Methanobacterium Thermoformicicum Research Articles

Formate production through carbon dioxide hydrogenation with recombinant whole cell biocatalysts.

The biological conversion of CO2 and H2 into formate offers a sustainable route to a valuable commodity chemical through CO2 fixation, and a chemical form of hydrogen fuel storage. Here we report the first example of CO2 hydrogenation utilising engineered whole-cell biocatalysts. Escherichia coli JM109(DE3) cells transformed for overexpression of either native formate dehydrogenase (FDH), the FDH from Clostridium carboxidivorans, or genes from Pyrococcus furiosus and Methanobacterium thermoformicicum predicted to express FDH based on their similarity to known FDH genes were all able to produce levels of formate well above the background, when presented with H2 and CO2, the latter in the form of bicarbonate. In the case of the FDH from P. furiosus the yield was highest, reaching more than 1 g L(-1)h(-1) when a hydrogen-sparging reactor design was used.

Biomethanation of Palm Oil Mill Effluent (POME) with a thermophilic mixed culture cultivated using POME as a substrate

Anaerobic systems operated under thermophilic condition often encounter longer start-up period and operating problems pertaining to temperature shift when mesophilic seed sludge were utilized as thermophilic sludge. Bioaugmentation products for thermophilic conditions were not readily available in the market. Hence, a thermophilic mixed culture has been cultivated specifically for Palm Oil Mill Effluent (POME) treatment at thermophilic conditions using a batch Continuous Stirred Tank Reactor (CSTR) where POME was utilized as a substrate for the growth of microbes. The thermophilic mixed culture managed to reduce at least 90% of Chemical Oxygen Demand (COD) in POME with a hydraulic retention time (HRT) of 6 days with a MLSS concentration of 14,000 mg/L. The biogas produced from the batch CSTR contained at least 64% of methane. The kinetic parameters for batch thermophilic POME treatment were obtained by fitting the Chen and Hashimoto's model to the experimental data. The maximum substrate utilization rate for this system was found to be 0.476 day −1, which was higher than the systems operated under mesophilic range, and dimensionless kinetic parameters k and Q were −1.365 and 0.0007 respectively. The mixed culture had a methanogenic population which consisted of Methanosaeta thermophila, Methanosarcina thermophila, Methanobacterium thermoautotrophicum, Methanobacterium thermoformicicum and Methanobacterium wolfei.

Anaerobic microbiology of an alkaline Icelandic hot spring

Samples of sediments and biofilm, defined as biomats or microbial mats, collected from a hot spring in Hveragerdi, Iceland at approximately 55°C and 75°C, were examined for anaerobic microbial activity and bacterial numbers. Generally more bacteria were present in the biomat at 75°C than at 55°C; 9.98×105 and 9.48×103 xylan degrading bacteria/gram volatile solids and 6.89×107 and 0 cellulose degrading bacteria/gram volatile solids were found at 75°C and 55°C, respectively. No proteolytic or lipolytic bacteria were present in the biomat at 75°C, but 1.38×103 proteolytic and 4.48×102 lipolytic cells/gram volatile solids were found in the biomat at 55°C. Acetate, propionate and butyrate were not degraded by the biomat either at 55°C or 75°C. No methanogenic activity was found at 75°C. However, most-probable number enumerations and indirect immunofluorescence using antibody probes against selected methanogenic archaea showed that methanogens were present at this temperature. H2 was the major electron donor for the methanogenic archaea at 55°C. No methane production was observed with acetate as substrate during short time (i.e. 24 h) incubation at 55°C. However, a significant amount of methane appeared after long periods of incubation at this temperature. Indirect immunofluorescence using antibody probes revealed a diverse methanogenic population in the spring. Cells immunologically related to Methanobacterium thermoautotrophicumΔH and Methanobacterium thermoformicicum CB 12 were the dominating methanogenic archaea at both temperatures. In addition, cells immunologically related to Methanoculleus thermophilus strain UCLA were present in high numbers at 75°C. Furthermore, cells immunologically related to Methanosaeta thermophila CALS-1 were found in low numbers in the biomat at 55°C.

