118 0.198 0.065 0.014 0.01 0.013 0.015 over  Sulphide (mg L- ) 1  0.00 0.26 42.29 75.91 135.24 162.68 348.23 309.69 308.27 30.53 301.32 240.19 81.29 80.13 56.01 0.50 0.81 0.10 0.11  Dil. 10 10 20 80 320 320 320 160 160 160 160 160 160 320 320 100 10 1 1 10  O.D. 0.001 0.003 over 0.75 0.222 0.279 0.513 0.897 0.874 0.055 0.701 0.645 0.244 0.08 0.069 0.012 0.021 0.032 0.022 0.026  Sulphide (mg L" ) 1  Dil.  1  1  0.00 0.00 109.23 125.26 158.90 356.44 314.23 306.08 16.00 244.81 255.29 81.79 67.77 59.15 4.52 0.67 0.09 0.07 0.80  Average Sulphide Sulphide (mg L- ) (mg L- ) 0.00 0.13 42.29 over 92.57 0.237 134.11 131.53 160.79 347.94 0.501 350.87 0.429 296.94 306.95 0.449 311.11 308.49 0.025 10.76 19.10 over 273.07 0.308 246.46 247.32 0.503 83.19 82.09 60.24 0.301 69.38 0.131 53.87 56.34 -0.002 2.26 1.75 0.018 0.60 0.69 0.09 0.09 0.056 0.76 0.78 O.D.  40 320 320 320 320 320 80 320 80 80 160 160 10  5  Agricultural Mesocosm B - 50 cm down the length of the mesocosm  —  Date 6-Jun 5-Jul 20-Jul 26-Jul 2-Aug 9-Aug 17-Aug  1  Time (Days) 182 211 226 232 239 246 254  1  1  1  Dil. 320 160 320 10 20 2 2  O.D. 0.172 0.285 0.058 0.021 0.009 0.021 0.012  *^  1  Sulphide (mg I/ )  Dil.  139.87 114.22 50.53 0.67 0.76 0.13 0.09  160 320 320 100 10 1 1  1  r  O.D.  Sulphide (mg I-") 1  0.341 0.09 0.052 0.014 0.024 0.039 0.031  136.16 75.61 45.82 5.01 0.75 0.11 0.09  Dil.  Average Sulphide Sulphide (mg L- ) (mg L' ) 0.672 132.94 136.32 0.511 97.07 101.39 0.124 51.13 49.16 -0.005 0.58 2.09 0.027 0.82 0.77 0.12 0.09 O.D.  1  1  80 80 160 160 10  Agricultural Mesocosm B -150 cm down the length of the mesocosm Date 20-Jun 5-Jul 20-Jul 26-Jul 2-Aug 9-Aug 17-Aug  to ON  Time (Days)  Dil.  196 211 226 232 239 246 254  320 160 320 10 20 2 2  O.D. 0.105 0.284 0.15 0.017 0.008 0.009 0.018  Sulphide (mg L )  Dil.  72.80 113.83 122.63 0.57 0.71 0.08 0.12  80 320 320 100 10 1 1  1  O.D. 0.431 0.113 0.121 0 0.025 0.014 0.039  Sulphide (mg L- ) 1  71.43 93.63 99.90 1.58 0.77 0.05 0.11  Dil.  O.D.  Average Sulphide Sulphide (mg L- ) (mg L- ) 73.96 72.73 109.87 122.16 116.04 125.58 2.93 1.70 1.97 1.15 0.06 0.12 1  1  160 80 160 160 10  0.22 0.617 0.314 0.001 0.074  A1-7c. pH in mesocosms while operated as batch systems Natural Treatment Mesocosm Water Column Mesocosm A Date Time PH (Days) 6-Dec 13-Dec 21-Dec 7-Jan 14-Jan 24-Jan 14-Feb 21-Feb 28-Feb 12-Apr 20-Jul  Mesocosm B PH  Agricultural Treatment Mesocosm Mesocosm A Date Time PH (Days)  Mesocosm B pH  0 7 15 32 39 49 70 77 84 127 226  7.41 7.37 7.24 7.24 7.31 7.55 7.40 7.57 8.43 8.50 9.10  7.43 7.45 7.19 7.22 7.45 7.58 7.31 7.62 8.32 8.43 9.01  7.32 7.45 7.58 7.32 7.28 7.77 7.62 7.10 8.94 8.48 9.30  7.27 7.41 7.59 7.33 7.12 7.56 7.65 7.12 8.68 8.52 9.35  10-Dec 13-Dec 21-Dec 7-Jan 14-Jan 24-Jan 14-Feb 21-Feb 2-Mar 7-Mar 9-Mar 14-Mar  0 3 11 28 35 45 66 73 82 87 89 94  7.50 6.99 6.58 6.95 7.11 7.31 7.36 7.44 7.50 5.51 7.02 7.16  7.45 7.05 6.64 6.85 7.03 7.37 7.36 7.29 7.55 5.50 7.06 7.22  7.50 6.04 6.52 6.97 7.20 7.38 7.51 7.51 7.77 5.38 6.96 7.18  Sediment 84 28-Feb 7-iViar 91 14-Mar 98 21-Mar 105 29-Mar 113 12-Apr 127 18-Apr 133 25-Apr 140 2-May 147 16-May 161 30-May 175  7.52 6.00 6.55 7.08 7.21 7.34 7.52 7.49 7.68 5.47 7.01 7.11  7.41 7.47 7.69 7.96 8.03 7.60 8.25 8.40 8.55 8.50 7.89  7.42 7.38 7.55 7.76 7.95 7.81 8.11 8.31 8.63 8.51 7.92  7.48 7.56 7.77 8.11 7.67 7.48 8.46 8.45 8.44 8.43 7.81  7.45 7.58 7.69 8.05 7.58 7.62 8.51 8.48 8.46 8.44 7.82  21-Mar 29-Mar 12-Apr 18-Apr 25-Apr 2-May 16-May 30-May 20-Jun 5-Jul 20-Jul  101 109 123 129 136 143 157 171 192 207 222  7.20 7.35 7.54 7.36 7.56 7.85 7.78 7.41 7.45 7.70 8.36  7.14 7.29 7.51 7.38 7.62 7.88 7.75 7.44 7.39 7.71 8.42  7.24 7.40 7.67 7.30 7.71 7.88 7.82 7.33 7.39 7.62 8.18  7.22 7.41 7.72 7.30 7.66 7.87 7.82 7.36 7.36 7.66 8.10  Date 20-Jun 5-Jul 20-Jul 26-Jul 2-Aug 9-Aug 17-Aug 13-Sep  Time (Days) 196 211 226 232 239 246 254 281  Mesocosm A pH 7.67 7.51 8.42 8.05 8.42 8.00 8.03 7.77  7.80 7.40 8.30 8.00 8.40 8.00 8.00 7.80  Mesocosm B PH 7.82 7.60 8.38 8.00 8.40 8.20 8.00 7.80  Date  7.88 7.64 8.21 7.98 8.03 8.22 8.05 7.69  26-Jul 2-Aug 9-Aug 17-Aug 13-Sep  Mesocosm A PH  Mesocosm B pH  Time (Days) 228 235 242 250 277  8.30 8.10 9.00 8.80 9.00  8.28 8.05 8.95 8.76 9.03  Dil.  