Anaerobic degradation of 4-hydroxybenzoate: Reductive dehydroxylation of 4-hydroxybenzoyl-CoA and ATP formation during 4-hydroxybenzoate decarboxylation by the phenol-metabolizing bacteria of a stable,strictly anaerobic consortium

4-Hydroxybenzoate was activated with coenzyme A by cells of a strictly anaerobic, phenol-degrading mixed culture to 4-hydroxybenzoyl-CoA, which was reductively dehydroxylated to benzoyl-CoA with reduced benzylviologen as an electron donor. The specific activity of the 4-hydroxybenzoyl-CoA ligase in cell-free extracts of the culture was 100–200 nmol min^–1 mg^–1, that of 4-hydroxybenzoyl-CoA reductase 14.5 nmol min^–1 mg^–1. An increased growth yield of the phenol-degrading mixed culture of 1.8 g/mol with 4-hydroxybenzoate in comparison to phenol as the substrate was found previously and indicated energy generation by decarboxylation of 4-hydroxybenzoate. Addition of 4-hydroxybenzoate to cell suspensions of the mixed culture resulted in a rapid increase of the cellular ATP level. The proton ionophore carbonylcyanidem-chlorophenylhydrazone and the H⁺-ATPase inhibitor dicyclohexylcarbodiimide prevented an increase of cellular ATP levels during 4-hydroxybenzoate decarboxylation, whereas the sodium ionophore monensin and the putative Na⁺-ATPase inhibitor ouabain revealed no effect. This was taken as good evidence for the generation of a proton gradient across the membrane by decarboxylation of 4-hydroxybenzoate and ATP formation by H+-ATPase.     

Degradation of phenol via carboxylation to benzoate by a defined,obligate syntrophic consortium of anaerobic bacteria

Summary An obligate syntrophic culture was selected in mineral medium with phenol as the only carbon and energy source. The consortium consisted of a short and a long rod-shaped bacterium and of low numbers of Desulfovibrio cells, and grew only in syntrophy with methanogens, e. g. Methanospirillum hungatei. Under N₂/CO₂, phenol was degraded via benzoate to acetate, CH₄ and CO₂, while in the presence of H₂/CO₂ benzoate was formed, but not further degraded. When 4-hydroxybenzoate was fed to the mixed culture, it was decarboxylated to phenol prior to benzoate formation and subsequent ring cleavage. Isolation of pure cultures of the two rod-shaped bacteria failed. Microscopic observations during feeding of either 4-hydroxybenzoate, phenol or benzoate implied an obligate syntrophic interdependence of the two different rod-shaped bacteria and of the methanogen. The non-motile rods formed phenol from 4-hydroxybenzoate and benzoate from phenol, requiring an as yet unknown co-substrate or co-factor, probably cross-fed by the short, motile rod. The short, motile rodshaped bacterium grew only in syntrophy with methanogens and degraded benzoate to acetate, CO₂ and methane. Desulfovibrio sp., present in low numbers, apparently could not contribute to the degradation of phenol or 4-hydroxybenzoate.     

Fermentative degradation of putrescine by new strictly anaerobic bacteria

Three strains of new strictly anaerobic, Grampositive, non-sporeforming bacteria were isolated from various anoxic sediment samples with putrescine as sole carbon and energy source. Optimal growth in carbonate-buffered defined medium occurred at 37°C at pH 7.2–7.6. The DNA base ratio of strain NorPut1 was 29.6±1 mol% guanine plus cytosine. In addition to a surface layer and the peptidoglycan layer, the cell wall contained a second innermost layer with a periodic arrangement of subunits. All strains fermented putrescine to acetate, butyrate, and molecular hydrogen; the latter originated from both oxidative putrescine deamination and 4-aminobutyraldehyde oxidation. In defined mixed cultures with methanogens or homoacetogenic bacteria, methane or additional acetate were formed due to interspecies hydrogen transfer. Also 4-aminobutyrate and 4-hydroxybutyrate were fermented to acetate and butyrate, but no hydrogen was released from these substrates. No sugars, organic acids, other primary amines or amino acids were used as substrates. Neither sulfate, thiosulfate, sulfur, nitrate nor fumarate was reduced. Most of the enzymes involved in putrescine degradation could be demonstrated in cell-free extracts. A pathway of putrescine fermentation via 4-aminobutyrate and crotonyl-CoA with subsequent dismutation to acetate and butyrate is suggested.     

