Hydrogen as a substrate for methanogenesis and sulphate reduction in anaerobic saltmarsh sediment

Hydrogen gas stimulated sulphate reduction in a saltmarsh sediment and the importance of H₂ transferred from organotrophic bacteria to the sulphate-reducers is discussed. -fluorolactate inhibited sulphate reduction whether lactate, ethanol or hydrogen was being used as growth substrate. When added to sediment -fluorolactate inhibited sulphate reduction with a consequent increase in methane production.Addition of H₂ stimulated methanogenesis in sediment and this stimulation was greater if CO₂ was also present. Hydrogen availability was the primary limitation of methanogenesis but the low concentration of dissolved CO₂ in seawater may limit methane production even if H₂ is available.The removal of inhibition of methanogenesis by the use of fluorolactate to suppress sulphate reduction or by the provision of hydrogen indicates competitive inhibition of methanogens by sulphate reducers utilizing transferred hydrogen.Abbreviations HSRB hydrogen utilizing sulphate reducing bacteria - HDO hydrogen donating organism     

Production of trace levels of carbon monoxide during methanogenesis on acetate and methanol

Summary Production of trace levels of carbon monoxide was consistently observed in the off-gas of a laboratory anaerobic digester fed Waste Activated Sludge. Inocula from this digester was enriched for acetate and methanol utilizing methanogenic populations. These enriched inocula were then monitored in batch assays for carbon monoxide and hydrogen production. Results demonstrated that carbon monoxide is produced during methanogenesis on both substrates. Subsequent utilization of CO was observed to occur after methane production was essentially complete for the assays conducted with methanol. Carbon monoxide evolution during methanogenesis on acetate displayed a markedly different trend from that observed from methanol.     

Thermophilic sulfate reduction and methanogenesis with methanol in a high rate anaerobic reactor

Sulfate reduction outcompeted methanogenesis at 65 degrees C and pH 7.5 in methanol and sulfate-fed expanded granular sludge bed reactors operated at hydraulic retention times (HRT) of 14 and 3.5 h, both under methanol-limiting and methanol-overloading conditions. After 100 and 50 days for the reactors operated at 14 and 3.5 h, respectively, sulfide production accounted for 80% of the methanol-COD consumed by the sludge. The specific methanogenic activity on methanol of the sludge from a reactor operated at HRTs of down to 3.5 h for a period of 4 months gradually decreased from 0. 83 gCOD. gVSS(-1). day(-1) at the start to a value of less than 0.05 gCOD. gVSS(-1). day(-1), showing that the relative number of methanogens decreased and eventually became very low. By contrast, the increase of the specific sulfidogenic activity of sludge from 0. 22 gCOD. gVSS(-1). day(-1) to a final value of 1.05 gCOD. gVSS(-1). day(-1) showed that sulfate reducing bacteria were enriched. Methanol degradation by a methanogenic culture obtained from a reactor by serial dilution of the sludge was inhibited in the presence of vancomycin, indicating that methanogenesis directly from methanol was not important. H(2)/CO(2) and formate, but not acetate, were degraded to methane in the presence of vancomycin. These results indicated that methanol degradation to methane occurs via the intermediates H(2)/CO(2) and formate. The high and low specific methanogenic activity of sludge on H(2)/CO(2) and formate, respectively, indicated that the former substrate probably acts as the main electron donor for the methanogens during methanol degradation. As sulfate reduction in the sludge was also strongly supported by hydrogen, competition between sulfate reducing bacteria and methanogens in the sludge seemed to be mainly for this substrate. Sulfate elimination rates of up to 15 gSO(4)(2-)/L per day were achieved in the reactors. Biomass retention limited the sulfate elimination rate.     

Effect of sulfate on carbon and electron flow during microbial methanogenesis in freshwater sediments.

The effect of sulfate on methane production in Lake Mendota sediments was investigated to clarify the mechanism of sulfate inhibition of methanogenesis. Methanogenesis was shown to be inhibited by the addition of as little as 0.2 mM sulfate. Sulfate inhibition was reversed by the addition of either H2 or acetate. Methane evolved when inhibition was reversed by H2 additions was derived from 14CO2. Conversely, when acetate was added to overcome sulfate inhibition, the evolved methane was derived from [2-14C]acetate. A competition for available H2 and acetate was proposed as the mechanism by which sulfate inhibited methanogenesis. Acetate was shown to be metabolized even in the absence of methanogenic activity. In the presence of sulfate, the methyl position of acetate was converted to CO2. The addition of sulfate to sediments did not result in the accumulation of significant amounts of sulfide in the pore water. Sulfate additions did not inhibit methanogenesis unless greater than 100 mug of free sulfide per ml was present in the pore water. These results indicate that carbon and electron flow are altered when sulfate is added to sediments. Sulfate-reducing organisms appear to assume the role of methanogenic bacteria in sulfate-containing sediments by utilizing methanogenic precursors.     