O.D.  sCOD (mg L- )  8.20 8.10 8.80 8.90 9.00  8.18 8.06 8.77 8.92 9.03  A1-7d. Soluble COD in mesocosms while operated as batch systems Natural Treatment Mesocosm A Date 6-Dec 21-Dec 29-Dec 7-Jan 14-Jan 24-Jan 4-Feb 14-Feb 21-Feb 21-Mar 12-Apr 20-Jun 13-Sep  ON  Time (Days)  Dil.  0 15 23 32 39 49 60 70 77 105 127 196 281  1 1 1 1 1 1 1 1 1 1 1 2 2  O.D. 0.058 0.017 0.108 0.021 0.021 0.023 0.025 0.03 0.028 0.061 0.053 0.041 0.04  sCOD (mg L- )  Dil.  143.46 42.05 267.13 51.94 51.94 56.89 61.84 74.20 69.26 150.88 131.09 202.82 197.87  2 2 2 1 1 1 1 1 1 2 1 4 4  1  O.D. 0.025 0.01 0.057 0.018 0.036 0.015 0.028 0.021 0.048 0.015 0.059 0.02 0.02  sCOD (mg L" ) 1  123.67 49.47 281.97 44.52 89.04 37.10 69.26 51.94 118.72 74.20 145.93 197.87 197.87  1  1 1 1 1 1 1 1 1 1  0.057 0.009 0.097 0.023 0.034 0.029 0.022 0.028 0.037  140.98 22.26 239.92 56.89 84.10 71.73 54.41 69.26 91.52  4  0.021  207.77  Average sCOD (mg L- ) 136.04 37.93 263.00 51.12 75.03 55.24 61.84 65.13 93.16 112.54 138.51 202.82 197.87  St. Dev.  1  10.78 14.06 21.32 6.22 20.14 17.37 7.42 11.69 24.78  4.95  Natural Treatment Mesocosm B Date 6-Dec 21-Dec 29-Dec 7-Jan 14-Jan 24-Jan 4-Feb 14-Feb 21-Feb 21-Mar 12-Apr 20-Jun 13-Sep  Time (Days)  Dil.  0 15 23 32 39 49 60 70 77 105 127 196 281  1 1 1 1 1 1 1 1 1 1 1 2 2  O.D. 0.008 0.012 0.057 0.024 0.041 0.036 0.034 0.021 0.054 0.032 0.047 0.041 0.038  sCOD (mg L- ) 1  19.79 29.68 140.98 59.36 101.41 89.04 84.10 51.94 133.56 79.15 116.25 202.82 187.98  Dil. 1 1 1 1 1 1 1 1 2 1 4 4  O.D. 0 0.014 0.025 0.033 0.035 0.021 0.027 0.023 0.033 0.012 0.054 0.021 0.031  sCOD (mg L' ) 1  0.00 34.63 123.67 81.62 86.57 51.94 66.78 56.89 81.62 59.36 133.56 207.77 306.70  Dil. 1 1  O.D.  sCOD (mg L- ) 1  1 1 1 1 1 1  0.015 0.008 0.029 0.018 0.042 0.048 0.021 0.023 0.049  37.10 19.79 143.46 44.52 103.88 118.72 51.94 56.89 121.20  4 10  0.022 0.012  217.66 296.81  Dil.  O.D.  sCOD (mg L- )  Average sCOD (mg L- ) 18.96 28.03 136.04 61.84 97.29 86.57 67.61 55.24 112.13 69.26 124.91 209.41 263.83  St. Dev.  1  18.56 7.56 10.78 18.67 9.36 33.46 16.09 2.86 27.13 13.99 12.24 7.56 65.87  Agricultural Treatment Mesocosm A (100 cm down the length of the mesocosm) Date 10-Dec 21-Dec 29-Dec 7-Jan 14-Jan 4-Feb 14-Feb 21-Feb 7-Mar 14-Mar 21-Mar 29-Mar  ON 45*  Time (Days)  Dil.  0 11 19 28 35 56 66 73 87 94 101 109  10 10 10 20 20 20 20 20 10 2 2 2  O.D. 0.151 0.131 0.113 0.056 0.04 0.055 0.031 0.026 0.078 0.279 0.289 0.279  sCOD (mg I-")  Dil.  3734.83 3240.15 2794.94 2770.21 1978.72 2720.74 1533.51 1286.17 1929.25 1380.16 1429.63 1380.16  20 20 20 20 40 40 40 40 20 10 10 10  1  O.D. 0.078 0.075 0.071 0.059 0.019 0.021 0.016 0.023 0.042 0.064 0.071 0.038  sCOD (mg I-") 1  3858.50 3710.10 3512.23 2918.61 1879.78 2077.66 1582.98 2275.53 2077.66 1582.98 1756.11 939.89  1  20 20 20 20 10 20 10 10  0.074 0.069 0.066 0.058 0.086 0.049 0.057 0.048  3660.63 3413.29 3264.89 2869.14 2127.12 2423.93 1409.84 1187.23  20 5 2  0.037 0.144  1830.32 1780.85 1335.64  o;27o  Average sCOD (mg L- ) 3751.32 3454.52 3190.69 2852.65 1995.21 2407.44 1508.77 1582.98 2003.45 1597.82 1655.53 1218.56  St. Dev.  1  99.96 237.67 364.35 75.56 124.49 321.86 89.18 601.80 225.45 196.03 242.36  Date 12-Apr 2-May 24-May 6-Jun . 