Assimilatory reduction of sulfate and sulfite by methanogenic bacteria

A variety of sulfur-containing compounds were investigated for use as medium reductants and sulfur sources for growth of four methanogenic bacteria. Sulfide (1 to 2 mM) served all methanogens investigated well. Methanococcus thermolithotrophicus and Methanobacterium thermoautotrophicum Marburg and delta H grew well with S0, SO3(2-), or thiosulfate as the sole sulfur source. Only Methanococcus thermolithotrophicus was able to grow with SO4(2-) as the sole sulfur source. 2-Mercaptoethanol at 20 mM was greatly inhibitory to growth of Methanococcus thermolithotrophicus on SO4(2-) or SO2(2-) and Methanobacterium thermoautotrophicum Marburg on SO3(2-) but not to growth of strain delta H on SO3(2-). Sulfite was metabolized during growth by Methanococcus thermolithotrophicus. Sulfide was produced in cultures of Methanococcus thermolithotrophicus growing on SO4(2-), SO3(2-), thiosulfate, and S0. Methanobacterium thermoautotrophicum Marburg was successfully grown in a 10-liter fermentor with S0, SO3(2-), or thiosulfate as the sole sulfur source.     

Anaerobic Transformation of Furfural by Methanococcus deltae (Delta)LH

Methanococcus deltae (Delta)LH was grown on H(inf2)-CO(inf2) in the presence of various concentrations of furfural. Furfural at higher concentrations, namely, 20 and 25 mM, inhibited growth of this organism. At concentration of 5 and 10 mM, no inhibition of growth was observed. The other methanogens in this study were not inhibited by 10 mM furfural. Among the methanogens tested, M. deltae was capable of transforming furfural, whereas Methanobacterium thermoautotrophicum Marburg, Methanosarcina barkeri 227, Methanococcus thermolithotrophicus, and Methanobrevibacter ruminantium lacked this capability. One hundred percent removal of furfural was observed within 48 h of incubation in M. deltae cultures. The end product observed during furfural metabolism was furfuryl alcohol. An almost stoichiometric amount of furfuryl alcohol was produced by M. deltae. This transformation is likely to be of value in the detoxification of furfural and in its ultimate conversion to methane and CO(inf2) by anaerobic digestion.     

Investigation of mercaptans,organic sulfides,and inorganic sulfur compounds as sulfur sources for the growth of methanogenic bacteria

A variety of compounds were investigated for use as sulfur sources for the growth of methanogenic bacteria.Methanococcus (Mc.) deltae, Mc. maripaludis, Methanobacterium (Mb.) speciesGC-2B, GC-3B, andMMY, Methanobrevibacter (Mbr.) ruminantium, andMethanosarcina (Ms.) barkeri strain 227 grew well with sulfide, S^o, thiosulfate, or cysteine as sole sulfur source.Mbr. ruminatium was able to grow on SO ₄ ⁼ or SO ₃ ⁼ , andMs. barkeri strain 227 was able to grow on SO ₃ ⁼ , but not on SO ₄ ⁼ as a sole sulfur source.Mc. jannaschii grew with sulfide, S^o, thiosulfate or SO ₃ ⁼ , but not on cysteine or SO ₄ ⁼ as sole surface source.Mc. thermolithotrophicus, Mc. jannaschii, Mc. deltae, andMb. thermoautotrophicum strains Marburg and H were able to grow with methanethiol, ethanethiol,n-propanethiol,n-butanethiol, methyl sulfide, dimethyl sulfoxide, ethyl sulfide, or CS₂ as a sulfur source, when very low levels (20–30 M) of sulfide were present; no growth occurred on 5–100 M sulfide alone. Methanethiol, ethanethiol, and methyl sulfide-using cultures produced sulfide during growth.     

