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 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.     

Degradation of hydroquinone,gentisate, and benzoate by a fermenting bacterium in pure or defined mixed culture

From a methanogenic fixed-bed reactor fed with hydroquinone as sole energy and carbon source, a rodshaped bacterium was isolated in pure culture which could degrade hydroquinone and gentisate (2,5-dihydroxybenzoate). In syntrophic coculture with either Desulfovibrio vulgaris or Methanospirillum hungatei, also benzoate could be degraded. Other substrates such as sugars, fatty acids, alcohols, and cyclohexane derivatives were not degraded. Sulfate, sulfite, or nitrate were not used as external electron acceptor. The isolate was a Gram-negative, non-motile, nonsporeforming strict anaerobe; the guanine-plus-cytosine content of the DNA was 53.2±1.0 mol%. In pure culture, hydroquinone was degraded to acetate and benzoate, probably via an intermediate carboxylation. In syntrophic mixed cultures, all three substrates were converted completely to acetate. Phenol was never detected as a fermentation product.     

Anaerobic degradation of 1,3-propanediol by sulfate-reducing and by fermenting bacteria

Three strains of strictly anaerobic Gram-negative, non-sporeforming, motile bacteria were enriched and isolated from freshwater sediments with 1,3-propanediol as sole energy and carbon source. Strain OttPdl was a sulfate-reducing bacterium which grew also with lactate, ethanol, propanol, butanol, 1,4-butanediol, formate or hydrogen plus CO₂, the latter only in the presence of acetate. In the absence of sulfate, most of these substrates were fermented to the respective fatty acids in syntrophic cooperation with Methanospirillum hungatei. Sulfur, thiosulfate, or sulfite were reduced, nitrate not. The other two isolates degraded propanediol only in coculture with Methanospirillum hungatei. Strain OttGlycl grew in pure culture with acetoin and with glycerol in the presence of acetate. Strain WoAcl grew in pure culture only with acetoin. Both strains did not grow with other substrates, and did not reduce nitrate, sulfate, sulfur, thiosulfate or sulfite. The isolates were affiliated with the genera Desulfovibrio and Pelobacter. The pathways of propanediol degradation and the ecological importance of this process are discussed.     

Isolation and characterization of an anaerobic syntrophic benzoate-degrading bacterium from sewage sludge

An anaerobic, motile, gram-negative, rod-shaped bacterium is described which degrades benzoate in coculture with an H₂-utilizing organism and in the absence of exogenous electron acceptors such as O₂, SO ₄ ⁼ or NO ₃ ^- . The bacterium was isolated from a municipal primary, anaerobic sewage digestor using anaerobic roll-tube medium with benzoate as the main energy source and in syntrophic association with an H₂-utilizing sulfate-reducing Desulfovibrio sp. which cannot utilize benzoate or fatty acids apart from formate as energy source. The benzoate utilizer produced acetate (3 mol/mol of substrate degraded) and presumably CO₂ and H₂, or formate from benzoate. In media without sulfate and with Methanospirillum hungatei (a methanogen that utilizes only H₂–CO₂ or formate as the energy source) added, 3 mol of acetate and 0.7 mol of methane were produced per mol of benzoate and CO₂ was probably formed. Low numbers of Desulfovibrio sp. were present in the methanogenic coculture and a pure coculture of the benzoate utilizer with M. hungatei was not obtained. The generation times for growth of the sulfate-reducing and methanogenic cocultures were 132 and 166h, respectively. The benzoate utilizer did not utilize other common aromatic compounds, C ₃ ^- –C₇ monocarboxylic acids, or C₄-C₆ dicarboxylic acids for growth, nor did it appear to use SO ₄ ⁼ , NO ₃ ^- or fumarate as alternative electron acceptors. Addition of H₂ inhibited growth and benzoate degradation.     

Anaerobic oxidation of fatty acids by Clostridium bryantii sp. nov., a sporeforming,obligately syntrophic bacterium

From marine and freshwater mud samples strictly anaerobic, Gram-positive, sporeforming bacteria were isolated which oxidized fatty acids in obligately syntrophic association with H₂-utilizing bacteria. Even-numbered fatty acids with up to 10 carbon atoms were degraded to acetate and H₂, odd-numbered fatty acids with up to 11 carbon atoms including 2-methylbutyrate were degraded to acetate, propionate and H₂. Neither fumarate, sulfate, thiosulfate, sullur, nor nitrate were reduced. A marine isolate, strain CuCal, is described as type strain of a new species, Clostridium bryantii sp. nov.     