20-Jun 5-Jul 20-Jul 13-Sep  Time (Days)  Dil.  123 143 165 178 192 207 222 277  2 2 2 5 2 2 4 2  O.D. 0.111 0.113 0.275 0.081 0.167 0.166 0.122 0.126  sCOD (mg L- )  Dil.  549.09 558.99 1360.37 1001.73 826.12 821.17 1207.02 623.2968  2 5 5 2 4 4 2 4  1  O.D. 0.168 0.046 0.110 0.188 0.072 0.089 0.249 0.067  sCOD (mg L' ) 1  831.06 568.88 1360.37 930.00 712.34 880.53 1231.75 662.87  Dil.  O.D.  sCOD (mg L- ) 1  10  0.031  766.75  10  0.057  1409.84  4 4 4  0.082 0.084 0.118  811.28 831.06 1167.44  Agricultural Treatment Mesocosm A (50 cm down the length of the mesocosm) 4 20-Jun 192 2 0.158 781.59 0.076 751.91 207 2 0.184 910.21 4 0.084 5-Jul 831.06 4 0.167 2 0.327 20-Jul 222 1652.23 1617.60  4 4 4  0.067  662.87  0.159  Agricultural Treatment Mesocosm A (150 cm down the length of the mesocosm) 20-Jun 192 4 0.087 860.74 2 821.17 0.166 207 2 0.177 4 5-Jul 875.58 0.087 860.74 4 20-Jul 222 0.131 1296.06 2 0.254 1256.49  4 4 4  sCOD (mg L" )  Dil.  3710.10 5688.82 3957.44 2720.74 2770.21 1879.78 1286.17 989.36  20 20 20 20 10 10 10 10  Average sCOD (mg L" ) 715.64 563.94 1376.86 965.86 783.24 844.25 1202.07 643.084  St. Dev.  1  147.77 28.56 61.85 31.80 32.44  61.79  1573.08  732.13 870.64 1614.31  0.081 0.079 0.13  801.38 781.59 1286.17  827.76 839.31 1279.57  30.23 50.53 20.60  O.D.  sCOD (mg L )  39.68  Agricultural Treatment Mesocosm B (100 cm down the length of the mesocosm) Date 10-Dec 21-Dec 29-Dec 7-Jan 14-Jan 4-Feb 14-Feb 21-Feb  to ON  Time (Days)  Dil.  0 11 19 28 35 56 66 73  10 10 10 20 20 20 20 20  O.D. 0.149 0.19 0.155 0.053 0.038 0.033 0.026 0.034  sCOD (mg L- )  Dil.  3685.37 4699.46 3833.77 2621.80 1879.78 1632.44 1286.17 1681.91  20 20 20 20 40 40 40 40  1  O.D. 0.075 0.115 0.08 0.055 0.028 0.019 0.013 0.01  1  0.074 0.105 0.075 0.056 0.103 0.077 0.054 0.044  1  3660.63 5194.14 3710.10 2770.21 2547.60 1904.52 1335.64 1088.30  Average sCOD (mg L" ) 3685.37 5194.14 3833.77 2704.25 2399.20 1805.58 1302.66 1253.19  St Dev  1  24.73 494.68 123.67 75.56 463.39 150.45 28.56 374.57  Date 7-Mar 14-Mar 21-Mar 29-Mar 12-Apr 2-May 24-May 6-Jun 5-Jul 20-Jul 13-Sep  Time (Days)  Dil.  87 94 101 109 123 143 165 178 207 222 277  10 2 2 2 2 2 2 5 2 4 2  O.D. 0.079 0.286 0.323 0.245 0.14 0.086 0.225 0.099 0.159 0.163 0.124  sCOD (mg I-")  Dil.  1953.99 1414.78 1597.82 1211.97 692.55 425.42 1113.03 1224.33 786.54 1612.66 613.40  20 10 10 2 2 5 5 2 4 2 4  1  Agricultural Treatment Mesocosm B (50 cm down the 20-Jun 192 2 0.146 722.23 207 5-Jul 2 0.153 756.86 4 0.102 20-Jul 222 1009.15  O.D. 0.045 0.099 0.063 0.228 0.15 0.044 0.11 0.207 0.081 0.348 0.068  sCOD (mg L- ) 1  2226.06 2448.67 1558.24 1127.87 742.02 544.15 1360.37 1023.99 801.38 1721.49 672.76  length of the mesocosm) 4 0.068 672.76 4 0.079 781.59 2 0.195 964.63  Agricultural Treatment Mesocosm B (150 cm down the length of the mesocosm) 0.072 20-Jun 192 2 0.136 672.76 4 712.34 207 2 0.148 4 0.071 5-Jul 732.13 702.45 222 4 0.124 2 20-Jul 1226.81 0.228 1127.87  OS  Dil.  O.D.  Average sCOD (mg L- ) 1879.78 2019.94 2275.53 2046.33 1578.03 1484.04 1274.63 890.424 775.00 484.79 1310.902 1261.43 1014.094 1087.47 793.96 1592.869 1642.34 643.08 sCOD (mg L- ) 1  St Dev  1  40 20  0.019 0.046  182.32 553.74  10 10  0.06 0.036  10 10  0.053 0.041  4  0.161  4 4 4  0.071 0.079 0.102  702.45 781.59 1009.15  699.15 773.35 994.31  24.90 14.28 25.70  4 4 4  0.071 0.074 0.119  702.45 732.13 1177.34  695.85 722.23 1177.34  20.60 17.14 49.47  186.17 102.98 130.88 118.63 69.26  A1-7e. Ammonia-nitrogen in mesocosms while operated as batch systems Natural Treatment Mesocosm A Date 6-Dec 13-Dec 21-Dec 29-Dec 7-Jan 14-Jan 24-Jan 4-Feb 14-Feb 21-Feb 21-Mar 2-May 5-Jul 13-Sep  Time (Days) 0 7 15 23 32 39 49 60 70 77 105 147 211 281  Dil. 10 11 11 11 . 11 5.5 5.5 2.04 2.04 1 10 10 10 20  O.D.  NH -N (mg LZ ) 3  1  Dil.  2.00 0.57 0.20 2.45 0.35 0.20 0.36 0.56 0.18 0.44 1.67 0.46 0.45 0.21  20.0 6.3 2.2 27.0 3.9 1.1 2.0 1.1 0.4 0.4 16.7 4.6 4.5 4.2  10 11 11 11 11 5.5 5.5 2.04 2.04 1 20 20 20 ' 10  O.D.  