Genomic markers of ancient anaerobic microbial pathways: sulfate reduction,methanogenesis, and methane oxidation

Genomic markers for anaerobic microbial processes in marine sediments-sulfate reduction, methanogenesis, and anaerobic methane oxidation-reveal the structure of sulfate-reducing, methanogenic, and methane-oxidizing microbial communities (including uncultured members); they allow inferences about the evolution of these ancient microbial pathways; and they open genomic windows into extreme microbial habitats, such as deep subsurface sediments and hydrothermal vents, that are analogs for the early Earth and for extraterrestrial microbiota.     

Hydrogenase activity and methanogenesis in anaerobic sewage sludge,in rumen liquid,and in freshwater sediments

Summary The applicability of hydrogenase determinations to the evaluation of hydrogen transfer reactions occurring within methanogenic environments was investigated. Enzymatic hydrogen production was determined in digester sludge, river sediments, and rumen liquid using reduced methyl viologen, formate, and pyruvate as hydrogen donors. Hydrogenase determinations turned out not to be inhibited by toxic compounds present in sediments of the polluted river Saar. Comparative kinetic studies of the conversion of acetate and of hydrogen to methane support the assumption that carbon dioxide reduction by hydrogen accounts for the major part of methane formed in river sediments. In rumen liquid and in river sediments similar enzyme patterns were observed which were different from that found in digester sludge. The rates of methanogenesis correlated well with hydrogenase activities in all ecosystems studied: Correlation coefficients ranged from 0.84 to 0.95. Rumen liquid and river sediments exhibited higher hydrogenase activities than digester sludge when compared at identical rates of methane production. According to these results, the hydrogenase determination is applicable to the evaluation of the hydrogen transfer, occurring within the microbial biomass of anaerobic ecosystems.     

Changes in bacterial communities from anaerobic digesters during petroleum hydrocarbon degradation

Anaerobic biodegradation of petroleum hydrocarbons (PHC) to methane has been recognized to occur in oil reservoirs and contaminated surface sites alike. This process could be employed efficiently for the treatment of contaminated materials, including petrochemical wastes and PHC-contaminated soil, since no external electron acceptor is required. Moreover, the controlled production of methane in digestion plants, similarly to the anaerobic digestion (AD) of energy crops or organic residues, would enable for energy recovery from these wastes. At present, little is known about the bacterial communities involved in and responsible for hydrocarbon fermentation, the initial step in PHC conversion to methane. In the present study, the fate of two different methanogenic communities derived from the AD of wastewater (WWT) and of biowaste, mixed with PHC-contaminated soil (SWT), was monitored during incubation with PHC using denaturing gradient gel electrophoresis (DGGE) of 16S rDNA genes amplified with Bacteria-specific primers. During 11 months of incubation, slight but significant degradation of PHC occurred in both sludges and distinct bacterial communities were developing. In both sludges, Bacteroidetes were found. In addition, in WWT, the bacterial community was found to be dominated by Synergistetes and Proteobacteria, while Firmicutes and unidentified members were abundant in SWT. These results indicate that bacterial communities from anaerobic digesters can adapt to and degrade petroleum hydrocarbons. The decontamination of PHC-containing waste via fermentative treatment appears possible.     

The effect of incubation temperature on steady-state concentrations of hydrogen and volatile fatty acids during anaerobic degradation in slurries from wetland sediments

Abstract Increasing the incubation temperature of two swamp slurries from 2°C to37°C resulted in a 8- to 18-fold increase in the H₂ partial pressure. The concentration of volatile fatty acids remained fairly constant except for butyrate, which decreased with increasing temperature. Calculation of Gibbs free energies of syntrophic degradation of butyrate and propionate, and of methanogenesis from acetate and H₂ revealed that these reactions were exergonic after the slurries had stabilized at the incubation temperatures. The changes in H₂ partial pressure and butyrate concentration with temperature were found important to render the processes exergonic within the tested temperature range.     