Utilization of mercapto-2-ethanol as medium reductant for determination of the metabolic response of methanogens towards inorganic sulfur compounds

Abstract Mercapto-2-ethanol was examined as a nontoxic and non-metabolizable reducing agent for growth of methanogens. It was used as a medium reductant to prove that Methanobacterium thermoautotrophicum and Methanobacterium strain ivanov grew with either sulfide or elemental sulfur as the sole source of nutrient sulfur but not with sulfate, thiosulfate, sulfite or dithionite. The later inorganic sulfur sources, except sulfate, were potent inhibitors of growth and methanogenesis at 5 mM. The practical utility of mercapto-2-ethanol as a reducing agent and the toxicity of inorganic sulfur sources on metabolic activity of the methanogens are discussed.     

Light sensitivity of methanogenic archaebacteria.

Representatives of four families of methanogenic archaebacteria (archaea), Methanobacterium thermoautotrophicum delta H, Methanobacterium thermoautotrophicum Marburg, Methanosarcina acetivorans, Methanococcus voltae, and Methanomicrobium mobile, were found to be light sensitive. The facultative anaerobic eubacteria Escherichia coli and Salmonella typhimurium, however, were tolerant of light when grown anaerobically under identical light conditions. Interference filters were used to show that growth of the methanogens is inhibited by light in the blue end of the visible spectrum (370 to 430 nm).     

Light sensitivity of methanogenic archaebacteria.

Representatives of four families of methanogenic archaebacteria (archaea), Methanobacterium thermoautotrophicum delta H, Methanobacterium thermoautotrophicum Marburg, Methanosarcina acetivorans, Methanococcus voltae, and Methanomicrobium mobile, were found to be light sensitive. The facultative anaerobic eubacteria Escherichia coli and Salmonella typhimurium, however, were tolerant of light when grown anaerobically under identical light conditions. Interference filters were used to show that growth of the methanogens is inhibited by light in the blue end of the visible spectrum (370 to 430 nm).     

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.     

Physiological and 15N-NMR analysis of molecular nitrogen fixation by Methanococcus thermolithotrophicus, Methanobacterium bryantii and Methanospirillum hungatei

Two mesophilic methanogenic bacteria, Methanobacterium bryantii strain MOH and Methanospirillum hungatei strain GP1 were demonstrated, using several different experimental approaches, to fix dinitrogen. Evidence includes (1) growth with N2 as the sole nitrogen source; (2) incorporation of 15N2 into cellular material (both soluble amino acid pools and insoluble cell protein and other macromolecules) detected by 15N-NMR spectroscopy; (3) acetylene reduction to ethylene by the cells, and inhibition of this reaction by bromoethanesulfonic acid (BES), a methanogen inhibitor. High-resolution 15N-NMR analysis of ethanol extracts of these organisms and cross-polarization magic-angle sample spinning analysis of the solid debris from these extracts are compared to labeled material from Methanococcus thermolithotrophicus, a methanogen previously determined to fix dinitrogen.     

Formaldehyde oxidation and methanogenesis

Formaldehyde oxidation by cell-free extracts of Methanobacterium thermoautotrophicum was shown to drive methanogenesis from CH3-S-coenzyme M or HCHO under a nonreductive atmosphere of N2. Under N2 when HCHO was the sole source of carbon and reducing equivalents in the reaction, it underwent oxidation and reduction events (disproportionation), the sum of the reactions being 3 HCHO + H2O----CH4 + 2 HCOO - + 2H+. This reaction predicts a CH4/HCHO ratio of 1/3, which is in agreement with the experimental finding of 1/2.9. In extracts of the mesophilic methanogen Methanococcus voltae and the extreme thermophile Methanococcus jannaschii , which exhibited formate dehydrogenase activity, the CH4/HCHO ratio was 1/2. NADPH stimulated methane formation from HCHO under N2. An unidentified, oxygen-labile cofactor, the formaldehyde activation factor, present in boiled-cell extract was discovered. Methanopterin , an oxygen-stable molecule, also substituted for boiled-cell extract.     

