Effect of Fall Turnover on Terminal Carbon Metabolism in Lake Mendota Sediments

The carbon and electron flow pathways and the bacterial populations responsible for the transformation of H₂-CO₂, formate, methanol, methylamine, acetate, ethanol, and lactate were examined in eutrophic sediments collected during summer stratification and fall turnover. The rate of methane formation averaged 1,130 μmol of CH₄ per liter of sediment per day during late-summer stratification versus 433 μmol of CH₄ per liter of sediment per day during the early portion of fall turnover, whereas the rate of sulfate reduction was 280 μmol of sulfate per liter of sediment per day versus 1,840 μmol of sulfate per liter of sediment per day during the same time periods, respectively. The sulfate-reducing population remained constant while the methanogenic population decreased by one to two orders of magnitude during turnover. The acetate concentration increased from 32 to 81 μmol per liter of sediment while the acetate transformation rate constant decreased from 3.22 to 0.70 per h, respectively, during stratification versus turnover. Acetate accounted for nearly 100% of total sedimentary methanogenesis during turnover versus 70% during stratification. The fraction of ¹⁴CO₂ produced from all ¹⁴C-labeled substrates examined was 10 to 40% higher during fall turnover than during stratification. The addition of sulfate, thiosulfate, or sulfur to stratified sediments mimicked fall turnover in that more CO₂ and CH₄ were produced. The addition of Desulfovibrio vulgaris to sulfate-amended sediments greatly enhanced the amount of CO₂ produced from either [¹⁴C]methanol or [2-¹⁴C]acetate, suggesting that H₂ consumption by sulfate reducers can alter methanol or acetate transformation by sedimentary methanogens. These data imply that turnover dynamically altered carbon transformation in eutrophic sediments such that sulfate reduction dominated over methanogenesis principally as a consequence of altering hydrogen metabolism.     

Evidence for Aceticlastic Methanogenesis in the Presence of Sulfate in a Gas Condensate-Contaminated Aquifer

The anaerobic metabolism of acetate was studied in sediments and groundwater from a gas condensate-contaminated aquifer in an aquifer where geochemical evidence implicated sulfate reduction and methanogenesis as the predominant terminal electron-accepting processes. Most-probable-number tubes containing acetate and microcosms containing either [2-¹⁴C]acetate or [U-¹⁴C]acetate produced higher quantities of CH₄ compared to CO₂ in the presence or absence of sulfate.¹⁴CH₄ accounted for 70 to 100% of the total labeled gas in the [¹⁴C]acetate microcosms regardless of whether sulfate was present or not. Denaturing gradient gel electrophoresis of the acetate enrichments both with and without sulfate using Archaea-specific primers showed identical predominant bands that had 99% sequence similarity to members of Methanosaetaceae. Clone libraries containing archaeal 16S rRNA gene sequences amplified from sediment from the contaminated portion of the aquifer showed that 180 of the 190 clones sequenced belonged to the Methanosaetaceae. The production of methane and the high frequency of sequences from the Methanosaetaceae in acetate enrichments with and without sulfate indicate that aceticlastic methanogenesis was the predominant fate of acetate at this site even though sulfate-reducing bacteria would be expected to consume acetate in the presence of sulfate.     

Metabolism of Acetate, Methanol, and Methylated Amines in Intertidal Sediments of Lowes Cove, Maine

The fates and the rates of metabolism of acetate, trimethylamine, methylamine, and methanol were examined to determine the significance of these compounds as in situ methane precursors in surface sediments of an intertidal zone in Maine. Concentrations of these potential methane precursors were generally <3 μM, with the exception of sediments containing fragments of the seaweed Ascophyllum nodosum, in which acetate was 96 μM. [2-¹⁴C]acetate turnover in all samples was rapid (turnover time <2 h), with ¹⁴CO₂ as the primary product. [¹⁴C]trimethylamine and methylamine turnover times were slower (>8 h) and were characterized by formation of both ¹⁴CH₄ and ¹⁴CO₂. Ratios of ¹⁴CH₄/¹⁴CO₂ from [¹⁴C]trimethylamine and methylamine in uninhibited sediments indicated that a significant fraction of these substrates were catabolized via a non-methanogenic process. Data from inhibition experiments involving sodium molybdate and 2-bromoethanesulfonic acid supported this interpretation. [¹⁴C]methanol was oxidized relatively slowly compared with the other substrates and was catabolized mainly to ¹⁴CO₂. Results from experiments with molybdate and 2-bromoethanesulfonic acid suggested that methanol was oxidized primarily through sulfate reduction. In Lowes Cove sediments, trimethylamine accounted for 35.1 to 61.1% of total methane production.     

