Biodegradation of atrazine under denitrifying conditions

Anaerobic biodegradation of atrazine by the bacterial isolate M91-3 was characterized with respect to mineralization, metabolite formation, and denitrification. The ability of the isolate to enhance atrazine biodegradation in anaerobic sediment slurries was also investigated. The organism utilized atrazine as its sole source of carbon and nitrogen under anoxic conditions in fixed-film (glass beads) batch column systems. Results of HPLC and TLC radiochromatography suggested that anaerobic biotransformation of atrazine by microbial isolate M91-3 involved hydroxyatrazine formation. Ring cleavage was demonstrated by ¹⁴CO₂ evolution. Denitrification was confirmed by detection of ¹⁵N₂ in headspace samples of K¹⁵NO₃-amended anaerobic liquid cultures. In aquatic sediments, mineralization of uniformly ring-labeled [¹⁴C]atrazine occurred in both M91-3-inoculated and uninoculated sediment. Inoculation of sediments with M91-3 did not significantly enhance anaerobic mineralization of atrazine as compared to uninoculated sediment, which suggests the presence of indigenous organisms capable of anaerobic atrazine biodegradation. Results of this study suggest that the use of M91-3 in a fixed-film bioreactor may have applications in the anaerobic removal of atrazine and nitrate from aqueous media. Received: 3 September 1997 / Received revision: 4 December 1997 / Accepted: 2 January 1998     

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.     

Anaerobic transformation of carbon monoxide by microbial communities of Kamchatka hot springs

Carbon monoxide (CO) is one of the common gaseous compounds found in hot volcanic environments. It is known to serve as the growth substrate for a number of thermophilic prokaryotes, both aerobic and anaerobic. The goal of this work was to study the process of anaerobic transformation of CO by microbial communities inhabiting natural thermal environments: hot springs of Uzon Caldera, Kamchatka. The anaerobic microbial community of Treshchinny Spring (80°C, pH 6.5) was found to exhibit two peaks of affinity for CO (K _S1 = 54 nM and K _S2 = 1 μM). The actual rate of anaerobic CO transformation by the microbial community of this spring, calculated after obtaining the concentration dependence curve and extrapolated to the natural concentration of CO dissolved in the hot spring water (20 nM), was found to be 120 μmol l⁻¹ of sediment day⁻¹. In all the hot springs studied, more than 90% of the carbon of ¹⁴CO upon anaerobic incubation was recovered as ¹⁴CO₂. From 1 to 5% of ¹⁴CO was transformed to volatile fatty acids (VFA). The number of microorganisms capable of anaerobic CO oxidation determined by dilution-to-extinction method reached 10⁶ cells ml⁻¹ of sediment. CO-transforming anaerobic thermophilic microorganisms isolated from the springs under study exhibited hydrogenogenic type of CO oxidation and belonged to the bacterial genera Carboxydocella and Dictyoglomus. These data suggest a significant role of hydrogenogenic carboxydotrophic prokaryotes in anaerobic CO transformation in Uzon Caldera hot springs.     

Anaerobic microbial activities including hydrogen mediated acetogenesis within natural gas transmission lines

Microbially influenced corrosion (MIC) is being increasingly recognised as a serious problem. To investigate the role of MIC, radiotracer activity and lipid biomass measurements were performed on samples from offshore and on‐shore natural gas transmission systems. These measurements evaluated the biomass and metabolism of microbial communities residing inside transmission pipelines. Aqueous and nonaqueous hydrocarbon samples from liquid separators, sludge catchers and nodules attached to pipe walls were aseptically recovered and inoculated into anaerobic tubes for radiotracer time course experiments or preserved with chloroform‐methanol for total lipid analyses. MPN enrichments and phospholipid biomass determinations estimated microbial populations of 10⁴—10⁷ cells per gram in several samples. General microbial metabolism was demonstrated by [l‐¹⁴C]acetate incorporation into lipids and by [¹⁴C]CO₂ production from [U‐¹⁴C]glucose. [¹⁴C]Acetate was slowly mineralised to ¹⁴CO₂ without significant methane production. [¹⁴C]Acetate was produced by fermentation of [¹⁴C]glucose, [¹⁴C]palmitate and by hydrogen mediated acetogenesis in the presence of [^I4C]CO₂. In one location acetogenesis from hydrogen and carbon dioxide accounted for 0–7 mmol.l^‐1 of acetate production per week. These results demonstrated that microorganisms could utilise natural gas impurities to produce organic acids. This activity could adversely affect the structural integrity (MIC) of high pressure natural gas pipelines.     

