Showing papers on "Methanosarcina barkeri published in 2011"

Effect of spatial differences in microbial activity, pH and substrate levels on methanogenesis initiation in refuse

Abstract: The initiation of methanogenesis in refuse occurs under high volatile fatty acid (VFA) concentration and low pH (5.5 to 6.25), which generally are reported to inhibit methanogenic Archaea. One hypothesized mechanism for the initiation of methanogenesis in refuse decomposition is the presence of pH-neutral niches within the refuse that act as methanogenesis initiation centers. To provide experimental support for this mechanism, laboratory-scale landfill reactors were operated and destructively sampled when methanogenesis initiation was observed. The active bacterial and archaeal populations were evaluated using RNA clone libraries, RNA terminal restriction fragment length polymorphism (T-RFLP), and reverse transcription-quantitative PCR (RT-qPCR). Measurements from 81 core samples from vertical and horizontal sections of each reactor showed large spatial differences in refuse pH, moisture content, and VFA concentrations. No pH-neutral niches were observed prior to methanogenesis. RNA clone library results showed that active bacterial populations belonged mostly to Clostridiales, and that methanogenic Archaea activity at low pH was attributable to Methanosarcina barkeri. After methanogenesis began, pH-neutral conditions developed in high-moisture-content areas containing substantial populations of M. barkeri. These areas expanded with increasing methane production, forming a reaction front that advanced to low-pH areas. Despite low-pH conditions in >50% of the samples within the reactors, the leachate pH was neutral, indicating that it is not an accurate indicator of landfill microbial conditions. In the absence of pH-neutral niches, this study suggests that methanogens tolerant to low pH, such as M. barkeri, are required to overcome the low-pH, high-VFA conditions present during the anaerobic acid phase of refuse decomposition.

Acetogens and Acetoclastic Methanosarcinales Govern Methane Formation in Abandoned Coal Mines

Abstract: In abandoned coal mines, methanogenic archaea are responsible for the production of substantial amounts of methane. The present study aimed to directly unravel the active methanogens mediating methane release as well as active bacteria potentially involved in the trophic network. Therefore, the stable-isotope-labeled precursors of methane, [ 13 C]acetate and H2- 13 CO2, were fed to liquid cultures from hard coal and mine timber from a coal mine in Germany. Guided by methane production rates, samples for DNA stable-isotope probing (SIP) with subsequent quantitative PCR and denaturing gradient gel electrophoretic (DGGE) analyses were taken over 6 months. Surprisingly, the formation of [ 13 C]methane was linked to acetoclastic methanogenesis in both the [ 13 C]acetate- and the H 2 - 13 CO 2 -amended cultures of coal and timber. H 2 - 13 CO 2 was used mainly by acetogens related to Pelobacter acetylenicus and Clostridium species. Active methanogens, closely affiliated with Methanosarcina barkeri, utilized the readily available acetate rather than the thermodynamically more favorable hydrogen. Thus, the methanogenic microbial community appears to be highly adapted to the low-H 2 conditions found in coal mines.

Genetic methods for methanosarcina species.

Abstract: Unlike most methanogenic microorganisms, Methanosarcina species are capable of utilizing a variety of growth substrates, a trait that greatly simplifies genetic analysis of the methanogenic process. The genetic tools and techniques discussed in this chapter form the basis for all genetic experiments in Methanosarcina acetivorans C2A and Methanosarcina barkeri Fusaro, two methanogens that are routinely used as model organisms for genetic experiments. Based on a number of reports, it is likely that they are portable to other Methanosarcina species, and perhaps to other methanogens as well. Here, we outline the procedures for high-efficiency transformation using liposomes, gene expression from a plasmid, and exploitation of homologous and site-specific recombination to add and delete genes from the chromosome. Finally, we outline the method for testing whether a gene is essential. These methods can be adapted and combined in any number of ways to design genetic experiments in Methanosarcina.

Investigation of the trophic relations between anaerobic microorganisms from an underground gas repository during methanol utilization

Abstract: Joint cultivation of the dominant strains of acetogenic, sulfate-reducing and methanogenic microorganisms isolated from water samples of the North Stavropol underground gas storage facility (UGSF) was carried out for revealing their probable trophic relationships. It was shown that acetogenic strains Eubacterium limosum AG12 and Sporomusa sphaeroides AG8-2 growing on methanol could form a considerable pool of hydrogen, which may support development of hydrogenotrophic cultures, the methanogen Methanobacterium formicicum MG134, or the sulfate reducer Desulfovibrio desulfuricans SR12. Growth of this sulfate-reducing strain was not stimulated under joint cultivation with Methanosarcina barkeri MGZ3 on methanol, probably due to its inability to take up low hydrogen concentrations observed during methanosarcina development. The results show that acetogens in the UGSF system are the most important consumers of methanol and hydrogen and after exhaustion of the latter and switching over to methanol utilization they can supply hydrogen to other microorganisms, including methanogens and sulfate reducers. The role of methanosarcina in the UGSF increases as the hydrogen and CO2 reserves are exhausted, and methanogenesis on methanol becomes the main way of its destruction.

