Application of the fluorescent-antibody technique to the study of a methanogenic bacterium in lake sediments.

Fluorescent antibody (FA) was prepared for a methanogenic bacterium isolated from Wintergreen Lake pelagic sediment. The isolate resembles Methanobacterium formicicum. The FA did not cross-react with 9 other methanogens, including M. formicicum strains, or 24 heterotrophs, 18 of which had been isolated from Wintergreen Lake sediment. FA-reacting methanogens were detected in heat-fixed smears of several different lake sediments and anaerobic sewage sludge. Pretreatment of all samples with either rhodamine-conjugated geletin or bovine serum albumin adequately controlled nonspecific absorption of the FA. Autofluorescent particles were observed in the sediment samples but, with experience, they could easily be distinguished from FA-reacting bacteria. 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 estimates of the total methanogenic population that was present in the same samples. The FA counts ranged from 3.1 X 10(6) to 1.4 X 10(7)/g of dry sediment. The highest most-probable-number estimates were at least an order ofmagnitude lower.     

Temperature limitation of methanogenesis in aquatic sediments.

Microbial methanogenesis was examined in sediments collected from Lake Mendota, Wisconsin, at water depths of 5, 10, and 18 m. The rate of sediment methanogenesis was shown to vary with respect to sediment site and depth, sampling date, in situ temperature, and number of methanogens. Increased numbers of methanogenic bacteria and rates of methanogenesis correlated with increased sediment temperature during seasonal change. The greatest methanogenic activity was observed for 18-m sediments throughout the sampling year. As compared with shallower sediments, 18-m sediment was removed from oxygenation effects and contained higher amounts of ammonia, carbonate, and methanogenic bacteria, and the population density of methanogens fluctuated less during seasonal change. Rates of methanogenesis in 18-m sediment cores decreased with increasing sediment depth. The optimum temperature, 35 to 42 C, for sediment methanogenesis was considerably higher than the maximum observed in situ temperature of 23 C. The conversion of H2 and [14C]carbonate to [14C]methane displayed the same temperature optimum when these substrates were added to sediments. The predominant methanogenic population had simple nutritional requirements and were metabolically active at 4 to 45 C. Hydrogen oxidizers were the major nutritional type of sediment methanogens; formate and methanol fermentors were present, but acetate fermentors were not observed. Methanobacterium species were most abundant in sediments although Methanosarcina, Methanococcus, and Methanospirillum species were observed in enrichment cultures. A chemolithotropic species of Methanosarcina and Methanobacterium was isolated in pure culture that displayed temperature optima above 30 C and had simple nutritional requirements.     

Characterization of anaerobic heterotrophic bacteria isolated from freshwater lake sediments.

Strict anaerobic culture techniques were used to quantitatively and qualitatively evaluate the anaerobic heterotrophic bacteria present at the sediment-water interface of hyperutrophic Wintergreen Lake (Augusta, Mich.). Anaerobic plate counts remained constant from March through December, 1973, ranging from 2.4 X 10(6) to 5.7 X 10(6) organisms/g (dry weight) of sediment. The isolatable bacteria represented a small percentage of the total microbial community, which was shown by direct microscopic counts to be 2.0 X 10' organisms/g (dry weight) of sediment during June and July. Bacteria of the genus Clostridium dominated the isolates obtained, accounting for 71.8% of the 960 isolates examined. A single species, Clostridium bifermentens, comprised 47.7% of the total. Additional bacterial groups and the percentage in which they were isolated included: Streptococcus sp. (10.8%), unidentified curved rods (9.5%y, gram-positive nonsporing rods (5.6%), and motile gram-negative rods (1.9%). Temperature growth studies demonstrated the ability of all the isolates to grow at in situ sediment temperatures. Gas-liqid radiochromatography was used to determine the soluble metabolic end products from [U-14C]glucose and a U-14C-labeled amino acid mixture by representative sedimentary clostridial isolates and by natural sediment microbial communities. At in situ temperatures the natural sediment microflora produced soluble fermentative end products characteristic of those elaborated by the clostridial isolates tested. These results are considered strong presumptive evidence that clostridia are actively metabolizing in the sediments of Wintergreen Lake.     

