LIBRARY Michigan State University This is to certify that the thesis entitled KINETIC PARAMETERS OF THE CONVERSION OF METHANE PRECURSORS TO METHANE IN A HYPEREUTROPHIC LAKE SEDIMENT presented by R. F. Strayer has been accepted towards fulfillment of the requirements for Ph . D degree in Microbiology 411524.44 \ ‘ ‘ . ajor professor 0 Datefliwf #2, 6‘}? 0-7639 \j) Mew METHANE PRODUCTION BY THE MICROFLORA OF THE ANAEROBIC PELAGIAL SEDIMENTS OF A HYPEREUTROPHIC LAKE BY Richard Floyd Strayer A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1977 ABSTRACT METHANE PRODUCTION BY THE MICROFLORA or THE ANAEROBIC PBLAGIAL SBDIMENTS or A HYPEREUTROPHIC LAKE By Richard Floyd Strayer Fluorescent antibody (PA) was prepared for a hydrogen—consuming methanogenic bacterium isolated from Wintergreen Lake pelagic sediment. The isolate resembles Methanobacterium formicicum. The FA did not cross react with nine other methanogens, including fl, formicicum strains, or 2H heterotrophs, 18 of which had been isolated from Wintergreen Lake sediment. In a qualitative survey of different anaerobic habitats, FA reacting methanogens were detected in heat fixed smears of several different lake sediments and anaerobic sewage sludge but not in bovine rumen fluid. FA direct counts of the specific methanogen in Wintergreen Lake sediments were made on four different sampling dates and compared with five tube most probable number (MPN) estimates of the total methano- genic population that was present in the same samples. The FA counts 6 to 1.4 x 107°g-l dry sediment. The highest MPN ranged from 3.1 x 10 estimates were at least an order of magnitude lower. These results indicate that a specific hydrogen-consuming methanogenic bacterium is among the predominant methane formers in this habitat. Michaelis-Menten kinetic analysis was used to determine the kinetic parameters for the conversion of methane precursors to methane by the sediment microflora. For these experiments I designed an incubation Richard Floyd Strayer system that was not easily contaminated by air, that gave a large gas atmosphere reservoir so that added hydrogen was not significantly depleted during incubation, and that maximized the phase transfer of gaseous substrates and products. Two independent methods were used to estimate the kinetic param- eters for the conversion of hydrogen to methane. Direct linear plots were used to estimate parameters from initial velocity versus initial substrate concentration (v vs. 8) experiments and an integrated solution to the Michaelis-Menten equation was used to estimate parameters from hydrogen and methane progress curves. Michaelis-Menten constants (Km) ranged from 0.13 to o.u7% hydrogen for v vs. S experiments and 0.21 to 0.48% hydrogen for progress curve experiments. The mean Km estimates for hydrogen consumption were 13 nmol I-I2°g'-1 dry sediment for sediment collected inJanuary and February and 29 nmol l-IQ'g-l for sediment collected in March. For the conversion of hydrogen to methane the average Km was 23 nmol Hz'g-l. For both v vs. S and progress curve experiments the maximum velocity (vmax) ranged from 1.1 to 5.9 nmol l-l2'gul°h-l for winter sediments. The vmax increased slightly from December to March. A new method was devised to quantitate the very low concentrations of dissolved hydrogen in the sediment. The method involves the trans- ferral of dissolved sediment gases to a carbon dioxide gas atmosphere. These sediment gases are then concentrated by absorption of the carbon dioxide by a high ionic-strength alkaline solution. For winter sedi- ments the in_§i£3_dissolved hydrogen concentration ranged from 6 to 10 l nmol'g- . The natural rates of hydrogen consumption and hydrogen con- version to methane were estimated from these dissolved H2 concentrations and the Km and Vmax estimates. For hydrogen consumption, the estimated Richard Floyd Strayer l. -1 natural rate ranged from #30 to 1500 nmol H2‘g— h . For the con- version of hydrogen to methane, the natural rate ranged from 72 to 290 nmol CH” produced'g-1'h-l. These results indicate that the methanogenic bacteria are capable of maintaining the low ig.gi£g_hydrogen concentra— tions that are a necessary prerequisite for interspecies hydrogen trans- fer. The rate of hydrogen consumption by sediment microflora was high enough to lower moderately high hydrogen levels to normal concentrations within several hours. Several potential hydrogen donors-~formate, lactate, propionate, valine, and leucine--were tested for the ability to stimulate sediment methanogenesis. Only formate significantly altered the rate. Rates of formate conversion to methane were so rapid that accurate kinetic estimates could not be made. For example, at an incubation time of only 10 min, at least u7% of the added formate had been converted to methane. Shorter incubation times were tried but kinetic parameters could not be estimated even though stimulation of methanogenesis occurred. The addition of unlabeled acetate had no effect on the rate of sediment methanogenesis but in other experiments [2-luC] acetate was converted to labeled methane. Kinetic experiments with labeled acetate were used to resolve these apparently conflicting results and to estimate . The the kinetic parameters Km + Sn’ V , and turnover time, Tt max results indicated that the conversion of acetate to methane was occurr- ing at rates very near the maximum. The minimum natural rate of acetate conversion to methane was estimated from the minimum in situ dissolved acetate concentration (measured by Molongoski and Klug (unpublished data) for Wintergreen Lake pelagic sediment in the summer of 1976) and Richard Floyd Strayer the average turnover time estimated from the kinetic experiments. The minimum rate was estimated to be 0.108 umol°g-l'h'l, which was 67% of. the vmax' Using the average sediment dissolved acetate concentration for the entire summer of 1976, the estimated natural rate was 88% of the Vmax' These results indicate that increases in heterotrophic acetate production in the sediment would lead to an increase in the acetate pool size since the acetate-using methanogenic bacteria are already convert- ing this substrate at or very near the maximum rate. A 15 min preincubation of sediment with 0.5% hydrogen in the gas phase had a pronounced effect on the kinetic parameters for the con- version of acetate to methane. When compared with parameters estimated from experiments with no hydrogen preincubation, the acetate pool size, as Km + Sn’ decreased by 37% and the turnover time for the acetate pool decreased by ”3%. The Vmax remained relatively constant. These results indicate that interspecies hydrogen transfer may be occurring in the sediment since less oxidized substrate, such as acetate, is produced in an uncoupled system. Further evidence that interspecies hydrogen transfer occurs was that a preincubation with hydrogen caused a 37% decrease in the amount of radioactive CO2 produced from the hetero- trophic metabolism of labeled valine by sediment microflora. To my wife, Diane, for her encouragement and patience. To my parents for their moral support. ii ACKNOWLEDGEMENTS I am deeply grateful to Dr. J. M. Tiedje for his expert guidance and encouragement. Special thanks are due to Dr. M. J. Klug for his helpful disucssions and for serving on my Guidance Committee. I am also thankful to Drs. C. A. Reddy, H. L. Sadoff, L. L. Bieber, and J. A. Breznak for serving on my Guidance Committee. My appreciation is extended to Drs. G. H. Lauff and M. J. Klug for making the facilities of the Kellogg Biological Station available. I would like to thank the following people for supplying cultures: Drs. M. P. Bryant, J. G. Zeikus, M. J. B. Paynter, J. G. Ferry, M. J. Klug, and C. A. Reddy, and J. J. Molongoski. I am also grateful for financial assistance from the Department of Microbiology and Public Health at Michigan State University, the National Institute of Health, and the National Science Foundation. I am grateful to Dr. M. P. Bryant for taking me into his laboratory to teach me the Hungate anaerobic technique. A special thanks is due to Sue Knoll for the task of typing the many revisions of this manuscript. I owe too a special thanks to my friends, past and present, in the quite informal "Institute of Microbial Ecology", better known as Dr. Tiedje's research group. Their discussions on a variety of topics made my educational experience a much more rewarding one. iii TABLE OF CONTENTS Page LIST OF TABLES O O O O O O O O O O O O O I O O O O O O O O O O 0 O 0 Vi LIST OF FIGURES O I O O O O O I I O O O O O O l O O O I O O O O O 0 viii CHAPTER I. REVIEW OF PERTINENT LITERATURE AND RATIONALE FOR EXPERIMENTAL APPROACH. . . . . . . . . . . . . . . . l [.4 INTRODUCTION . . . . . . . . . . . . . . SUBSTRATBS FOR MBTHANOGENIC BACTERIA IN PURE CULTURE. . . . . . . . . . . . . . MBTHANB PRODUCTION IN ANABROBIC HABITATS . . . . Hydrogen as a substrate. . . . . . . . Acetate as a substrate . . . . . . . . . . INTERSPECIES H2 TRANSFER . . . . . . . . . . . . . . 1” RESEARCH REPORTED IN THIS DISSERTATION . . . . . . . 15 LITERATURE CITED 0 O O O O O O O O O O O O O O O O O 21 '44-'43: CHAPTER II. APPLICATION OF THE FLUORESCENT ANTIBODY TECHNIQUE TO STUDY A METHANOGENIC BACTERIUM IN LAKE SEDIMENTS . . 24 INTRODUCTION . . . . . . . . . . . . . . . . . . . . 24 MATERIALS AND METHODS. . . . . . . . . . . . . . . . 25 Media. . . . . . . . . . . . . . . . . . . . 25 Anaerobic methodology. . . . . . . . . . . . 26 Antigen preparation. . . . . . . . . . . . . 26 Antibody preparation . . . . . . . . . . . . 27 FA stain of cultures and natural samples . . 27 RESULTS. . . . . . . . . . . . . . . . . . . . . . . 29 Description of methanogenic organism purified from the sediment. . . . . . . . . . . . . 29 Tests on antibody prepared with the methanogen . . . . . . . . . . . . . . . . . 30 Qualitative survey of various habitats for PA reacting bacteria . . . . . . . . . . . 3n Direct count FA and MPN estimates of the methanogenic bacteria in sediments . . . . . 3n DISCUSSION . . . . . . . . . . . . . . . . . . . . . 39 LITERATURE CITED . . . . . . . . . . . . . . . . . . H3 iv CHAPTER III. Page KINETIC PARAMETERS OF THE CONVERSION OF METHANE PRECURSORS TO METHANE IN A HYPEREUTROPHIC LAKE SBDIMENT C O C C O O O C C O O C . O C O O C I O O ”6 INTRODUCTION . . . . . . . . . . . . . . . . . . . #6 MATERIALS AND METHODS. . . . . . . . . . . . . . . #9 Sampling . . . . . . . . . . . . . . . . . M9 Incubation system. . . . . . . . . . . . . #9 Estimation of dissolved hydrogen . . . . . 