严格厌氧菌铁氧还蛋白的研究进展

铁氧还蛋白(ferredoxin,Fd)是一类含有铁硫簇的小分子蛋白质,广泛存在于自然界中,参与生物体内的呼吸、发酵、固氮、二氧化碳固定和制氢等生理过程.Fd对于严格厌氧的细菌尤为重要,是因为这类细菌的能量代谢高度依赖于低氧化还原电势的生物组分,而Fd能够利用铁硫中心灵活调节其氧还电势,适应低电势需求.本文选取厌氧细菌...     

Degradation of cinnamate via beta-oxidation to benzoate by a defined,syntrophic consortium of anaerobic bacteria

A syntrophic consortium was enriched in a basal medium containing cinnamate as the carbon and energy source. It was found to consist of three morphologically distinct microbes, viz., a short, rod-shaped, non-motile bacterium with distinctly pointed ends, Papillibacter cinnamivorans; a rod-shaped, motile bacterium with rounded ends, Syntrophus sp.; and a methanoarchaeon, Methanobacterium sp. This methanogen was then replaced by a collection strain of Methanobacterium formicicum. A syntrophic interdependency of the three partners of the consortium was observed during growth on cinnamate. In the presence of bromoethanesulfonic acid (BESA), cinnamate was transformed to benzoate, whereas under methanogenic conditions without BESA, cinnamate was first transformed to benzoate via beta-oxidation and subsequently completely degraded into acetate, CH(4), and CO(2). Papillibacter cinnamivorans was responsible for benzoate production from cinnamate, whereas a syntrophic association between Syntrophus sp. and the methanogen degraded benzoate to acetate, CH(4), and CO(2). A new anaerobic degradation pathway of cinnamate into benzoate via beta-oxidation by a pure culture of P. cinnamivorans is proposed.     

Hydrogen evolution by strictly aerobic hydrogen bacteria under anaerobic conditions.

When strains and mutants of the strictly aerobic hydrogen-oxidizing bacterium Alcaligenes eutrophus are grown heterotrophically on gluconate or fructose and are subsequently exposed to anaerobic conditions in the presence of the organic substrates, molecular hydrogen is evolved. Hydrogen evolution started immediately after the suspension was flushed with nitrogen, reached maximum rates of 70 to 100 mumol of H2 per h per g of protein, and continued with slowly decreasing rates for at least 18 h. The addition of oxygen to an H2-evolving culture, as well as the addition of nitrate to cells (which had formed the dissimilatory nitrate reductase system during the preceding growth), caused immediate cessation of hydrogen evolution. Formate is not the source of H2 evolution. The rates of H2 evolution with formate as the substrate were lower than those with gluconate. The formate hydrogenlyase system was not detectable in intact cells or crude cell extracts. Rather the cytoplasmic, NAD-reducing hydrogenase is involved by catalyzing the release of excessive reducing equivalents under anaerobic conditions in the absence of suitable electron acceptors. This conclusion is based on the following experimental results. H2 is formed only by cells which had synthesized the hydrogenases during growth. Mutants lacking the membrane-bound hydrogenase were still able to evolve H2. Mutants lacking the NAD-reducing or both hydrogenases were unable to evolve H2.     

Sulfide removal from livestock biogas by Azospirillum-like anaerobic phototrophic bacteria consortium

Biogas production from anaerobic biodegradation of livestock waste is a potential source of renewable energy. In addition to methane, biodegradation of this high-strength waste also produces sulfide that must be removed in order to prevent costly corrosive impacts on infrastructure. In this work, an anaerobic, phototrophic microbial community enriched from the native population in a swine waste lagoon was evaluated for its potential to remove sulfide from swine waste biogas. Batch experiments with the consortium attained removal efficiencies greater than 97% for sulfide concentrations above 1200 ppm. 16S rRNA gene sequencing revealed that the dominant population was most closely related to the isolate Azospirillum strain C5 (similarity index of 99%). Photomicrograph of the enriched consortium revealed the presence of cells with intracellular globules resembling sulfur storage. The enrichment of Azospirillum-like and the concomitant sulfide consumption suggest that this microorganism played an important role in sulfide removal in the bioreactor.     