Methanogenesis from Methanol and Methylamines and Acetogenesis from Hydrogen and Carbon Dioxide in the Sediments of a Eutrophic Lake

¹⁴C-tracer techniques were used to examine the metabolism of methanol and methylamines and acetogenesis from hydrogen and carbon dioxide in sediments from the profundal and littoral zones of eutrophic Wintergreen Lake, Michigan. Methanogens were primarily responsible for the metabolism of methanol, monomethylamine, and trimethylamine and maintained the pool size of these substrates below 10 μM in both sediment types. Methanol and methylamines were the precursors for less than 5 and 1%, respectively, of the total methane produced. Methanol and methylamines continued to be metabolized to methane when the sulfate concentration in the sediment was increased to 20 mM. Less than 2% of the total acetate production was derived from carbon dioxide reduction. Hydrogen consumption by hydrogen-oxidizing acetogens was 5% or less of the total hydrogen uptake by acetogens and methanogens. These results, in conjunction with previous studies, emphasize that acetate and hydrogen are the major methane precursors and that methanogens are the predominant hydrogen consumers in the sediments of this eutrophic lake.     

Estimation of methanogen biomass by quantitation of coenzyme M

Determination of the role of methanogenic bacteria in an anaerobic ecosystem often requires quantitation of the organisms. Because of the extreme oxygen sensitivity of these organisms and the inherent limitations of cultural techniques, an accurate biomass value is very difficult to obtain. We standardized a simple method for estimating methanogen biomass in a variety of environmental matrices. In this procedure we used the thiol biomarker coenzyme M (CoM) (2-mercaptoethanesulfonic acid), which is known to be present in all methanogenic bacteria. A high-performance liquid chromatography-based method for detecting thiols in pore water (A. Vairavamurthy and M. Mopper, Anal. Chim. Acta 78:363-370, 1990) was modified in order to quantify CoM in pure cultures, sediments, and sewage water samples. The identity of the CoM derivative was verified by using liquid chromatography-mass spectroscopy. The assay was linear for CoM amounts ranging from 2 to 2,000 pmol, and the detection limit was 2 pmol of CoM/ml of sample. CoM was not adsorbed to sediments. The methanogens tested contained an average of 19.5 nmol of CoM/mg of protein and 0.39 +/- 0.07 fmol of CoM/cell. Environmental samples contained an average of 0.41 +/- 0.17 fmol/cell based on most-probable-number estimates. CoM was extracted by using 1% tri-(N)-butylphosphine in isopropanol. More than 90% of the CoM was recovered from pure cultures and environmental samples. We observed no interference from sediments in the CoM recovery process, and the method could be completed aerobically within 3 h. Freezing sediment samples resulted in 46 to 83% decreases in the amounts of detectable CoM, whereas freezing had no effect on the amounts of CoM determined in pure cultures. The method described here provides a quick and relatively simple way to estimate methanogenic biomass.     

Microbial community structure and biomass estimates of a methanogenic Antarctic Lake ecosystem as determined by phospholipid analyses

Phospholipid analyses were performed on water column particulate and sediment samples from Ace Lake, a meromictic lake in the Vestfold Hills, Antarctica, to estimate the viable microbial biomass and community structure in the lake. In the water column, methanogenic bacterial phospholipids were present below 17 m in depth at concentrations which converted to a biomass of between 1 and 7×10⁸ cells/liter. Methanogenic biomass in the sediment ranged from 17.7×10⁹ cells/g dry weight of sediment at the surface to 0.1×10⁹ cells/g dry weight at 2 m in depth. This relatively high methanogenic biomass implies that current microbial degradation of organic carbon in Ace Lake sediments may occur at extremely slow rates. Total microbial biomass increased from 4.4×10⁸ cells/ liter at 2 m in depth to 19.4×10⁸ cells/liter at 23 m, near the bottom of the water column. Total nonarchaebacterial biomass decreased from 4.2 ×10⁹ cells/g dry weight in the surface sediment (1/4 the biomass of methanogens) to 0.06×10⁸ cells/g dry weight at 2 m in depth in the sediment. Phospholipid fatty acid profiles showed that microeukaryotes were the major microbial group present in the oxylimnion of the lake, while bacteria dominated the lower, anoxic zone. Sulfate-reducing bacteria (SRB) comprised 25% of the microbial population at 23 m in depth in the water column particulates and were present in the surface sediment but to a lesser extent. Biomass estimates and community structure of the Ace Lake eco-system are discussed in relation to previously measured metabolic rates for this and other antarctic and temperate ecosystems. This is the first instance, to our knowledge, in which the viable biomass of methanogenic and SRB have been estimated for an antarctic microbial community.     