Syntrophorhabdus aromaticivorans gen. nov., sp. nov., the first cultured anaerobe capable of degrading phenol to acetate in obligate syntrophic associations with a hydrogenotrophic methanogen

Phenol degradation under methanogenic conditions has long been studied, but the anaerobes responsible for the degradation reaction are still largely unknown. An anaerobe, designated strain UI(T), was isolated in a pure syntrophic culture. This isolate is the first tangible, obligately anaerobic, syntrophic substrate-degrading organism capable of oxidizing phenol in association with an H(2)-scavenging methanogen partner. Besides phenol, it could metabolize p-cresol, 4-hydroxybenzoate, isophthalate, and benzoate. During the degradation of phenol, a small amount of 4-hydroxybenzoate (a maximum of 4 microM) and benzoate (a maximum of 11 microM) were formed as transient intermediates. When 4-hydroxybenzoate was used as the substrate, phenol (maximum, 20 microM) and benzoate (maximum, 92 microM) were detected as intermediates, which were then further degraded to acetate and methane by the coculture. No substrates were found to support the fermentative growth of strain UI(T) in pure culture, although 88 different substrates were tested for growth. 16S rRNA gene sequence analysis indicated that strain UI(T) belongs to an uncultured clone cluster (group TA) at the family (or order) level in the class Deltaproteobacteria. Syntrophorhabdus aromaticivorans gen. nov., sp. nov., is proposed for strain UI(T), and the novel family Syntrophorhabdaceae fam. nov. is described. Peripheral 16S rRNA gene sequences in the databases indicated that the proposed new family Syntrophorhabdaceae is largely represented by abundant bacteria within anaerobic ecosystems mainly decomposing aromatic compounds.     

Degradation of Cinnamate via β-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 β-oxidation and subsequently completely degraded into acetate, CH₄, and CO₂. Papillibacter cinnamivorans was responsible for benzoate production from cinnamate, whereas a syntrophic association between Syntrophus sp. and the methanogen degraded benzoate to acetate, CH₄, and CO₂. A new anaerobic degradation pathway of cinnamate into benzoate via β-oxidation by a pure culture of P. cinnamivorans is proposed. Received: 27 December 2001 / Accepted: 28 March 2002     

Acetate Inhibition of Methanogenic, Syntrophic Benzoate Degradation

Acetate inhibited benzoate degradation by a syntrophic coculture of an anaerobic benzoate degrader (strain BZ-2) and Methanospirillum strain PM-1; the apparent K_i for acetate was approximately 40 mM. The addition of acetate resulted in a decrease in the hydrogen concentration in the coculture, indicating that phenomena related to interspecies hydrogen transfer affected this value and that the effect of acetate on the benzoate-degrading partner was probably greater than the apparent K_i for the coculture suggests.     

Sporomusa malonica sp. nov., a homoacetogenic bacterium growing by decarboxylation of malonate or succinate

A new strictly anaerobic bacterium was isolated from an enrichment culture with glutarate as sole substrate and freshwater sediment as inoculum, however, glutarate was not metabolized by the pure culture. The isolate was a mesophilic, spore-forming, Gram-negative, motile curved rod. It fermented various organic acids, alcohols, fructose, acetoin, and H₂/CO₂ to acetate, usually as the only product. Other acids were fermented to acetate and propionate or acetate and butyrate. Succinate and malonate were decarboxylated to propionate or acetate, respectively, and served as sole sources of carbon and energy for growth. No inorganic electron acceptors except CO₂ were reduced. Yeast extract (0.05% w/v) was required for growth. Small amounts of cytochrome b were detected in membrane fractions. The guanine-plus-cytosine content of the DNA was 44.1±2 mol%. The isolate is described as a new species of the genus Sporomusa, S. malonica.     