Terminal Processes in the Anaerobic Degradation of an Algal-Bacterial Mat in a High-Sulfate Hot Spring

The algal-bacterial mat of a high-sulfate hot spring (Bath Lake) provided an environment in which to compare terminal processes involved in anaerobic decomposition. Sulfate reduction was found to dominate methane production, as indicated by comparison of initial electron flow through the two processes, rapid conversion of [2-¹⁴C]acetate to ¹⁴CO₂ and not to ¹⁴CH₄, and the lack of rapid reduction of NaH¹⁴CO₃ to ¹⁴CH₄. Sulfate reduction was the dominant process at all depth intervals, but a marked decrease of sulfate reduction and sulfate-reducing bacteria was observed with depth. Concurrent methanogenesis was indicated by the presence of viable methanogenic bacteria and very low but detectable rates of methane production. A marked increased in methane production was observed after sulfate depletion despite high concentrations of sulfide (>1.25 mM), indicating that methanogenesis was not inhibited by sulfide in the natural environment. Although a sulfate minimum and sulfide maximum occurred in the region of maximal sulfate reduction, the absence of sulfate depletion in interstitial water suggests that methanogenesis is always severely limited in Bath Lake sediments. Low initial methanogenesis was not due to anaerobic methane oxidation.     

Influence of pH on Terminal Carbon Metabolism in Anoxic Sediments from a Mildly Acidic Lake

The carbon and electron flow pathways and the bacterial populations responsible for transformation of H₂-CO₂, formate, methanol, methylamine, acetate, glycine, ethanol, and lactate were examined in sediments collected from Knaack Lake, Wis. The sediments were 60% organic matter (pH 6.2) and did not display detectable sulfate-reducing activity, but they contained the following average concentration (in micromoles per liter of sediment) of metabolites and end products: sulfide, 10; methane, 1,540; CO₂, 3,950; formate, 25; acetate, 157; ethanol, 174; and lactate, 138. Methane was produced predominately from acetate, and only 4% of the total CH₄ was derived from CO₂. Methanogenesis was limited by low environmental temperature and sulfide levels and more importantly by low pH. Increasing in vitro pH to neutral values enhanced total methane production rates and the percentage of CO₂ transformed to methane but did not alter the amount of ¹⁴CO₂ produced from [2-¹⁴C]acetate (~24%). Analysis of both carbon transformation parameters with ¹⁴C-labeled tracers and bacterial trophic group enumerations indicated that methanogenesis from acetate and both heterolactic- and acetic acid-producing fermentations were important to the anaerobic digestion process.     

Carbon and Electron Flow in Mud and Sandflat Intertidal Sediments at Delaware Inlet, Nelson, New Zealand

An investigation of carbon and electron flow in mud and sandflat intertidal sediments showed that the terminal electron acceptor was principally sulfate and that the carbon flow was mainly to CO₂. Studies with thin layers of sediment exposed to H₂ showed that methane production accounted for virtually none of the H₂ utilized, whereas sulfate reduction accounted for a major proportion of the gas uptake. At all sampling sites except one (site B7), rates of methanogenesis were low but sulfate concentrations in the interstitial water were high (>18 mM). At site B7, the sulfate concentrations declined with depth from 32 mM at 2 cm to <1 mM at 10 cm or below, and active methanogenesis occurred in the low-sulfate zone. Sulfate-reducing activity at this site initially decreased and then increased with depth so that elevated rates occurred in both the active and nonactive methanogenic zones. The respiratory index (RI) [RI = ¹⁴CO₂/(¹⁴CO₂ + ¹⁴CH₄)] for [2-¹⁴C]acetate catabolism at site B7 ranged from 0.98 to 0.2 in the depth range of 2 to 14 cm. Addition of sulfate to sediment from the low-sulfate zone resulted in an increase in RI and a decrease in methanogenesis. At all other sites examined, RI ranged from 0.97 to 0.99 and was constant with depth. The results suggested that although methanogenesis was inhibited by sulfate (presumably through the activity of sulfate-reducing bacteria), it was not always limited by sulfate reduction.     