Molecular diagnostics and chemical analysis for assessing biodegradation of polychlorinated biphenyls in contaminated soils

Summary The microbial populations in PCB-contaminated electric power substation capacitor bank soil (TVA soil) and from another PCB-contaminated site (New England soil) were compared to determine their potential to degrade PCB. Known biphenyl operon genes were used as gene probes in colony hybridizations and in dot blots of DNA extracted from the soil to monitor the presence of PCB-degrading organisms in the soils. The microbial populations in the two soils differed in that the population in New England soil was enriched by the addition of 1000 p.p.m. 2-chlorobiphenyl (2-CB) whereas the population in the TVA capacitor bank soil was not affected. PCB degradative activity in the New England soil was indicated by a 50% PCB disappearance (gas chromatography), accumulation of chlorobenzoates (HPLC), and¹⁴CO₂ evolution from¹⁴C-2CB. The PCB-degrading bacteria in the New England soil could be identified by their positive hybridization to thebph gene probes, their ability to produce the yellowmeta-cleavage product from 2,3-dihydroxybiphenyl (2,3-DHB), and the degradation of specific PCB congeners by individual isolates in resting cell assays. Although the TVA capacitor bank soil lacked effective PCB-degrading populations, addition of a PCB-degrading organism and 10 000 p.p.m. biphenyl resulted in a >50% reduction of PCB levels. Molecular characterization of soil microbial populations in laboratory scale treatments is expected to be valuable in the design of process monitoring and performance verification approaches for full scale bioremediation.     

Patterns of carbon flow from glucose to methane in a thermophilic anaerobic bioreactor

[U-¹⁴C]Glucose, added carrier-free to sludge from a thermophilic anaerobic bioreactor being fed a lignocellulose waste, was rapidly turned over with less than one-third of the original radiolabel remaining as glucose after 5 s of incubation. The primary labeled products found were [¹⁴C]acetate and ¹⁴CO₂, which were in a ratio near 2:1. Further incubation resulted in the disappearance of [¹⁴C]acetate and the appearance of an equivalent amount of label as ¹⁴CH₄ and ¹⁴CO₂. No significant production of [¹⁴C]propionate, butyrate, lactate, or ethanol was detected from [¹⁴C]glucose, even if these potential intermediates (unlabeled) were added to the sludge at a concentration of 1 mM to trap any label entering their pools. Addition of 0.8 atm (80 kPa) of H₂ to the headspace over sludge resulted in some accumulation of [¹⁴C]lactate and a corresponding decrease in [¹⁴C]acetate produced from [¹⁴C]glucose. Production of [¹⁴C]propionate, butyrate and ethanol were still not significant in the presence of H₂. Incubation of sludge for 1 h in the presence of hydrogen resulted in increases in the lactate and formate concentrations, but not those of propionate, butyrate, or ethanol. These results demonstrate that glucose was metabolized directly to acetate, CO₂, and H₂ by the microbial populations in the bioreactor with little carbon from glucose flowing through other intermediates, indicating a high degree of coupling between glucose fermentation and hydrogen uptake. The short-term response of these microbial populations to elevated H₂ partial pressures was to increase lactate production.     