Fermentative degradation of glutarate via decarboxylation by newly isolated strictly anaerobic bacteria

Two strains of new strictly anaerobic, gramnegative bacteria were enriched and isolated from a freshwater (strain WoG13) and a saltwater (strain CuG11) anoxic sediment with glutarate as sole energy source. Strain WoG13 formed spores whereas strain CuG11 did not. Both strains were rod-shaped, motile bacteria growing in carbonate-buffered, sulfide-reduced mineral medium supplemented with 2% of rumen fluid. Both strains fermented glutarate to butyrate, isobutyrate, CO₂, and small amounts of acetate. With methylsuccinate, the same products were formed, and succinate was fermented to propionate and CO₂. No sugars, amino acids or other organic acids were used as substrates. Molar growth yields (Ys) were very small (0.5–0.9 g cell dry mass/mol dicarboxylate). Cells of strain WoG13 contained no cytochromes, and the DNA base ratio was 49.0±1.4 mol% guanine-plus-cytosine. Enzyme activities involved in glutarate degradation could bedemonstrated in cell-free extracts of strain WoG13. A pathway of glutarate fermentation via decarboxylation of glutaconyl-CoA to crotonyl-CoA is suggested which forms butyrate and partly isobutyrate by subsequent isomerization.     

Effect of specific inhibitors on the anaerobic reductive dechlorination of 2,4,6-trichlorophenol by a stable methanogenic consortium

The transformation of 2,4,6-trichlorophenol (TCP) into 4-chlorophenol (4CP) was studied using a stable methanogenic enrichment culture derived from an anaerobic fixed bed reactor. Using acetate as a growth substrate, different inhibitors of methanogenesis exhibited distinct effects on TCP dechlorination. Whereas reductive dechlorination activity was not affected by 2% ethylene in the gas phase, 25 mM bromoethanesulfonic acid (BESA) had a direct inhibitory effect on this process. The choice of BESA as a specific inhibitor for identifying the subpopulations involved in reductive dechlorination of chloroaromatics is thus questionable. Inhibitors of sulfate reduction such as molybdate (20 mM) and selenate (20 mM) had a direct inhibitory effect on reductive dechlorination independently of the presence of sulfate in the medium supplemented with acetate as growth substrate. Consequently much more care must also be taken with these inhibitors to prove that reductive chlorination is coupled to sulfate reduction.     

A novel mode of lactate metabolism in strictly anaerobic bacteria

Lactate is a common substrate for major groups of strictly anaerobic bacteria, but the biochemistry and bioenergetics of lactate oxidation is obscure. The high redox potential of the pyruvate/lactate pair of E₀′ = ?190 mV excludes direct NAD⁺ reduction (E₀′ = ?320 mV). To identify the hitherto unknown electron acceptor, we have purified the lactate dehydrogenase (LDH) from the strictly anaerobic, acetogenic bacterium Acetobacterium woodii. The LDH forms a stable complex with an electron‐transferring flavoprotein (Etf) that exhibited NAD⁺ reduction only when reduced ferredoxin (Fd^2?) was present. Biochemical analyses revealed that the LDH/Etf complex of A. woodii uses flavin‐based electron confurcation to drive endergonic lactate oxidation with NAD⁺ as oxidant at the expense of simultaneous exergonic electron flow from reduced ferredoxin (E₀′ ≈ –500 mV) to NAD⁺ according to: lactate + Fd^2? + 2 NAD⁺ → pyruvate + Fd + 2 NADH. The reduced Fd^2? is regenerated from NADH by a sequence of events that involves conversion of chemical (ATP) to electrochemical and finally redox energy (Fd^2? from NADH) via reversed electron transport catalysed by the Rnf complex. Inspection of genomes revealed that this metabolic scenario for lactate oxidation may also apply to many other anaerobes.     

The isolation of tetracycline-resistant strains of strictly anaerobic bacteria from the rumen

Tetracycline resistant (Tc^R) strains of three of the major species of strictly anaerobic rumen bacteria Megasphaera elsdenii, Selenomonas ruminantium and Butyrivibrio fibrisolvens , were recovered with an isolation medium containing 20 μg/ml tetracycline. Only two of 14 strains of these species from other sources, isolated without antibiotic selection, showed tetracycline resistance. Evidence was found for the presence of plasmids in two tetracycline-resistant strains of M. elsdenii , and in some strains of S. ruminantium.     