Phylogenetic analysis of 18 thermophilic Methanobacterium isolates supports the proposals to create a new genus, Methanothermobacter gen. nov., and to reclassify several isolates in three species, Methanothermobacter thermautotrophicus comb. nov., Methanothermobacter wolfeii comb. nov., and Methanothermobacter marburgensis sp. nov.

Using a combination of 16S rRNA analysis and antigenic fingerprinting consisting of new and published data, the phylogenetic position of 18 thermophilic isolates currently classified as Methanobacterium species was reinvestigated. The results were verified by independent methods, including, where applicable, plasmid and phage typing. Comparative analysis of 16S rRNA data for 30 strains belonging to the order Methanobacteriales strongly suggested that mesophilic and thermophilic Methanobacterium isolates are distantly related and should be assigned to separate genera. For the thermophilic strains the genus Methanothermobacter was initially proposed by Boone, Whitman and Rouvière. Furthermore, the results support a reclassification of 15 isolates in three species within the proposed genus: (i) Methanothermobacter thermautotrophicus comb. nov., containing eight isolates, six of which are able to utilize formate (type strain deltaHT); (ii) Methanothermobacter wolfeii comb. nov., containing four formate-utilizing isolates (type strain DSM 2970T); (iii) Methanothermobacter marburgensis sp. nov., containing three obligately autotrophic isolates (type strain MarburgT). Of the nine isolates formerly referred to as Methanobacterium thermoformicicum, six were reclassified as Methanothermobacter thermautotrophicus and three as Methanothermobacter wolfeii.

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

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 g-methylaspartase, a key enzyme of the mesaconate pathway leading to acetate. The presence of two enzymes (alanine-f-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.

Methanobacterium thermoformicicum thymine DNA mismatch glycosylase: conversion of an N-glycosylase to an AP lyase.

The thymine DNA mismatch glycosylase from Methanobacterium thermoformicicum, a member of the endonuclease III family of repair proteins, excises the pyrimidine base from T-G and U-G mismatches. Unlike endonuclease III, it does not cleave the phosphodiester backbone by a beta-elimination reaction. This cleavage event has been attributed to a nucleophilic attack by the conserved Lys120 of endonuclease III on the aldehyde group at C1' of the deoxyribose and subsequent Schiff base formation. The inability of TDG to perform this beta-elimination event appears to be due to the presence of a tyrosine residue at the position equivalent to Lys120 in endonuclease III. The purpose of this work was to investigate the requirements for AP lyase activity. We replaced Tyr126 in TDG with a lysine residue to determine if this replacement would yield an enzyme with an associated AP lyase activity capable of removing a mismatched pyrimidine. We observed that this replacement abolishes the glycosylase activity of TDG but does not affect substrate recognition. It does, however, convert the enzyme into an AP lyase. Chemical trapping assays show that this cleavage proceeds through a Schiff base intermediate and suggest that the amino acid at position 126 interacts with C1' on the deoxyribose sugar.