NH -N (mg I-")  Dil.  26.2 5.3 1.7 7.3 4.5 1.0 4.3 1.1 0.4  10 11 11 11 11 5.5 5.5 2.04 2.04  NH -N (mg L" )  Average NH -N (mg L/ )  1.32 0.56 0.21 2.38 0.37 0.25 0.26 0.46 0.18 0.46 0.87 0.25 0.24 0.43  13.2 6.2 2.3 26.2 4.1 1.4 1.4 0.9 0.4 0.5 17.4 5.0 4.8 4.3  16.6 6.2 2.3 26.6 4.0 1.2 1.7 1.0 0.4 0.5 17.1 4.8 4.7 4.3  O.D.  NH -N (mg L" )  O.D.  3  1  3  1  Natural Treatment Mesocosm B Date 6-Dec 13-Dec 21-Dec 29-Dec 7-Jan 14-Jan 24-Jan 4-Feb 14-Feb  to ON  ^0  Time (Days)  Dil.  0 7 15 23 32 39 49 60 70  10 11 11 11 11 5.5 5.5 2.04 2.04  2.62 0.48 0.15 0.66 0.41 0.19 0.79 0.54 0.18  3  1  1.6 0.56 0.14 0.66 0.37 0.16 0.59 0.33 0.16  3  1  16.0 6.2 1.5 7.3 4.1 0.9 3.2 0.7 0.3  Average NH -N (mg L' ) 21.1 5.7 1.6 7.3 4.3 1.0 3.8 0.9 0.3 3  1  Date  Time (Days)  Dil.  O.D.  NH -N (mg I-")  Dil.  O.D.  NH -N (mg I-")  21-Feb 21-Mar 2-May 5-Jul 13-Sep  77 105 147 211 281  1 10 10 20 20  0.27 1.17 0.48 0.36 0.38  0.3 11.7 4.8 7.2 7.6  1 20 20 10 10  0.37 0.62 0.91 0.6 0.63  0.4 12.4 18.2 6.0 6.3  NH -IM (mg I-")  Dil.  O.D.  NH -N (mg L" )  120.6 120.6 129.0 119.4 111.7 122.1 125.0 142.8 130.3 185.0 185.0 131.0 94.0 130.5 116.5 >150 31.0  41.6 41.6 41.6 55 55 55 51 51 50.1 100 100 100 50 100 100 100 50  3  1  3  1  Average NH -N (mg L" ) 0.3 12.1 11.5 6.6 7.0 3  1  Agricultural Treatment Mesocosm A Date 10-Dec 21-Dec 29-Dec 7-Jan 14-Jan 24-Jan 4-Feb 14-Feb 21-Feb 7-Mar 21-Mar 12-Apr 2-May 24-May 6-Jun 20-Jun 13-Sep  as oo  Time (Days)  Dil.  0 11 19 28 35 45 56 66 73 87 101 123 143 165 178 192 277  41.6 41.6 41.6 55 55 55 51 51 50.1 100 100 100 100 50 50 50 100  O.D. 2.9 2.9 3.1 2.17 2 2.22 2.45 2.8 2.6 1.85 1.85 1.31 0.94 2.61 2.33 over 0.31  3  1  2.81 2.89 3.1 2 2 2.01 2.51 2.8 2.66 1.8 1.9 1.42 1.89 1.24 0.89 1.4 0.59  3  1  116.9 120.2 129.0 110.0 107.3 110.6 128.0 142.8 133.3 180.0 190.0 142.0 94.5 124.0 89.0 140 29.5  Average NH -N (mg L- ) 118.8 120.4 129.0 114.7 109.5 116.3 126.5 142.8 131.8 182.5 187.5 136.5 94.3 127.3 102.8 140 30.3 3  1  Agricultural Treatment Mesocosm B Date 10-Dec 21-Dec 29-Dec 7-Jan 14-Jan 24-Jan 4-Feb 14-Feb 21-Feb 7-Mar 21-Mar 12-Apr 2-May 24-May 6-Jun 20-Jun 13-Sep  Time (Days)  Dil.  0 11 19 28 35 45 56 66 73 87 101 123 143 165 178 192 277  41.6 41.6 41.6 55 55 55 51 51 50.1 100 100 100 100 100 50 50 100  O.D. 2.70 3.00 3.10 2.10 2.14 2.34 2.55 2.81 2.67 1.83 1.71 1.34 1.00 1.41 2.66 over 0.3  NH -N (mg I-")  Dil.  112.3 124.8 129.0 115.5 117.7 128.7 130.1 143.3 133.8 183.0 171.0 134.0 100.0 141.0 133.0 >150 30.0  41.6 41.6 41.6 55 55 55 51 51 50.1 100 100 100 50.0 50.0 100 100.0 50  3  1  NH -N (mg L- )  O.D.  3  1  2.30 3:00 3.10 2.15 2.30 2.19 2.35 2.79 2.66 1.84 1.84 1.37 2.03 2.66 1.05 1.32 0.53  95.7 124.8 129.0 118.3 126.5 120.5 119.9 142.3 133.3 184.0 184.0 137.0 101.5 133.0 105.0 132 26.5  Average NH -N (mg I-") 104.0 124.8 129.0 116.9 122.1 124.6 125.0 142.8 133.5 183.5 177.5 135.5 100.8 137.0 119.0 132 28.3 3  1  A1-7f. Ortho-phosphate in mesocosms while operated as batch systems Natural Treatment Mesocosm A Date 6-Dec 13-Dec 21-Dec 29-Dec 7-Jan 14-Jan  OS VO  P0 ' (mg L- )  Dil.  12.9 18.5 1.4 23.5 7.5 8.5  10 11 11 11 11 11  3  Time (Days)  Dil.  0 7 15 23 32 39  10 11 11 11 11 11  O.D. 1.29 1.68 0.13 2.14 0.68 0.77  4  1  O.D. 1.35 1.61 0.15 2.2 0.64 0.8  (mg L' )  Average P0 " (mg L' )  13.5 17.7 1.7 24.2 7.0 8.8  13.2 18.1 1.5 23.9 7.3 8.6  PO4 ' 3  3  1  4  1  Date 24-Jan 4-Feb 14-Feb 21-Feb 21-Mar 12-Apr 2-May 13-Sep  P0 (mg L' )  Dil.  0.09 1.01 1.62 2.23 1.57 0.9 0.8 over  1.0 10.1 16.2 22.3 15.7 9.0 8.0 >6  11 10 10 10 20 10 20 20  0.07 0.88 1.7 2.09 0.74 0.88 0.389 0.253  O.D.  P0 ' (mg L- )  Dil.  