Transformations of chloroguaiacols, chloroveratroles, and chlorocatechols by stable consortia of anaerobic bacteria

Metabolically stable consortia of anaerobic bacteria obtained by enrichment of sediment samples with 3,4,5-trimethoxybenzoate (TMBA), 3,4,5-trihydroxybenzoate (gallate [GA]), or 5-chlorovanillin (CV) were used to study the anaerobic transformation of a series of chloroveratroles, chloroguaiacols, and chlorocatechols used as cosubstrates. Experiments were carried out with growing cultures, and the following pathways were demonstrated for metabolism of the growth substrates: (i) TMBA produced GA, which was further degraded without the formation of aromatic intermediates; (ii) GA formed pyrogallol, which was stable to further transformation; and (iii) CV was degraded by a series of steps involving de-O-methylation, oxidation of the aldehyde group, and decarboxylation to 3-chlorocatechol before ring cleavage. Mono-de-O-methylation of the cosubstrates occurred rapidly in the order 4,5,6-trichloroguaiacol greater than 3,4,5-trichloroguaiacol approximately 3,4,5-trichloroveratrole approximately tetrachloroveratrole greater than tetrachloroguaiacol and was concomitant with degradation of the growth substrates. For the polymethoxy compounds--chloroveratroles, 1,2,3-trichloro-4,5,6-trimethoxybenzene, and 4,5,6-trichlorosyringol--de-O-methylation took place sequentially. The resulting chlorocatechols were stable to further transformation until the cultures had exhausted the growth substrates; selective dechlorination then occurred with the formation of 3,5-dichlorocatechol from 3,4,5-trichlorocatechol and of 3,4,6-trichlorocatechol from tetrachlorocatechol. 2,4,5-, 2,4,6-, and 3,4,5-trichoroanisole and 2,3,4,5-tetrachloroanisole were de-O-methylated, but the resulting chlorophenols were resistant to dechlorination. These results extend those of a previous study with spiked sediment samples and their endogenous microflora and illustrate some of the transformations of chloroguaiacols and chlorocatechols which may be expected to occur in anaerobic sediments.     

Variability in survival of Pectinatus cerevisiiphilus, strictly anaerobic bacteria, under different oxygen conditions

Survival of Pectinatus cerevisiiphilus DSM 20466 in pure culture at variable temperatures under different oxygen concentrations was measured. Survival of P. cerevisiiphilus in co-culture with Saccharomyces cerevisiae under both saturated oxygen and brewing conditions was also studied. The survival of strictly anaerobic bacteria to oxygen seems to follow the classical laws of heat resistance. The D(oxy) values of P. cerevisiiphilus , calculated as a function of oxygen level, shows that the oxygen level is important for the survival duration of the bacteria. The temperature greatly influences the oxygen resistance of P. cerevisiiphilus, which increases when the temperature decreases. P. cerevisiiphilus resists better in co-culture than in pure culture under saturated oxygen conditions. Therefore, the oxygenation of the wort does not totally eliminate the risk of beer contamination by this bacterium. Under brewing conditions in co-culture at 8 degrees C, P. cerevisiiphilus grows slowly to reach a final cell concentration up to 10(6) cells/mL in beer, which is undrinkable. Pectinatus is a strictly anaerobic bacterium; however, it is resistant under certain oxygen conditions of incubation. This resistance is considerably higher in the presence of Saccharomyces cerevisiae .     

Transformations of chloroguaiacols, chloroveratroles, and chlorocatechols by stable consortia of anaerobic bacteria.

Metabolically stable consortia of anaerobic bacteria obtained by enrichment of sediment samples with 3,4,5-trimethoxybenzoate (TMBA), 3,4,5-trihydroxybenzoate (gallate [GA]), or 5-chlorovanillin (CV) were used to study the anaerobic transformation of a series of chloroveratroles, chloroguaiacols, and chlorocatechols used as cosubstrates. Experiments were carried out with growing cultures, and the following pathways were demonstrated for metabolism of the growth substrates: (i) TMBA produced GA, which was further degraded without the formation of aromatic intermediates; (ii) GA formed pyrogallol, which was stable to further transformation; and (iii) CV was degraded by a series of steps involving de-O-methylation, oxidation of the aldehyde group, and decarboxylation to 3-chlorocatechol before ring cleavage. Mono-de-O-methylation of the cosubstrates occurred rapidly in the order 4,5,6-trichloroguaiacol greater than 3,4,5-trichloroguaiacol approximately 3,4,5-trichloroveratrole approximately tetrachloroveratrole greater than tetrachloroguaiacol and was concomitant with degradation of the growth substrates. For the polymethoxy compounds--chloroveratroles, 1,2,3-trichloro-4,5,6-trimethoxybenzene, and 4,5,6-trichlorosyringol--de-O-methylation took place sequentially. The resulting chlorocatechols were stable to further transformation until the cultures had exhausted the growth substrates; selective dechlorination then occurred with the formation of 3,5-dichlorocatechol from 3,4,5-trichlorocatechol and of 3,4,6-trichlorocatechol from tetrachlorocatechol. 2,4,5-, 2,4,6-, and 3,4,5-trichoroanisole and 2,3,4,5-tetrachloroanisole were de-O-methylated, but the resulting chlorophenols were resistant to dechlorination. These results extend those of a previous study with spiked sediment samples and their endogenous microflora and illustrate some of the transformations of chloroguaiacols and chlorocatechols which may be expected to occur in anaerobic sediments.     