Alcohol conversion by Desulfobulbus propionicus Lindhorst in the presence and absence of sulfate and hydrogen

Ethanol was rapidly degraded to mainly acetate in anaerobic freshwater sediment slurries. Propionate was produced in small amounts. Desulfovibrio species were the dominant bacteria among the ethanol-degrading organisms. The propionate-producing Desulfobulbus propionicus came to the fore under iron-limited conditions in an ethanol-limited chemostat with excess sulfate inoculated with anaerobic intertidal freshwater sediment. In the absence of sulfate, ethanol was fermented by D. propionicus Lindhorst to propionate and acetate in a molar ratio of 2.0.l-Propanol was intermediately produced during the fermentation of ethanol. In the presence of H₂ and CO₂, ethanol was quantitatively converted to propionate. H₂-plus sulfate-grown cells of D. propionicus Lindhorst were able to oxidize l-propanol and l-butanol to propionate and butyrate respectively with the concomitant reduction of acetate plus CO₂ to propionate. Growth was also observed on acetate alone in the presence of H₂ and CO₂ D. propionicus was able to grow mixotrophically on H₂ plus an organic compound. Finally, a brief discussion has been given of the ecological niche of D. propionicus in anaerobic freshwater sediments.     

Fermentation of trihydroxybenzenes by Pelobacter acidigallici gen. nov. sp. nov., a new strictly anaerobic,non-sporeforming bacterium

Five strains of rod-shaped, Gram-negative, non-sporing, strictly anaerobic bacteria were isolated from limnic and marine mud samples with gallic acid or phloroglucinol as sole substrate. All strains grew in defined mineral media without any growth factors; marine isolates required salt concentrations higher than 1% for growth, two freshwater strains only thrived in freshwater medium. Gallic acid, pyrogallol, 2,4,6-trihydroxybenzoic acid, and phloroglucinol were the only substrates utilized and were fermented stoichiometrically to 3 mol acetate (and 1 mol CO₂) per mol with a growth yield of 10g cell dry weight per mol of substrate. Neither sulfate, sulfur, nor nitrate were reduced. The DNA base ratio was 51.8% guanine plus cytosine. A marine isolate, Ma Gal 2, is described as type strain of a new genus and species, Pelobacter acidigallici gen. nov. sp. nov., in the family Bacteroidaceae. In coculture with Acetobacterium woodii, the new isolates converted also syringic acid completely to acetate. Cocultures with Methanosarcina barkeri converted the respective substrates completely to methane and carbon dioxide.     

Isolation and physiological characterization of Syntrophus buswellii strain GA from a syntrophic benzoate-degrading,strictly anaerobic coculture

A stable, syntrophic benzoate-degrading bacterial consortium was enriched from sewage sludge. It oxidized benzoate or 3-phenylpropionate to acetate, H₂ and CO₂. As hydrogen scavengers Methanospirillum hungatei and Desulfovibrio sp. were present. The benzoate-degrading bacteria of this syntrophic culture and of Syntrophus buswelli were able to grow with benzoate/crotonate or crotonate alone in the absence of a hydrogen-utilizing partner organism. If crotonate was the only substrate, acetate and butyrate were produced, while during growth on benzoate or 3-phenylpropionate crotonate served as a reducible co-substrate and was exclusively converted to butyrate. In the presence of crotonate interspecies hydrogen transfer was not necessary as a hydrogen sink. The benzoate degrader was isolated as a pure culture with crotonate as the only carbon source. The pure culture could also grow with benzoate/crotonate or 3-phenylpropionate/crotonate. The effect of high concentrations of crotonate and of acetate or butyrate on growth of the benzoate degrader was investigated. The benzoate degrader was compared with S. buswellii for its morphology, physiology and DNA base composition. Except for the fact that S. buswellii was also able to grow on cinnamate, no differences between the two organisms were detected. The isolate is named S. buswelli, strain GA.     