Analysis of sample preparation procedures for enumerating fecal coliforms in coarse southwestern U.S. bottom sediments by the most-probable-number method

The determination of bacterial densities in aquatic sediments generally requires that a dilution-mixing treatment be used before enumeration of organisms by the most-probable-number fermentation tube method can be done. Differential sediment and organism settling rates may, however, influence the distribution of the microbial population after the dilution-mixing process, resulting in biased bacterial density estimates. For standardization of sample preparation procedures, the influence of settling by suspended sediments on the fecal coliform distribution in a mixing vessel was examined. This was accomplished with both inoculated (Escherichia coli) and raw, uninoculated freshwater sediments from Saguaro Lake, Ariz. Both test sediments were coarse (greater than 90% gravel and sand). Coarse sediments are typical of southwestern U.S. lakes. The distribution of fecal coliforms, as determined by the most-probable-number method, was not significantly influenced by sediment settling and remained homogenous over a 16-min postmix period. The technique developed for coarse sediments may be useful for standardizing sample preparation techniques for other sediment types.     

Estimation of Methanogen Biomass by Quantitation of Coenzyme M

Determination of the role of methanogenic bacteria in an anaerobic ecosystem often requires quantitation of the organisms. Because of the extreme oxygen sensitivity of these organisms and the inherent limitations of cultural techniques, an accurate biomass value is very difficult to obtain. We standardized a simple method for estimating methanogen biomass in a variety of environmental matrices. In this procedure we used the thiol biomarker coenzyme M (CoM) (2-mercaptoethanesulfonic acid), which is known to be present in all methanogenic bacteria. A high-performance liquid chromatography-based method for detecting thiols in pore water (A. Vairavamurthy and M. Mopper, Anal. Chim. Acta 78:363–370, 1990) was modified in order to quantify CoM in pure cultures, sediments, and sewage water samples. The identity of the CoM derivative was verified by using liquid chromatography-mass spectroscopy. The assay was linear for CoM amounts ranging from 2 to 2,000 pmol, and the detection limit was 2 pmol of CoM/ml of sample. CoM was not adsorbed to sediments. The methanogens tested contained an average of 19.5 nmol of CoM/mg of protein and 0.39 ± 0.07 fmol of CoM/cell. Environmental samples contained an average of 0.41 ± 0.17 fmol/cell based on most-probable-number estimates. CoM was extracted by using 1% tri-(N)-butylphosphine in isopropanol. More than 90% of the CoM was recovered from pure cultures and environmental samples. We observed no interference from sediments in the CoM recovery process, and the method could be completed aerobically within 3 h. Freezing sediment samples resulted in 46 to 83% decreases in the amounts of detectable CoM, whereas freezing had no effect on the amounts of CoM determined in pure cultures. The method described here provides a quick and relatively simple way to estimate methanogenic biomass.     

Analysis of sample preparation procedures for enumerating fecal coliforms in coarse southwestern U.S. bottom sediments by the most-probable-number method.

The determination of bacterial densities in aquatic sediments generally requires that a dilution-mixing treatment be used before enumeration of organisms by the most-probable-number fermentation tube method can be done. Differential sediment and organism settling rates may, however, influence the distribution of the microbial population after the dilution-mixing process, resulting in biased bacterial density estimates. For standardization of sample preparation procedures, the influence of settling by suspended sediments on the fecal coliform distribution in a mixing vessel was examined. This was accomplished with both inoculated (Escherichia coli) and raw, uninoculated freshwater sediments from Saguaro Lake, Ariz. Both test sediments were coarse (greater than 90% gravel and sand). Coarse sediments are typical of southwestern U.S. lakes. The distribution of fecal coliforms, as determined by the most-probable-number method, was not significantly influenced by sediment settling and remained homogenous over a 16-min postmix period. The technique developed for coarse sediments may be useful for standardizing sample preparation techniques for other sediment types.     