Anaerobic microbiology of an alkaline Icelandic hot spring

Samples of sediments and biofilm, defined as biomats or microbial mats, collected from a hot spring in Hveragerdi, Iceland at approximately 55°C and 75°C, were examined for anaerobic microbial activity and bacterial numbers. Generally more bacteria were present in the biomat at 75°C than at 55°C; 9.98×10 5 and 9.48×10 3 xylan degrading bacteria/gram volatile solids and 6.89×10 7 and 0 cellulose degrading bacteria/gram volatile solids were found at 75°C and 55°C, respectively. No proteolytic or lipolytic bacteria were present in the biomat at 75°C, but 1.38×10 3 proteolytic and 4.48×10 2 lipolytic cells/gram volatile solids were found in the biomat at 55°C. Acetate, propionate and butyrate were not degraded by the biomat either at 55°C or 75°C. No methanogenic activity was found at 75°C. However, most-probable number enumerations and indirect immunofluorescence using antibody probes against selected methanogenic archaea showed that methanogens were present at this temperature. H 2 was the major electron donor for the methanogenic archaea at 55°C. No methane production was observed with acetate as substrate during short time (i.e. 24 h) incubation at 55°C. However, a significant amount of methane appeared after long periods of incubation at this temperature. Indirect immunofluorescence using antibody probes revealed a diverse methanogenic population in the spring. Cells immunologically related to Methanobacterium thermoautotrophicum ΔH and Methanobacterium thermoformicicum CB 12 were the dominating methanogenic archaea at both temperatures. In addition, cells immunologically related to Methanoculleus thermophilus strain UCLA were present in high numbers at 75°C. Furthermore, cells immunologically related to Methanosaeta thermophila CALS-1 were found in low numbers in the biomat at 55°C.

Cloning and expression of the gene for serine-glyoxylate aminotransferase from an obligate methylotroph Hyphomicrobium methylovorum GM2.

The gene encoding serine-glyoxylate aminotransferase, one of key enzymes for the assimilation of one-carbon compounds in methylotrophs, and its flanking regions were isolated from an obligate methylotrophic bacterium, Hyphomicrobium methylovorum GM2. Nucleotide sequencing of the recombinant plasmids revealed that the serine-glyoxylate aminotransferase gene encodes a 405-amino-acid protein with a calculated molecular mass of 43880 Da. The amino acid sequence of the enzyme showed identity to the sequences of serine-glyoxylate aminotransferase of Methylobacterium extorquens AM1 (57%), aspartate aminotransferase of Methanobacterium thermoformicicum (31%), human peroxisomal alanine-glyoxylate aminotransferase (27%), and serine-pyruvate aminotransferase of rat liver mitochondria (33%). The recombinant plasmid, which was constructed by ligation of the cloned gene and an expression vector pKK223-3, was introduced into Escherichia coli HB101. The recombinant enzyme was purified from transformed E. coli cells and analyzed by immunological and enzymological methods. The overexpressed enzyme was indistinguishable from the wild-type enzyme isolated from H. methylovorum GM2.

Methanogenic Fermentation of Formate by an Upflow Anaerobic Sludge Blanket (UASB) Reactor under Thermophilic Conditions

The performance of a thermophilic (65 °C) upflow anaerobic sludge blanket (UASB) reactor was investigated using wastewater consisting of formate as a main carbon source for a period of 126 days. The removal efficiencies on the basis of influent TOC vs. effluent DOC were above 90% at loading rates of 10–23 kg TOC m−3 d−1. No granulation of the biomass retained in the reactor was observed throughout the experimental period, and the sludge concentration was found to be exceptionally low (around 3000 mg VSS l−1 throughout the reactor column at the termination of the experiment). The methanogenic activity of the biomass for formate was calculated to be 9.0 kg CH4-COD kg VSS−1 d−1 according to the results of methane production from the reactor and the biomass volume. This high methanogenic activity led to the relatively high performance of the reactor despite the low sludge concentration. Microscopic examinations indicated that the biomass mostly comprised cylindrical, and slightly curved rod-shaped bacterium, which was presumably Methanobacterium thermoformicicum.

Aspartate aminotransferase from a thermophilic formate-utilizing methanogen, Methanobacterium thermoformicicum strain SF-4: relation to serine and phosphoserine aminotransferases, but not to the aspartate aminotransferase family.