52 Gas analysis . . . . . . . . . . . . . . . 53 Radioactive isotopes and analysis of radioactive gases. . . . . . . . . . . . . 53 Theory . . . . . . . . . . . . . . . . . . 5” RESULTS. . . . . . . . . . . . . . . . . . . . . . 56 Kinetics of hydrogen conversion to methane 56 Other hydrogen donors and the stimulation of methanogenesis. . . . . . . . . . . . . 6H , Kinetics of acetate conversion to methane. 6# DISCUSSION . . . . . . . . . . . . . . . . . . . . 83 LITERATURE CITED . . . . . . . . . . . . . . . . . 9O Table LIST OF TABLES CHAPTER I Summary of reported examples of pure culture studies in which interspecies hydrogen transfer has been shown, and the effect of this metabolic coupling on the end products. The hypothesized interaction for natural sediments is included for comparative purposes . . . . . . . . . . . . . . . . . CHAPTER II Control tests needed for the application of FA (from SChHlidt (20)). o o o o o o o o o o o o o o o o Microorganisms tested for cross-reactivity with FA preparation: no cross-reaction observed. . . . Number of methane producing bacteria in Wintergreen ‘Lake sediment on different sampling dates as estimated by FA direct counts and by a five tube MPN technique using three different media. MPN tubes were scored as positive if methane was produced within u weeks. . . . . . . . . . . . . . . . CHAPTER III Summary of kinetic experiments of the consumption of hydrogen and the conversion of hydrogen to methane by Wintergreen Lake sediments. . . . . . . . . Ratio of hydrogen consumed to methane produced with decreasing hydrogen concentration calculated from the progress curves of Fig. u. . . . . . . . . . . . Effect of potential hydrogen donors on the rate of sediment methanogenesis. Incubation temperature was 12 C . . . . . . . . . . . . . . . . . Effect of dissolved formate and dissolved acetate concentrations on the rate of sediment methanogenesis. Incubation time was 10 min at 23 C. Sediment was collected in the late winter . . . . . . . . . . . . . . . . . . . . . . . . vi Page 18 33 35 38 59 67 68 72 Table Page Kinetic parameters estimated from the regression of t/f versus A, as in Fig. 8. Experiment l.-run within u days of collecting sediment in early July. Experiment 2.-run within 2 weeks of collecting sediment in late June . . . . . . . . . . . 80 vii Figure LIST OF FIGURES Page CHAPTER I Generalized scheme of the anaerobic decomposition of organic matter . . . . . . . . . . . . . . . . . . . . 3 Effect of the fraction of the total volume occupied by the aqueous phase on the transfer of dissolved hydrogen to the vapor phase for incubation temperatures of 10 and 35 C. Arrows denote the percent aqueous phase in the incubation systems of Smith and Mah (27) and Cappenberg and Prins (7). Curves calculated from the equation of Flett et al. (12) . . . . . . . . . . . . . . . . . . . . . . . 13 Comparison of the products formed and energy generated in the fermentation of glucose by Ruminococcus albus grown in mono-culture and in mixed culture with a methanogen. The figure is derived from Thauer et al. (29) but with the hydrogen utilizing Vibrio succinggenes replaced by a hydrogen-using methanogen. . . . . . . . . . . . . . l7 CHAPTER II Phase contrast photomicrograph of a short filament and single cells of a methanogenic bacterium isolated from Wintergreen Lake sediment and used to prepare the FA. Bar represents 10 um. . . . . . . . . 32 Photomicrograph of an FA-stained smear of the methanogenic isolate. Bar represents 10 um . . . . . . . 32 Photomicrographs of immunofluorescing cells in FA stained smears of various habitats. (a) Wintergreen Lake pelagic sediment. (b) Sludge from an anaerobic sewage digestor. (c) Bovine rumen fluid. Bars represent 10 um . . . . . . . . . . . 37 viii Figure Incubation system used to study the kinetics of methane production in lake sediments. was specially made from a 20 x 200 mm culture tube with the top modified to accept a flange type butyl rubber stopper and Hungate-type screw cap. Tubes with 5 to 10 ml sediment were incubated in a near horizontal position on a rotary mixer as shown. Effect of increasing hydrogen concentration , [S], on the rate of sediment methane production, v. The rates have been corrected for the control Incubation time was 1.25 h Sediment was collected in the early (no hydrogen added). at 10 C. winter. . Direct linear plot of the substrate, velocity CHAPTER III The tube data pairs plotted in Figure 2. . . . . . . . . Progress curves of the headspace hydrogen and methane concentrations with time. Methane concentrations have been corrected for the control which had no hydrogen added. Headspace (vapor) volume was about was 12 C. 65 ml. Sediment was collected in late winter . Effect of dissolved formate concentration of the rate of sediment methanogenesis. rates have been corrected for the rate of the control, which received an identical volume of oxygen free water but no formate. time was 1 h at 12 C. in mid Winter 0 O O O O I O O O O O O O O 0 Amount of methane produced by sediment with time at different concentrations of added dissolved acetate . Incubation temperature was 12 C. Sediment was collected in mid winter. . . . Productii of labeled methane and carbon dioxide C] acetate (1 uCi). O - labeled methane produced in the absence of hydrogen 0 - labeled from [2- Incubation Sediment was collected Methane production Incubation temperature carbon dioxide produced in the absence of hydrogen. A - labeled methane produced in the presence of 0.5% Hydrogen was added 15 min prior to the Incubation temperature hydrogen. addition of labeled acetate. was 12 C. Sediment was collected in June . ix Page 51 61 63 66 70 71+ 77 Figure Page Effect of acetate conifintration and hydrogen on the conversion of [2- C] acetate to methane by sediment. 0 - labeled methane produced within 15 min in the absence of 0.5% hydrogen. 0 - labeled methane produced within 10 min in the presence of 0.5% hydrogen. The hydrogen was added 15 min prior to the acetate addition. Incubation temperature was 12 C. Sediment was collected in June . . . . . . . . . . . . . . . . . . . . 79 Effefit of acetatiuconcentration on the production of CH from 2- C acetate by lake sediments. Each acetate concentration had the same specific activity of labeled acetate. Incubation time was 15 min at 12 C. Sediment was collected in June . . . . . 82 CHAPTER I REVIEW OF PERTINENT LITERATURE AND RATIONALE FOR EXPERIMENTAL APPROACH INTRODUCTION The major end products of the fermentation of organic compounds by 2, H2, short chain fatty acids, and other reduced molecules. In the anaerobic pure cultures of anaerobic heterotrophic bacteria are CO decomposition of organic matter in habitats such as the bovine rumen (l4), sewage sludge digestors (18), and sediments of hypereutrophic lakes (33,5), some of these heterotrophic products are converted to methane. A generalized scheme of this decomposition chain is shown in Fig. 1. Typically, anaerobic decomposition is viewed as a two-stage process, with acid formation the initial phase and methane production the terminal phase. Bryant (2), however, has hypothesized a third phase which consists of "acetogenic" bacteria that convert short chain organic acids (and alcohols) to acetate and H Although artifically separated 2. into phases which are carried out by physiologically distinct microbial populations, anaerobic decomposition is better viewed as a continuum with metabolic interactions among and between the separate physiological groups. The most extensively studied metabolic interaction between anaerobic populations is interspecies hydrogen transfer (4,9,16,22,31, 35). This process has been studied with pure and mixed cultures isolat— ed from a variety of anaerobic habitats, but no work has been done with Figure l. Generalized scheme of the anaerobic decomposition of organic matter. Proteins Polysaccharides Amino acids Monosaccharides "Acid—forming" bacteria nfCaproate . iso-Butyratel " ° n (Butyrate) r --------- ) Acetogenic Pro i n te , . ) P O a ): ,v” bacteria 4? a '.,-- l',-’ Acetate k " , ’ Formate ,v"’ co , ’ 2 .r’ L’ //[H2 Methane producing bacteria \ H4 samples from natural habitats. The purpose of this chapter is to selectively review the pertinent literature related to the subjects of sediment methanogenesis and interspecies hydrogen transfer. I then discuss my hypothesis and the research approach and experiments designed to test this hypothesis. Substrates for methanogenic bacteria in pure culture Until 1967, methanogenic bacteria were thought to metabolize a variety of compounds to methane (1,28). These substrates included straight chain volatile fatty acids from formic to caproic, iso-and n- alcohols from methanol to pentanol, and the gases H CO, and CO 2’ 2 (1,28). Then, Bryant et. al. (3) discovered that the supposedly pure culture of Methanobacterium omelianskii was actually a mixture of two distinct species; an ethanol-degrading bacterium (S organism) that formed acetate and hydrogen, and a methanogenic organism (Methano- bacterium strain MoH) that could only use H and CO2 to produce methane. 2 Elucidation of this mixed culture interaction has led to the observation by Wolfe (36), ...."