Evidence that the heterodisulfide of coenzyme M and 7-mercaptoheptanoylthreonine phosphate is a product of the methylreductase reaction in Methanobacterium

When 7-mercaptoheptanoylthreonine phosphate (HS-HTP) was used as the sole source of electrons for reductive demethylation of 2-(methylthio)-ethanesulfonic acid (CH3-S-CoM) by cell extracts of Methanobacterium thermoautotrophicum strain delta H, the heterodisulfide of coenzyme M and HS-HTP (CoM-S-S-HTP) was quantitatively produced: HS-HTP + CH3-S-CoM----CH4 + CoM-S-S-HTP. CH4 and CoM-S-S-HTP were produced stoichiometrically in a ratio of 1:1. Coenzyme M (HS-CoM) inhibited HS-HTP driven methanogenesis indicating that CH3-S-CoM rather than HS-CoM was the substrate for CoM-S-S-HTP formation.     

Free amino acid turnover in methanogens measured by 15N NMR spectroscopy

Turnover of the nitrogen moiety from free amino acid pools in two thermophilic methanogens, Methanobacterium thermautotrophicum delta H and Methanococcus thermolithotrophicus SN1, has been monitored with 15N NMR spectroscopy. In cells growing exponentially on 15NH4Cl, glutamate was the major soluble 15N-labeled species in both organisms. When the Mb. thermoautotrophicum cells were harvested, washed, and resuspended into medium containing 14NH4Cl, the resonance for [15N]glutamate decreased with a half-life of 0.5 h. This is considerably faster than the turnover rate for the carbon side chain of glutamate (7 h) obtained when a 13CO2 pulse followed by a 12CO2 chase was incorporated into the 15N/14N-labeling experiment. Such behavior is consistent with recycling of the glutamate carbon skeleton via alpha-ketoglutarate after transamination reactions remove the 15N for biosynthesis of other amino acids, nucleic acids, etc. When the cells were in stationary phase, 15N turnover was considerably slower indicating that transaminase activity had also decreased. Mc. thermolithotrophicus has a much more fragile cell wall and easily lyses. To avoid cell loss in the 15N/14N experiment, 15NH+4 growth followed by 14NH4+ dilution was used. In this organism the glutamate-labeled nitrogen turns over quite rapidly (t1/2 approximately 9 min), at a rate comparable to that for the carbon skeleton (t1/2 approximately 10 min). Beta-Glutamate, the second major carbon and nitrogen pool in this organism, turns over its 15N label very slowly. Therefore, this beta-amino acid does not appear to serve as a nitrogen donor in Mc. thermolithotrophicus.     

Simultaneous utilization of cysteine and thiosulfate for growth ofMethanobacterium thermoautotrophicum strain ΔH

Summary The sulfur-containing compounds cysteine, thiosulfate and dithionite, were investigated to substitute for sulfide as a sulfur source for cultivation ofMethanobacterium thermoautotrophicum. although none of the three compounds was suitable as the sole sulfur source, a combination of thiosulfate and cysteine supported growth and methanogenesis of the methanogen in batch and continuous culture as efficiently as sulfide.     

A plasmid in the archaebacterium Methanobacterium thermoautotrophicum

The archaebacterium Methanobacterium thermoautotrophicum Marburg (DSM 2133) was found to contain a plasmid (pME2001) in covalently closed circular form. It was isolated by CsCl gradient centrifugation of total DNA in the presence of ethidium bromide. Multimers up to the hexamer were observed upon agarose gel electrophoresis and electron microscopy of a purified plasmid preparation. A restriction map was constructed. The length of plasmid pME2001 was determined to be approximately 4,500 bp. Southern hybridization of plasmid DNA to DNA extracted from Methanobacterium thermoautotrophicum delta H (DSM1053) revealed the presence of a plasmid with homologous sequences in the delta H strain.     