The primary structure of the aspartate aminotransferase (AspAT) of an archaebacterium, Methanobacterium thermoformicicum strain SF-4, has been determined by cloning and sequencing of the gene for the enzyme. The gene had a consensus promoter and a ribosome binding sequence of methanogens in the 5' untranslated region, followed by an open reading frame starting with ATG and terminating with TGA. The deduced amino acid sequence was identical with the partial amino acid sequences of the enzyme including the N-terminal sequence, and the deduced molecular weight of 41,684 was virtually identical to that reported earlier for this enzyme [Tanaka, T., Yamamoto, S., Taniguchi, M., Hayashi, H., Kuramitsu, S., Kagamiyama, H., & Oi, S. (1992) J. Biochem. 112, 811-815]. The gene was expressed in Escherichia coli by inserting it into an expression vector just downstream of the lacZ promoter, and this verified that the cloned gene really encodes the Methanobacterium AspAT. The primary structure of the Methanobacterium AspAT showed extremely low homology, 5%, with AspATs of eubacteria, eukaryotes, and a thermoacidophilic arachaebacterium, Sulfolobus solfataricus. On the other hand, the Methanobacterium AspAT showed remarkable amino acid sequence homology, 31.5%, with rat serine:pyruvate aminotransferase and, 13.5%, with E. coli phosphoserine aminotransferase. Thus, the Methanobacterium AspAT apparently belongs to subgroup IV of the aminotransferases [Mehta, P.K., Hale, T.I., & Christen, P. (1993) Eur. J. Biochem. 214, 549-561], but not to subgroup I, in which all the AspATs known so far are included.

Formate production and utilization by methanogens and by sewage sludge consortia — interference with the concept of interspecies formate transfer

Pure cultures of H2/CO2- and formate-utilizing methanogens or mixed consortia of sewage sludge generated some formate from H2/CO2 at H2 partial pressure in the gas phase above 200 kPa. At decreasing H2 partial pressure the formate was taken up again and converted to methane. If methanogenesis was inhibited by bromoethanesulphonic acid (BESA) or a high redox potential (−180 to −200 mV), formate-utilizing methanogens produced high amounts of formate from H2/CO2. No formate was excreted by the species, which could only utilize H2/CO2 for methanogenesis. In contrast, H2 formation from formate was observed in cultures of Methanobacterium thermoformicicum and M. formicicum. Measurable amounts were, however, only formed if its immediate utilization for methane production was inhibited by BESA. In the light of the data on formate formation from H2/CO2 and its re-utilization by all formate-utilizing methanogens, the concept of interspecies formate transfer of Thiele and Zeikus should be reconsidered. In pure cultures of methanogens or complex ecosystems with excess H2, formate formation seemed to serve more as a means of disposal of surplus reducing power than for H2 transfer.

Potassium-stimulating mechanism of geranylgeranyl diphosphate synthase of Methanobacterium thermoformicicum SF-4.

The catalytic properties of geranylgeranyl diphosphate (GGPP) synthase [EC 2.5.1.29] purified from Methanobacterium thermoformicicum SF-4 were studied by kinetic procedures. The plots of 1/v versus 1/[S] and inhibition patterns by enzyme reaction products, PPi and GGPP, showed that the GGPP synthase reaction mechanism is an ordered-sequential Bi Bi one. Monovalent cations at low concentration (0.05 M) enhanced the enzyme activity, but at high concentration (0.4 M) they were inhibitory, except for K+. The K+ ion was found to be a modifier forming a parallel reaction pathway and accelerated the binding of substrates to the enzyme, especially the binding of isopentenyl diphosphate (IPP). When substrate concentrations are near the Km values, the rate-limiting step of the GGPP synthase reaction may be the substrate-binding step, probably the IPP-binding step, rather than the conversion step of the enzyme-farnesyl diphosphate-IPP complex to the enzyme-PPi-GGPP complex.