Cultivation of methanogens from shallow marine sediments at Hydrate Ridge, Oregon

Little is known about the methanogenic degradation of acetate, the fate of molecular hydrogen and formate or the ability of methanogens to grow and produce methane in cold, anoxic marine sediments. The microbes that produce methane were examined in permanently cold, anoxic marine sediments at Hydrate Ridge (44 degrees 35' N, 125 degrees 10' W, depth 800 m). Sediment samples (15 to 35 cm deep) were collected from areas of active methane ebullition or areas where methane hydrates occurred. The samples were diluted into enrichment medium with formate, acetate or trimethylamine as catabolic substrate. After 2 years of incubation at 4 degrees C to 15 degrees C, enrichment cultures produced methane. PCR amplification and sequencing of the rRNA genes from the highest dilutions with growth suggested that each enrichment culture contained a single strain of methanogen. The level of sequence similarity (91 to 98%) to previously characterized prokaryotes suggested that these methanogens belonged to novel genera or species within the orders Methanomicrobiales and Methanosarcinales. Analysis of the 16S rRNA gene libraries from DNA extracted directly from the sediment samples revealed phylotypes that were either distantly related to cultivated methanogens or possible anaerobic methane oxidizers related to the ANME-1 and ANME-2 groups of the Archaea. However, no methanogenic sequences were detected, suggesting that methanogens represented only a small proportion of the archaeal community.     

Methanogens revealed immunologically in granules from five Upflow Anaerobic Sludge Blanket (UASB) bioreactors grown on different substrates

Abstract Methanogens were identified and quantified using antibody probes and the antigenic fingerprinting method in five different kinds of granular sludge taken from five Uplow Anaerobic Sludge Blanket (UASB) bioreactors maintained on different substrates. The methanogenic flora present in each bioreactor was elucidated and expressed in cells per garm dry weight. Autofluorescence, phase-contrast and bright field-microscopy of unstained and Gram-stained preparations were used in parallel with immunotechnology to characterize each methanogenic subpopulation. Ten different methanogens were prevalent in the five bioreactor systems. Methanogens antigenically related to Methanobacterium formicicum MF, Methanobrevibacter arboriphilus AZ and Methanothrix soehngenii Opfikon were found in all five granules, while other methanogens were present in only some. A trend was observed towards a wider diversity of methanogenic subpopulations parallelling an increase in the complexity of the bioreactor's substrate.     

Composition and Role of Extracellular Polymers in Methanogenic Granules

Methanobacterium formicicum and Methanosarcina mazeii are two prevalent species isolated from an anaerobic granular consortium grown on a fatty acid mixture. The extracellular polysaccharides (EPS) were extracted from Methanobacterium formicicum and Methanosarcina mazeii and from the methanogenic granules to examine their role in granular development. The EPS made up approximately 20 to 14% of the extracellular polymer extracted from the granules, Methanobacterium formicicum, and Methanosarcina mazeii. The EPS produced by Methanobacterium formicicum was composed mainly of rhamnose, mannose, galactose, glucose, and amino sugars, while that produced by Methanosarcina mazeii contained ribose, galactose, glucose, and glucosamine. The same sugars were also present in the EPS produced by the granules. These results indicate that the two methanogens, especially Methanobacterium formicicum, contributed significantly to the production of the extracellular polymer of the anaerobic granules. Growth temperature, substrates (formate and H(inf2)-CO(inf2)), and the key nutrients (nitrogen and phosphate concentrations) affected polymer production by Methanobacterium formicicum.     

Evidence for para Dechlorination of Polychlorobiphenyls by Methanogenic Bacteria

When microorganisms eluted from upper Hudson River sediment were cultured without any substrate except polychlorobiphenyl (PCB)-free Hudson River sediment, methane formation was the terminal step of the anaerobic food chain. In sediments containing Aroclor 1242, addition of eubacterium-inhibiting antibiotics, which should have directly inhibited fermentative bacteria and thereby should have indirectly inhibited methanogens, resulted in no dechlorination activity or methane production. However, when substrates for methanogenic bacteria were provided along with the antibiotics (to free the methanogens from dependence on eubacteria), concomitant methane production and dechlorination of PCBs were observed. The dechlorination of Aroclor 1242 was from the para positions, a pattern distinctly different from, and more limited than, the pattern observed with untreated or pasteurized inocula. Both methane production and dechlorination in cultures amended with antibiotics plus methanogenic substrates were inhibited by 2-bromoethanesulfonic acid. These results suggest that the methanogenic bacteria are among the physiological groups capable of anaerobic dechlorination of PCBs, but that the dechlorination observed with methanogenic bacteria is less extensive than the dechlorination observed with more complex anaerobic consortia.     