O.D.  12.1 17.5 0.6 1.3 6.2 0.0 0.8 5.9 18.2 16.3 10.4 8.1 7.3 >6  10 11 11 11 11 11 11 10.2 10.2 10.02 10 10 20 20  3_  Time (Days)  Dil.  49 60 70 77 105 127 147 281  11 10 10 10 10 10 10 2  O.D.  4  1  P0 " (mg L- ) 3  O.D.  4  1  0.8 8.8 17.0 20.9 14.8 8.8 7.8 5.1  Average P0 " (mg I.") 0.9 9.5 16.6 21.6 15.3 8.9 7.9 3  4  1  Natural Treatment Mesocosm B Date 6-Dec 13-Dec 21-Dec 29-Dec 7-Jan 14-Jan 24-Jan 4-Feb 14-Feb 21-Feb 21-Mar 12-Apr 2-May 13-Sep  to o  3  Time (Days)  Dil.  0 7 15 23 32 39 49 60 70 77 105 127 147 281  10 11 11 11 11 11 11 10.2 10.2 10.02 10 10 10 2  1.21 1.59 0.05 0.12 0.56 0 0.07 0.58 1.78 1.63 1.04 0.81 0.73 over  4  1  PO 3  1.18 1.62 0.1 0.15 0.5 0.01 0.06 0.45 1.7 1.54 1.14 0.8 0.34 0.3  4  11.8 17.8 1.1 1.7 5.5 0.1 0.7 4.6 17.3 15.4 11.4 8.0 6.8 6.0  Average P0 (mg I-") 12.0 17.7 0.8 1.5 5.8 0.1 0.7 5.3 17.7 15.9 10.9 8.1 7.1 3_  (mg L- ) 1  4  1  Agricultural Treatment Mesocosm A Date 10-Dec 21-Dec 29-Dec 7-Jan 14-Jan 24-Jan 4-Feb 14-Feb 21-Feb 7-Mar 21-Mar 12-Apr 2-May 24-May 6-Jun 20-Jun 13-Sep  P0 " (mg I-")  Dil.  100.5 96.5 82.8 85.8 88.6 95.7 76.5 61.7 137.8 28.5 44.5 39.0 25.0 55.0 61.0 65.5 26.0  100 41.6 41.6 55 55 55 51 51 50.1 100 50 50 50 100 100 100 100  P0 ' (mg L- )  Dil.  98.5 82.8 70.7 165.0 69.3 62.7 102.5 129.5  100 41.6 41.6 55 55 55 51 51  P0 " (mg L- )  Average P0 " (mg L- )  1.03 2.4 1.89 1.6 1.49 1.66 1.66 1.26 2.83 0.24 0.94 0.85 0.57 0.49 0.53 0.63 0.26  103.0 99.8 78.6 88.0 82.0 91.3 84.7 64.3 141.8 24.0 47.0 42.5 28.5 49.0 53.0 63.0 26.0  101.8 98.2 80.7 86.9 85.3 93.5 80.6 63.0 139.8 26.3 45.8 40.8 26.8 52.0 57.0 64.3 26.0  O.D.  P0 " (mg L- )  3  Time (Days)  Dil.  0 11 19 28 35 45 56 66 73 87 101 123 143 165 178 192 277  50 41.6 41.6 55 55 55 51 51 50.1 50 50 50 100 50 50 50 50  O.D. 2.01 2.32 1.99 1.56 1.61 1.74 1.5 1.21 2.75 0.57 0.89 0.78 0.25 1.1 1.22 1.31 0.52  4  1  3  O.D.  4  1  3  4  1  Agricultural Treatment Mesocosm B Date 10-Dec 21-Dec 29-Dec 7-Jan 14-Jan 24-Jan 4-Feb 14-Feb  to  3  Time (Days)  Dil.  0 11 19 28 35 45 56 66  50 41.6 41.6 55 55 55 51 51  O.D. 1.97 1.99 1.7 3 1.26 1.14 2.01 2.54  4  1  3  0.96 1.89 1.8 2.56 1.3 1 1.75 2.68  4  1  96.0 78.6 74.9 140.8 71.5 55.0 89.3 136.7  Average P0 " (mg L- ) 97.3 80.7 72.8 152.9 70.4 58.9 95.9 133.1 3  4  1  Date  Dil.  73 87 101 123 143 165 178 192 277  50.1 50 50 50 100 100 50 50 50  21-Feb 7-Mar 21-Mar 12-Apr 2-May 24-May 6-Jun 20-Jun 13-Sep  P0 " (mg L" )  Dil.  41.6 37.5 40.5 34.5 27.0 51.0 63.0 63.5 29.5  50.1 100 50 50 50 50 100 100 100  3  Time (Days)  O.D. 0.83 0.75 0.81 0.69 0.27 0.51 1.26 1.27 0.59  Average P0 " (mg L' ) 42.3 35.8 41.8 34.8 27.5 51.0 60.0 63.8 29.3  P0 " (mg L' ) 3  4  1  O.D. 0.86 0.34 0.86 0.7 0.56 1.02 0.57 0.64 0.29  4  3  1  4  1  43.1 34.0 43.0 35.0 28.0 51.0 57.0 64.0 29.0  A1-8a. S0 " concentration in Lake 52 in situ experiment 2  4  Location 4-Oct-04 Site 1 Site 1 Site 1 Site 1 Site 1 Site 1 Site 2 Site 2 Site 2 Site 2 Site 2 Site 2  rO ro  Depth (cm)  Dil.  5 10 15 20 25 30 5 10 15 20 25 30  40 80 50 80 80 50 40 40 80 50 80 80  O.D.  0.794 0.434 0.727 0.419 0.393 0.731 0.795 0.833 0.422 0.705 0.389 0.411  so 2  SO4 ' 2  (mg L- ) 1  3827.97 4540.04 4422.52 4410.21 4185.18 4444.16 3832.30 3996.75 4436.18 4303.51 4150.56 4340.97  Dil.  80 40 80 50 40 40 80 80 50 40 40 50  O.D.  0.375 0.898 0.377 0.686 0.813 0.903 0.375 0.354 0.684 0.891 0.882 0.711  4  (mg L- ) 1  4029.38 4278.04 4046.69 4200.73 3910.19 4299.68 4029.38 3847.62 4189.91 4247.75 4208.80 4335.97  S0 " (mg L )  Average S0 ' (mg L' )  St. Dev.  3978.94 4054.67 3827.97 4122.25 4303.51 4514.08 4081.72 3876.16 4208.80 4410.21 4038.44 4273.71  3945.43 4290.92 4099.06 4244.40 4132.96 4419.31 3981.13 3906.84 4278.30 4320.49 4132.60 4316.88  104.80 242.94 300.72 148.87 201.79 109.34 131.53 79.16 137.06 82.55 86.59 37.47  2  Dil.  50 50 40 40 50 80 50 50 40 80 50 40  O.D.  0.645 0.659 0.794 0.862 0.705 0.431 0.664 0.626 0.882 0.419 0.656 0.897  4  1  2  4  1  Location 11-Aug-05 Site 1 Site 1 Site 1 Site 1 Site 1 Site 1 Site 2 Site 2 Site 2 Site 2 Site 2 