(that methanogenic) organisms which use propionate, butyrate or higher fatty acids and alcohols are (likewise) not rep- resented in pure culture at the present time." All known pure cultures of methanogenic bacteria can use H2 and C02 to form methane (36,37). In addition, some species can use formate or CO and Methanosarcina barkeri can also utilize methanol and acetate (36). Methane production in anaerobic habitats Hydrogen as a substrate. Hydrogen and carbon dioxide are the principle substrates for methanogenesis in the bovine rumen (15). Formate, which is converted to H and C02, accounts for 18% of the 2 hydrogen formed (15). The rest of the H2 comes from the split of pyruvate into acetyl CoA, H2 andCO2 and from NADH via hydrogenase (l4). Kinetic studies of the Conversion of hydrogen to methane indicated that the KS (substrate half-saturation constant) was about 1 umole dissolved H2°l-1 and that the dissolved H2 pool size was 1 umole‘l-l(15). Thus, H2 was converted to methane at half the maximal rate. In a much earlier study (8), the maximal rate was determined to be 1.42 umole H2'g-l'min-1. Hungate (14) has used this maximum rate to calculate that the turnover 2 (equivalent to 178 nmol CHu produced 'g-l°min-l) and that the pool rate for H conversion to methane in the rumen was 710 nmol H2'g-l°min-l turnover rate constant was 710 min-1. However, the studies determining KS and pool size were published 14 years after the study which determin- ed the maximum rate, so these estimates of turnover rate and turnover rate constant may not be accurate. I have estimated the vmax from the l/v vs. l/s plots of Hungate's 1970 study (15) and obtained an average value of 2.9 umoles CH1+ produced 'g-l'h-l, which would be equivalent to 196 nmoles H consumed -g-l-min-l. At an in situ H concentration of 2 2 luM, the turnover rate would be 98 nmoles H2'g"l°min-l and the turnover rate constant would be 98 min-l. Thus Hungate's published rate estimate is 7-fold higher than my corrected value for his data. For comparative purposes this rumen value is 42 times higher than the sediment H2 turnover rate which I measured (Chapter III), when both are compared on a per g wet wt. basis. Very little work has been done on the conversion of H2 to CHl+ in anaerobic sewage sludge digestors because of the conviction that acetate is the predominent substrate for methanogenesis. However, one study does deserve critical discussion. Shea et a1. (26) isolated a hydrogen utilizing methanogen from sludge and studied the kinetics of its growth in continuous culture. Hydrogen and liquid flow rates were varied but the two rates were kept proportional. The lowest hydrogen concentration was about 1.9% (PH2 = 14.3 mm Hg) and the highest was greater than 25% (extrapolated from their Fig. 4). Their results show a KS of 569 mm Hg [based on hydrogen COD (chemical oxygen demand) removal basis] and 724 mm Hg (based on total COD removal). The maximum velocities were 24.8 mg H2-COD removal ° mg"l volatile suspended solids (VSS) ' day-1 and 31.7 mg total COD removed ' mg-lVSS'day-l. Their Km valves are equivalent to 75 and 95% H2, respectively. The authors then note that hydrogen was generally less than 3% of the product gases in normal sludge digestion and consequently that the substrate removal activity of the hydrogen assimilating bacteria was less than 3% of the maximum. Their Km estimates are 560 times greater than those of Hungate e£_gl, (15), and are at values which would make many of the heterotrophic fermentations a thermodynamic impossibility. Clearly, something is amiss. A minimum concentration of 1.9% H2 should place the system near maximum velocity. Perhaps this work should be disregarded because of obvious errors, however, it is the only published work on H2 with sewage sludge organ- isms and it was felt that its probable inaccuracies should be brought to light. Conversion of H2 to methane by the microflora in lake sediments has also received little attention, but due more to a lack of investigations on this ecosystem than to interest in the fate of other methanogenic precursors. The majority of the current research has been done in the laboratory of J.G. Zeikus at Wisconsin. Winfrey et al. (33) have shown that the addition of hydrogen caused a significant stimulation in sediment methanogenesis and that the amount of methane formed was proportional to the concentration of hydrogen added. The hydrogen concentrations that they added were rather high, 4.0% to 71%, and the shortest incubation time at which H2 or CH“ were measured was 24 b. However these investigators were not attempting a kinetic analysis but rather simply demonstrating the effects of H Hydrogen was also shown 2. to stimulate the conversion of 1”C labeled bicarbonate, formate, methanol and 1 and 2 labeled l”C-acetate to methane (32). In a study of the inhibitory effects of sulfate on methane production in lake sediments, Winfrey and Zeikus (34) showed that the sulfate inhibition could be overcome by the addition of either H2 or acetate. The authors proposed that competition for these substrates was the mechanism by which sulfate inhibited methanogenesis. In a series of articles on sulfate-reducing and methane-producing bacteria in Lake Vechten sediment, Cappenberg (5,6,7) has neglected to report on hydrogen conversion to methane, but has determined that the majority of formate and hydrogen - C0 utilizing methanogens are located 2 at a sediment depth of 2 to 3 cm, as opposed to acetate utilizing methanogens which are at 4 to 6 cm. Acetate as a substrate. Methanosarcina barkeri is the only methano- genic bacterium in pure culture that can use acetate as its sole carbon and energy source (36). The morphological shape of the predominant bacterium in acetate enrichments that produce methane is often not a sarcina (ll,18,21,30,39), but all attempts to isolate this bacterium(ia) have failed. The following reaction for acetate fermentation to methane (29) CH3COO + H2O + CH,4 + HCO3 o' - shows that not enough energy is available for bacterial growth from this 1 reaction (39). In a re-examination of acetate conversion to methane by Methanosarcina barkeri, Zeikus et a1. (39) found that methane formation from acetate required the presence of hydrogen in the gas atmosphere and that methane was not formed when the bacteria were grown in a 100% N2 or CO2 atmosphere. Similar results were obtained with cultures of Methano- bacterium thermoautotrOphicum, which had not been previously shown to use acetate. The authors imply that both acetate and C0 are used by 2 these bacteria to accept electrons and both substrates are reduced to methane in the presence of hydrogen. They did not determine, however, if the acetate was directly reduced to methane, or if it was first oxidized to C02 and then reduced. Either mechanism could explain their results. Zeikus et al. (39) have proposed the following reaction to explain their results enacoo‘ + 4H + H+ + 2 can + 2H 0 2 o' - AG = -39 kcal mol. 2 1 Other methanogens converting acetate to methane in the presence of H2 include M, arbophilicum (37) and Methanospirillum hungatii strain GPl (19). The anaerobic habitat that has received the most attention with regard to acetate conversion to methane is the sewage sludge digestor. Jeris and McCarty (17) studied the fermentation of fatty acids, carbohy- drates and proteins in order to determine the importance of acetic acid as an intermediate in sludge digestion. They concluded that, for all _substrates tested, about 70% of the methane produced came from the degradation of acetate. However, each experimental digestor was accli- mated to the test substrate for a period of at least three months prior to use in a test run. Mah et al. (unpublished data cited in Chynoweth and Mah (10)), have criticized this practice since they found that euryoxic organisms which comprised less than 2.4% of the original sludge population became the most numerous types only 10 to 12 h after the addition of carbohydrate. Certainly a three month acclimation period would also lead to microbial populations quite different from those in the original sludge. Since single substrates were used by Jeris and McCarty (17) for each test run, the species diversity after three months would also be less than in the original sludge. These criticisms lead one to doubt the validity of their conclusion. Smith and Mah (27) have also estimated that about 70% of the methane formed during sludge digestion comes from acetate. These invest- igators determined the acetate turnover rate constant by an isotope dilution technique. This turnover rate constant was multiplied by the acetate pool size to get the acetate turnover rate. They then deter- mined the rate of total methane production for a separate sludge sample and compared this with the acetate turnover rate to obtain their con- clusion that 70% of the methane came from acetate. Their assumption that all of the acetate is converted to methane is probably not valid. Any other fates of acetate such as oxidation by sulfate and sulfur reducers (20,32) were not considered. The presence of these bacteria in sludge has not been investigated but a strain of the sulfate—reducing, acetate oxidizing bacterium, Desulfotomaculum acetoxidans, has been isolated from piggery waste (32). A second alternative fate of acetate could be the reduction of this molecule and a second molecule of either degradation of acetate. However, each experimental digestor was accli— mated to the test substrate for a period of at least three months prior to use in a test run. Mah et al. (unpublished data cited in Chynoweth and Mah (10)), have criticized this practice since they found that euryoxic organisms which comprised less than 2.4% of the original sludge population became the most numerous types only 10 to 12 h after the addition of carbohydrate. Certainly a three month acclimation period would also lead to microbial populations quite different from those in the original sludge. Since single substrates were used by Jeris and McCarty (17) for each test run, the species diversity after three months would also be less than in the original sludge. These criticisms lead one to doubt the validity of their conclusion. Smith and Mah (27) have also estimated that about 70% of the methane formed during sludge digestion comes from acetate. These invest- igators determined the acetate turnover rate constant by an isotope dilution technique. This turnover rate constant was multiplied by the acetate pool size to get the acetate turnover rate. They then deter- mined the rate of total methane production for a separate sludge sample and compared this with the acetate turnover rate to obtain their con- clusion that 70% of the methane came from acetate. Their assumption that all of the acetate is converted to methane is probably not valid. Any other fates of acetate such as oxidation by sulfate and sulfur reducers (20,32) were not considered. The presence of these bacteria in sludge has not been investigated but a strain of the sulfate-reducing, acetate oxidizing bacterium, Desulfotomaculum acetoxidans, has been isolated from piggery waste (32). A second alternative fate of acetate could be the reduction of this molecule and a second molecule of either 10 acetate, propionate, or butyrate to the respective products butyrate, valerate, or caproate (29). The change in the Gibb's free energy for each of these three reactions is —1l.5 kcal/reaction (29). Evidence for the condensation and reduction of two acetate molecules was presented by Chynoweth and Mah (10) who found that radioactively labeled acetate was converted to butyrate in sludge. Conversion of acetate to methane in lake sediments has been studied