Methanolobus zinderi sp. nov., a methylotrophic methanogen isolated from a deep subsurface coal seam.

A methanogenic organism from the domain Archaea (SD1(T)) was isolated from saline water released from a coal seam located 926 m below the surface via a methane-producing well near Monroe, Louisiana, USA. Growth and methanogenesis were supported with methanol, monomethylamine, dimethylamine or trimethylamine, but not with dimethylsulfide, formate, acetate or H(2)/CO(2). Cells grew in high-salt minimal medium but growth was stimulated with yeast extract or tryptone. Cells were single, non-motile, irregular coccoids 0.5-1.0 microm in diameter and the cell wall contained protein. Conditions for the maximum rate of growth were 40-50 degrees C, 0.2-0.6 M NaCl, 100->or=200 mM MgCl(2), and pH 7.0-8.0. The G+C content of the genomic DNA was 42+/-1mol %. A comparison of 16S rRNA gene sequences indicated that strain SD1(T) was most closely related to Methanolobus oregonensis DSM 5435(T) with 96 % gene sequence similarity. It is proposed that strain SD1(T) represents a novel species, Methanolobus zinderi sp. nov. The type strain is SD1(T) (=ATCC BAA-1601(T)=DSM 21339(T)).

Characterization of the acetate binding pocket in the Methanosarcina thermophila acetate kinase.

Acetate kinase catalyzes the reversible magnesium-dependent synthesis of acetyl phosphate by transfer of the ATP gamma-phosphoryl group to acetate. Inspection of the crystal structure of the Methanosarcina thermophila enzyme containing only ADP revealed a solvent-accessible hydrophobic pocket formed by residues Val(93), Leu(122), Phe(179), and Pro(232) in the active site cleft, which identified a potential acetate binding site. The hypothesis that this was a binding site was further supported by alignment of all acetate kinase sequences available from databases, which showed strict conservation of all four residues, and the recent crystal structure of the M. thermophila enzyme with acetate bound in this pocket. Replacement of each residue in the pocket produced variants with K(m) values for acetate that were 7- to 26-fold greater than that of the wild type, and perturbations of this binding pocket also altered the specificity for longer-chain carboxylic acids and acetyl phosphate. The kinetic analyses of variants combined with structural modeling indicated that the pocket has roles in binding the methyl group of acetate, influencing substrate specificity, and orienting the carboxyl group. The kinetic analyses also indicated that binding of acetyl phosphate is more dependent on interactions of the phosphate group with an unidentified residue than on interactions between the methyl group and the hydrophobic pocket. The analyses also indicated that Phe(179) is essential for catalysis, possibly for domain closure. Alignments of acetate kinase, propionate kinase, and butyrate kinase sequences obtained from databases suggested that these enzymes have similar catalytic mechanisms and carboxylic acid substrate binding sites.

H2-producing bacterial communities from a heat-treated soil inoculum.

Hydrogen gas (approximately 60% H(2)) was produced in a continuous flow bioreactor inoculated with heat-treated soil, and fed synthetic wastewater containing glucose (9.5 g l(-1)). The pH in the bioreactor was maintained at 5.5 to inhibit consumption of H(2) by methanogens. The objective of this study was to characterize bacterial communities in the reactor operated under two different hydraulic retention times (HRTs of 30-h and 10-h) and temperatures (30 degrees C and 37 degrees C). At 30-h HRT, the H(2) production rate was 80 ml h(-1) and yield was 0.91 mol H(2)/mol glucose. At 10-h HRT, the H(2) production rate was more than 5 times higher at 436 ml h(-1), and yield was 1.61 mol H(2)/mol glucose. Samples were removed from the reactor under steady-state conditions for PCR-based detection of bacterial populations by ribosomal intergenic spacer analysis (RISA). Populations detected at 30-h HRT were more diverse than at 10-h HRT and included representatives of Bacillaceae, Clostridiaceae, and Enterobacteriaceae. At 10-h HRT, only Clostridiaceae were detected. When the temperature of the 10-h HRT reactor was increased from 30 degrees C to 37 degrees C, the steady-state H(2) production rate increased slightly to 463 ml h(-1) and yield was 1.8 mol H(2)/mol glucose. Compared to 30 degrees C, RISA fingerprints at 37 degrees C from the 10-h HRT bioreactor exhibited a clear shift from populations related to Clostridium acidisoli (subcluster Ic) to populations related to Clostridium acetobutylicum (subcluster Ib).