Structures and activities of archaeal members of the LigD 3'-phosphoesterase DNA repair enzyme superfamily.

Abstract: LigD 3'-phosphoesterase (PE) is a component of the bacterial NHEJ apparatus that performs 3'-end-healing reactions at DNA breaks. The tertiary structure, active site and substrate specificity of bacterial PE are unique vis-a-vis other end-healing enzymes. PE homologs are present in archaea, but their properties are uncharted. Here, we demonstrate the end-healing activities of two archaeal PEs - Candidatus Korarchaeum cryptofilum PE (CkoPE; 117 amino acids) and Methanosarcina barkeri PE (MbaPE; 151 amino acids) - and we report their atomic structures at 1.1 and 2.1 {angstrom}, respectively. Archaeal PEs are minimized versions of bacterial PE, consisting of an eight-stranded {beta} barrel and a 3{sub 10} helix. Their active sites are located in a crescent-shaped groove on the barrel's outer surface, wherein two histidines and an aspartate coordinate manganese in an octahedral complex that includes two waters and a phosphate anion. The phosphate is in turn coordinated by arginine and histidine side chains. The conservation of active site architecture in bacterial and archaeal PEs, and the concordant effects of active site mutations, underscore a common catalytic mechanism, entailing transition state stabilization by manganese and the phosphate-binding arginine and histidine. Our results fortify the proposal that PEs comprise a DNA repair superfamily distributedmore » widely among taxa.« less

Isolation and investigation of anaerobic microorganisms involved in methanol transformation in an underground gas storage facility

Abstract: High methanol and acetate concentrations (up to 12 and 14 g l−1, respectively) were found in water samples collected at different objects of the North Stavropol underground gas storage facility (UGSF), and significant seasonal variations in the content of these compounds were revealed The dominant anaerobic microorganisms isolated from these samples during the study belonged to acetogens, methanogens, and sulfate reducers The results of 16S rRNA gene sequencing and analysis of the physiological properties showed that the isolates were close to the species of Eubacterium limosum, Sporomusa sphaeroides, Methanosarcina barkeri, Methanobacterium formicicum, and Desulfovibrio desulfuricans The isolated organisms, except for Methanobacterium formicicum, were capable of methylotrophic growth All strains were characterized by resistance to high methanol concentrations (up to 40–50 g l−1) Their other energy substrate was hydrogen The combination of the growth characteristics of these strains (pH, temperature, and salinity ranges) was shown to correspond to the ecological situation observed in the UGSF The results of investigation of the isolated strains suggest that organic acids (acetate, butyrate) found in high concentrations in the initial samples are metabolic products of the revealed acetogens Based on the established biological peculiarities of the isolated strains of methanogens, acetogens, and sulfate-reducing bacteria, these microorganisms may be considered as the main agents of anaerobic transformation of methanol and some other organic and inorganic compounds in UGSFs

Pre-transfer Editing of Serine Hydroxamate within the Active Site of Methanogenic-type Seryl-tRNA Synthetase

Abstract: Aminoacyl-tRNA synthetases (aaRSs) maintain fidelity of protein synthesis by matching only cognate amino acid-tRNA pairs. Aminoacylation occurs through activation of amino acid to yield aminoacyl-adenylate followed by transfer of acyl-moiety to tRNA. Error-prone aaRSs achieve high level of accuracy using inherent hydrolytic activities towards noncognate aminoacyl-adenylate or misacylated tRNA (pre- and post-transfer editing).Seryl-tRNA synthetases can be divided into two structurally different types: canonical and methanogenic-type. Both types have been shown to efficiently activate serine analogue serine hydroxamate (SerHX). Moreover, this analogue has been also eliminated by pre-transfer editing within the canonical synthetic site of yeast SerRS. Here we show that methanogenic-type SerRS from Methanosarcina barkeri clears misactivated SerHX similarly as the yeast enzyme: SerHX-adenylate is not expelled into solution, but is enzymatically hydrolyzed in a tRNA-independent manner. Since the enzyme lacks domain specialized in editing, this shows that methanogenic-type catalytic core is also capable to perform pre-transfer editing.

Sodium Ion Translocation and ATP Synthesis in Methanogens

Abstract: Methanogens are the only significant biological producers of methane A limited number of C(1) substrates, such as methanol, methylamines, methyl sulfate, formate, H(2)+CO(2) or CO, and acetate, serve as carbon and energy source During degradation of these compounds, a primary proton as well as a primary sodium ion gradient is established, which is a unique feature of methanogens This raises the question about the coupling ion for ATP synthesis by the unique A(1)A(o) ATP synthase Here, we describe how to analyze and determine the Na(+) dependence of two model methanogens, the hydrogenotrophic Methanothermobacter thermautotrophicus and the methylotrophic Methanosarcina barkeri Furthermore, the determination of important bioenergetic parameters like the ΔpH, ΔΨ, or the intracellular volume in M barkeri is described For the analyses of the A(1)A(O) ATP synthase, methods for measurement of ATP synthesis as well as ATP hydrolysis in Methanosarcina mazei Go1 are described