Radioisotopic tracing of carbon monoxide conversion by anaerobic thermophilic prokaryotes

The rate of CO conversion by a pure culture of a thermophilic CO-oxidizing, H₂-producing bacterium Carboxydocella sp. strain 1503 was determined by the radioisotopic method. The overall daily uptake of ¹⁴CO by the bacterium was estimated at 38–56 μmol CO per 1 ml of the culture. A radioisotopic method was developed to separate and quantitatively determine the products of anaerobic CO conversion by microbial communities in hot springs. The new method was first tested on the microbial community from a sample obtained from a hot spring in Kamchatka. The potential rate of CO conversion by the anaerobic microbial community was found to be 40.75 nmol CO/cm³ sediment per day. 85% of the utilized ¹⁴CO was oxidized to carbon dioxide; 14.5% was incorporated into dissolved organic matter, including 0.2% that went into volatile fatty acids; 0.5% was used for cell biomass production; and only just over 0.001% was converted to methane.     

Consumption of Methane and CO2 by Methanotrophic Microbial Mats from Gas Seeps of the Anoxic Black Sea

The deep anoxic shelf of the northwestern Black Sea has numerous gas seeps, which are populated by methanotrophic microbial mats in and above the seafloor. Above the seafloor, the mats can form tall reef-like structures composed of porous carbonate and microbial biomass. Here, we investigated the spatial patterns of CH₄ and CO₂ assimilation in relation to the distribution of ANME groups and their associated bacteria in mat samples obtained from the surface of a large reef structure. A combination of different methods, including radiotracer incubation, beta microimaging, secondary ion mass spectrometry, and catalyzed reporter deposition fluorescence in situ hybridization, was applied to sections of mat obtained from the large reef structure to locate hot spots of methanotrophy and to identify the responsible microbial consortia. In addition, CO₂ reduction to methane was investigated in the presence or absence of methane, sulfate, and hydrogen. The mat had an average δ¹³C carbon isotopic signature of −67.1‰, indicating that methane was the main carbon source. Regions dominated by ANME-1 had isotope signatures that were significantly heavier (−66.4‰ ± 3.9 ‰ [mean ± standard deviation; n = 7]) than those of the more central regions dominated by ANME-2 (−72.9‰ ± 2.2 ‰; n = 7). Incorporation of ¹⁴C from radiolabeled CH₄ or CO₂ revealed one hot spot for methanotrophy and CO₂ fixation close to the surface of the mat and a low assimilation efficiency (1 to 2% of methane oxidized). Replicate incubations of the mat with ¹⁴CH₄ or ¹⁴CO₂ revealed that there was interconversion of CH₄ and CO_2. The level of CO₂ reduction was about 10% of the level of anaerobic oxidation of methane. However, since considerable methane formation was observed only in the presence of methane and sulfate, the process appeared to be a rereaction of anaerobic oxidation of methane rather than net methanogenesis.     

Dechlorination of polychlorinated biphenyl congeners by an anaerobic microbial consortium

  An anaerobic methanogenic microbial consortium, developed in a granular form, exhibited extensive dechlorination of defined polychlorinated biphenyl (PCB) congeners. A 2,3,4,5,6-pentachlorobiphenyl was dechlorinated to biphenyl via 2,3,4,6-tetrachlorobiphenyl, 2,4,6-trichlorobiphenyl, 2,4-dichlorobi-phenyl and 2-chlorobiphenyl (CB). Removal of chlorine atoms from all three positions of the biphenyl ring, i.e., ortho, meta and para, was observed during this reductive dechlorination process. Biphenyl was identified as one of the end-products of the reductive dechlorination by GC-MS. After 20 weeks, the concentrations of the dechlorination products 2,4,6-CB, 2,4-CB, 2-CB and biphenyl were 8.1, 41.2, 3.0 and 47.8 μM respectively, from an initial 105 μM 2,3,4,5,6-CB. The extent and pattern of the dechlorination were further confirmed by the dechlorination of lightly chlorinated congeners including 2-CB, 3-CB, 4-CB, 2,4-CB and 2,6-CB individually. This study indicates that the dechlorination of 2,3,4,5,6-CB to biphenyl is due to ortho, meta and para dechlorination by this anaerobic microbial consortium. Received: 30 April 1996 / Received revision: 26 July 1996 / Accepted: 5 August 1996     