Occurrence of beta-glutamate, a novel osmolyte, in marine methanogenic bacteria

The unusual compound beta-aminoglutaric acid (beta-glutamate) has been identified by 13C nuclear magnetic resonance spectroscopy in soluble extracts of marine methanogenic bacteria. We examined several methanogen species representing nine genera and found that beta-glutamate occurred in methanococci and two methanogenium strains (Methanogenium cariaci JR1 and "Methanogenium anulus" AN9). The presence of this compound in the methanococci examined was further restricted to thermophilic members of the genus Methanococcus, including Methanococcus thermolithotrophicus strains, Methanococcus jannaschii, and "Methanococcus igneus." The two Methanogenium strains examined were mesophiles. Studies using Methanococcus thermolithotrophicus showed that levels of beta-glutamate in cells of that species were not affected by variation in growth temperature (40 to 65 degrees C), NH4+ (2 to 80 mM), Mg2+ (10 to 50 mM), or K+ (2 to 10 mM) in the medium. In contrast, soluble pools of beta-glutamate and L-alpha-glutamate (the other major free amino acid in all the methanococci) were proportional to NaCl levels in the growth medium. This dependence of beta-glutamate and L-alpha-glutamate concentrations on salt levels in the medium suggests that they function as osmolytes in these cells.     

Occurrence of beta-glutamate, a novel osmolyte, in marine methanogenic bacteria.

The unusual compound beta-aminoglutaric acid (beta-glutamate) has been identified by 13C nuclear magnetic resonance spectroscopy in soluble extracts of marine methanogenic bacteria. We examined several methanogen species representing nine genera and found that beta-glutamate occurred in methanococci and two methanogenium strains (Methanogenium cariaci JR1 and "Methanogenium anulus" AN9). The presence of this compound in the methanococci examined was further restricted to thermophilic members of the genus Methanococcus, including Methanococcus thermolithotrophicus strains, Methanococcus jannaschii, and "Methanococcus igneus." The two Methanogenium strains examined were mesophiles. Studies using Methanococcus thermolithotrophicus showed that levels of beta-glutamate in cells of that species were not affected by variation in growth temperature (40 to 65 degrees C), NH4+ (2 to 80 mM), Mg2+ (10 to 50 mM), or K+ (2 to 10 mM) in the medium. In contrast, soluble pools of beta-glutamate and L-alpha-glutamate (the other major free amino acid in all the methanococci) were proportional to NaCl levels in the growth medium. This dependence of beta-glutamate and L-alpha-glutamate concentrations on salt levels in the medium suggests that they function as osmolytes in these cells.     

Ferrihydrite-dependent growth of Sulfurospirillum deleyianum through electron transfer via sulfur cycling

Observations in enrichment cultures of ferric iron-reducing bacteria indicated that ferrihydrite was reduced to ferrous iron minerals via sulfur cycling with sulfide as the reductant. Ferric iron reduction via sulfur cycling was investigated in more detail with Sulfurospirillum deleyianum, which can utilize sulfur or thiosulfate as an electron acceptor. In the presence of cysteine (0.5 or 2 mM) as the sole sulfur source, no (microbial) reduction of ferrihydrite or ferric citrate was observed, indicating that S. deleyianum is unable to use ferric iron as an immediate electron acceptor. However, with thiosulfate at a low concentration (0.05 mM), growth with ferrihydrite (6 mM) was possible and sulfur was cycled up to 60 times. Also, spatially distant ferrihydrite in agar cultures was reduced via diffusible sulfur species. Due to the low concentrations of thiosulfate, S. deleyianum produced only small amounts of sulfide. Obviously, sulfide delivered electrons to ferrihydrite with no or only little precipitation of black iron sulfides. Ferrous iron and oxidized sulfur species were produced instead, and the latter served again as the electron acceptor. These oxidized sulfur species have not yet been identified. However, sulfate and sulfite cannot be major products of ferrihydrite-dependent sulfide oxidation, since neither compound can serve as an electron acceptor for S. deleyianum. Instead, sulfur (elemental S or polysulfides) and/or thiosulfate as oxidized products could complete a sulfur cycle-mediated reduction of ferrihydrite.     

Component A3 of the methylcoenzyme M methylreductase system of Methanobacterium thermoautotrophicum delta H: resolution into two components.

Component A3 of the methylcoenzyme M methylreductase system of Methanobacterium thermoautotrophicum (strain delta H) has been resolved into two fractions. One, named component A3a, was defined as the fraction required along with components A2 and C to produce methane from 2-(methylthio)ethanesulfonate when titanium(III) citrate was used as the sole source of electrons. The second one, named component A3b, was required when H2 and 7-mercapto-N-heptanoyl-O-phospho-L-threonine were provided as the dual source of electrons. Component A3a was a large iron-sulfur protein aggregate (Mr 500,000) and is most likely involved in providing electrons at a low potential for the reductive activation of component C.     

