Showing papers on "Methanogen published in 1999"

Methane-consuming archaebacteria in marine sediments

Abstract: Large amounts of methane are produced in marine sediments but are then consumed before contacting aerobic waters or the atmosphere1. Although no organism that can consume methane anaerobically has ever been isolated, biogeochemical evidence indicates that the overall process involves a transfer of electrons from methane to sulphate and is probably mediated by several organisms, including a methanogen (operating in reverse) and a sulphate-reducer (using an unknown intermediate substrate)2. Here we describe studies of sediments related to a decomposing methane hydrate. These provide strong evidence that methane is being consumed by archaebacteria that are phylogenetically distinct from known methanogens. Specifically, lipid biomarkers that are commonly characteristic of archaea are so strongly depleted in carbon-13 that methane must be the carbon source, rather than the metabolic product, for the organisms that have produced them. Parallel gene surveys of small-subunit ribosomal RNA (16S rRNA) indicate the predominance of a new archael group which is peripherally related to the methanogenic orders Methanomicrobiales and Methanosarcinales.

Reducing rumen methane emissions through elimination of rumen protozoa

Abstract: Methanogens living on and within rumen ciliate protozoa may be responsible for up to 37% of the rumen methane emissions. In the absence of protozoa, rumen methane emissions are reduced by an average of 13% but this varies with diet. Decreased methane emissions from the protozoa-free rumen may be a consequence of: (1) reduced ruminal dry matter digestion; (2) a decreased methanogen population; (3) an altered pattern of volatile fatty acid production and hydrogen availability; or (4) increased partial pressure of oxygen in the rumen. The decline in methanogenesis associated with removal of protozoa is greatest on high concentrate diets and this is in keeping with protozoa being relatively more important sources of hydrogen on starch diets, because many starch-fermenting bacteria do not produce H2. Because protozoa also decrease the supply of protein available to the host animal, their elimination offers benefits in both decreasing greenhouse gas emissions and potentially increasing livestock production. Strategies for eliminating protozoa are reviewed. None of the available techniques is considered practical for commercial application and this should be addressed.

Isolation and characterization of Methanomethylovorans hollandica gen. nov., sp. nov., isolated from freshwater sediment, a methylotrophic methanogen able to grow on dimethyl sulfide and methanethiol

Abstract: Dimethyl sulfide (DMS) has an impact on global warming and acid precipitation and on the global sulfur cycle because of its oxidation products (e.g., methanesulfonic acid and SO2), which are released into the atmosphere. For this reason transformations of volatile organic sulfur compounds have been intensively studied during the past few decades. Microbial formation and degradation of DMS and methanethiol (MT) have been shown to have a significant effect on the total flux of sulfur compounds in the atmosphere (13, 14, 21). In freshwater sediments, formation of MT and DMS is balanced by degradation of these compounds, which results in low steady-state concentrations (18–20). In contrast to marine and estuarine systems, in which DMS originates mainly from dimethylsulfoniumpropionate, volatile organic sulfur compounds in freshwater sediments are derived mainly from methylation of sulfide and MT (7, 15, 18). In systems with high salt contents, degradation of MT and DMS has been attributed to members of various groups of bacteria, including aerobes (e.g., thiobacilli and Methylophaga spp.) (4, 34–36) and anaerobes (anoxygenic phototrophs, sulfate reducers, and methanogens) (8, 16, 17, 23, 25, 26, 33, 39). The activity of members of these trophic groups depends on the light intensity and the availability of oxygen or alternative electron acceptors, such as sulfate and nitrate. Due to oxygen limitation in freshwater sediments, DMS and MT are degraded mainly anaerobically by means of methanogenic activity (19). Methanogenic conversion of MT and DMS in sediment slurries was first demonstrated by Zinder and Brock (40, 41). Since then, various methanogens have been isolated with DMS or MT from marine, estuarine, salt marsh, and salt lake sediments (8, 12, 16, 17, 23, 25). These methanogens belong to the genera Methanosarcina, Methanolobus, and Methanosalsus. Although methanogens have been identified as the dominant consumers of DMS and MT in sulfate-poor freshwater sediments (18–20, 40, 41), previous attempts to isolate methanogens which are able to grow on DMS or MT from such sediments were unsuccessful (33, 41). Moreover, production of methane or carbon dioxide (or [14C]methane and [14C]carbon dioxide) from MT or DMS (or [14C]MT and [14C]DMS) was not detected in pure cultures of methanogens isolated from nonsaline systems (e.g., Methanobacterium ruminantium, Methanobacterium thermautotrophicum, and Methanosarcina barkeri cultures) (28, 41). In this paper we describe the isolation of a nonhalophilic methylotrophic methanogen, strain DMS1T, from the sediment of a eutrophic freshwater pond on the campus of the Dekkerswald Institute, Nijmegen, The Netherlands. This strain has the salt tolerance characteristic of freshwater bacteria and is able to use DMS, MT, methanol, and methylamines for growth and methanogenesis. Characteristics of strain DMS1T are discussed in relation to its ecological niche. Phylogenetic analysis revealed that this strain represents a novel genus in the family Methanosarcinaceae.