by Winfrey et al. (33) and Cappenberg and Prins (7). Winfrey et al. (33) found that [2 l”Cl-acetate added to sediment was quickly converted to methane and carbon dioxide. They did not mention their label re— covery in these products, so I have extrapolated these data from their Fig. 4D and calculated that after 12 h of incubation 15% of the label was converted to methane and 9.4% was converted to carbon dioxide. The amount of label left in acetate or converted to other products should have been determined. Cappenberg and Prins (7) used the same approach as Smith and Mah (27) to determine the turnover rate for acetate and the total rate at which methane was produced in the sediment. A difference was that Cappenberg and Prins (7) made corrections for the conversion of luCO2 to methane. The rate at which acetate was converted to methane by the sediment microflora was estimated to be 70% of the total methane pro- duction rate. I believe the estimate that approximately 70% of the methane produced in sediment and sludge comes from acetate (7,27) is not valid and is probably too high. Both Smith and Mah (27) and Cappenberg and Prins (7) employed a manometric method to determine the total rate of methane production. Smith and Mah (27) used a 100 ml Warburg vessel 11 with 50 ml of sludge and an incubation temperature of 35 C. Cappenberg and Prins (7) used a 50 ml Warburg vessel and 3-4 g wet mud (3-4 ml?) at 10 C. The percent of the initial dissolved hydrogen transferred from the aqueous phase to the gaseous phase in these experimental vessels can be calculated from the equation of Flett et al. (12) 31 = ———1——- x100 [1] M (1+aA/B) where X = volume of gas in the vapor phase, M = volume of gas in the aqueous phase, a = the Bunsen absorption coefficient at the incubation temperature, A volume of the aqueous phase and B = volume of the vapor phase. I have used this equation to generate the 10 C and 35 C curves plotted in Fig. 2, and have designated the points where the values for the two experimental systems would be. Greater than 98% of the initial dissolved hydrogen in both systems should have been transferred to the headspace. This calculation means that the total methane production determined by Smith and Mah (27) and Cappenberg and Prins (7) does not include H2 conversion to methane since less than 2% of the hydrogen concentration (original plus any H2 produced during the incubation period) remains in the sediment. The revelation that acetate may not contribute 70% of the methane carbon is extremely important for understanding anaerobic fermentations. With the new interest in replacement of fossil fuels with renewable energy sources (24), such an understanding is important to the design of systems for the optimum conversion of solid wastes (paper) and plant materials to methane. I suggest that a re-evaluation of the role of acetate and hydrogen in the production of methane in natural and man- made habitats is necessary. Figure 2. 12 Effect of the fraction of the total volume occupied by the aqueous phase on the transfer of dissolved hydrogen to the vapor phase for incubation temperatures of 10 and 35 C. Arrows denote the percent aqueous phase in the incubation systems of Smith and Mah (27) and Cappenberg and Prins (7). Curves calculated from the equation of Flett et a1. (12). 13 was: mos; E ammmmumzqfi A: 3383 s cc. _ on on mm o q Tim. £55 2:. 9.2535 All mom. :32 23 £25 \o.o_ 0 com mm on 2. oo. 3SVHd SflOSflOV % 14 From my review of the literature, the information on lake sediment methanogenesis was largely introductory and descriptive. Kinetic parameters are central to understanding flow and mechanisms in mixed culture systems and no such information for both hydrogen and acetate existed. Previous studies were typified by lengthy incubations at substrate concentrations far greater than natural levels, and sometimes at temperatures far above ambient, thus confounding the interpretation due to de_ngyg synthesis or to other effects of an altered assay environ- ment. Thus, an overall goal of my thesis research has been to introduce a greater level of sophistication into studies of sediment methano- genesis in order to understand the critical parameters controlling the process in nature. Interspecies H,1 Transfer The energy—yielding oxidation reactions of fermentative bacteria generate electrons in the form of reduced nicotinamide adenine dinucleo- tide (NADH). Since further fermentation can continue only if oxidation of NADH occurs, disposition of these electrons is necessary. In pure cultures, fermentation intermediates, such as pyruvate or electron acceptors derived from pyruvate, are usually used as electron sinks for this disposal, but an alternative for many bacteria is the formation of molecular hydrogen (13). But, the formation of H from NADH is not 2 thermodynamically favorable, with an apparent equilibrum constant of 6.7x10"1+ at standard conditions of 1 atmosphere pressure for H2 (35). This reaction does become favorable, however, when the partial pressure of hydrogen is below 10"3 to 10-" atmospheres. The concept of inter- species hydrogen transfer is based on the maintenance of low partial 15 pressures of hydrogen by H2 consuming microorganisms, such as the methanogenic bacteria (35). An hypothetical example of the effects of interspecies hydrogen transfer is presented in Fig. 3. This figure is adapted from Thauer, et a1. (29), who constructed it using the data of Ianotti et al. (16) with Vibrio succinogenes as the H -consuming organ- 2 ism. I have replaced this organism with a H2-consuming methanogenic bacterium. In mono-culture Ruminococcus albus produces appreciable amounts of ethanol in order to oxidize NADH (Fig. 3). This deprives the micro- organism of ATP regeneration via acetyl CoA. In co-culture with a methanogenic bacterium all of the NADH is used to produce H2, which the methanogen keeps at a low partial pressure by reducing carbon dioxide to methane. When compared to the uncoupled fermentation, the coupled fermentation is characterized by: 1. an increase in substrate utilization (not evident in Fig. 3, but see Reddy et al. (22)) 2. a decrease in reduced fermentation products (ethanol) 3. an increase in oxidized fermentation products (acetate) 4. an increase in hydrogen produced as methane 5. an increase in energy production (3, glbg§_3.3 to 4.0 ATP, methanogen ? ATP) and thus enhanced growth of the interacting bacteria. (35) A summary of reported examples of pure and mixed culture studies in which interspecies H2 transfer has been shown is presented in Table 1. Research reported in this dissertation In the preceeding review, I have stressed the importance of the production of hydrogen and its subsequent utilization for methane Figure 3. 16 Comparison of the products formed and energy generated in the fermentation of glucose by Ruminococcus albus grown in mono—culture and in mixed culture with a methanogen. 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My hypo- thesis has been that interspecies hydrogen transfer is operative in lake sediments and of importance to the methanogenic and heterotrophic hydrogen-producing bacteria, and thus to the carbon and electron cycle of the lake. My first objective was to determine if hydrogen consuming methano- genic bacteria were among the predominant methane formers. I used the autecological tool, the fluorescent antibody technique (25), to in- vestigate this objective (Chapter II). My second objective was to establish not only that hydrogen was a rate limiting substrate for methanogenesis, but that the methanogenic population was capable of maintaining the low partial pressures of hydrogen that are a necessary prerequisite for interspecies hydrogen transfer. A Michaelis-Menten kinetic analysis was selected (Chapter III), since the Michaelis-Menten constant, or Km, is an indication of the affinity of an enzyme for its substrate. Also needed was infor- mation on the natural rates of substrate utilization, substrate pool sizes and turnover times. An important part of this objective was to design an incubation system that was not easily contaminated by air, that gave a large substrate reservoir so that substrate concentration would not greatly change with time, and that maximized phase transfer of gaseous substrates and products. My third objective was to determine the effects of hydrogen on various heterotrophic metabolic processes. Previous investigators have made the errors noted above because of their failure to perform experi- mentation with both of the prominent substrates involved in methane production. My approach was to use 1nC-labeled compounds in order to 20 study both the products and substrates involved (Chapter III). Acetate was selected not only because of its implied importance to methano— genesis, but also because its production is expected to increase if interspecies hydrogen transfer occurs. Valine was selected because of the possibility that the Stickland reaction, or the coupled oxidation of amino acids like valine with the reduction of amino acids like glycine and proline could be coupled with methane production instead. 10. LITERATURE CITED Barker, H.A. 1956. Bacterial fermentations. Wiley. New York. Bryant, M.P. 1976. The microbiology of anaerobic degradation and methanogenesis with special reference to sewage, p. 107-117. IE_H.L. Schlegel and J. Barnes (eds.) Microbiol energy conversion. Goltze, Gottingen. Bryant, M.P., E.A. WOlin, M.J. Wblin, and R.S. Wolfe. 1967. Methanobacillus omelianskii, a symbiotic association of two Bryant, M.P., L.L. Campbell, C.A.Reddy, and M.R. Crabill. 1977. Growth of desulfovibrio in lactate or ethanol media low in sulfate in association with H -utilizing methanogenic bacteria. Appl. Environ. Microbiol. 33:1162-1169. Cappenberg, T.E. 1974. Interrelationships between sulfate- reducing and methane-producing bacteria in bottom deposits of a freshwater lake. I. Field observations. Antonie van Leeuwenhoek J. Microbiol. Serology 495285-295. Cappenberg, T.E. 1974. Interrelationships between sulfate- reducing and methane-producing bacteria in bottom deposits of a freshwate lake. II. Inhibition experiments. Antonie van Leeuwenhoek J. Microbiol. Serol. 493297-306. Cappenberg, T.E. and R.A. Prins. 1974. Interrelationships between sulfate-reducing and methane-producing bacteria in bpttom deposits of a freshwater lake. III. Experiments with C labeled substrates. Antonie van Leeuwenhoek J. Microbiol. Serol. 495457-469. Carroll, E.J., and R.E. Hungate. 