Ferrihydrite-Dependent Growth of Sulfurospirillum deleyianum through Electron Transfer via Sulfur Cycling

Observations in enrichment cultures of ferric iron-reducing bacteria indicated that ferrihydrite was reduced to ferrous iron minerals via sulfur cycling with sulfide as the reductant. Ferric iron reduction via sulfur cycling was investigated in more detail with Sulfurospirillum deleyianum, which can utilize sulfur or thiosulfate as an electron acceptor. In the presence of cysteine (0.5 or 2 mM) as the sole sulfur source, no (microbial) reduction of ferrihydrite or ferric citrate was observed, indicating that S. deleyianum is unable to use ferric iron as an immediate electron acceptor. However, with thiosulfate at a low concentration (0.05 mM), growth with ferrihydrite (6 mM) was possible and sulfur was cycled up to 60 times. Also, spatially distant ferrihydrite in agar cultures was reduced via diffusible sulfur species. Due to the low concentrations of thiosulfate, S. deleyianum produced only small amounts of sulfide. Obviously, sulfide delivered electrons to ferrihydrite with no or only little precipitation of black iron sulfides. Ferrous iron and oxidized sulfur species were produced instead, and the latter served again as the electron acceptor. These oxidized sulfur species have not yet been identified. However, sulfate and sulfite cannot be major products of ferrihydrite-dependent sulfide oxidation, since neither compound can serve as an electron acceptor for S. deleyianum. Instead, sulfur (elemental S or polysulfides) and/or thiosulfate as oxidized products could complete a sulfur cycle-mediated reduction of ferrihydrite.     

Characterization of RNase P holoenzymes from Methanococcus jannaschii and Methanothermobacter thermoautotrophicus

The partial purification and basic biochemical characterization of the RNase P holoenzymes of two species of methanogenic Archaea, Methanothermobacter thermoautotrophicus (previously Methanobacterium thermoautotrophicum strain deltaH) and Methanococcus jannaschii, are described. The properties of these enzymes, particularly buoyant density in Cs2SO4 and recent information about the subunit composition of the archaeal enzymes, suggest that RNase P enzymes in Archaea are much more alike than earlier studies in Sulfolobus acidocaldarius and Haloferax volcanii suggested.     

Isolation, characterization, and in situ detection of a novel chemolithoautotrophic sulfur-oxidizing bacterium in wastewater biofilms growing under microaerophilic conditions

We successfully isolated a novel aerobic chemolithotrophic sulfur-oxidizing bacterium, designated strain SO07, from wastewater biofilms growing under microaerophilic conditions. For isolation, the use of elemental sulfur (S(0)), which is the most abundant sulfur pool in the wastewater biofilms, as the electron donor was an effective measure to establish an enrichment culture of strain SO07 and further isolation. 16S rRNA gene sequence analysis revealed that newly isolated strain SO07 was affiliated with members of the genus Halothiobacillus, but it was only distantly related to previously isolated species (89% identity). Strain SO07 oxidized elemental sulfur, thiosulfate, and sulfide to sulfate under oxic conditions. Strain SO07 could not grow on nitrate. Organic carbons, including acetate, propionate, and formate, could not serve as carbon and energy sources. Unlike other aerobic sulfur-oxidizing bacteria, this bacterium was sensitive to NaCl; growth in medium containing more than 150 mM was negligible. In situ hybridization combined with confocal laser scanning microscopy revealed that a number of rod-shaped cells hybridized with a probe specific for strain SO07 were mainly present in the oxic biofilm strata (ca. 0 to 100 micro m) and that they often coexisted with sulfate-reducing bacteria in this zone. These results demonstrated that strain SO07 was one of the important sulfur-oxidizing populations involved in the sulfur cycle occurring in the wastewater biofilm and was primarily responsible for the oxidation of H(2)S and S(0) to SO(4)(2-) under oxic conditions.