Molecular Ecological Analysis of Methanogens and Methanotrophs in Blanket Bog Peat.

Abstract: Methane production and methane oxidation potential were measured in a 30 cm peat core from the Moorhouse Nature Reserve, UK. The distribution of known groups of methanogens and methane oxidizing bacteria throughout this peat core was assessed. Using 16S rRNA gene retrieval and functional gene probing with genes encoding key proteins in methane oxidation and methanogenesis, several major groups of microorganisms were detected. Methane production and oxidation was detected in all depths of the peat core. PCR amplification and oligonucleotide probing experiments using DNA isolated from all sections of the peat core detected methanotrophs from the groups Methylosinus and Methylococcus and methanogens from the groups Methanosarcinaceae, Methanococcaceae, and Methanobacteriaceae. 16S rDNA sequences amplified with the Methylosinus-specific primer were shown to have a high degree of identity with 16S rDNA sequences previously detected in acidic environments. However, no methanogen sequences were detected by the probes available in this study in the sections of the peat core (above 7 cm) where the majority of methanogenesis occurred, either because of low methanogen numbers or because of the presence of novel methanogen sequences.

Phylogenetic diversity of methanogenic archaea in swine waste storage pits.

Abstract: Total DNA was isolated from swine feces and a swine waste storage pit and used as templates for PCR amplification of archaeal 16 rDNA using specific primers Only the sample from the center of the waste pit produced a PCR product DNA sequence analyses of random clones demonstrated a variety of methanogenic archaea Six groups of sequences were identified, including those similar to Methanobrevibacter sp, Methanocorpusculum sp, and Methanoculleus sp Three groups of sequences represented unidentified organisms These data suggest that swine waste storage pits may represent an untapped source of novel methanogenic archaea

Degradation of proteins and amino acids by Caloramator proteoclasticus in pure culture and in coculture with Methanobacterium thermoformicicum Z245

Abstract: This study investigated the degradation of proteins and amino acids by Caloramator proteoclasticus, an anaerobic thermophilic (55 °C) fermentative bacterium isolated from an anaerobic bioreactor. Experiments were performed in the presence and absence of Methanobacterium thermoformicicum Z245, a methanogen that can use both hydrogen and formate for growth. Higher production rates and yields of the principal fermentation products from gelatin were observed in methanogenic coculture. The specific proteolytic activity in coculture tripled the value obtained in pure culture. C. proteoclasticus fermented glutamate to acetate, formate, hydrogen and alanine. In methanogenic coculture, a shift towards higher amounts of acetate and hydrogen with no alanine production was observed. Extracts of glutamate-grown cells possessed high activities of β-methylaspartase, a key enzyme of the mesaconate pathway leading to acetate. The presence of two enzymes (alanine-α-ketoglutarate aminotransferase and NADH-dependent alanine dehydrogenase) usually involved in the biosynthesis of alanine from pyruvate was also detected. The fermentation of amino acids known to be oxidatively deaminated (leucine and valine) was improved in the presence of both methanogenesis and glycine, a known electron acceptor in the Stickland reaction. Culture conditions seem to be very important in the way C. proteoclasticus disposes of reducing equivalents formed during the degradation of amino acids.