Site 2 #1 Control #1 Control #1 Control #1 Control #1 Control #1 Control #3 Inoculated #3 Inoculated #3 Inoculated #3 Inoculated #3 Inoculated #4 Amended #4 Amended #4 Amended  S0 ' (mg L- )  Dil.  0.983 0.890 0.471 0.969 0.770 0.466 0.508 0.720 0.390 0.499 0.727 0.491 0.804 0.787 0.469 0.807 0.513 0.787 0.518  4470.60 4076.56 4602.46 4411.28 4460.14 4560.09 4916.00 4195.32 4242.40 4436.43 4232.40 4771.94 4640.21 4550.18 4585.51 4656.10 4958.37 4550.18 5000.75  80 80 40 80 80 50 40 80 50 50 80 50 80 80 50 80 50 80 50  80  0.448  4407.56  15  50  0.815  20  50  25 5 10 15  S0 ' (mg L- )  Dil.  0.532 0.460 0.914 0.516 0.497 0.746 0.960 0.433 0.640 0.797 0.445 0.776 0.486 0.514 0.751 0.516 0.754 0.489 0.802  5119.38 4509.25 4178.25 4983.80 4822.79 4333.03 4373.15 4280.45 4085.92 4219.54 4382.14 4491.92 4729.57 4966.85 4359.51 4983.80 4375.40 4755.00 4629.62  50 50 50 50 40 40 50 40 40 40 40 40 40 40 40 40 40 40 60  50  0.727  4232.40  4698.47  80  0.496  0.786  4544.88  80  80  0.473  4619.41  80 50 50  0.619 0.907 0.993  5856.63 5185.73 5641.22  2  Depth (cm)  Dil.  5 10 15 20 25 30 5 10 15 20 25 30 5 10 15 20 25 30 5  40 40 80 40 50 80 80 50 80 80 50 80 50 50 80 50 80 50 80  10  O.D.  4  1  4  1  so 2  2  O.D.  O.D.  4  Average  SO/  St. Dev.  (mg L- )  (mg L- )  0.881 0.742 0.744 0.773 0.999 1.000 0.746 0.931 0.810 1.050 0.928 0.988 1.030 1.050 0.989 1.020 1.030 1.030 0.669  5048.03 4311.84 4322.43 4476.03 4538.40 4542.63 4333.03 4250.28 4049.06 4358.28 4237.57 4491.79 4669.75 4754.49 4496.03 4627.37 4669.75 4669.75 4710.25  4879.34 4299.22 4367.71 4623.70 4607.11 4478.58 4540.73 4242.01 4125.79 4338.08 4284.03 4585.22 4679.84 4757.17 4480.35 4755.76 4667.84 4658.31 4780.21  355.77 216.62 215.70 313.53 190.84 126.36 325.62 43.16 102.65 109.84 85.00 161.71 45.53 208.35 113.82 198.01 291.49 102.89 195.20  60  0.600  4271.72  4303.89  91.91  4814.31  60  0.675  4748.39  4753.72  58.11  0.461  4517.72  60  0.641  4532.30  4531.63  13.59  50  0.782  4523.69  60  0.651  4595.85  4579.65  49.87  50 80 80  0.920 0.597 0.668  5254.59 5670.20 6271.86  60 60 60  0.780 0.770 0.810  5415.72 5352.17 5606.39  5508.98 5402.70 5839.82  311.67 246.15 374.56  1  1  Location #4 Amended #4 Amended #5 Control #5 Control #5 Control #5 Control #5 Control #5 Control #5 Control #6 Amended #6 Amended #6 Amended #6 Amended #6 Amended #6 Amended  S0 ' (mg L- ) 2  Depth (cm)  Dil.  20 25 5 10 15 20 25 30 35 5 10 15 20 25 30  80 50 50 80 80 50 80 50 80 50 80 80 50 50 50  O.D.  4  1  S0 ' (mg L" ) 2  Dil.  O.D.  4  1  S0 " (mg L- ) 2  Dil.  O.D.  4  1  Average  so/  St. Dev.  (mg L- ) 5340.91 5408.00 5106.81 5073.62 5069.03 5327.49 5329.96 5486.38 4775.97 4953.93 4334.96 4175.01 4755.49 4497.03 4364.62 1  0.590 0.918 0.843 0.557 0.547 0.898 0.610 0.918 0.513 0.834 0.442 0.426 0.814 0.768 0.733  5610.88 5243.99 4846.77 5331.24 5246.49 5138.07 5780.36 5243.99 4958.37 4799.10 4356.71 4221.13 4693.18 4449.55 4264.17  50 80 80 50 50 80 50 80 50 80 50 50 80 80 80  0.918 0.583 0.554 0.829 0.840 0.603 0.863 0.619 0.816 0.528 0.717 0.688 0.516 0.466 0.453  5243.99 5551.56 5305.81 4772.62 4830.88 5721.04 4952.70 5856.63 4703.77 5085.49 4179.43 4025.84 4983.80 4560.09 4449.93  60 60 60 60 60 60 60 60 60 60 60 60 60 60 60  0.741 0.782 0.741 0.733 0.735 0.734 0.755 0.771 0.662 0.711 0.631 0.601 0.650 0.633 0.617  Sulphide (mg L )  Dil.  O.D.  Sulphide (mg L- )  Dil.  