1955. Formate dissimilationi and methane production in bovine rumen contents. Arch. Biochem. Biophys. 56:525-536. Chung, K.-T. 1976. Inhibitory effects of H on growth of Clostridium cellobiopgrum. Appl. Environ. Microbiol. 313342-348. Chynoweth, D.P. and R.A. Mah. 1971. Volatile acid formation in sludge digestion. IE_F.G. Pohland (ed.) Anaerobic biological treatment processes, Advances in Chemistry Series 105. American Chemical Society, Washington, D.C. 21 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 22 Ferry, J.G., and R.S. Wolfe. 1976. Anaerobic degradation of benzoate to methane by a microbial consortium. Arch. Microbiol. 107:33-40. Flett, R.J., R.D. Hamilton, and N.E.R. Campbell. 1976. Aquatic acetylene-reduction techniques: Solutions to several problems. Can. J. Microbiol. ggf43-Sl. Gray, C.T. and H. Gest. 1965. Biological formation of molecular hydrogen. Science 148:186-192. Hungate, R.E. 1975. The rumen microbial ecosystem. Ann. Rev. Ecol. Systematics. 6:39-66. Hungate, R.E., W. Smith, T. Bauchop, I. Yu, and J.C. Rabinowitz. 1970. Formate as an intermediate in the bovine rumen fermentation. J. Bacteriol. 102:389-397. Ianotti, E.L., D. Kafkewitz, M.J. Wolin and M.P. Bryant. Glucose fermentation products of Ruminococcus albus grown in continuous culture with Vibrio succinggenes: Changes caused by interspecies transfer at H2. J. Bacteriol. 114:1231-1240. Jeris, J ., and P.L. McCarty. 1965. The biochemistry of methane using C tracers. Water Poll. Control Fed. J. 31fl78-192. Mylroie, R.L., and R.E. Hungate. 1954. Experiments on the methane bacteria in sludge. Can. J. Microbiol. 1:55-64. Patel, G.B., L.A. Roth, L. vanden Berg, and D.S. Clark. 1976. Characterization of a strain of Methanobacterium hungatii. Can. J. Microbiol. 22:1404-1410. Pfenning, N., and H. Biebl. 1976. Desulfuromonas acetoxidans gen. nov. and sp. nov., a new anaerobic, sulfur-reducing, acetate-oxidizing bacterium. Arch. Microbiol. 110:3-12. Pretorius, W.A. 1972. The effect of formate on the growth of acetate utilizing bacteria. Water Res. 6:1213—1217. Reddy, C.A., M.P. Bryant, and M.J. Wolin. 1972. Characteristics of S organism isolated from Methanobacillus omelianskii. J. Bacteriol. 109:539-545. Scheifinger, C.C., B. Linehan, and M.J. Wolin. 1975. H2 production by Selenomonas ruminantium in the absence and presence of methanogenic bacteria. Appl. Microbiol. 29:480—483. Schlegel, H.L., and J. Barnea (ed). 1976. Microbiol energy conversion. Goltze, Gottingen. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 23 Schmidt, E.L., R.0. Bankole, and 8.3. Bohlool. 1968. Fluorescent antibody approach to the study of rhizobia in soil. J. Bacteriol. 95:1987-1992. Shea, T.G., W.A. Pretorius, R.D. Cole, and E.A. Pearson. 1968. Kinetics of hydrogen assimilation in the methane fermentation. Water Research. 2:833-848. Smith, P.H., and R.A. Mah. 1966. Kinetics of acetate metabolism during sludge digestion. Appl. Microbiol. 14:368-371. Stadtman, T.C. 1967. Methane fermentation. Ann. Rev. Microbiol. 21:121-142. Thauer, R.K., K. Jungermann, and K. Decker. 1977. Energy conservation in chemotrophic bacteria. Bacterial. Rev. 41:100-180. van den Berg, L., G.B. Patel, D.S. Clark, and C.P. Lentz. 1976. Factors affecting methane formation from acetic acid by enriched methanogenic cultures. Can. J. Microbiol. 22:1312—1319. Weimer, P.J. and J.G. Zeikus. 1977. Fermentation of cellulose and cellobiose by Clostridium thermocellum in the absence and presence of Methanobacterium thermoautotrophicum. Appl. Environ. Microbiol. 33:289-297} Widdel, F., and N. Pfennig. 1977. A new anaerobic, sparing, acetate-oxidizing, sulfate-reducing bacterium, Desulfotomaculum (emend.) acetoxidans. Arch. Microbiol. 112:119-122. Winfrey, M.R., D.R. Nelson, S.C. Klevickis, and J.G. Zeikus. 1977. Association of hydrogen metabolism with methanogenesis in Lake Mendota sediments. Appl. Environ. Microbiol. 33:312-318. Winfrey, M.R. and J.G. Zeikus. 1977. Effect of sulfate on carbon and electron flow during microbial methanogenesis in freshwater sediments. 33:275-281. Wolin, M.J. 1974. Metabolic interactions among intestinal microorganisms. Amer. J. Clin. Nutr. 27:1320-1328. Wolfe, R.S. 1971. Microbial formation of methane. Adv. Microbial Zeikus, J.G., and D.L. Henning. 1975. Methanobacterium arbophilicum sp. nov. An obligate anaerobe isolated from wetwood of living trees. Antonie van Leeuwenhoek. 41:543-552. Zeikus, J.G., and M.R. Winfrey. 1976. Temperature limitation of methanogenesis in aquatic sediments. Appl. Environ. Microbiol. 31:99-107. Zeikus, J.G., P.J. Weimer, D.R. Nelson, and L. Daniels. 1975. Bacterial methanogenesis: Acetate as a methane precursor in pure culture. Arch. Microbiol. 104:129-134. CHAPTER II APPLICATION OF THE FLUORESCENT ANTIBODY TECHNIQUE TO STUDY A METHANOGENIC BACTERIUM IN LAKE SEDIMENTS INTRODUCTION Methods for enumerating methane producing bacteria in anaerobic habitats require anaerobic culture techniques, prolonged incubation times, detection of methane (19, 26) and, in the case of roll tube methods, some way of determining which colonies are responsible for methane production (8, 19). While these methods represent the state-of- the-art for estimating the total methanogenic population, autecological studies of specific species of methanogens are either extremely tedious, or cannot be done at all. The fluorescent antibody (FA) technique, which is one of the most useful methods available for studying microbial autecology (ll, 12, 21), has not been investigated with methane bacteria and has not been used in the anaerobic lake sediment habitat. One purpose of this paper is to report my evaluation of these applications of the FA technique. The second purpose is to compare two methods of enumeration - a fluorescent antibody direct count of a specific hydrogen-consuming methanogen and a most probable number (MPN) estimate of total methanogens in lake sedi- ment. This comparison was made in order to determine if hydrogen— consuming methanogenic bacteria were among the predominant methane farmers in this habitat. 24 25 MATERIALS AND METHODS Media. The enrichment-purification (E?) medium for the methanogen contained the following per liter of distilled water: K2HP04, 0, 0.076 g; 0.225 g; KH2P0u, 0.225 g; NHuCl, 0.185 g; NaCl, 0.45 g; MgCl2-6H2 CaClz-2H20, 0.060 g; trace elements solution (24), 10 ml; vitamin solution (24), 10 ml; 0.01% (w/v) resazurin solution, 1.0 m1; C02 25 ml; cysteine-HCl, 0.5 g; Na2S-9H20, 0.5 g. The gas atmosphere of all anaerobically prepared culture tubes was equilibrated 8% (w/v) Na2C03, oxygen-free H -CO2 (50:50, v/v). An incubation temperature of 28°C was 2 used for enrichment and isolation. Counts of FA reacting methanogens in the sediemnt were compared with estimates of the total methanogenic population made by the five tube MPN technique (1). Three different MPN media were employed. All had a mineral composition that was identical to that of the EP medium. In addition the following organic substrates were added per liter of medium: trypticase, 2.0 g; yeast extract, 2.0 g; Na formate, 2.0 g; Na acetate, 2.0 g. The three media differed in the reducing agent and/or growth factors that were added, per liter of medium; (i) cysteine-HCl, 1.0 g; (ii) a 24 h culture of Escherichia coli grown aerobically at 37°C in trypic soy broth (Difco), 40 ml; or (iii) Na28-9H20, 0.1 g; Ti(III) citrate (25), 0.6 mM_Ti(III); 2-methyl butyric acid, isovaleric acid, and isobutyric acid, 0.1 mg of each. These reductants were chosen to avoid possible sulfide toxicity which has been noted by Cappenberg (8). A11 reducing agents were added 24 h prior to inoculation with sediment. Preliminary experiments showed that the added E, coli would reduce methyl viologen within this time. The method of preparing 26 Ti(III) citrate (25) was modified such that all solutions were boiled under 02-free nitrogen prior to mixing and all manipulations were done anaerobically. Anaerobic methodolggy, The anaerobic culture and isolation tech- niques used were those described by Hungate (15) with the modifications of either Bryant and Robinson (7) or Macy, et_al, (17). For some purification attempts I used an anaerobic glove chamber (2) (Cay Manu- facturing Co., Ann Arbor, MI) with a gas atmosphere of H2:N2 (10:90, v/v). Petri plate agar (EP medium plus 1.5% agar w/v) streaks were incubated in anaerobic containers within the glove chamber. These containers were similar to those described by Edwards and McBride (10) and had a gas atmosphere of H :CO2 (50:50, v/v). Exposure of the plates 2 to the glove box atmosphere occurred only during inspection for growth and culture transfer to fresh medium. Antigen preparation. The methanogenic culture that I used as the antigen in the preparation of fluorescent antibody (FA) was isolated from a dilution series of EP medium that had been inoculated with lake sediment. The sediment was collected by a gravity carer from the 6 m depth of Wintergreen Lake (Kalamazoo County, Michigan, R 9W, T 2N, Sec 8) in July. The medium was inoculated from a ten-fold dilution series of a homogeneous mixture of the upper 5 cm of the sediment care. All atempts to obtain an axenic methanogenic culture were unsuccess- ful. However, contaminant levels could be kept at a minimum (less than 1% of the presumptive methanogen) by keeping the headspace H concen- 2 trations high and by transferring the culture to fresh medium (lo-fold dilution) every 2 days. Cells to be used as antigen were grown in 200 ml of EP medium, harvested by centrifugation and killed by suspending in 4% (v/v) formalin in 0.85% (w/v) sterile saline for 72 h. 27 Antibody preparation. The antigen suspension was injected into young adult male rabbits according to the schedule described by Schmidt (22). Blood was collected every two weeks from a puncture of the marginal vein of the ear. At each bleeding 30 ml of blood was collected in an equal volume of anticoagulant solution. Purification of the serum antibody and conjugation with fluorescein isothiocyanate (FITC) followed the precedure of Schmidt (22). The FITC to protein ratio was 1/50 (w/w) and protein was determined by the method of Lowry (16) with bovine serum albumin (BSA) as the standard. Excess FITC was removed from conjugated PA by passage through a Sephadex G-25 column that had been equilibrated with 0.12 M_phosphate buffered saline, pH 7.2. The FA was collected in 5 m1 portions, preserved with 0.01% thimerosal (w/v, Sigma Chemical Co.) and frozen until needed. FA stain