Methanogenic activity and diversity in the centre of the Amsterdam Mud Volcano, Eastern Mediterranean Sea

Marine mud volcanoes are geological structures emitting large amounts of methane from their active centres. The Amsterdam mud volcano (AMV), located in the Anaximander Mountains south of Turkey, is characterized by intense active methane seepage produced in part by methanogens. To date, information about the diversity or the metabolic pathways used by the methanogens in active centres of marine mud volcanoes is limited. (14)C-radiotracer measurements showed that methylamines/methanol, H(2)/CO(2) and acetate were used for methanogenesis in the AMV. Methylotrophic methanogenesis was measured all along the sediment core, Methanosarcinales affiliated sequences were detected using archaeal 16S PCR-DGGE and mcrA gene libraries, and enrichments of methanogens showed the presence of Methanococcoides in the shallow sediment layers. Overall acetoclastic methanogenesis was higher than hydrogenotrophic methanogenesis, which is unusual for cold seep sediments. Interestingly, acetate porewater concentrations were extremely high in the AMV sediments. This might be the result of organic matter cracking in deeper hotter sediment layers. Methane was also produced from hexadecanes. For the most part, the methanogenic community diversity was in accordance with the depth distribution of the H(2)/CO(2) and acetate methanogenesis. These results demonstrate the importance of methanogenic communities in the centres of marine mud volcanoes.     

Methanogenic diversity and activity in hypersaline sediments of the centre of the Napoli mud volcano, Eastern Mediterranean Sea

Submarine mud volcanoes are a significant source of methane to the atmosphere. The Napoli mud volcano, situated in the brine-impacted Olimpi Area of the Eastern Mediterranean Sea, emits mainly biogenic methane particularly at the centre of the mud volcano. Temperature gradients support the suggestion that Napoli is a cold mud volcano with moderate fluid flow rates. Biogeochemical and molecular genetic analyses were carried out to assess the methanogenic activity rates, pathways and diversity in the hypersaline sediments of the centre of the Napoli mud volcano. Methylotrophic methanogenesis was the only significant methanogenic pathway in the shallow sediments (0-40 cm) but was also measured throughout the sediment core, confirming that methylotrophic methanogens could be well adapted to hypersaline environments. Hydrogenotrophic methanogenesis was the dominant pathway below 50 cm; however, low rates of acetoclastic methanogenesis were also present, even in sediment layers with the highest salinity, showing that these methanogens can thrive in this extreme environment. PCR-DGGE and methyl coenzyme M reductase gene libraries detected sequences affiliated with anaerobic methanotrophs (mainly ANME-1) as well as Methanococcoides methanogens. Results show that the hypersaline conditions in the centre of the Napoli mud volcano influence active biogenic methane fluxes and methanogenic/methylotrophic diversity.     

Sediment Distribution of Methanogenic Bacteria in Lake Erie and Cleveland Harbor

The direct fluorescent-antibody technique was employed to determine the distribution patterns of four species of methanogens in the sediments of Lake Erie and Cleveland Harbor. Methanobacterium ruminantium was the most numerous methanogen found in regions of high-organic-silt sediments. The population of this species ranged from 10⁶ to 10⁹ cells/g of dry sediment. Methanobacterium strain MoH and Methanosarcina barkeri were identified in sand-silt, clay, or sand sediments. These methanogens ranged in density from 10⁶ to 10⁷ cells/g of dry sediment. Methanospirillum hungatii was observed only after an organic enrichment was performed on Cleveland Harbor sediments. The seasonal and selective sediment distribution of these methanogens appears to be related to the type and concentration of carbon as substrate as well as to the activities of heterotrophic and sulfate-reducing bacteria.     

Microbiological Comparisons within and across Contiguous Lacustrine, Paleosol, and Fluvial Subsurface Sediments

Twenty-six subsurface samples were collected from a borehole at depths of 173.3 to 196.8 m in the saturated zone at the Hanford Site in south-central Washington State. The sampling was performed throughout strata that included fine-grained lacustrine (lake) sediments, a paleosol (buried soil) sequence, and coarse-grained fluvial (river) sediments. A subcoring method and tracers were used to minimize and quantify contamination to obtain samples that were representative of subsurface strata. Sediment samples were tested for total organic carbon, inorganic carbon, total microorganisms by direct microscopic counts, culturable aerobic heterotrophs by plate counts, culturable anaerobes by most-probable-number enumeration, basal respiration rates, and mineralization of (sup14)C-labeled glucose and acetate. Total direct microscopic counts of microorganisms were low, ranging from below detection to 1.9 x 10(sup5) cells g (dry weight)(sup-1). Culturable aerobes and anaerobes were below minimum levels of detection in most samples. Direct microscopic counts, basal respiration rates, and (sup14)C-glucose mineralization were all positively correlated with total organic carbon and were highest in the lacustrine sediments. In contrast to previous subsurface studies, these saturated-zone samples did not have higher microbial abundance and activities than unsaturated sediments sampled from the same borehole, the fine-textured lacustrine sediment had higher microbial numbers and activities than the coarse-textured fluvial sands, and the paleosol samples did not have higher biomass and activities relative to the other sediments. The results of this study expand the subsurface microbiology database to include information from an environment very different from those previously studied.     