Methane oxidation and methane driven redox process during sequential reduction of a flooded soil ecosystem

A laboratory incubation study conducted to assess the temporal variation of CH₄ oxidation during soil reduction processes in a flooded soil ecosystem. A classical sequence of microbial terminal electron accepting process observed following NO₃ ^? reduction, Fe³⁺ reduction, SO₄ ^2? reduction and CH₄ production in flooded soil incubated under initial aerobic and helium-flushed anaerobic conditions. CH₄ oxidation in the slurries was influenced by microbial redox process during slurry reduction. Under aerobic headspace condition, CH₄ oxidation rate (k) was stimulated by 29 % during 5 days (NO₃ ^? reduction) and 32 % during both 10 days (Fe³⁺) and 20 days (early SO₄ ^2? reduction) over unreduced slurry. CH₄ oxidation was inhibited at the later methanogenic period. Contrastingly, CH₄ oxidation activity in anaerobic incubated slurries was characterized with prolonged lag phase and lower CH₄ oxidation. Higher CH₄ oxidation rate in aerobically incubated flooded soil was related to high abundance of methanotrophs (r?=?0.994, p?<?0.01) and ammonium oxidizers population (r?=?0.184, p?<?0.05). Effect of electron donors NH₄ ⁺, Fe^2+, S^2? on CH₄ oxidation assayed to define the interaction between reduced inorganic species and methane oxidation. The electron donors stimulated CH₄ oxidation as well as increased the abundance of methanotrophic microbial population except S^2? which inhibited the methanotrophic activity by affecting methane oxidizing bacterial population. Our result confirmed the complex interaction between methane-oxidizing microbial groups and redox species during sequential reduction processes of a flooded soil ecosystem.     

Glucose metabolism in anaerobic rice seedlings

More than 80% of the radioactivity from [U-¹⁴C]glucose metabolised by anaerobic rice seedlings or by excised roots or coleoptiles was recovered as ethanol plus CO₂; less than 5% was recovered as water-soluble acidic components. Rates of ¹⁴CO₂ formation from [U-¹⁴C]glucose were similar in roots and coleoptiles in both N₂ and air atmospheres. More ¹⁴CO₂ was formed from [U-¹⁴C]glucose than could be accounted for by ethanolic fermentation, and the specific yields of ¹⁴CO₂ from [6-¹⁴C]glucose and [1-¹⁴C]glucose gave unusually high C-6/C-1 ratios (1.7) in the anaerobic coleoptile. The results may indicate that appreciable pentan synthesis occurs in the anaerobic coleoptile.     

Methylmercury Decomposition in Sediments and Bacterial Cultures: Involvement of Methanogens and Sulfate Reducers in Oxidative Demethylation