Biological hydrogen production using a membrane bioreactor.

A cross-flow membrane was coupled to a chemostat to create an anaerobic membrane bioreactor (MBR) for biological hydrogen production. The reactor was fed glucose (10,000 mg/L) and inoculated with a soil inoculum heat-treated to kill non-spore-forming methanogens. Hydrogen gas was consistently produced at a concentration of 57-60% in the headspace under all conditions. When operated in chemostat mode (no flow through the membrane) at a hydraulic retention time (HRT) of 3.3 h, 90% of the glucose was removed, producing 2200 mg/L of cells and 500 mL/h of biogas. When operated in MBR mode, the solids retention time (SRT) was increased to SRT = 12 h producing a solids concentration in the reactor of 5800 mg/L. This SRT increased the overall glucose utilization (98%), the biogas production rate (640 mL/h), and the conversion efficiency of glucose-to-hydrogen from 22% (no MBR) to 25% (based on a maximum of 4 mol-H(2)/mol-glucose). When the SRT was increased from 5 h to 48 h, glucose utilization (99%) and biomass concentrations (8,800 +/- 600 mg/L) both increased. However, the biogas production decreased (310 +/- 40 mL/h) and the glucose-to-hydrogen conversion efficiency decreased from 37 +/- 4% to 18 +/- 3%. Sustained permeate flows through the membrane were in the range of 57 to 60 L/m(2) h for three different membrane pore sizes (0.3, 0.5, and 0.8 microm). Most (93.7% to 99.3%) of the membrane resistance was due to internal fouling and the reversible cake resistance, and not the membrane itself. Regular backpulsing was essential for maintaining permeate flux through the membrane. Analysis of DNA sequences using ribosomal intergenic spacer analysis indicated bacteria were most closely related to members of Clostridiaceae and Flexibacteraceae, including Clostridium acidisoli CAC237756 (97%), Linmingia china AF481148 (97%), and Cytophaga sp. MDA2507 AF238333 (99%). No PCR amplification of 16s rRNA genes was obtained when archaea-specific primers were used.

Crystal structure of phosphotransacetylase from the methanogenic archaeon Methanosarcina thermophila.

Phosphotransacetylase (Pta) [EC 2.3.1.8] is ubiquitous in the carbon assimilation and energy-yielding pathways in anaerobic prokaryotes where it catalyzes the reversible transfer of the acetyl group from acetyl phosphate to CoA forming acetyl CoA and inorganic phosphate. The crystal structure of Pta from the methane-producing archaeon Methanosarcina thermophila, representing the first crystal structure of any Pta, was determined by multiwavelength anomalous diffraction at 2.7 A resolution. In solution and in the crystal, the enzyme forms a homodimer. Each monomer consists of two alpha/beta domains with a cleft along the domain boundary, which presumably contains the substrate binding sites. Comparison of the four monomers present in the asymmetric unit indicates substantial variations in the relative orientation of the two domains and the structure of the putative active site cleft. A search for structural homologs revealed the NADP(+)-dependent isocitrate and isopropylmalate dehydrogenases as the only homologs with a similar two-domain architecture.

Expression, purification, crystallization and preliminary X-ray analysis of phosphotransacetylase from Methanosarcina thermophila.

Phosphotransacetylase (Pta) from the anaerobic archaeon Methanosarcina thermophila has been heterologously expressed in a soluble form which facilitated crystallization using the hanging-drop vapor-diffusion method with ammonium sulfate as a precipitant. This is the first report of the crystallization of any Pta. While the M. thermophila Pta has high sequence identity to Ptas from other organisms, it has no homology to any previously crystallized proteins. The protein crystallized in space group I4(1), with unit-cell parameters a = b = 114.8, c = 127.8 A, alpha = beta = gamma = 90 degrees. The crystals diffracted to 2.5 A resolution using Cu Kalpha radiation. The enzyme had previously been reported to exist as a monomer; however, the self-rotation function showed the presence of a non-crystallographic symmetry axis at psi = 90, phi = 90, kappa = 180 degrees, suggesting oligomerization. Dynamic light-scattering analysis supported a dimeric state for Pta in solution.