Population Dynamics of Polychlorinated Biphenyl-Dechlorinating Microorganisms in Contaminated Sediments

The growth dynamics of polychlorinated biphenyl (PCB)-dechlorinating microorganisms were determined for the first time, along with those of sulfate reducers and methanogens, by using the most-probable-number technique. The time course of Aroclor 1248 dechlorination mirrored the growth of dechlorinators; dechlorination ensued when the dechlorinating population increased by 2 orders of magnitude from 2.5 x 10(sup5) to 4.6 x 10(sup7) cells g of sediment(sup-1), at a specific growth rate of 6.7 day(sup-1) between 2 and 6 weeks. During this period, PCB-dechlorinating microorganisms dechlorinated Aroclor 1248 at a rate of 3.9 x 10(sup-8) mol of Cl g of sediment(sup-1) day(sup-1), reducing the average number of Cl molecules per biphenyl from 3.9 to 2.8. The growth yield was 4.2 x 10(sup13) cells mol of Cl dechlorinated(sup-1). Once dechlorination reached a plateau, after 6 weeks, the number of dechlorinators began to decrease. On the other hand, dechlorinators inoculated into PCB-free sediments decreased over time from their initial level, suggesting that PCBs are required for their selective enrichment. The numbers of sulfate reducers and methanogens increased in both PCB-free and contaminated sediments, showing little difference between them. The maximum population size of sulfate reducers was about an order of magnitude higher than that of dechlorinators, whereas that of methanogens was slightly less. Unlike those of dechlorinators, however, numbers of both sulfate reducers and methanogens remained high even when dechlorination ceased. The results of this study imply that PCB concentrations may have to exceed a certain threshold to maintain the growth of PCB dechlorinators.     

Survival of Escherichia coli in lake bottom sediment.

The survival of Escherichia coli in bottom sediment (Lake Onalaska, navigation pool no. 7, Mississippi River) was studied by using in situ dialysis culture of sterile (autoclaved) and unsterile sediment samples. Bags made from dialysis tubing were filled with either course sand sediment (28.8% fine) or organic, silty clay sediment (77.2% fine) and placed at the sediment-water interface. Bags representing sterile controls, unsterile uninoculated controls, autoclaved inoculated sediment, and unsterile inoculated sediment were studied during a 5-day period for each sediment type. Daily most-probable-number determinations indicated that E. coli populations in unsterile inoculated sediment fluctuated between 5.3 X 10(2) and 2.2 X 10(3) bacteria per g of silty clay and between 3.0 X 10(3) and 1.4 X 10(4) bacteria per g of sand. Autoclaved silty clay sediment inoculated with 1.0 X 10(6) bacteria per g increased to 2.2 X 10(8) bacteria per g in 3 days. During the same period, autoclaved sand sediment inoculated with 1.2 X 10(5) cells per g increased to 5.4 X 10(7) bacteria per g. By day 5, populations in both cultures had decreased by 1 log. The ability of E. coli to survive for several days in aquatic sediment in situ suggests that fecal coliforms in water may not always indicate recent fecal contamination of that water but rather resuspension of viable sediment-bound bacteria.     

Carbon isotope effects associated with aceticlastic methanogenesis

The carbon isotope effects associated with synthesis of methane from acetate have been determined for Methanosarcina barkeri 227 and for methanogenic archaea in sediments of Wintergreen Lake, Michigan. At 37 degrees C, the 13C isotope effect for the reaction acetate (methyl carbon) --> methane, as measured in replicate experiments with M. barkeri, was - 21.3% +/- 0.3%. The isotope effect at the carboxyl portion of acetate was essentially equal, indicating participation of both positions in the rate-determining step, as expected for reactions catalyzed by carbon monoxide dehydrogenase. A similar isotope effect, - 19.2% +/- 0.3% was found for this reaction in the natural community (temperature = 20 degrees C). Given these observations, it has been possible to model the flow of carbon to methane within lake sediment communities and to account for carbon isotope compositions of evolving methane. Extension of the model allows interpretation of seasonal fluctuations in 13C contents of methane in other systems.