The influence of sulfate and nitrate on the methane formation by methanogenic archaea in freshwater sediments

Abstract: In this thesis the effect of inorganic electron acceptors (sulfate and nitrate) on methane emission from freshwater sediments in the Netherlands was investigated. The chosen study area was a polder located between Leiden and Utrecht, and is representative for similar polders in The Netherlands (Chapter 3). The polder contains peat grasslands in which ditches are lying used for maintaining stable water levels. The ditches contain sediment which is a potential source of CH 4 . In freshwater environments, sulfate can be introduced by infiltration water, supply water or due to the oxidation of S-rich organic matter and iron sulfide. Also high nitrate concentrations can occur in the groundwater as a result of intensive agricultural activities. Therefore, in The Netherlands, sulfate and nitrate concentrations in the water may control the methane emission from methanogenic environments. The influence of sulfate and nitrate on methanogenesis Methane is produced by methanogenic archaea (methanogenesis) living in syntrophic association with fermentative and acetogenic bacteria. In presence of sulfate and nitrate, sulfate- and nitrate-reducing populations may successfully compete with these methanogenic consortia. In Chapter 4 the sediment was investigated for its potential methanogenic and syntrophic activity and the influence of sulfate and nitrate on these potential activities. Addition of acetate stimulated both methane formation and sulfate reduction, indicating that an active acetate-utilizing population of methanogens and sulfate reducers was present in the sediment. When inorganic electron acceptors were absent, substrates like propionate and butyrate were converted by syntrophic methanogenic consortia. However, addition of sulfate or nitrate resulted in the complete inhibition of these consortia. Our results showed that propionate and butyrate were directly used by the sulfate and nitrate reducers. This indicated that the syntrophic methanogenic consortia could not compete with nitrate and sulfate reducers. Acetate, a key intermediate in the anaerobic degradation of organic matter In Chapter 5 the importance of methanogenesis and sulfate reduction in a freshwater sediment was investigated by using (non) specific inhibitors. Only the combined inhibition of methanogenesis and sulfate reduction resulted in the accumulation of intermediates (acetate, propionate and valerate). Acetate was the most important compound in the accumulation (93 mole %) and thereby confirming its role as a key intermediate in the terminal step of organic matter mineralization. Furthermore, the inhibition studies showed that about 70-80% of the total carbon flow to CH 4 was through acetate. This clearly demonstrated that acetate was quantitatively the most important substrate for methanogens in the sediment. Addition of chloroform (CHCl 3 ) inhibited methanogens and acetate-utilizing sulfate reducers in the sediment. Pure culture studies showed that CHCl 3 was an inhibitor of growth and product formation by methanogenic archaea, homoacetogenic bacteria, a syntrophic bacterium ( Syntrophobacter fumaroxidans ) and the sulfate-reducing bacterium ( Desulfotomaculum acetoxidans ) operating the acetylCoA-pathway.In the sediment acetate is quantitatively the most important substrate for methanogens (chapter 5). Therefore, the anaerobic conversion of [2- 13 C] acetate in the presence of sulfate or nitrate was investigated (Chapter 6). Aceticlastic methanogenesis was the dominant acetate-utilizing process when the sulfate concentration was below 70 m M. At higher sulfate concentrations the formation of 13 C-labeled CH 4 decreased significantly, indicating that methanogens and sulfate reducers were competing for the same substrate. When sufficient sulfate (>500 m M) was present the outcome of the competition was in favor of the sulfate reducers. Unexpectedly, nitrate-reducing bacteria hardly competed with methanogens and sulfate reducers for the available acetate. The electron-acceptor/acetate ratio indicated that denitrification was coupled to the oxidation of reduced sulfur compounds or other electron donors rather than to the oxidation of acetate. Furthermore, nitrate reduction seemed to have a direct inhibitory effect on methanogenesis, and an indirect effect as a consequence of the oxidation of reduced sulfur-compounds to sulfate. The inhibition of methanogenesis by nitrate was probably not the result of competition for substrate but was due to the formation of toxic intermediates of the denitrification processes. The fact that acetate-utilizing nitrate reducers were outnumbered by the methanogens and