Anaerobic degradation of o-phenylphenol by mixed and pure cultures

Summary An anaerobic enrichment culture that degraded 0.4 mmol/l per day of o-phenylphenol was selected from sediment of a waste water pond of a sugar factory. From the consortium an o-phenylphenol-degrading bacterium, strain B10, was isolated. Strain B10 could not degrade other aromatic substances, including phenylacetic acid, benzoate, o-hydroxybenzoate, p-hydroxybenzoate and phenol. Best growth was observed with glucose, pyruvate, lactate, methanol and H₂/CO₂ as substrates. o-Phenylphenol was slowly degraded if supplied as the only carbon source and was cometabolized in the presence of >5 mmol/l glucose. Strain B10 has not yet been assigned to a known species or family.     

Fermentative toluene degradation in anaerobic defined syntrophic cocultures

A syntrophic coculture of a new sulfate-reducing isolate, strain TRM1, with Wolinella succinogenes degraded toluene with either fumarate or NO3- as the terminal electron acceptor. Neither strain TRM1 nor W. succinogenes could metabolise toluene under these conditions in pure culture. Syntrophic degradation was 2-3 times slower than toluene utilisation by strain TRM1 in pure culture with sulfate as electron acceptor. The culture did not produce benzoate or fatty acids like acetate or propionate in detectable amounts. An increase in biomass of the syntrophic toluene-degrading culture was shown in a growth curve with nitrate as the terminal electron acceptor. Both partner organisms were detected microscopically at the end of the growth experiment. Syntrophic degradation of toluene with W. succinogenes and fumarate as the terminal electron acceptor was also demonstrated with the iron reducer Geobacter metallireducens. The results provide the first example of a fermentative oxidation of an aromatic hydrocarbon in a defined coculture.     

Fermentation of mandelate to benzoate and acetate by a homoacetogenic bacterium

A strictly anaerobic, Gram-positive, rod-shaped bacterium, strain AmMan1, was isolated from freshwater sediment with mandelate (-hydroxy-phenylacetate) as sole carbon and energy source, and was assigned to the genus Acetobacterium. Only the d-enantiomer of mandelate was degraded, and was fermented to acetate and benzoate. Non-aromatic growth substrates (pyruvate, lactate, malate, glycerol, ethylene glycol, and H₂/CO₂) were fermented to acetate as sole product. Methoxylated aromatics were demethoxylated to the corresponding phenols. The guanine-plus-cytosine content of the DNA was 36.5±1.5%. Carbon monoxide dehydrogenase, dichlorophenol indophenol-reducing lactate dehydrogenase, NAD-dependent mandelate dehydrogenase, phosphate acetyl transferase, acetate kinase, and pyruvate- or phenylglyoxylate-dependent benzylviologen reductase were measured in mandelate-and/or lactate-grown cells, respectively. A pathway of the homoacetogenic fermentation of mandelate is suggested as another example of incomplete substrate oxidation by homoacetogenic bacteria.     

Fermentative degradation of resorcinol and resorcylic acids

Anaerobic fermentative degradation of resorcinol and resorcylates was studied in enrichment cultures inoculated with marine or freshwater sediments or digested sludge. -Resorcylate (3,5-dihydroxybenzoate) was degraded very rapidly to acetate and methane by enrichment cultures inoculated with freshwater sediment or sewage sludge, but degradation was slow in enrichments from marine habitats. The freshwater cultures did not degrade any other related phenolic substrates. Inhibition of methanogenic bacteria by bromoethanesulfonate and acetylene led to enhanced acetate formation indicating homoacetogenic hydrogen oxidation. With resorcinol (1,3-dihydroxybenzene) and - and -resorcylate (2,4- and 2,6-dihydroxybenzoate), two different types of Gram-positive spore-forming strict anaerobes were isolated, which both did not grow with -resorcylate. Both were assigned to the genus Clostridium. From freshwater enrichments, six strains were isolated in defined coculture with Campylobacter sp. They fermented resorcinol and - and -resorcylate stoichiometrically to acetate and butyrate. No interspecies hydrogen transfer to methanogenic or other anaerobic bacteria was found. None out of numerous organic nutrients tested substituted for Campylobacter sp. as partner in defined cultures; the nutritive dependence of this bacterium could not be elucidated. Isolates from marine sediments formed acetate and hydrogen from resorcyclic compounds, and depended on syntrophic association with hydrogenscavenging anaerobes such as methanogens.     