The role of formylmethanofuran: tetrahydromethanopterin formyltransferase in methanogenesis from carbon dioxide

Formylmethanofuran: tetrahydromethanopterin formyltransferase was purified to electrophoretic homogeneity from cells of Methanobacterium thermoautotrophicum. The enzyme is a tetramer of similar or identical subunits (Mr = 41,000). The equilibrium favors transfer of the formyl group to tetrahydromethanopterin (H4MPT) at physiological pH. The product of formyl transfer by the purified enzyme was shown by a number of criteria to be 5-formyl-H4MPT, as opposed to 10-formyl-H4MPT or 5,10-methenyl-H4MPT. Reconstitution of a portion of the methanogenic C1 cycle was effected by combining purified formyltransferase, methenyl-H4MPT cyclohydrolase, formylmethanofuran, and H4MPT to give methenyl-H4MPT. Additional reconstitution experiments established that the formyltransferase is an essential enzyme for the conversion of carbon dioxide to methane. In conjunction with previously published data (Donnelly, M.I., Escalante-Semerena, J.C., Rinehart, K. L., Jr., and Wolfe, R.S. (1985) Arch. Biochem. Biophys. 242, 430-439), these data substantiate the role of 5-formyl-H4MPT as an intermediate of methanogenesis.     

The anaerobic degradation of l-serine and l-threonine in enterobacteria: networks of pathways and regulatory signals

The mechanisms controlling the biosynthesis and degradation of l-serine and l-threonine are remarkably complex. Their metabolism forms a network of pathways linking several amino acids, central primary metabolites such as pyruvate, oxaloacetate and 3-phosphoglycerate, and C₁ metabolism. Studies on the degradation of these amino acids in Escherichia coli have revealed the involvement of fascinating enzymes that utilise quite diverse catalytic mechanisms. Moreover, it is emerging that both environmental and metabolic signals have a major impact in controlling enzyme synthesis. This is exemplified by the anaerobically regulated tdc operon, which encodes a metabolic pathway for the degradation of serine and threonine. Studies on this pathway are beginning to provide insights into how an organism adapts its genetic makeup to meet the physiological demands of the cell. Received: 30 August 1998 / Accepted: 9 October 1998     

Interrelation between the pathways of isoprenoid biosynthesis and carbon source catabolism in anaerobic and facultatively anaerobic bacteria

Data on the interrelation between the pathways of the carbon source catabolism and isoprenoid biosynthesis in anaerobic and facultatively anaerobic bacteria were obtained. Two pathways of isoprenoid biosynthesis (nonmevalonate and mevalonate) were revealed in the representatives of the genus Clostridium. The nonmevalonate pathway of isoprenoid biosynthesis and the glycolytic pathway of substrate oxidation are typical of glucose-grown bacteria, whereas the pentose phosphate cycle operates in xylose-grown bacteria. The mevalonate pathway of isoprenoid biosynthesis was revealed in strain Clostridium thermosaccharolyticum DSM 571 grown in the presence of mevinolin, as well as in a number of lactic acid bacteria. Mevinolin is known to react with the lactate dehydrogenase complex, preventing reduction of pyruvate. The nonmevalonate pathway of isoprenoid biosynthesis was revealed in Bifidobacterium bifidum. The role of different metabolic pathways in isoprenoid biosynthesis is discussed.     

Sources of organic carbon in mangrove sediments: variability and possible ecological implications

Mangrove sediments from three different mangrove ecosystems (Coringa Wildlife Sanctuary in the Godavari Delta, Andhra Pradesh, India, and Galle and Pambala, south-west Sri Lanka) were analysed for their organic carbon content, elemental ratios (C:N) and carbon stable isotope composition. Organic carbon content (0.6 – 31.7% dry weight), C/N ratios (7.0 – 27.3) and ¹³C (between –29.4 and –20.6) showed a wide range of values. Lower stocks of organic carbon coincided with low C/N (atom) ratios and less negative ¹³C values, indicating import of marine or estuarine particulate suspended matter. High organic carbon stocks coincided with high C/N ratios and ¹³C values close, but not equal, to those of the mangrove vegetation. The variations observed in this study and published literature data could be adequately described by a simple two-end mixing model, whereby marine/estuarine suspended matter and mangrove litter were taken as end members. Thus, while in some mangrove ecosystems or vegetation zones, organic carbon stocks can be very high and are almost entirely of mangrove origin, there also appear to be cases in which deposited estuarine or marine suspended matter is the dominant source of organic carbon and nitrogen in mangrove sediments. This situation is remarkably similar to that observed in temperate salt marsh ecosystems where the importance of local vascular plant production to the sediment organic carbon pool is equally variable. The observed high variability in organic matter origin is thought to have a major impact on the overall carbon dynamics in intertidal mangrove ecosystems.     