Microbial Methanogenesis and Acetate Metabolism in a Meromictic Lake

Methanogenesis and the anaerobic metabolism of acetate were examined in the sediment and water column of Knaack Lake, a small biogenic meromictic lake located in central Wisconsin. The lake was sharply stratified during the summer and was anaerobic below a depth of 3 m. Large concentrations (4,000 μmol/liter) of dissolved methane were detected in the bottom waters. A methane concentration maximum occurred at 4 m above the sediment. The production of ¹⁴CH₄ from ¹⁴C-labeled HCOOH, HCO₃⁻, and CH₃OH and [2-¹⁴C]acetate demonstrated microbial methanogenesis in the water column of the lake. The maximum rate of methanogenesis calculated from reduction of H¹⁴CO₃⁻ by endogenous electron donors in the surface sediment (depth, 22 m) was 7.6 nmol/h per 10 ml and in the water column (depth, 21 m) was 0.6 nmol/h per 10 ml. The methyl group of acetate was simultaneously metabolized to CH₄ and CO₂ in the anaerobic portions of the lake. Acetate oxidation was greatest in surface waters and decreased with water depth. Acetate was metabolized primarily to methane in the sediments and water immediately above the sediment. Sulfide inhibition studies and temperature activity profiles demonstrated that acetate metabolism was performed by several microbial populations. Sulfide additions (less than 5 μg/ml) to water from 21.5 m stimulated methanogenesis from acetate, but inhibited CO₂ production. Sulfate addition (1 mM) had no significant effect on acetate metabolism in water from 21.5 m, whereas nitrate additions (10 to 14,000 μg/liter) completely inhibited methanogenesis and stimulated CO₂ formation.     

Partitioning Effects during Terminal Carbon and Electron Flow in Sediments of a Low-Salinity Meltwater Pond near Bratina Island, McMurdo Ice Shelf, Antarctica

A study of anaerobic sediments below cyanobacterial mats of a low-salinity meltwater pond called Orange Pond on the McMurdo Ice Shelf at temperatures simulating those in the summer season (<5°C) revealed that both sulfate reduction and methane production were important terminal anaerobic processes. Addition of [2-¹⁴C]acetate to sediment samples resulted in the passage of label mainly to CO₂. Acetate addition (0 to 27 mM) had little effect on methanogenesis (a 1.1-fold increase), and while the rate of acetate dissimilation was greater than the rate of methane production (6.4 nmol cm⁻³ h⁻¹ compared to 2.5 to 6 nmol cm⁻³ h⁻¹), the portion of methane production attributed to acetate cleavage was <2%. Substantial increases in the methane production rate were observed with H₂ (2.4-fold), and H₂ uptake was totally accounted for by methane production under physiological conditions. Formate also stimulated methane production (twofold), presumably through H₂ release mediated through hydrogen lyase. Addition of sulfate up to 50-fold the natural levels in the sediment (interstitial concentration, ~0.3 mM) did not substantially inhibit methanogenesis, but the process was inhibited by 50-fold chloride (36 mM). No net rate of methane oxidation was observed when sediments were incubated anaerobically, and denitrification rates were substantially lower than rates for sulfate reduction and methanogenesis. The results indicate that carbon flow from acetate is coupled mainly to sulfate reduction and that methane is largely generated from H₂ and CO₂ where chloride, but not sulfate, has a modulating role. Rates of methanogenesis at in situ temperatures were four- to fivefold less than maximal rates found at 20°C.     