of cultures and natural samples. The microscope slide staining procedure of Schmidt (22) was used to stain heat fixed cultures or natural samples. Nan-specific absorption of the PA was controlled by the prior addition of either rhodamine-conjugated gelatin (5) or 2% BSA. The FA stained smears were observed under a Leitz Ortholux microscope with incident ultraviolet (uv) light illumination. A 150 W Xenon lamp was used as the uv light source. The excitation filters were two KP490. The dichroic beam splitting mirror was a TK510 and the barrier filters were a K515 and a K510. Two thicknesses of Kodak Wratten gelatin filter No. 12 were added to the barrier filter assembly when photomicrOgraphs were taken. A modification of the soil procedure of Bohlool and Schmidt (6) was used to obtain direct counts of FA reacting bacteria in lake sediments. Ten ml of sediment, 90 m1 of filter sterilized distilled water and 0.1 m1 Tween 80 were mixed for 5 min in a blender. A flocculent (0.5 g 28 Ca(0H)2, 1.25 g MgCO dried overnight at 90 C) was added to this dis- 3! persed sediment which was then vigorously shaken by hand for 2 additional minutes. Two drops of Antifoam B emulsion (Sigma Chemical Co.) were added during the last 10 sec of shaking. The floc was allowed to settle, undisturbed, for 15 min. The supernatant liquid was then carefully removed by aspiration and a sample or, when necessary, dilu— tion of the supernatant fluid was immediatley filtered through a black Sartorius 25 mm diameter membrane filter, 0.45 um pore diameter. The procedure of Bohlool and Schmidt (6) as modified by Schmidt (2) was used to stain the bacteria on the filters with FA, except that 2% BSA was used to control non-specific absorption. Stained bacteria were observed under the Leitz microscope with incident uv illumination as described above. Since the number of FA reactingbacteria per microscope field followed a Poisson distribution, these data were normalized with a square root transformation so that 95% confidence limits could be calculated. The bacteria on a minimum of three membrane filters were stained with FA on each sampling date and 100 microscope fields per filter were counted. The number of FA react- ing organisms per gram dry weight of sediment was calculated using the equation of Bohlool and Schmidt (6). Since microorganisms are removed by flocculation (21) I attempted to correct for this by determining the percentage of total sediment bacteria lost during this step. Total bacteria present before and after flocculation were determined by the acridine orange-epifluorescence, direct counting method of Daley and Hobbie (9). Only green fluorescing bacteria were counted. 29 For comparison with the FA direct counts, five tube MPN estimates of the sediment methanogenic bacterial population were made using the three MPN media described above. A lO-fold serial dilution of the sediment was made in EP medium with l min of vortex mixing between each dilution. MPN tubes were inoculated from this dilution series. The tubes were incubated for 4 weeks at 37°C. At the end of this time, each tube was analyzed for methane production as determined by flame ioniza- tion gas chromatography. RESULTS Description of methanogenic organism purified from the sediment. Fig. l is a phase contrast photomicrograph of single cells and a fila- ment of the methanogen. The organism is gram positive and produces methane from hydrogen and carbon dioxide, and from formate. It does not produce methane from acetate or methanol in the absence of hydrogen. The optimum growth temperature is 370C. Chains and filaments are common, often exceeding 60 um in length. Cellular dimensions are 0.5 um in diameter by 3 to 6 um. Surface colonies are pale yellow to tan and flat. These colonies fluoresce blue-green when exposed to long wave uv light. This fluorescence is characteristic of methanogens and is due to oxidized factor 420, a compound which has only been found in methano- gens (10). Individual cells of an actively growing culture exhibited a faint autofluorescence when observed microscopically under incident uv illumination. This allowed us to be certain that the morphotype react- ing with the FA was a methanogen. 30 Tests an antibody prepared with the methanpgep, Agglutination titers of serum collected from injected rabbits never exceeded 800. Although this value is low, Fig. 2 shows that FA prepared from this serum adequately stained the antigen. An FA stain of a culture in which the contaminants were deliberately allowed to proliferate showed that the contaminants did not react with the FA. Table 1 lists the control tests which Schmidt (20) states are necessary for the application of FA. My results from these tests are listed in the right hand column. Several tests were run to satisfy the requirement that the FA should stain antigen cells grown under various conditions. The FA reactions of the methanogenic culture at different stages of the growth cycle and after different periods of starvation were all +4. An FA reaction of +4 was arbitrarily defined as the FA reaction of the original antigen preparation. A freshly stained smear of this antigen was used as a reference for each stain. The FA reaction of the methanogen culture grown at different temperatures ranged from +2 for cells exposed to 4°C to +4 for cells grown at 28 and 370C. The morphology of the methanogenic bacteria changed at the different growth temperatures with single cells the predominant morphotype at 4°C and filaments the predominant morphotype at the higher temperatures. Tests on the antibody (Table 1) included staining related and unrelated organisms to test the specificity of the FA preparation. The organisms tested are listed in Table 2. None were stained by the FA. Of particular importance to this study was the finding that no other organism isolated from Wintergreen Lake sediment reacted with the FA. 31 Figure 1. Phase contrast photomicrograph of a short filament and single cells of a methanogenic bacterium isolated from Winter- green Lake sediment and used to prepare the FA. Bar rep- resents 10 um. Figure 2. Photomicrograph of an FA-stained smear of the methanogenic isolate. Bar represents 10 um. 32 33 AN oaame momv mEmHammso oopmaosaa mo swarm saws: azosw maaoo w< wo Casum maaoo w< Iva: wonpomom coma mm: pace «I mSOMOHOEOI zuwz swapm passwoom mxmq coopwsopawz pcoefioam oxmq soosmsouawz mawooam macawpapomm m pcoenowsco wafioaoos nopmmaam m mo pmoEm amasopomn pcoenowsam owaowoamnuae m anm popmaomw moaosomcm m AMA .msospam pcoENoom oxmg coosmpauawz mcwmsum waH>MNsuaoo : maax .h.z paoENQom oxmq cowswsopafl3 monosomcm m>eumuaaomm o madx .w.z paovaom oxmq Goosmsopaaz oaosomcm oumwwaao ponpo a maax .n.I paoefipom axon soosmsoucwz mcwmopm Hmwpwspmoao : mEchmmso caumaosca pam>sm .m.z paaasowhaa oumonaon no :«mspm .omoaam amazon ownosoma< «wwmwwan eaaawswmwoamnpoz maxflom .o.w paoswoom meooaoz axmq .m.m.D mamasmmacmnpoz pammsm .m.z amoaam amazon ownosoam< m2 maHoLMmocmcpaz pouchmm .m.w.z uaoEwoom gnome uamm .am anpapomaoamnpoz waxwoN .m.w mommy mo noosuox anfiawnmaasm um uaoecowsao opmouaoa Iago» .m.n .mmpaaw amazon ownosomc< In cashew eaowoflahom am pammsm .N.I spasm Hz awmspm eawpamaweas um pammpm .m.z mmoaam amazon owoosoma< mm awaspm eawpamaweas.dm “spasm .m.z was ham oowwocmsm com .I.o.z camera aaflsouomaoamzuoz mamwcmmno popmaom nowanmspaoo opmaomw mo monaom Emwamwmo .vo>somoo nowvomosummoso as mcoflumnmaopa 9w>flwomosnmmoso pom powwow mamwamwsoosoflz .N canoe 36 Figure 3. Photomicrographs of immunofluorescing cells in FA stained smears of various habitats. (a) Wintergreen Lake pelagic sediment. (b) Sludge from an anaerobic sewage digestor. (c) Bovine rumen fluid. Bars represent 10 um. 37 38 oocwesopoo poz u oz A Hm>houaw monopwmcoo n .H.om Assns.mv we Adm-s.mv Ha A:.o-m.ov o.m Aosmuommv oam same so: ma Qz Aonauva mo Analm.Nv m.o Aomonommv 0mm ohma woo ca 92 az Ammns.ov :N Aommnoomv ONm osma mow m Q2 nmz ANNnm.mv 0H Aoomanoomav oosa osma can mN A.H.o wmmv powwoaoos A.H.o wmmv pcmuoamwm A.H.o wmmv pamuoaoos Am.H.o wmmv uaaoo cusp AHHHvHB «Hoe .m ocfiopmmo pooswo wpflmoa mm posoom one: moody 2m: .mwpoe econommwo mossy moans madficnoop zmz snap o>wm a ma cam muaaoo Hoopwo 950 N 0000 0000.0 00.0 000.0 00.0 00.0-0 mmmnmopa 00.0 0 0>g0 N 0000 0000.0 00.0 000.0 00.0 00.0-0 mmmnwoam 00.0 00 00 002 00 00: 00 000.0 00.0 000.0 00.0 00 0>0HSU N 0000 0000.0 00.0 000.0 00.0 0.0-0 00000000 00.0 0 00 00: 00 em: 00 000.0 00.0 000.0 00.0 0:0 00 000.0 00.0 000.0 00.0 00.0 0 .00 > 00.0-00.0 0: 00 an: 00 00: 00 000.0 00.0 000.0 00.0 000 00 0000.0 00.0 000.0 00.0 00.0 0 .0> > 00.0-00.0 00 00 00: 00 00: 00 000.0 00.0 000.0 00.0 000 00 0000.0 00.0 000.0 00.0 00.0 0 .0> > 00.0-00.0 00 00 00: 00 00: 00 0>0H5U N 0000 0000.0 00.0 000.0 00.0 0-0 00000000 00.0.00.0 : ”>950 N 000 0000.0 00.0 000.0 00.0 0-0 mmwemoea 00.0.00.0 0 00 000 00 am: 0 A ”>30 0 000 0000.0 00.0 000.0 00.0 0-0 mumpmopa 00.0 0 AH Q>§U N 0000 0000.0 00.0 000.0 00.0 0-0 mmupmopa 00.0 m 00 000 00 000 00 000 000.0 000.0 000.0 00.0 00.0 0 .00 > 00.0 00.0 000 00 poo 00 000 00 x N In an .n A x x I D. I S a a m 0 .. 0 0 0 m m 0 w s 0. 0 0 .0. C U. \l J..- T. X E D. m. 8 an .4 S 8 8 O U. S S r T. 1 3 A p ( a H H D. O O D.— ..“ 0 S O m HIV 7v .flu % 1. T3 T3 T P. TL 3 T: e n u . m t. e 1 T. D. x s I a 00 a nu o x. u.nu u a a t. as e d o .T. 70 u I. 370 E o m X P J u T. u D. T. C 1+ 9 d o S p 1* a p 3 K J a u a 0. .00 0. 0. 0. ,0 0 0 d p .. 0. D a S a a D. D D. J M m a 3 p O 3 3 0 D. a S p T.. \.I a a u p S u 1.. o O A U; X a n .4 m h.- p a D— D. 10 1 T... u. s u. a . w m u... u T. 1. 60 Figure 2. Effect of increasing hydrogen concentration, [8], on the rate of sediment methane production, v. The rates have been corrected for the control (no hydrogen added). Incubation time was 1.25 h at 10 C. Sediment was collected in the early winter. 61 $260333... 0. «I a: Hm”— O.N m._ 0.. ad - I q q i) $06 0.0 VN .O de Ovd (.-qx.-6x ”HO swam?!“ 62 Figure 3. Direct linear plot of the substrate, velocity data pairs plotted in Figure 2. 63 .5. 21.000 0.0 O 0.0.- 0.7 0._l O.NI -ON.O , M I" w m . m x o I . \ . H 9.0 \ \ .7 \\ s X . x .3 \\ T.E.-outta ao_oein¢.o-xoe> 02.002 x . 0: 0000.0 . .5. 5.0.: 0.. 