O.D.  0.29 0.13 0.25 0.12 0.04 0.03 0.04 0.05 0.03 0.04  1 2 1 2 1 2 1 1 1 2  0.30 0.09 0.23 0.05 0.07 0.05 0.05 0.09 0.04 0.03  2 1 2 1 1 1 2 1 2 1  5167.85 5428.43 5167.85 5117.01 5129.72 5123.37 5256.83 5358.52 4665.76 4977.19 4468.74 4278.07 4589.50 4481.45 4379.76  236.88 154.80 235.53 281.82 214.35 340.91 418.65 325.72 159.11 144.60 145.87 132.29 204.40 56.90 93.80  A1-8b. Total sulphide in Lake 52 in situ experiment  Location 4-Oct-04 Site 1 Site 1 Site 1 Site 1 Site 1 Site 1 Site 2 Site 2 Site 2 Site 2  4^  Depth (cm) 5 10 15 20 25 30 5 10 15 20  Dil.  1 1 1 1 1 1 1 1  O.D.  0.139 0.068 0.122 0.064 0.018 0.023 0.028 0.021 0.022 0.029  1  0.144 0.031 0.115 0.021 0.040 0.020 0.034 0.050 0.026 0.017  1  Average Sulphide Sulphide (mg L' ) (mg L" ) 1  St. Dev.  1  0.068 0.061 0.072 0.058 0.033 0.040 0.024 0.045 0.018 0.025  0.26 0.11 0.28 0.11 0.05 0.07 0.06 0.08 0.04 0.03  0.28 0.11 0.25 0.09 0.05 0.05 0.05 0.07 0.03 0.04  0.02 0.02 0.02 0.04 0.02 0.02 0.01 0.02 0.01 0.01  Location 11-Aug-05 Site 1 Site 1 Site 1 Site 1 Site 1 Site 1 Site 2 Site 2 Site 2 Site 2 Site 2 Site 2 #1 Control #1 Control #1 Control #1 Control #1 Control #1 Control #3 Inoculated #3 Inoculated #3 Inoculated #3 Inoculated #3 Inoculated #4 Amended #4 Amended  to - J Ul  Depth (cm)  Sulphide (mg L- )  Dil.  1  0.020 0.050 0.043 0.039 0.083 0.038 0.057 0.041 0.030 0.045 0.037 0.045 0.437 0.419 0.318 0.343 0.190 0.113 0.435  0.06 0.14 0.12 0.11 0.22 0.11 0.16 0.12 0.10 0.12 0.11 0.13 1.09 1.04 0.79 0.86 0.48 0.29 1.08  2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2  10  1  0.415  1.03  15  1  0.343  20  1  25 5 10  5 10 15 20 25 30 5 10 15 20 25 30 5 10 15 20 25 30 5  Average Sulphide Sulphide (mg L- ) (mg L- )  Sulphide (mg L- )  Dil.  0.000 0.010 0.019 0.015 0.030 0.015 0.020 0.022 0.000 0.018 0.015 0.015 0.167 0.170 0.176 0.135 0.071 0.055 0.208  0.03 0.08 0.12 0.11 0.18 0.11 0.13 0.14 0.03 0.11 0.11 0.11 0.85 0.86 0.89 0.69 0.38 0.30 1.05  1 1 1 1 1 1 1 1 1 1 1 1 5 5 5 5 5 5 5  0.030 0.041 0.022 0.028 0.077 0.041 0.042 0.037 0.038 0.061 0.037 0.046 0.068 0.055 0.055 0.057 0.041 0.015 0.079  0.09 0.12 0.07 0.08 0.20 0.12 0.12 0.11 0.12 0.15 0.11 0.13 0.91 0.75 0.75 0.78 0.58 0.26 1.05  0.06 0.11 0.11 0.10 0.20 0.11 0.13 0.12 0.08 0.13 0.11 0.12 0.95 0.89 0.81 0.78 0.48 0.29 1.06  0.03 0.03 0.03 0.01 0.02 0.01 0.02 0.02 0.04 0.02 0.00 0.01 0.12 0.15 0.07 0.08 0.10 0.02 0.02  2  0.234  1.18  5  0.081  1.07  1.09  0.08  0.86  2  0.175  0.89  5  0.061  0.83  0.86  0.03  0.106  0.28  2  0.062  0.34  5  0.017  0.29  0.30  0.03  1  0.092  0.24  2  0.010  0.08  5  0.000  0.08  0.13  0.09  1 1  0.923 1.007  2.28 2.48  2 2  0.683 0.674  3.38 3.33  5 5  0.200 0.210  2.53 2.65  2.73 2.82  0.58 0.45  Dil.  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  O.D.  1  O.D.  1  O.D.  1  St. Dev.  1  Location #4 Amended #4 Amended #4 Amended #5 Control #5 Control #5 Control #5 Control #5 Control #5 Control #5 Control #6 Amended #6 Amended #6 Amended #6 Amended #6 Amended #6 Amended  Depth (cm)  Dil.  15 20 25 5 10 15 20 25 30 35 5 10 15 20 25 30  1 1 1 1 1 1 1 1 1 10 1 1 1 1 1 5  O.D.  Sulphide (mg L )  Dil.  2.25 3.08 1.85 1.30 0.83 0.81 0.78 0.79 0.42 37.63 0.34 0.27 0.12 0.06 0.20 7.97  2 2 2 2 2 2 2 2 2 50 2 2 2 2 2 10  1  0.911 1.252 0.747 0.526 0.333 0.324 0.314 0.316 0.163 1.530 0.131 0.105 0.043 0.016 0.075 0.644  O.D. 0.631 0.764 0.487 0.339 0.247 0.222 0.253 0.238 0.099 0.308 0.084 0.029 0.018 0.004 0.022 0.26  Sulphide (mg L- )  Dil.  3.12 3.77 2.42 1.69 1.24 1.12 1.27 1.20 0.52 38.51 0.44 0.17 0.12 0.05 0.14 6.53  5 5 5 5 5 5 5 5 5 20 5 5 5 5 5 20  1  O.D.  Average Sulphide Sulphide (mg L- ) (mg L- ) 3.17 2.84 3.03 3.30 1.93 2.06 1.44 1.48 0.95 1.01 0.88 0.93 0.96 1.01 0.95 0.98 0.46 0.46 39.99 38.71 0.39 0.39 0.26 0.24 0.08 0.11 0.08 0.06 0.20 0.18 7.71 7.40 1  St. Dev.  1  0.252 0.241 0.151 0.111 0.071 0.065 0.072 0.071 0.031 0.810 0.025 0.015 0.000 0.000 0.010 0.151  A1-8c. Temperature, pH, DO in Lake 52 in situ experiment 4-Oct-04  Depth (cm)  Site 1  0-5 5-10 10-15 15-20 20-25 25-30 0-5 5-10 10-15 15-20 20-25 25-30  Site 2  Temperature (°C) 11.9 11.9 11.9 11.9 11.9 11.9 14.5 14.2 13.7 13.7 13.7 13.7  PH  DO % Saturation  11-Aug-05  Depth (cm)  Temperature (°C)  PH  9.65 9.67 9.62 9.68 9.69 9.72 9.36 9.32 