Stimulation of methanol degradation in UASB reactors: in situ versus pre-loading cobalt on anaerobic granular sludge

The effect of pre-loading and in situ loading of cobalt onto a cobalt-limited granular sludge on the performance of methanol fed bioreactors was investigated. One upflow anaerobic sludge bed (UASB) reactor was inoculated with cobalt pre-loaded sludge (24h; 30 degrees C; 1 mM CoCl2) and a second UASB with unloaded sludge. The UASB reactors (30 degrees C; pH 7) were operated for 77 days at 8 h hydraulic retention time and organic loading rates ranging from 5 to 20 g COD.L reactor(-1).d(-1). Cobalt pre-loading clearly stimulated the methanogenic activity of the sludge with methanol as the substrate, e.g., after 30 days of reactor operation this activity was 5.8 times higher than that of the cobalt unloaded sludge. During the experiment, part of the cobalt leached from the pre-loaded sludge, i.e., 54% of the cobalt content was lost during the 77 days of reactor operation. Sequential metal extraction showed that losses mainly occurred from the exchangeable and carbonate fraction and in the sludge remaining cobalt was mainly present in the organic/sulfide fraction of the sludge. In situ loading of cobalt in the unloaded UASB reactor on day 57 by adding 31 microM cobalt to the influent for a 24-h period (16% of the cobalt present in the loaded sludge at day 11) resulted in a 4 time increase of the methanogenic activity of the sludge with methanol as the substrate at the end of the reactor experiment, while the accumulated amount of cobalt in the sludge only amounted to 6% of the cobalt accumulated in the loaded sludge (on day 11). This study showed that both pre-loading sludge and in situ loading are adequate for achieving an increased reactor performance of methanol fed UASB reactors operating under cobalt limitation. However, the in situ dosing procedure needs substantially lower amounts of cobalt, while it also gives significantly smaller losses of cobalt with the effluent.     

Competition between methanogenesis and quinone respiration for ecologically important substrates in anaerobic consortia

Anaerobic consortia obtained from a wide variety of environments were tested for oxidizing several ecologically significant substrates with the humic model compound, anthraquinone-2,6-disulfonate (AQDS), as terminal electron acceptor. All the substrates, including hydrogen, acetate, propionate, methanol and lactate, were completely or partially converted to methane when bicarbonate was the only electron acceptor available. Addition of AQDS (20 mM) to the cultures prevented methanogenesis in most cases and AQDS reduction became the preferred pathway. AQDS was shown to be toxic for methanogenesis and this effect played an important role in enabling quinone-respiring bacteria to outcompete methanogens. Furthermore, AQDS respiration is thermodynamically more favorable than methanogenesis. All the consortia evaluated were capable of oxidizing hydrogen linked to the reduction of AQDS. Most inocula tested were also able to oxidize acetate and lactate in the same way. When methanol was provided as an electron donor competition between methanogenesis and acetogenesis occurred. Acetate accumulated from the latter process was responsible for quinone respiration. These results suggest that quinone-respiring bacteria are ubiquitous and that quinones in humus may significantly contribute to carbon cycling process by serving as a terminal electron acceptor for the anaerobic microbial oxidation of a wide variety of ecologically important substrates.     

Acetate oxidation in a thermophilic anaerobic sewage-sludge digestor: the importance of non-aceticlastic methanogenesis from acetate

Abstract Acetate conversion to methane in a steady-state, thermophilic (60°C) anaerobic sewage-sludge digestor and in a thermophilic (60°C) acetate chemostat inoculated with anaerobic thermophilic sewage sludge, was investigated by use of radiotracer methodology. When the acetate pool in the sewage-sludge digestor was 1–2 mM, 4.1% of 2-labeled acetate was converted to CO₂. However, when acetate was consumed to less than 1.0 mM, prior to isotopic examinations, this increased to 14.1%. Microscopic observations showed a shift in the acetate-degrading populations during start-up of the acetate-limited chemostat inoculated from the sewage-sludge digestor. Large numbers of Methanosarcina -aggregates were seen during the first 100–150 days of operation, while Methanosaeta -like rods were not observed. The Methanosarcina -aggregates disappeared concurrently with a decrease in the acetate concentration to approx. 0.4 mM, and the culture consisted mainly of a large number of autofluorescent, short rods together with fewer and longer, non-fluorescent, rods. Non-aceticlastic oxidation of acetate to methane was the mechanism of the acetate conversion in the chemostat after 7 months of operation. Our results indicate that the concentration of acetate can influence the mechanism of acetate conversion during thermophilic anaerobic digestion of organic matter.     