Anaerobic degradation of 3-aminobenzoate by a newly isolated sulfate reducer and a methanogenic enrichment culture

A new rod-shaped, gram-negative, non-sporing sulfate reducer, strain mAB1, was enriched and isolated from marine sediment samples with 3-aminobenzoate as sole electron and carbon source. Strain mAB1 degraded 3-aminobenzoate completely to CO₂ and NH₃ with stoichiometric reduction of sulfate to sulfide. Cells contained carbon monoxide dehydrogenase, cytochromes, and sulfite reductase P582. Strain mAB1 degraded also benzoate, 4-aminobenzoate, hydroxybenzoates, and some aliphatic compounds. Besides sulfates, also sulfite was reduced with 3-aminobenzoate as electron donor, but not thiosulfate, sulfur, nitrate, or fumarate. The strain grew in sulfide-reduced mineral medium supplemented with 7 vitamins. Strain mAB1 was tentatively affiliated with the genus Desulfobacterium. Experiments with dense cell supsensions showed benzoate accumulation during 3-aminobenzoate degradation under conditions of sulfate limitation or cyanide inhibition. 3-Aminobenzoate was activated to 3-aminobenzoyl-CoA by cell extracts in the presence of ATP, coenzyme A, and Mg²⁺. Acitivity of 3-aminobenzoyl-CoA synthetase was 16 nmol per min and mg protein, with a K_M for 3-aminobenzoate lower than 50 M. Cell extract of 3-aminobenzoate-grown cells activated also 3-hydroxybenzoate (31.7 nmol per min and mg protein) and benzoate (2.3 nmol per min and mg protein), but not 2-aminobenzoate or 4-aminobenzoate. In the presence of NADH of NADPH, 3-aminobenzoyl-CoA was further metabolized to a not yet identified reduced product.Freshwater enrichments with 3-aminobenzoate in the absence of an extenal electron acceptor led to a stable methanogenic enrichment culture consisting of three types of bacteria. 3-Aminobenzoate was degraded completely to CO₂ and stoichiometric amounts of CH₄, with intermediary acetate accumulation.     

Fructose degradation byDesulfovibrio sp. in pure culture and in coculture withMethanospirillum hungatei

In a mineral medium containing sulfate, the sulfate-reducing bacteriumDesulfovibrio sp. strain JJ degraded 1 mol of fructose stoichiometrically to 1 mol of H₂S, 2 mol of acetate, and presumably 2 mol of CO₂. The doubling time was 10 h, and the yield was 41.6 g dry weight/mol fructose degraded. In the absence of sulfate, the hydrogenophilic methanogenMethanospirillum hungatei replaced sulfate as hydrogen sink. In such cocultures, 1 mol of fructose was converted to acetate, methane, succinate, and presumably CO₂ in varying concentrations. The growth yield of the H₂-transferring association was 33 g dry weight/mol fructose. In the absence of sulfate,Desulfovibrio strain JJ slowly fermented 1 mol of fructose to 1 mol of succinate, 0.5 mol of acetate, and 0.5 mol of ethanol. The results are compared with those of other anaerobic hexose-degrading bacteria.     

Oxidation of primary aliphatic alcohols by Acetobacterium carbinolicum sp. nov., a homoacetogenic anaerobe

Four strains of new homoacetogenic bacteria were enriched and isolated from freshwater sediments and sludge with ethanol, propanol, 1,2-propanediol, or 1,2-butanediol as substrates. All strains were Gram-positive nonsporeforming rods and grew well in carbonate-buffered defined media under obligately anaerobic conditions. Optimal growth occurred at 27° C around pH 7.0. H₂/CO₂, primary aliphatic alcohols C₃–C₅, glucose, fructose, lactate, pyruvate, ethylene glycol, 1,2-propanediol, 2,3-butanediol, acetoin, glycerol, and methyl groups of methoxylated benzoate derivates and betaine were fermented to acetate or, in case of primary alcohols C₃–C₅ and 1,2-propanediol, to acetate and the respective fatty acid. In coculture with methanogens methane was formed, probably due to interspecies hydrogen transfer. Strain WoProp 1 is described as a new species, Acetobacterium carbinolicum. It had a DNA base composition of 38.5±1.0% guanine plus cytosine, and contained murein of crosslinkage type B similar to A. woodii.