Intermediary Metabolism of Organic Matter in the Sediments of a Eutrophic Lake

The rates, products, and controls of the metabolism of fermentation intermediates in the sediments of a eutrophic lake were examined. ¹⁴C-fatty acids were directly injected into sediment subcores for turnover rate measurements. The highest rates of acetate turnover were in surface sediments (0- to 2-cm depth). Methane was the dominant product of acetate metabolism at all depths. Simultaneous measurements of acetate, propionate, and lactate turnover in surface sediments gave turnover rates of 159, 20, and 3 μM/h, respectively. [2-¹⁴C]propionate and [U-¹⁴C]lactate were metabolized to [¹⁴C]acetate, ¹⁴CO₂, and ¹⁴CH₄. [¹⁴C]formate was completely converted to ¹⁴CO₂ in less than 1 min. Inhibition of methanogenesis with chloroform resulted in an immediate accumulation of volatile fatty acids and hydrogen. Hydrogen inhibited the metabolism of C₃-C₅ volatile fatty acids. The rates of fatty acid production were estimated from the rates of fatty acid accumulation in the presence of chloroform or hydrogen. The mean molar rates of production were acetate, 82%; propionate, 13%; butyrates, 2%; and valerates, 3%. A working model for carbon and electron flow is presented which illustrates that fermentation and methanogenesis are the predominate steps in carbon flow and that there is a close interaction between fermentative bacteria, acetogenic hydrogen-producing bacteria, and methanogens.     

Methane production in Santa Barbara basin sediments

In vitro incubation of Santa Barbara Basin sediments indicated that methane production occurs at all depths sampled, including those in which sulfate reduction occurs. Methane production in the sulfate zone decreases with depth. U‐¹⁴C‐lactate is readily metabolized in the sulfate‐reducing zone, with ¹⁴CO₂ production being greater than ¹⁴CH₄ production. However, if sulfate is added to incubated sediments that have become depleted in sulfate, the ¹⁴CH₄ production increases dramatically at the expense of ¹⁴CO₂ production. Contrary to what has been observed in other ecosystems, sulfate stimulated methane production, especially from lactate. Experiments using 2‐¹⁴C‐acetate or H¹⁴CO₃ ^‐ have indicated that bicarbonate is the principal source of methane and acetate is oxidized to CO₂ in sediments from the methane‐producing zone.     

Fate of Immediate Methane Precursors in Low-Sulfate, Hot-Spring Algal-Bacterial Mats

The fates of acetate and carbon dioxide were examined in several experiments designed to indicate their relative contributions to methane production at various temperatures in two low-sulfate, hot-spring algal-bacterial mats. [2-¹⁴C]acetate was predominantly incorporated into cell material, although some ¹⁴CH₄ and ¹⁴CO₂ was produced. Acetate incorporation was reduced by dark incubation in short-term experiments and severely depressed by a 2-day preincubation in darkness. Autoradiograms showed that acetate was incorporated by long filaments resembling phototrophic microorganisms of the mat communities. [³H]acetate was not converted to C³H₄ in samples from Octopus Spring collected at the optimum temperature for methanogenesis. NaH¹⁴CO₃ was readily converted to ¹⁴CH₄ at temperatures at which methanogenesis was active in both mats. Comparisons of the specific activities of methane and carbon dioxide suggested that of the methane produced, 80 ± 6% in Octopus Spring and 71 ± 21% in Wiegert Channel were derived from carbon dioxide. Addition of acetate to 1 mM did not reduce the relative importance of carbon dioxide as a methane precursor in samples from Octopus Spring. Experiments with pure cultures of Methanobacterium thermoautotrophicum suggested that the measured ratio of specific activities might underestimate the true contribution of carbon dioxide in methanogenesis.     

Mesophilic syntrophic acetate oxidation during methane formation by a triculture at high ammonium concentration

In a mesophilic (37°C) triculture at a high ammonium concentration and pH8, methanogenesis from acetate occurred via syntrophic acetate oxidation. Studies with ¹⁴C-labelled substrates showed that the amount of labelled methane formed from 1-¹⁴C-labelled acetate was equal to that formed from 2-¹⁴C-labelled acetate. Labelled methane was also formed from H¹⁴CO₃ ^-. These results clearly showed that both the methyl and carboxyl groups of acetate were oxidized to CO₂ and that CO₂ was reduced to CH₄ through hydrogenotrophic methanogenesis. During growth of the triculture, a significant isotopic exchange between the carboxyl group of acetate and bicarbonate occurred. As a result, there was a decrease in the specific activity of 1-¹⁴C-acetate, and the production of ¹⁴CO₂ was slightly higher from 1-¹⁴C- than from 2-¹⁴C-acetate. For each mole acetate degraded, 0.94 mol methane was formed; 9.2 mmol acetate was metabolized during the 294 days of incubation.     