Lipid-phase transitions of the strictly anaerobic bacteria Veillonella parvula and Anaerovibrio lipolytica.

As a basis for physicochemical studies on the membranes of the strictly anaerobic bacteria Veillonella parvula, Anaerovibrio lipolytica, and Megasphaera elsdenii, the fatty acyl and alk-1-enyl moieties on the phosphoglycerides of these organism were characterized. Uncommon is the high proportion of a heptadecenoic acyl and alk-1-enyl moiety in these three lactate-fermenting bacteria. In contrast to V. parvula and A. lipolytica, M. elsdenii contains high amounts of branched-chain acyl and alk-1-enyl moieties. Freeze-etching electron microscopy showed that the lipids of the plasma membranes of V. parvula and A. lipolytica go from the liquid crystalline to the gel state upon lowering of the temperature, indicating that the membrane lipids are predominantly in the fluid state. No lipid-protein segregation could be detected in the plasma membrane of M. elsdenii. This can be explained by the abundance of branched-chain fatty acyl and alk-1-enyl residues in the membranes of this organism which may prevent lipid-protein segregation during the lipid-phase transition.     

Microbial bioassay of fungal compounds that stimulate the growth of a consortium of anaerobic cellulolytic bacteria

Summary A bioassay is developed for testing growth factors present in fungal extracts and acting on a consortium of cellulolytic bacteria produced in a continuous anaerobic digester inoculated with rumen liquor and fed with carboxymethylcellulose. Fungal extracts stimulated overall biomass production of the cellulolytic bacterial consortium without changing specific cellulolytic activity.     

Fermentative degradation of glyoxylate by a new strictly anaerobic bacterium

A new strictly anaerobic, gram-negative, nonsporeforming bacterium, Strain PerGlx1, was enriched and isolated from marine sediment samples with glyoxylate as sole carbon and energy source. The guanineplus-cytosine content of the DNA was 44.1±0.2 mol %. Glyoxylate was utilized as the only substrate and was stoichiometrically degraded to carbon dioxide, hydrogen, and glycolate. An acetyl-CoA and ADP-dependent glyoxylate converting enzyme activity, malic enzyme, and pyruvate synthase were found at activities sufficient for growth (0.25 U x mg protein^-1). These findings allow to design a new degradation pathway for glyoxylate: glyoxylate is condensed with acetyl-CoA to form malyl-CoA; the free energy of the thioester linkage in malyl-CoA is conserved by substrate level phosphorylation. Part of the electrons released during glyoxylate oxidation to CO₂ reduce a small fraction of glyoxylate to glycolate.     

Fermentative degradation of dipicolinic acid (pyridine-2,6-dicarboxylic acid) by a defined coculture of strictly anaerobic bacteria

Degradation of dipicolinic acid (pyridine-2,6-dicarboxylic acid) under strictly anaerobic conditions was studied in enrichment cultures from marine and freshwater sediments. In all cases, dipicolinic acid was completely degraded. From an enrichment culture from a marine sediment, a defined coculture of two bacteria was isolated. The dipicolinic acid-fermenting bacterium was a Gram-negative, non-sporeforming strictly anaerobic short rod which utilized dipicolinic acid as sole source of carbon, energy, and nitrogen, and fermented it to acetate, propionate, ammonia, and 2CO₂. No other substrate was fermented. This bacterium could be cultivated only in coculture with another Gram-negative, non-sporeforming rod from the same enrichment culture which oxidized acetate to CO₂ with fumarate, malate, or elemental sulfur as electron acceptor, similar to Desulfuromonas acetoxidans. Since this metabolic activity is not important in substrate degradation by the coculture, the basis of the dependence of the dipicolinic acid-degrading bacterium on the sulfur reducer may be sought in the assimilatory metabolism.