Taxonomic Analysis of Thermophilic Strains of the Genus Methanobacterium: Reclassification of Methanobacterium thermoalcaliphilum as a Synonym of Methanobacterium thermoautotrophicum

DNA reassociation, a comparative analysis of whole-cell protein patterns, and indirect immunofluorescence methods were used to determine the taxonomic relationships of thermophilic Methanobacterium strains. On the basis of the results dendrograms showing degrees of similarity among the organisms were constructed. The organisms studied are members of three different groups. One group, whose members exhibit 10 to 40% cross-hybridization, includes Methanobacterium wolfeii, “Methanobacterium defluvium” ADZ, and “Methanobacterium thermoflexum” IDZ. The second group contains Methanobacterium thermophilum MT (T = type strain) and Methanobacterium thermoaggregans (levels of DNA relatedness, 30 to 45%). The third group, whose members exhibit 65 to 99% cross-hybridization, includes Methanobacterium thermoautotrophicum ΔHT, F-1, and DV; Methanobacterium thermoformicicum Z-245T; and Methanobacterium thermoalcaliphilum AC60T. The combination of three independent taxonomic methods showed that the group of strains studied is phenotypically, genotypically, and antigenically diverse. The most distinct organisms are M. wolfeii, M. thermoaggregans, M. thermophilum, “M. defluvium,”“M. thermoflexum,” and M. thermoautotrophicum. These species names should be adopted. The results of a DNA-DNA hybridization study (level of hybridization, 99%), an immunological analysis (+3 reaction), and a protein similarity study (level of similarity, 75%) indicate that M. thermoalcaliphilum should be reclassified as a synonym of M. thermoautotrophicum.

Purification and Characterization of Geranylgeranyl Diphosphate Synthase from Methanobacterium thermoformicicum SF-4.

Geranylgeranyl diphosphate (GGPP) synthase [EC 2.5.1.29] was purified to homogeneity from Methanobacterium thermoformicicum SF-4. The enzyme was a dimeric protein consisting of two identical subunits (Mr = 39,000) and catalyzed prenyl transfer reactions using isopentenyl diphosphate (Km = 30.8 μM) and either dimethylallyl diphosphate (Km=16.8 μM), geranyl diphosphate (Km=12.6 μM), or farnesyl diphosphate (Km=14.7 μM) as allylic partners. During a sequential elongation, C5→C10→C15→C20' intermediates were accumulated with various ratios to the final product GGPP. In the presence of 0.8 M KCl, GGPP synthease activity was greatly enhanced, stabilized to heat treatment at 65°C for 30 min, and protected from inhibition by p-chloromercuribenzoic acid. No other prenyltransferase synthesizing C20 or shorter prenyl diphosphate was observed in M. thermoformicicum SF-4. These suggest that GGPP synthase alone is important in the biosynthetic pathways to squalene and membrane polar lipids at a chain elongation stage in this strain.

DNA relatedness among some thermophilic members of the genus Methanobacterium: emendation of the species Methanobacterium thermoautotrophicum and rejection of Methanobacterium thermoformicicum as a synonym of Methanobacterium thermoautotrophicum.

DNA reassociation was used to determine levels of relatedness among four thermophilic Methanobacterium strains that are able to use formate and between these organisms and two representative strains of Methanobacterium thermoautotrophicum, strain delta HT (= DSM 1053T = ATCC 29096T) (T = type strain) and strain Marburg (= DSM 2133). Three homology groups were delineated, and these groups coincided with the clusters identified by antigenic fingerprinting. The first group, which had levels of cross hybridization that ranged from 73 to 99%, included M. thermoautotrophicum delta HT, Methanobacterium thermoformicicum Z-245, Methanobacterium sp. strain THF, and Methanobacterium sp. strain FTF. The second and third groups were each represented by only one strain, Methanobacterium sp. strain CB-12 and M. thermoautotrophicum Marburg, respectively (cross-hybridization levels, 13 to 30 and 29 to 33%, respectively). Our results indicate that the name M. thermoformicicum should be rejected as it is a synonym of M. thermoautotrophicum. The taxonomic positions of strains Marburg and CB-12 need further investigation.