Demethylation of monomethylmercury in freshwater and estuarine sediments and in bacterial cultures was investigated with ¹⁴CH₃HgI. Under anaerobiosis, results with inhibitors indicated partial involvement of both sulfate reducers and methanogens, the former dominating estuarine sediments, while both were active in freshwaters. Aerobes were the most significant demethylators in estuarine sediments, but were unimportant in freshwater sediments. Products of anaerobic demethylation were mainly ¹⁴CO₂ as well as lesser amounts of ¹⁴CH₄. Acetogenic activity resulted in fixation of some ¹⁴CO₂ produced from ¹⁴CH₃HgI into acetate. Aerobic demethylation in estuarine sediments produced only ¹⁴CH₄, while aerobic demethylation in freshwater sediments produced small amounts of both ¹⁴CH₄ and ¹⁴CO₂. Two species of Desulfovibrio produced only traces of ¹⁴CH₄ from ¹⁴CH₃HgI, while a culture of a methylotrophic methanogen formed traces of ¹⁴CO₂ and ¹⁴CH₄ when grown on trimethylamine in the presence of the ¹⁴CH₃HgI. These results indicate that both aerobes and anaerobes demethylate mercury in sediments, but that either group may dominate in a particular sediment type. Aerobic demethylation in the estuarine sediments appeared to proceed by the previously characterized organomercurial-lyase pathway, because methane was the sole product. However, aerobic demethylation in freshwater sediments as well as anaerobic demethylation in all sediments studied produced primarily carbon dioxide. This indicates the presence of an oxidative pathway, possibly one in which methylmercury serves as an analog of one-carbon substrates.     

Methodological Modifications for Accurate and Efficient Determination of Contaminant Biodegradation in Unsaturated Calcareous Soils

Many techniques for quantifying microbial biodegradation of ¹⁴C-labeled compounds use soil-water slurries and trap mineralization-derived ¹⁴CO₂ in solution wells suspended within the incubation flasks. These methods are not satisfactory for studies of arid-region soils that are highly calcareous and unsaturated because (i) slurries do not simulate unsaturated conditions and (ii) the amount of CO₂ released from calcareous soils exceeds the capacity of the suspended well. This report describes simple, inexpensive methodological modifications for quantifying microbial degradation of [¹⁴C]benzene and 1,2-dichloro[U-¹⁴C]ethane in calcareous soils under unsaturated conditions. Soils at 50% water holding capacity were incubated with labeled contaminants for periods up to 10 weeks, followed by acidification of the soil and trapping of the evolved CO₂ in a separate container of 2 N NaOH. The CO₂ was transferred from the incubation flask to the trap solution by a gas transfer shunt containing activated charcoal to remove any volatilized labeled organics. The amount of ¹⁴CO₂ in the trap solution was measured by scintillation counting (disintegrations per minute). The method was tested by using two regional unamended surface soils, a sandy aridisol and a clay-rich riparian soil. The results demonstrated that both [¹⁴C]benzene and 1,2-dichloro[U-¹⁴C]ethane were mineralized to release substantial amounts of ¹⁴CO₂ within 10 weeks. Levels of mineralization varied with contaminant type, soil type, and aeration status (anaerobic vs. aerobic); no significant degradation was observed in abiotic control samples. Methodological refinements of this technique resulted in total ¹⁴CO₂ recovery efficiency of approximately 90%.     

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.     

Linking Belowground Carbon Allocation to Anaerobic CH4 and CO2 Production in a Forested Peatland,New York State

Heterotrophic soil microorganisms rely on carbon (C) allocated belowground in plant production, but belowground C allocation (BCA) by plants is a poorly quantified part of ecosystem C cycling, especially, in peat soil. We applied a C balance approach to quantify BCA in a mixed conifer-red maple (Acer rubrum) forest on deep peat soil. Direct measurements of CH₄ and CO₂ fluxes across the soil surface (soil respiration), production of fine and small plant roots, and aboveground litterfall were used to estimate respiration by roots, by mycorrhizae and by free-living soil microorganisms. Measurements occurred in two consecutive years. Soil respiration rates averaged 1.2 bm μmol m^{? 2} s^{? 1} for CO₂ and 0.58 nmol m^{? 2} s^{? 1} for CH₄ (371 to 403 g C m^{? 2} year^{? 1}). Carbon in aboveground litter (144 g C m^{? 2} year^{? 1}) was 84% greater than C in root production (78 g C m^{? 2} year^{? 1}). Complementary in vitro assays located high rates of anaerobic microbial activity, including methanogenesis, in a dense layer of roots overlying the peat soil and in large-sized fragments within the peat matrix. Large-sized fragments were decomposing roots and aboveground leaf and twig litter, indicating that relatively fresh plant production supported most of the anaerobic microbial activity. Respiration by free-living soil microorganisms in deep peat accounted for, at most, 29 to 38 g C m^{? 2} year^{? 1}. These data emphasize the close coupling between plant production, ecosystem-level C cycling and soil microbial ecology, which BCA can help reveal.     