Role of Sulfate Reduction Versus Methanogenesis in Terminal Carbon Flow in Polluted Intertidal Sediment of Waimea Inlet, Nelson, New Zealand

An investigation of the terminal anaerobic processes occurring in polluted intertidal sediments indicated that terminal carbon flow was mainly mediated by sulfate-reducing organisms in sediments with high sulfate concentrations (>10 mM in the interstitial water) exposed to low loadings of nutrient (equivalent to <10² kg of N · day⁻¹) and biochemical oxygen demand (<0.7 × 10³ kg · day⁻¹) in effluents from different pollution sources. However, in sediments exposed to high loadings of nutrient (>10² kg of N · day⁻¹) and biochemical oxygen demand (>0.7 × 10³ kg · day⁻¹), methanogenesis was the major process in the mediation of terminal carbon flow, and sulfate concentrations were low (≤2 mM). The respiratory index [¹⁴CO₂/(¹⁴CO₂ + ¹⁴CH₄)] for [2-¹⁴C]acetate catabolism, a measure of terminal carbon flow, was ≥0.96 for sediment with high sulfate, but in sediments with sulfate as little as 10 μM in the interstitial water, respiratory index values of ≤0.22 were obtained. In the latter sediment, methane production rates as high as 3 μmol · g⁻¹ (dry weight) · h⁻¹ were obtained, and there was a potential for active sulfate reduction.     

Interrelations between sulfate-reducing and methane-producing bacteria in bottom deposits of a fresh-water lake. III. Experiments with 14C-labeled substrates

An ecological substrate relationship between sulfate-reducing and methane-producing bacteria in mud of Lake Vechten has been studied in experiments using ¹⁴C-labeled acetate and lactate as substrates. Fluoroacetate strongly inhibited the formation of ¹⁴CO₂ from [U-¹⁴C]-acetate and β-fluorolactate gave an inhibition of similar magnitude of the breakdown of [U-¹⁴C]-l-lactate to ¹⁴CO₂ thus confirming earlier results on the specific action of these inhibitors. The turnover-rate constant of l-lactate was 2.37 hr^-1 and the average l-lactate pool size was 12.2 μg per gram of wet mud, giving a turnover rate of 28.9 μg of lactate/gram of mud per hr. The turnover-rate constant of acetate was 0.35 hr^-1 and the average pool size was 5.7 μg per gram of wet mud, giving a rate of disappearance of 1.99 μg of acetate/gram of mud per hr. Estimations of the acetate turnover rate based upon the formation of ¹⁴CO₂ from [U-¹⁴C]-acetate or [1-¹⁴C]-acetate yielded figures of the same magnitude (range 0.45 to 1.74). These and other results suggest that only a portion of the lactate dissimilated is turned over through the acetate pool. The ratio of ¹⁴CO₂/¹⁴CH₄ produced from [U-¹⁴C]-acetate by mud was 1.32; indicating that 0.862 moles of CH₄ and 1.138 moles of CO₂ are formed per mole of acetate. From the rate of disappearance of acetate (0.027 μmoles/gram wet mud per hr) and the rate of methane production (0.034 μmoles/gram wet mud per hr), it may be concluded that acetate is an important precursor of methanogenesis in mud (approximately 70%). A substrate relationship between the two groups of bacteria is likely since ¹⁴CH₄ was formed from [U-¹⁴C]-l-lactate.     