sulfate reducers and hardly competed with these types of microorganisms for the available acetate indicated that acetate-utilizing nitrate reducers played a minor role in the degradation of acetate in the sediment (Chapter 6). Anaerobic acetate-utilizing microorganisms Enumeration of acetate-utilizing anaerobes gave insight into the different groups of microorganisms involved in the acetate metabolism in the sediment. In Chapter 7 the physiological properties of the acetate-utilizing anaerobes obtained by direct serial dilution of freshwater sediment are described. An acetate-utilizing methanogen (culture AMPB-Zg) was enriched and appeared to be closely related to Methanosaeta concilii . The most dominant acetate-utilizing sulfate reducer (strain ASRB-Zg) in the sediment was related to Desulfotomaculum nigrificans and Desulfotomaculum thermosapovorans . This result supported our hypothesis that acetate is a competitive substrate for methanogens and sulfate reducers in the sediment (Chapter 5 and 6). An acetate-utilizing nitrate reducer (strain ANRB-Zg) was isolated which showed to be related to Variovorax paradoxus . In the presence of acetate and nitrate, strain ANRB-Zg was capable of oxidizing reduced sulfur compounds to sulfate. Strain ANRB-Zg may have been involved in the oxidation of reduced sulfur compounds to sulfate in the sediment (Chapter 6). However, at this moment too little information is available to understand the exact role of strain ANRB-Zg in the sulfur and carbon cycle of the sediment. The degradation of acetate in the absence and presence of SO 4 2- and NO 3 - is depicted in Fig. 1. The dominant acetate-utilizing anaerobes and their metabolic interactions are given as well. Figure 1: The influence of sulfate and nitrate on aceticlastic methanogenesis in freshwater sediment. AMPB: aceticlastic methanogen, ASRB: acetate-utilizing sulfate reducer, ANRB: acetate-utilizing nitrate reducer. Thick stripped lines represent competition for acetate between AMPB and ASRB. Thick dotted lines represent inhibition caused by toxic intermediates. Finally, the conversion of acetate by methanogenic and sulfidogenic communities under acetate-limited conditions was studied in Chapter 8. Our results showed that the acetate-utilizing methanogens were able to compete efficiently with the sulfate reducers for the available acetate in an acetate-limited chemostat with sulfate in excess during a long-term experiment (1 year). From the chemostat studies it became clear that the kinetic properties of the acetate-utilizing methanogen and sulfate reducer were almost similar. Unfortunately, exact values for these kinetic properties are still lacking. Therefore predictions based on these parameters about the outcome of the competition of methanogens and sulfate reducers for acetate could not be made. In Chapter 2 a review of the physiological, ecological and biochemical aspects of acetate-utilizing anaerobes and their metabolic interactions are presented. Concluding remarks The results which are presented in this thesis advanced our knowledge of the effect of sulfate and nitrate on methane formation in sediments which are found in a typical Dutch polder. The sediment is a potential source of methane but it remains unclear if the sediment emits high quantities of methane. It was assumed that the methane emission is in the same order of magnitude (42-225 kg CH 4 ha -1 yr -1 ) as reported for another sediment. The presence of sulfate appeared to be a major factor in controlling the formation of methane. This is due to the competition between acetate-utilizing methanogens and sulfate reducers. Nevertheless, the origin of sulfate and its effect on methane emission on the long-term is not fully understood. The inhibitory effect of nitrate on methanogenesis appears to be the result of the formation of toxic intermediates of the denitrification processes but tangible proof is still lacking at this moment. Also the physiology and ecophysiology of some of the dominant acetate-utilizing anaerobes, and the metabolic interactions among them are not completely resolved. Further investigations of these topics are needed to get a better understanding of the environment as a source of methane and the emission from it. Intriguingly, measurements of CH 4 emissions from grasslands near the location of the sediments have shown that a net methane consumption in the area is possible. This indicates that methane produced in the ditches and originating from other sources may be oxidized again by the grassland soils. To determine a methane budget for Dutch polders the potential sink and/or source capacity of the grasslands should be included to get insight in the contribution to the emission of methane to the atmosphere.