50.0 64 the ratio of hydrogen consumed to methane produced increased as the hydrogen concentration in the headspace decreased (Table 2). Other hydrogen donors and the stimulation of methanogenesis. Table 3 shows a survey of the effect of various potential hydrogen donors on the rate of methanogenesis. Since only formate immediately stimulated methanogenesis, I attempted to determine the kinetic para- meters for the conversion of this substrate to methane (Fig. 5). In this experiment, the response approaches a hyperbolic relationship. However, assuming that four moles of formate are needed to produce one mole of methane, at the lowest concentration of 1.16 umol formate'g-l, 87% of the formate was converted to methane within the l h incubation period. This percentage does not include other non—methane fates of formate. In an attempt to achieve a more valid temporal resolution, progress curves at several formate concentrations and with 2 min sam- pling intervals were employed. The experiment was, of necessity, per- formed at room temperature (23 C) next to the gas chromatograph. The rates of methane production, calculated for the 10 min sampling time (Table 4), still indicated that at least 47% of the formate had been consumed within this short time. At sampling times under 10 min the rates were variable but suggested that maximum velocities were reached somewhere between 260 and 400 nmol formate'g"l sediment . Km values could not be estimated. Kinetics of acetate conversion to methane. The addition of between 1.23 to 12.1 umol acetate'g-l sediment did not stimulate the rate of sediment methanogenesis beyond the rate of the control with no addition (Fig. 6). This rate, calculated from the slope of the linear portion of the lines plotted in Fig. 6 averaged 0.307 umol CH4 produced 'gnl'h-l Figure 4. 65 Progress curves of the headspace hydrogen and methane con— centrations with time. Methane concentrations have been corrected for the control which had no hydrogen added. Incubation temperature was 12 C. Headspace (vapor) volume was about 65 ml. Sediment was collected in late winter. 66 :uewgpes .-6 x ”HO SO|OUJ 1’ N O Q ‘9. "I 9! _: ...: O O O O r I I U I I 4 . I e V I O L... I 1 J l l N O a) (D d’ N luetugpes .-6 x 3H sa|ow 1’ Time (hours) 67 Table 2. Ratio of hydrogen consumed to methane produced with decreasing hydrogen concentration calculated from the progress curves of Fig. 4. Time H2 in H2 consumed (h) headspace CH produced A (96) 0.00 0.50 --— 0.75 0.43 4.1 1.50 0.33 5.7 2.25 0.24 7.2 3.00 0.18 8.1 3.75 0.12 8.1 4.50 0.09 8.8 68 - o.© omm-o: mcflosmq - 0.3m n o.m ommlo: maflam> - 0.3m - o.m ommno: mpmcowaopm n 0.3m - 0.9 ommuo: mpmpomq +++ o.a oooanooa +++ mm.o oma-mm momegou A- no +v Anv Aana.mmHoEnv mEHu owcmg COMPMHUEwwm cofipmASUCH cowpmgpcmocoo mpmhpmosm .0 mm mm: myopmpmanp coflpmnsozH .mwmwcowocwzpoe pcmeflwmm mo mymm mLp co mQOCOU somoavmz Hmwpcovoa mo pomwmm .m manmh Figure 5. 69 Effect of dissolved formate concentration on the rate of sediment methanogenesis. Methane production rates have been corrected for the rate of the control, which received an identical volume of oxygen free water but no formate. Incubation time was 1 h at l2 C. Sediment was collected in mid winter. 70 VIC 0.6 - TE .70 .510 3.9:: nmoles formate - g“ 71 (t 4% s,n = 11). This lack of stimulation prompted me to attempt short term progress curves at lower concentrations and with methane measure— ments every 2 min. The results for the 10 min sampling time are presen- ted in Table 4. Even under these conditions I could not detect signifi- cant acetate stimulation of methanogenesis. In order to determine if the lack of acetate stimulation was due to the absence of acetate utilizing methanogens, I monitored the production of radioactively labeled methane and CO from [2—luC] acetate with time 2 (Fig. 7). Also plotted is the production of labeled methane by sediment that had been preincubated with 0.5% H for 15 min prior to the addition 2 of labeled acetate. In both the presence and absence of added H2, acetate was rapidly converted to methane, though the addition of H2 caused an immediate conversion. Because labeled methane appeared so rapidly after the addition of labeled acetate I ran kinetic experiments that were similar in approach to the uptake kinetic experiments described by Wright and Hobbie (33). A different amount of unlabeled acetate was added to each tube of sediment. Each tube then received the same amount (1 uCi) of [2-luC] labeled acetate. After an incubation time of 10 to 15 min the amount of label in the headspace methane was determined. This radioactivity was used to calculate the term t/f of Hobbie and Crawford (17), which was plotted against the amount of labeled plus unlabeled acetate added to each tube. This plot is equivalent to a kinetic plot of s/v versus 8. The results of this experiment, for the two cases of plus and minus pre- incubation with 0.5% H2, are presented in Fig. 8. The kinetic param- eters estimated from this plot and a similar experiment are presented in Table 5. 72 Table 4. Effect of dissolved formate and dissolved acetate concentrations on the rate of sediment methanogenesis. Incubation time was 10 min at 23 C. Sediment was collected in the late winter. Rate of methane formation Substrate Formate -l Acetatel -1 concentration (nmol CHu°g 'h ) (nmol CHu'g 'h ) (nmol ' g ) 0 220 250 130 340 170 270 520 330 400 776 280 530 850 300 660 ND* 310 800 950 330 *ND- not determined 73 Figure 6. Amount of methane produced by sediment with time at different concentrations of added dissolved acetate. Incubation temperature was 12 C. Sediment was collected in mid winter. l6 7n nmoles acetate-g" o 7.3 . 4.9 l2 0 3.7 T o 2.4 U. . :V/ . l2 3? //' 0.0 0 A 0' a”. 8 3/ ‘e’ //,:/ 3. . / 4 O 1 4 1 1 J 1 1 J 0 l2 24 36 48 Time (hours) 75 Since all results for acetate conversion to methane now indicated that the natural rate of conversion was at or very close to the Vmax’ a second type of labeled experiment was run, where each tube received the same specific activity of acetate but a different total acetate concen- tration. A plot of the amount of radioactive methane produced versus the acetate concentration should be in the form of a Michaelis—Menten rectangular hyperbola. The results of this experiment (Fig. 9) confirm that the rate of acetate conversion to methane is zero order with respect to acetate concentration. Since the addition of hydrogen had a pronounced effect on the on lLiCO production conversion of acetate to methane, the effect of H2 2 from [U-luC] valine, a potential H donor (Table 2) was examined. Four 2 tubes, each containing l0 ml sediment, were incubated at 12 C for 15 min prior to the addition of l uCi labeled valine. Hydrogen (0.5% v/v) was added to two of the tubes at the start of the preincubation period. The 4 . . . amount of 1 CO2 present in the headspace gas was determined 15 min after the addition of the label. In the absence of hydrogen, 6.3 x 104 dpm 4 . . 1 CO was detected 1D the headspace. The sediment exposed to 0.5% 2 4 l4 . 0 hydrogen produced only 4.0 x l0 dpm C02. ThlS represents a 376 decrease in the production of labeled carbon dioxide from uniformly labeled valine. Figure 7. 76 Production of labeled methane and carbon dioxide from [2-luC] acetate (1 uCi). O — labeled methane produced in the absence of hydrogen. 0 - labeled carbon dioxide produced in the absence of hydrogen. A — labeled methane produced in the presence of 0.5% hydrogen. Hydrogen was added 15 min prior to the addition of labeled acetate. Incubation temperature was 12 C. Sediment was collected in June. 77 4.0 _ O 30 - A .- '4CH4 I?“ \ Q o .. *“2 NoH ‘U 8 -_-, 2.0 - 3. O. U | o . ”002 o/. O 1 1 4 1 0 IO 20 Time (minutes) Figure 8. 78 Effect of acetate concentration and hydrogen on the conversion of [2-luC] acetate to methane by sediment. 0 - labeled methane produced within 15 min in the absence of 0.5% hydrogen. 0 - labeled methane produced within l0 min in the presence of 0.5% hydrogen. The hydrogen was added 15 min prior to the acetate addition. Incubation temperature was 12 C. Sediment was collected in June. 79 NO :66888 3.063 < who NO no Nd _ .0 0 mad u N ._ . o o 0 bad u N. (smuun 80 Table 5. Kinetic parameters estimated from the regression of t/f versus A, as in Fig. 8. Experiment l-run within 4 days of collecting sediment in early July. Experiment 2-run within 2 weeks of collecting sediment in late June. Preincubation K + S V T m n -1 maxl —l t with 0.5% H2 (umol acetate°g ) (umol CHu'g 'h ) (h) Expt. 1 - 0.281 0.181 1.56 + 0.177 0.181 0.98 Bxpt. 2 - 0.258 0.145 1.78 + 0.165 0.189 0.91 81 . . . . 14 Figure 9. Effect of acetate concentration on the production of CH4 from [2—1HC] acetate by lake sediments. Each acetate con centration had the same specific activity of labeled acetate. Incubation time was 15 min at 12 C. Sediment was collected in June. 82 (.-U!w 9| ~9-0I x wdp) 17H3.” 0.6 0.4 Acetate (nmoles - g") 83 DISCUSSION The Km estimates for the conversion of hydrogen to methane (Table 1) demonstrate that, if present in sufficient numbers, the methanogenic bacteria are capable of maintaining low concentrations of hydrogen in the sediment. Evidence that the methanogenic population is sufficient is indicated by the progress curve (Fig. 4) where moderately high initial hydrogen levels were decreased by 83% within 4.5 h. This rapid rate of consumption suggests that hydrogen limits methanogenesis and that hydrogen concentrations that may inhibit heterotrophic hydrogen pro- duction are quickly lowered to more favorable concentrations. In spite of the heterogeneity of sediments, I was able to obtain fairly reproduc— ible Km estimates among experiments employing different methods of analysis and with sediments collected on different dates (Table 1). These estimates must be considered the upper limit since transfer of hydrogen from the vapor phase to the aqueous sediment phase may still be rate limiting despite the design to optimize the transfer. The mean Km for hydrogen consumption for the January and February sediment was 0.013 umol‘g"l (or 1.6 umole ' 1.1 wet sediment) and for the March sediment it was 0.029 umole'g-l (or 3.5 umole'fl wet sediment). This compares favorably to the Km value of 1 umole'l—l determined by Hungate et al. (16) for bovine rumen fluid and for Methanobacterium ruminantium. The mean Km for methane production (Table l) was 0.023 umol'g-l which does not vary significantly from the one for H consumption. 