Methanogenesis in Big Soda Lake, Nevada: an Alkaline, Moderately Hypersaline Desert Lake

Incubated sediment slurries from Big Soda Lake, Nevada, produced significant levels of CH₄, and production was inhibited by 2-bromoethanesulfonic acid and by autoclaving. Methane production was stimulated by methanol, trimethylamine, and, to a lesser extent, methionine. Surprisingly, hydrogen, acetate, and formate amendments provided only slight or no stimulation of methanogenesis. Methane production by sediment slurries had a pH optimum of 9.7. A methanol-grown enrichment culture containing a small, epifluorescent coccus as the predominant organism was recovered from sediments. The enrichment grew best when FeS or autoclaved sediment particles were included in the media, had a pH optimum of 9.7, and produced ¹⁴CH₄ from ¹⁴CH₃OH. The methane formed by methanolgrown enrichment cultures was depleted in ¹³C by 72 to 77‰ relative to the methanol.     

Methanogenesis and microbial lipid synthesis in anoxic salt marsh sediments

In anoxic salt marsh sediments of Sapelo Island, GA, USA, the vertical distribution of CH₄ production was measured in the upper 20 cm of surface sediments in ten locations. In one section of high marsh sediments, the concentration and oxidation of acetate in sediment porewaters and the rate and amount of¹⁴C acetate and¹⁴CO₂ incorporation into cellular lipids of the microbial population were investigated. CH₄ production rates ranged from <1 to 493 nM CH₄ gram sediment⁻¹ day⁻¹ from intact subcores incubated under nitrogen. Replacement with H₂ stimulated the rate of methane release up to nine fold relative to N₂ incubations. Rates of lipid synthesis from CO₂ averaged 39.2 ×10⁻²nanomoles lipid carbon cm³ sediment⁻¹ hr⁻¹, suggesting that CO₂ may be an important carbon precursor for microbial membrane synthesis in marsh sediments under anoxic conditions. Qualitative measurements of lipid synthesis rates from acetate were found to average 8.7 × 10⁻² nanomoles. Phospholipids were the dominant lipids synthesized by both substrates in sediment cores, accounting for an average of 76.6% of all lipid radioactivity. Small amounts of ether lipids indicative of methanogenic bacteria were observed in cores incubated for 7 days, with similar rates of synthesis for both CO₂ and acetate. The low rate of ether lipid synthesis suggests that either methanogen lipid biosynthesis is very slow or that methanogens represent a small component of total microbial lipid synthesis in anoxic sediments. present address: The University of Maryland,, Chesapeake Biological Laboratory, Box 38, Solomons, MD 20688, USA     

Methanogenesis and microbial lipid synthesis in anoxic salt marsh sediments

In anoxic salt marsh sediments of Sapelo Island, GA, USA, the vertical distribution of CH₄ production was measured in the upper 20 cm of surface sediments in ten locations. In one section of high marsh sediments, the concentration and oxidation of acetate in sediment porewaters and the rate and amount of¹⁴C acetate and¹⁴CO₂ incorporation into cellular lipids of the microbial population were investigated. CH₄ production rates ranged from <1 to 493 nM CH₄ gram sediment⁻¹ day⁻¹ from intact subcores incubated under nitrogen. Replacement with H₂ stimulated the rate of methane release up to nine fold relative to N₂ incubations. Rates of lipid synthesis from CO₂ averaged 39.2 ×10⁻²nanomoles lipid carbon cm³ sediment⁻¹ hr⁻¹, suggesting that CO₂ may be an important carbon precursor for microbial membrane synthesis in marsh sediments under anoxic conditions. Qualitative measurements of lipid synthesis rates from acetate were found to average 8.7 × 10⁻² nanomoles. Phospholipids were the dominant lipids synthesized by both substrates in sediment cores, accounting for an average of 76.6% of all lipid radioactivity. Small amounts of ether lipids indicative of methanogenic bacteria were observed in cores incubated for 7 days, with similar rates of synthesis for both CO₂ and acetate. The low rate of ether lipid synthesis suggests that either methanogen lipid biosynthesis is very slow or that methanogens represent a small component of total microbial lipid synthesis in anoxic sediments. present address: The University of Maryland,, Chesapeake Biological Laboratory, Box 38, Solomons, MD 20688, USA     