土壤和沉积物中多氯代有机化合物厌氧降解研究进展

多氯代有机化合物(PCOCs)是土壤和沉积物中的典型污染物,厌氧条件下PCOCs能够发生脱氯发应,从而使其毒性大大降低,脱氯后形成的低氯代化合物可以进一步好氧降解,直至完全矿化。从PCOCs的降解过程出发,重点综述了几种典型PCOCs的厌氧脱氯机理以及几种重要影响因素;阐明了脱氯反应是PCOCs厌氧降解的关键步骤,反应的发生必须有还原剂提供电子,微生物的参与尤为重要;同时展望了同位素示踪法在研究PCOCs降解机制上的应用,以及开发高效降解PCOCs微生物的必要性等。     

Complementary pathways of dissolved organic carbon removal pathways in clear-water Amazonian ecosystems: photochemical degradation and bacterial uptake

Dissolved organic carbon (DOC) photochemical reactions establish important links between DOC and planktonic bacteria. We hypothesize that seasonal changes in DOC quality, related to the flood pulse, drive the effects of light-DOC interactions on uptake by planktonic bacteria uptake in clear-water Amazonian ecosystems. Water samples from two ecosystems (one lake and one stream) were incubated in sunlight during different hydrological periods and were then exposed to bacterial degradation. Photochemical and bacterial degradation were driven by seasonal DOC inputs. Bacterial mineralization was the main degradation pathway of autochthonous DOC in the lake, while allochthonous DOC was more available for photochemical oxidation. We suggest that sunlight enhances the bacterial uptake of refractory DOC but does not alter uptake of labile forms. We also observed a positive relationship between sunlight and bacterial degradation of DOC, instead of competition. We conclude that photochemical reactions and bacteria complementarily degrade the different sources of DOC during the flood pulse in Amazonian clear-water aquatic ecosystems.     

Damage and degradation rates of extracellular DNA in marine sediments: implications for the preservation of gene sequences

The extracellular DNA pool in marine sediments is the largest reservoir of DNA of the world oceans and it potentially represents an archive of genetic information and gene sequences involved in natural transformation processes. However, no information is at present available for the gene sequences contained in the extracellular DNA and for the factors that influence their preservation. In the present study, we investigated the depurination and degradation rates of extracellular DNA in a variety of marine sediment samples characterized by different ages (up to 10 000 years) and environmental conditions according to the presence, abundance and diversity of prokaryotic gene sequences. We provide evidence that depurination of extracellular DNA in these sediments depends upon the different environmental factors that act synergistically and proceeds at much slower rates than those theoretically predicted or estimated for terrestrial ecosystems. These findings suggest that depurination in marine sediments is not the main process that limits extracellular DNA survival. Conversely, DNase activities were high suggesting a more relevant role of biologically driven processes. Amplifiable prokaryotic 16S rDNA sequences were present in most benthic systems analysed, independent of depurination and degradation rates and of the ages of the sediment samples. Additional molecular analyses revealed that the extracellular DNA pool is characterized by relatively low-copy numbers of prokaryotic 16S rDNA sequences that are highly diversified. Overall, our results suggest that the extracellular DNA pool in marine sediments represents a repository of genetic information, which can be used for improving our understanding of the biodiversity, functioning and evolution of ecosystems over different timescales.     

Use of a bromobenzoate for cross-adaptation of anaerobic bacteria in Lake Ontario sediments for degradation of chlorinated aromatics

The capacity of anaerobic micro-organisms in the sediment of a freshwater lake to degrade halogenated benzoates was investigated. Sediments collected from Lake Ontario along the Toronto waterfront (Ontario, Canada) were incubated with halogenated benzoates and dehalogenation was measured by high pressure liquid chromatography (HPLC). Following adaptation to monohalogenated benzoates (3-bromobenzoate, 3-chlorobenzoate), cross-adaptation to complex halogenated aromatics (3,5-dichlorobenzoate, 4-amino-3,5-dichlorobenzoate), was assessed by monitoring their depletion by HPLC. Prior adaptation to 3-bromobenzoate resulted in a more rapid depletion of the complex halogenated aromatics (3,5-dichlorobenzoate and 4-amino-3,5-dichlorobenzoate) than prior adaptation to 3-chlorobenzoate. The results suggest that cross-adaptation may be an approach to a more rapid biodegradation of complex pollutants in lake sediments or in wastewater treatment systems, with the 3-bromobenzeate preferred over the 3-chlorobenzoate as the adaptation substrate.