Isolation and characterization of plasmids from different strains of Methanobacterium thermoformicicum

Seven strains of the formate-utilizing thermophilic methanogen Methanobacterium thermoformicicum were screened for the presence of extrachromosomal DNA. Covalently closed circular plasmid DNA was detected in three strains, Z-245, FTF and THF. No plasmids were found in strains CB12 and SF-4, nor in two new isolates, FF1 and FF3. The plasmid from strain Z-245, designated pFZ1, and that from strain FTF, designated pFZ2, were approximately 10·5 kb in size and contained homologous and similarly sized restriction fragments. A physical map of plasmid pFZ1 was constructed and three KpnI-fragments comprising the entire plasmid were cloned in Escherichia coli using pUC19. Plasmid pFV1 isolated from strain THF was approximately 14 kb in size and contained regions with strong homology to pFZ1 DNA. Using the Methanobacterium formicicum fdhA gene as a hybridization probe the strains of the species M. thermoformicicum could be classified into two major groups.

Effects of ammonia on growth and morphology of thermophilic hydrogen-oxidizing methanogenic bacteria

The effects of ammonia on pure cultures of thermophilic hydrogen-utilizing methanogens were studied. The bacteria used were Methanobacterium thermoautotrophicum strain ΔH, Methanobacterium thermoformicicum, a Methanogenium sp., and a putative Methanobacterium thermoautotrophicum strain BA. For all strains tested initial inhibition occurred at 3.0–4.0 g NH4+−N1 (214–286 mM), and at approximately 6.0 g NH4+−N1 (428 mM), a 50% decrease of the growth rate was seen. At 9.0 g NH4+−N1 (643 mM), growth of Methanobacterium thermoformicum was observed, whereas the other strains showed either very unstable growth or were totally inhibited. For some strains ammonia had a pronounced effect on cell morphology, inducing formation of large aggregates.

Purification and Some Properties of Aspartate Aminotransferase ofMethanobacterium thermoformicicumSF-4

Aspartate aminotransferase (AAT) of Methanobacterium thermoformicicum SF-4 was purified to a homogeneous state on polyacrylamide gel electrophoresis (PAGE) by DEAE-cellulose, Polybuffer Exchanger (PBE), Phenyl-Sepharose, and hydroxyapatite column chromatography. The molecular weight of the enzyme was estimated to be 57,000 by both gel filtration and PAGE on a gradient gel. Subunit analysis by sodium dodecyl sulfate (SDS)-PAGE suggested its existence as a single polypeptide with a molecular weight of 50,000. The enzyme showed its maximum activity at 60°C and at pH 8.5. The activity did not decrease to any extent after heat treatment at 70°C for 1 hr at pH 7.5. Transamination activity on l-glutamate was about 4 times higher than that on l-aspartate and was also observed weakly on l-cysteine. Maleate was the most effective inhibitor, while glutarate and fumarate were less effective inhibitors.

Purification and some properties of aspartate aminotransferase of Methanobacterium thermoformicicum SF-4.

Aspartate aminotransferase (AAT) of Methanobacterium thermoformicicum SF-4 was purified to a homogeneous state on polyacrylamide gel electrophoresis (PAGE) by DEAE-cellulose, Polybuffer Exchanger (PBE), Phenyl-Sepharose, and hydroxyapatite column chromatography. The molecular weight of the enzyme was estimated to be 57,000 by both gel filtration and PAGE on a gradient gel. Subunit analysis by sodium dodecyl sulfate (SDS)-PAGE suggested its existence as a single polypeptide with a molecular weight of 50,000. The enzyme showed its maximum activity at 60°C and at pH 8.5. The activity did not decrease to any extent after heat treatment at 70°C for 1 hr at pH 7.5. Transamination activity on l-glutamate was about 4 times higher than that on l-aspartate and was also observed weakly on l-cysteine. Maleate was the most effective inhibitor, while glutarate and fumarate were less effective inhibitors.