Tracer Analysis of Methanogenesis in Salt Marsh Soils

Differences in paths of carbon flow have been found in soils of the tall (TS) and short (SS) Spartina alterniflora marshes of Sapelo Island, Ga. Gaseous end products of [U-¹⁴C]glucose metabolism were ¹⁴CO₂ and ¹⁴CH₄ in the SS region and primarily ¹⁴CO₂ in the TS region. Sulfate concentration did not demonstrably affect glucose catabolism or the distribution of end products in either zone. [U-¹⁴C]acetate was converted to ¹⁴CO₂ and ¹⁴CH₄ in the SS soils and almost exclusively to ¹⁴CO₂ in the TS soils. Sulfate concentration did not affect acetate metabolism in the SS soils; however, a noticeable effect of sulfate dilution was seen in TS soils. Sulfate dilution in TS samples resulted in increased methane formation. Total glucose and acetate metabolism were similar in TS and SS soils despite differences in end products. A microbial community characterized by fermentative/sulfate-reducing processes has developed in TS soils as opposed to the fermentative/methanogenic/sulfate-reducing community found in SS soils.     

Autotrophic synthesis of activated acetic acid from two CO2 in Methanobacterium thermoautotrophicum

In a previous study with Methanobacterium thermoautotrophicum evidence was presented that methanogenesis and autotrophic synthesis of activated acetic acid from CO₂ are linked processes. In this study one-carbon metabolism was investigated with growing cultures and in vitro.Serine was shown to be converted into glycine and activated formaldehyde, but only traces of label from [¹⁴C-3] of serine appeared in biosynthetic one-carbon positions. This seeming discrepancy could be explained if the same activated formaldehyde is an intermediate in biosynthesis and in methanogenesis from CO₂. This hypothesis was supported by demonstrating that [¹⁴C-3] of serine and [¹⁴C] formaldehyde were rapidly converted into methane, but a small portion of the label was also specifically incorporated into the methyl group of acetate. Methane and acetate synthesis in vitro were similarly stimulated by various compounds. These experiments indicate that the methyl of acetate and methane share common one-carbon precursor(s), i.e. methylene tetrahydromethanopterin, which can also be formed enzymatically from C-3 of serine or chemically from formaldehyde.Propyl iodide 20–40 M) and methyl iodide (1–3 M) completely inhibited growth in the dark. This effect was abolished by light. Methane formation was hardly affected. When ¹⁴CH₃I was applied at an only slightly inhibitory concentration, ¹⁴C was incorporated into the methyl of acetate. In vitro, similar effects on [¹⁴C] acetate formation from ¹⁴CO₂ or from [¹⁴C-3] of serine were observed, except that methyl iodide did not inhibit, but even stimulated acetate synthesis. These experiments indicate that a corrinoid is involved in acetate synthesis and probably not in methanogenesis from CO₂; the metal is light-reversibly alkylated and functions in methyl transfer to the acetate methyl.     

Methane production in littoral sediment of Lake Constance

Maximum rates of CH₄ production in the littoral sediment were observed in 2–5 cm depth. The CH₄ production rates increased during the year from about 5 mmol m⁻²d⁻¹ in December to a maximum of about 95 mmol m⁻²d⁻¹ in September. CH₄ production rates showed a temperature optimum at 30°C and an apparent activation energy of 76 kJ mol⁻¹. A large part of the seasonality of CH₄ production could be ascribed to the change of the sediment temperature. Most of the produced CH₄ was lost by ebullition. Gas bubbles contained about 60–70% CH₄ with an average δ¹³C of −56.2% and δD of −354%, and 2% CO₂ with an average δ¹³C of −14.1% indicating that CH₄ was produced from methyl carbon, i.e. mainly using acetate as methanogenic substrate. This result was confirmed by inhibition of methanogenesis with chloroform which resulted in an accumulation rate of acetate equivalent to 81% of the rate of CH₄ production. Most probable numbers of methanogenic bacteria were in the order of 10⁴ bacteria g⁻¹d.w. sediment for acetate-, methanol- or formate-utilizing, and of 10⁵ for H₂-utilizing methanogens. The turnover times of acetate were in the order of 2.3–4.8 h which, with in situ acetate concentrations of about 25–50 μM, resulted in rates of acetate turnover which were comparable to the rates of CH₄ production. The respiratory index (RI) showed that [2−¹⁴C]acetate was mainly used by methanogenesis rather than by respiratory processes, although the zone of CH₄ production in the sediment overlapped with the zone of sulfate reduction.