Medium factors on anaerobic production of rhamnolipids by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> SG and a simplifying medium for in situ microbial enhanced oil recovery applications

Aerobic production of rhamnolipid by Pseudomonas aeruginosa was extensively studied. But effect of medium composition on anaerobic production of rhamnolipid by P. aeruginosa was unknown. A simplifying medium facilitating anaerobic production of rhamnolipid is urgently needed for in situ microbial enhanced oil recovery (MEOR). Medium factors affecting anaerobic production of rhamnolipid were investigated using P. aeruginosa SG (Genbank accession number KJ995745). Medium composition for anaerobic production of rhamnolipid by P. aeruginosa is different from that for aerobic production of rhamnolipid. Both hydrophobic substrate and organic nitrogen inhibited rhamnolipid production under anaerobic conditions. Glycerol and nitrate were the best carbon and nitrogen source. The commonly used N limitation under aerobic conditions was not conducive to rhamnolipid production under anaerobic conditions because the initial cell growth demanded enough nitrate for anaerobic respiration. But rhamnolipid was also fast accumulated under nitrogen starvation conditions. Sufficient phosphate was needed for anaerobic production of rhamnolipid. SO₄ ^2? and Mg²⁺ are required for anaerobic production of rhamnolipid. Results will contribute to isolation bacteria strains which can anaerobically produce rhamnolipid and medium optimization for anaerobic production of rhamnolipid. Based on medium optimization by response surface methodology and ions composition of reservoir formation water, a simplifying medium containing 70.3 g/l glycerol, 5.25 g/l NaNO₃, 5.49 g/l KH₂PO₄, 6.9 g/l K₂HPO₄·3H₂O and 0.40 g/l MgSO₄ was designed. Using the simplifying medium, 630 mg/l of rhamnolipid was produced by SG, and the anaerobic culture emulsified crude oil to EI₂₄ = 82.5 %. The simplifying medium was promising for in situ MEOR applications.     

Monochloro- and dichloroacetic acids as carbon and energy sources for a stable,methanogenic mixed culture

A stable methanogenic mixed culture was enriched from an industrial environment to utilize chloroacetate as sole carbon and energy source for growth. It immobilized spontaneously on activated charcoal and grew reproducibly on this carrier in a fluidized bed reactor when supplied with an anaerobic mineral salts medium. Substrate disappearance was complete. Methane, CO₂ and chloride ions were conclusively identified as the metabolic products and quantified. The growth yield from chloroacetate was about 1 g of protein/mol of carbon. The calculated degradation rate in the fluidized bed reactor was 0.2 to 0.8 mmol/l·h. The first metabolic intermediate from [2–¹³C]monochloroacetate in portions of biofilm-coated carrier was shown by ¹³C-NMR to be glycolate, from which ¹³CO₂ and ¹³CH₄ were formed. Glycolate was formed in an oxygen-insensitive hydrolysis, but its conversion to CO₂ and CH₄ was strictly anaerobic and sensitive to inhibition by bromoethanesulfonate. Degradation of [1-¹⁴C]-and [2-¹⁴C]-chloroacetate each yielded the same amount of [¹⁴C]-methane. We thus presume glycolate to be cleaved to CO₂ and H₂, which were the substrates for methanogenesis. Dehalogenation was limited to chlorobromo-, iodo- and dichloroacetate. These four compounds and glycolate were utilized as the sole carbon and energy sources by the methanogenic mixed culture.     