9.2 9.3 9.38 9.37  95.6 94 89.9 91  Site 1  5 12.5 20 5 12.5 20 5 12.5 20 5 12.5 20  23.7 23.6 23.4 21.2 21.2 21.2 20.9 21.0 21.0 21.0 21.0 21.0  9.34 9.32 9.33 9.38 9.4 9.39 9.40 9.40 9.40 9.40 9.40 9.40  94.8 94.8 94.3 92.3 91.7 93.6 93.9  Site 2  #1 Control  #3 Inoculated  0.52 0.42 0.31 0.20 0.21 0.16 0.25 0.21 0.05 1.19 0.05 0.05 0.02 0.02 0.04 0.77  11-Aug-05 #4 Amended  #5 Control  #6 Amended  —J  Depth (cm) 5 12.5 20 5 12.5 20 5 12.5 20  Temperature (°C) 20.6 20.5 20.5 20.1 20.3 20.2 20.1 20.0 20.0  PH 8.92 8.91 8.90 9.34 9.32 9.32 9.34 9.34 9.33  A1-8d. Soluble COD in Lake 52 in situ experiment  Sample 4-Oct-04 Site 1 Site 2 11-Aug-05 Site 1  Site 2  #1 Control  #3 Inoculated  #4 Amended  #5 Control  #6 Amended  Depth (cm)  Dil.  15 15  1 1  5 10 20 5 10 20 5 10 20 5  1 1 1 1 1 1 1 1 1  10 20 5  1 1  10 20 5 10 20 5  1 1 1 1 1  10 20  1 1  1  1  sCOD (mg L- )  Dil.  0.058 0.057  143.46 140.98  1 1  0.059 0.058 0.055 0.054 0.058 0.058 0.054 0.055 0.051 0.057  145.93 143.46 136.04 133.56 143.46 143.46 133.56 136.04 126.14 140.98  1 1 1 1 1 1 1 1 1  0.071 0.057 0.087  175.61 140.98 215.19  1 1  0.079 0.078 0.086 0.08 0.08 0.066  195.40 192.93 212.71 197.87 197.87 1.63.24  1 1 1 1 1  0.063 0.064  155.82 158.30  O.D.  1  sCOD (mg L" )  Average sCOD (mg L )  0.063 0.054  155.82 133.56  145.93 140.98  0.061 0.056 0.054 0.051 0.057 0.05 0.055 0.056 0.051 0.057  150.88 138.51 133.56 126.14 140.98 123.67 136.04 138.51 126.14 140.98  149.23 139.33 134.39 126.14 138.51 131.09 137.69 140.98 130.27 139.33  0.06 0.061 0.079  148.40 150.88 195.40  155.00 158.30 208.59  1  0.079 0.081 0.086 0.08 0.08 0.061  195.40 200.35 212.71 197.87 197.87 150.88  195.40 209.41 214.36 195.40 198.70 155.00  1 1  0.065 0.059  160.77 145.93  159.95 146.76  sCOD (mg L- )  Dil.  0.056 0.06  138.51 148.40  1 1  0.061 0.055 0.054 0.048 0.053 0.051 0.058 0.06 0.056 0.055  150.88 136.04 133.56 118.72 131.09 126.14 143.46 148.40 138.51 136.04  1 1 1 1 1 1 1 1 1  0.057 0.074 0.087  140.98 183.03 215.19  1 1  195.40 234.97 217.66 190.45 200.35 150.88  1 1 1 1 1  1  0.079 0.095 0.088 0.077 0.081 0.061  1 1  0.066 0.055  163.24 136.04  1  1  O.D.  1  1  1  O.D.  1  1  A1-8e. Ortho-phosphate and ammonia-nitrogen in Lake 52 in situ experiment 4-Oct-04 2 1 3 Average 0.04 0.04 0.10 0.06 PCV" (mg L" ) 1  N0 "-N (mg L" )  0.06  0.04  0.05  0.05  NH -N (mg L- )  0.38  0.42  0.37  0.39  Depth (cm)  P0 " (mg L- )  P0 " (mg L- )  Average P0 ' (mg L" )  5 12.5 20 5 12.5 20 5 12.5 20 5  0.00 0.00 0.01 0.04 0.00 0.00 0.01 0.02 0.02 0.01  0.00 0.01 0.00 0.01 0.00 0.00 0.00 0.01 0.01 0.00  0.00 0.01 0.01 0.03 0.00 0.00 0.01 0.02 0.02 0.01  12.5 20 5  0.01 0.06 0.30  0.00 0.04 0.21  0.01 0.05 0.26  12.5 20  0.34 0.26  0:54 0.27  0.44 0.27  3  1  3  1  11-Aug-05 Site 1  Site 2  #1 Control  #3 Inoculated  #4 Amended  SO  3  4  1  3  4  1  3  4  Depth (cm)  NH -N (mg L- )  NH -N (mgL )  #3 Inoculated  5 12.5 20 5 12.5 20 5 12.5 20 5  0.39 0.34 0.46 0.54 0.43 0.43 0.45 0.45 0.44 0.45  0.35 0.36 0.38 0.50 0.45 0.43 0.46 0.44 0.46 0.38  Average NH -N (mg L" ) 0.37 0.35 0.42 0.52 0.44 0.43 0.46 0.45 0.45 0.42  #4 Amended  12.5 20 5  0.39 0.46 1.20  0.42 0.44 1.19  0.41 0.45 1.20  12.5 20  1.18 1.19  1.17 1.21  1.18 1.20  11-Aug-05  1  Site 1  Site 2  #1 Control  3  1  3  1  3  1  11-Aug-05 #5 Control  #6 Amended  ro  00 O  Depth (cm)  P0 ' (mg L- )  P0 " (mg L' )  5 12.5 20 5  0.00 0.09 0.09 0.23  0.04 0.05 0.04 0.23  Average P0 ' (mg L- ) 0.02 0.07 0.07 0.23  12.5 20  0.23 0.21  0.24 0.19  0.24 0.20  3  4  1  3  4  1  3  4  11-Aug-05  1  #5 Control  #6 Amended  Average  Depth (cm)  NH -N (mg L )  NH -N (mg L- )  5 12.5 20 5  0.82 0.80 0.84 0.76  0.81 0.80 0.82 0.72  (mg L- ) 0.82 0.80 0.83 0.74  12.5 20  0.72 0.76  0.71 0.75  0.72 0.76  3  1  3  1  NH3-N 1  

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