2 The Vmax for both the consumption of hydrogen and for the conver— sion of hydrogen to methane generally increased from December to March (Table 1). Since Vmax is proportional to enzyme concentration this finding suggests that the population or activity of hydrogen consumers 84 and/or methane producers increased slightly throughout the Winter. The in_§i£2_rate of hydrogen consumption can be calculated from the in_§itg_hydrogen concentration, which was 6 to 10 nmol'g-l, and the Km and Vmax (Table 1) according to equation [1]. The range in estimated . . . . — —1 natural veloc1t1es of H consumption was 430 to 1530 nmol H2 g 1'h 2 and for H2 conversion to methane it was 72 to 290 nmol CHu°g-l'h-l. The non—methanogenic utilization of hydrogen by sediment microflora indicated in Table 2 has also been suggested by Winfrey et al. (29). A possible explanation is that the H2 (and C02) are also being converted to acetate by an organism similar to the Clostridium isolated by Ohwaki and Hungate (20) from sewage sludge. A second possibility is that, with short term incubations, hydrogen consumption may be due to heterotrophic bacteria and the reaction H2 + NAD+ + NADH + H+. However, this reaction will be limited by the availability of organic electron acceptors, which are needed to recycle the catalytic amounts of NAD+. The potential hydrogen donors listed in Table 3 were selected for the following reasons. Formate was chosen because some methanogens can use it directly (31) and because it can be converted to hydrogen and carbon dioxide by a variety of heterotrophs (ll). Lactate was selected because many heterotrophs can ferment it (11) and because Bryant et al. (3) have shown that with lactate, and in the absence of sulfate, inter— species hydrogen transfer occurs between Desulfovibrio and Methanobact--8 erigm_strain MoH. Propionate was selected as a representative volatile fatty acid because Bryant (2) has hypothesized that there is an aceto— genic population in anaerobic habitats that can convert these compounds and alcohols to acetate and hydrogen. The amino acids valine and leucine were selected because Molongoski and Klug (19) have shown that proteo— 85 lytic clostridia are the predominant isolatable heterotrophs in Winter— green Lake sediments. The lack of significant stimulation of methane production by all but formate (Table 2) may indicate that either these substrates are unimportant hydrogen donors, or that they are already being turned over at maximum rates. Although kinetic constants could not be estimated for the conver— sion of formate to methane, the almost immediate stimulation by low levels of formate (Table 4) suggests that this substrate could also be important in methane production. The rapid stimulation of sediment methanogenesis by formate has also been observed by Winfrey et al. (29). Formate concentrations in Wintergreen Lake sediments have been measured by Molongoski and Klug (unpublished data) and range from 13 to 63 nmol‘g-l during May to October. As with H2, the methanogens may be responsible, in part, for maintaining these low concentrations. During short incubations and at added formate concentrations in excess of 130 nmol'g-l, I have detected only very low concentrations of hydrogen in the headspace gas. Since these hydrogen levels were not high enough to account for the observed stimulation of methanogenesis, I believe that formate may be directly used by the methanogenic bacteria present. This finding is consistent with the fluorescent antibody studies (Chapter II) which showed a formate and H2 utilizing organism to predominate in these sediments. The rate of conversion of acetate to methane appears to be quite close to the Vmax for this reaction (Fig. 9, Table 4), which would explain the apparent lack of stimulation by additions of unlabeled acetate (Fig. 6). Natural concentrations of acetate in these sediments have been determined by Molongoski and Klug (unpublished data) to range 86 from 180-550 nmol'g_l over the summer of 1976. Using the minimum measur- ed acetate pool size and an average turnover time of 1.67 h (Table 5) then an estimate of the minimum natural velocity (equation 6) would be 108 nmol CHL+ produced ‘g-l‘h-l. This rate is 66% of the average Vmax (Table 5) of 163 nmol CH4 produced 'g-l'h-l, which further indicates that acetate is being converted to methane at rates very near the maxi- mum. This discovery implies that the only way for an increase in the rate of acetate conversion to methane to occur would be by an increase in the population of acetate utilizing methanogens. But these bacteria have very slow growth rates (A. Zehnder, personal communication, and 22), so an increase in the rate of heterotrophic acetate production would lead to an increase in the acetate pool size. My estimated minimum natural rate of acetate conversion to methane is approximately equivalent to 17 nmol ' g.1 wet sediment'h-l. Cappen— berg (6) has estimated that acetate accounts for a rate of methane production of about 23 nmol'g_l wet mud'h_l in Lake Vechten sediments. This rate was about 70% of the apparent rate of total sediment methane production. Comparisons between the rates at which methane is formed from H2 and from acetate by Wintergreen Lake sediments can not be made since the rate estimates were for samples collected at very different times of the year. The minimum rate at which hydrogen was converted to methane by winter sediment was about 67% of the rate at which acetate was converted to methane by sediments collected in June. The rate of H2 conversion to methane by June sediments is unknown, but it probably greater than the minimum winter rate. Cappenberg's (6) estimate that 70% of the methane comes from acetate is probably too high because the method he used to determine the 87 total rate of methane production underestimates the actual in_§itg_ratv. Cappenberg used a manometric method which involved placing 3—4 g wet sediment in a 50 ml Warburg vessel (6). I have used the equation of Flett et al. (13) and the Bunsen absorption coefficient for hydrogen at 100C to calculate that, under Cappenberg's experimental conditions, 99.8% of the hydrogen initially dissolved in the sediment would have been transferred to the gas phase at equilibrium. Thus Cappenberg's estimate of the total methane production rate (0.034 umol'g_lwet sediment'h-l) (6) does not include conversion of dissolved hydrogen. Note that this same criticism applies to the results of Smith and Mah (26), who estimated that 70% of the methane produced in sludge came from acetate. For their experimental system I have calculated that 98.4% of the initial dissolved hydrogen was transferred to the gas phase. Methane may not be the only fate of acetate in the sediment. Only 15% of the labeled acetate appeared as methane by the time labeled methane production ceased (Fig. 7). In other experiments (not shown) more of the label was converted to methane, 23% and 32%, but production also terminated at these values regardless of the length of incubation. Additional evidence for another fate of acetate comes from Winfrey et al. (29) in their study of Lake Mendota sediments. I have calculated that only 12% of the methyl labeled acetate that they added was converted to methane. Other possible acetate consuming organisms are the recently described sulfur—reducer, Desulfuromonas acetoxidans (21), and sulfate- reducer, Desulfotomaculum acetoxidans (28). However, only 6% of the labeled acetate I added was converted to carbon dioxide (Fig. 7), which still leaves nearly 80% to an unknown fate. 88 Hydrogen appears to stimulate the conversion of acetate to methane (Fig. 7), an observation also made by Winfrey et al. (29) for Lake Mendota sediments. However, the kinetic experiments (Fig. 8, Table 5) show that the preincubation period with 0.5% hydrogen caused a decrease in the acetate pool, as Km + Sn’ and a concomittant decrease in the acetate turnover time. The apparent stimulation in Fig. 7 is more likely due to a lower acetate pool and thus higher specific activity of the acetate than to an increase in the methanogenic conversion of acetate. This explanation fits with the hypothesis that interspecies hydrogen transfer occurs in the sediment since, in an uncoupled system, less oxidized substrate such as acetate is produced (32). Thus a decrease in the acetate pool would occur if the hydrogen that I added inhibited the heterotrophic production of acetate. Further evidence that interspecies hydrogen transfer occurs in the lake sediment is that preincubation with hydrogen causes a 37% decrease in the amount of radioactive C02 produced from the heterotrophic metab— olism of labeled valine. Under anaerobic conditions, valine is normally metabolized via the Stickland reaction to isobutyrate, with NAD+ being reduced to NADH+(1). The NADH+ is oxidized back to NAD+ by transferring the electrons to an electron accepting amino acid such as glycine or proline. The high hydrogen concentrations that I added to the sediment may have caused most of the NAD+ to be in the reduced form and unable to accept electrons from valine, thus decreasing the production of 1”C02. A second possibility is that the hydrogen had not effect on the Stickland reaction, but instead inhibited the further degradation of isobutyrate, the product of valine degradation via the Stickland reaction. 8 9 Cleland (9) has stated "... it is not practical to have the sub— strate which is an intermediate in a metabolic pathway present in levels much above its apparent Michaelis constant. Maintenance of a stable flow through a metabolic pathway is favored if each enzyme operates in the proportional region of its velocity versus concentration curve, so that a momentary rise in reactant concentration results in an increased reaction rate and tends to prevent further increase in concentration." (underline mine.) My experimental evidence shows that, for the anerobic sediment habitat, short term increases in hydrogen levels lead to increased reaction rates and thus prevention of further increase in dissolved hydrogen levels. However, for acetate, a short term increase in the pool size cannot cause a significant increase in the reaction rate since the rate is already near the maximum. Therefore, further increases in acetate concentration are not prevented. 10. 11. 12. LITERATURE CITED Barker, H.A. 1961. Fermentations of nitrogenous organic compounds. 12_I.C. Gunsalus and R.Y. Stanior (eds.) The Bacteria. Vol. II: Metabolism. Academic Press, New York. Bryant, M.P. 1976. The microbiology of anaerobic degradation and methanogenesis with Special reference to sewage, p. lO7-ll7. In H.L. Schlegel and J. Barnea (eds.) Microbial energy conversion. Caltze, Gottingen. Bryant, M.P., L.L. Campbell, C.A. Reddy, and M.R. Crabill. 1977. 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