Nitrogen Fixation Dynamics of Two Diazotrophic Communities in Mono Lake, California

Two types of diazotrophic microbial communities were found in the littoral zone of alkaline hypersaline Mono Lake, California. One consisted of anaerobic bacteria inhabiting the flocculent surface layers of sediments. Nitrogen fixation (acetylene reduction) by flocculent surface layers occurred under anaerobic conditions, was not stimulated by light or by additions of organic substrates, and was inhibited by O₂, nitrate, and ammonia. The second community consisted of a ball-shaped association of a filamentous chlorophyte (Ctenocladus circinnatus) with diazotrophic, nonheterocystous cyanobacteria, as well as anaerobic bacteria (Ctenocladus balls). Nitrogen fixation by Ctenocladus balls was usually, but not always, stimulated by light. Rates of anaerobic dark fixation equaled those in the light under air. Fixation in the light was stimulated by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea and by propanil [N-(3,4-dichlorophenyl)propanamide]. 3-(3,4-Dichlorophenyl)-1,1-dimethyl urea-elicited nitrogenase activity was inhibited by ammonia (96%) and nitrate (65%). Fixation was greatest when Ctenocladus balls were incubated anaerobically in the light with sulfide. Dark anaerobic fixation was not stimulated by organic substrates in short-term (4-h) incubations, but was in long-term (67-h) ones. Areal estimates of benthic N₂ fixation were measured seasonally, using chambers. Highest rates (~29.3 μmol of C₂H₄ m⁻² h⁻¹) occurred under normal diel regimens of light and dark. These estimates indicate that benthic N₂ fixation has the potential to be a significant nitrogen source in Mono Lake.     

Anaerobic dechlorination and mineralization of pentachlorophenol and 2,4,6-trichlorophenol by methanogenic pentachlorophenol-degrading granules

Anaerobic granules developed for the treatment of pentachlorophenol (PCP) completely minearilized¹⁴C-labeled PCP to¹⁴CH₄ and¹⁴CO₂. Release of chloride ions from PCP was performed by live cells in the granules under anaerobic conditions. No chloride ions were released under aerobic conditions or by autoclaved cells. Addition of sulfate enhanced the initial chloride release rate and accelerated the process of mineralization of¹⁴C-labeled PCP. Addition of molybdate (10 mM) inhibited the chloride release rate and severely inhibited PCP mineralization. This suggests involvement of sulfate-reducing bacteria in PCP dechlorination and mineralization. Addition of 2-bromoethane sulfonate slightly decreased the chloride release rate and completely stopped production of¹⁴CH₄ and¹⁴CO₂ from [¹⁴C]PCP. 2,4,6-trichlorophenol was observed as an intermediate during PCP dechlorination. On the basis of experimental results, dechlorination of 2,4,6-trichlorophanol by the granules was conducted through 2,4-dichlorophenol, 4-chlorophenol or 2-chlorophenol to phenol at pH 7.0–7.2.     

The use of multiple-vessel,open flow systems to investigate carbon flow in anaerobic microbial communities

Five vessels, connected in series, were used for a continuous flow system to model carbon flow in anaerobic microbial communities. Two such 5-vessel systems were constructed, the inflows containing 10 mM sulfate and either 10 mM glucose or benzoate. Dilution was slow (D=0.0018 h^?1 for the whole system). Analyses of dissolved organic and inorganic carbon, and of CO₂ and CH₄, showed that the systems attained steady states in which biomass was constant, although there was net biosynthesis in the early vessels and net mineralization in succeeding vessels. Examination of the distributions of sulfate reduction, methanogenesis, and of H₂+CO₂-utilizing fatty acid-forming bacteria revealed spatial separation of these functional groups of bacteria in different vessels of the array, resembling the vertical spatial separation found in many natural sediments. Such model systems should, therefore, prove valuable in investigating the many microbial activities that contribute to the flow of carbon in anaerobic microbial communities.