Analysis of the anaerobic microbial community capable of degrading p-toluene sulphonate

Three strains of Clostridium sp., 14 (VKM B-2201), 42 (VKM B-2202), and 21 (VKM B-2279), two methanogens, Methanobacterium formicicum MH (VKM B-2198) and Methanosarcina mazei MM (VKM B-2199), and one sulfate-reducing bacterium, Desulfovibrio sp. SR1 (VKM B-2200), were isolated in pure cultures from an anaerobic microbial community capable of degrading p-toluene sulfonate. Strain 14 was able to degrade p-toluene sulfonate in the presence of yeast extract and bactotryptone and, like strain 42, to utilize p-toluene sulfonate as the sole sulfur source with the production of toluene. p-Toluene sulfonate stimulated the growth of Ms. mazei MM on acetate. The sulfate-reducing strain Desulfovibrio sp. SR1 utilized p-toluene sulfonate as an electron acceptor. The putative scheme of p-toluene sulfonate degradation by the anaerobic microbial community is discussed.     

Enantiomeric degradation of 2-(4-Sulfophenyl)Butyrate via 4-sulfocatechol in Delftia acidovorans SPB1

Enrichment cultures with enantiomeric 2-(4-sulfophenyl)butyrate (SPB) as the sole added source(s) of carbon and energy for growth yielded a pure culture of a degradative bacterium, which was identified as Delftia acidovorans SPB1. The organism utilized the enantiomers sequentially. R-SPB was utilized first (specific growth rate [mu] = 0.28 h(-1)), with transient excretion of an unknown intermediate, which was identified as 4-sulfocatechol (4SC). Utilization of S-SPB was slower (mu = 0.016 h(-1)) and was initiated only after the first enantiomer was exhausted. Suspensions of cells grown in S-SPB excreted 4SC, so metabolism of the two enantiomers converged at 4SC. The latter was degraded by ortho cleavage via 3-sulfo-cis,cis-muconate. Strain SPB1 grew with 4SC and with 1-(4-sulfophenyl)octane (referred to herein as model LAS) but not with commercial linear alkylbenzenesulfonate (LAS) surfactant, which is subterminally substituted but nontoxic. It would appear that metabolism of the model LAS does not represent metabolism of commercial LAS.     

Enantiomeric Degradation of 2-(4-Sulfophenyl)Butyrate via 4-Sulfocatechol in Delftia acidovorans SPB1

Enrichment cultures with enantiomeric 2-(4-sulfophenyl)butyrate (SPB) as the sole added source(s) of carbon and energy for growth yielded a pure culture of a degradative bacterium, which was identified as Delftia acidovorans SPB1. The organism utilized the enantiomers sequentially. R-SPB was utilized first (specific growth rate [μ] = 0.28 h⁻¹), with transient excretion of an unknown intermediate, which was identified as 4-sulfocatechol (4SC). Utilization of S-SPB was slower (μ = 0.016 h⁻¹) and was initiated only after the first enantiomer was exhausted. Suspensions of cells grown in S-SPB excreted 4SC, so metabolism of the two enantiomers converged at 4SC. The latter was degraded by ortho cleavage via 3-sulfo-cis,cis-muconate. Strain SPB1 grew with 4SC and with 1-(4-sulfophenyl)octane (referred to herein as model LAS) but not with commercial linear alkylbenzenesulfonate (LAS) surfactant, which is subterminally substituted but nontoxic. It would appear that metabolism of the model LAS does not represent metabolism of commercial LAS.     

Sulfonate-sulfur can be assimilated for fermentative growth

Abstract Bacterial assimilation of sulfonate-sulfur under anaerobic conditions has been demonstrated. Two different bacteria able to grow fermentatively using sulfonate-sulfur as sole sulfur source were isolated by enrichment culture; neither were able to utilize sulfonates as sole source of carbon and energy for growth. The isolate of Clostridium pasteurianum assimilated the sulfur of isethionate (2-hydroxyethanesulfonate), taurine (2-aminoethanesulfonate), or p -toluenesulfonate. A facultatively fermentative Klebsiella strain did not utilize the sulfur of any of these sulfonates, but assimilated cysteate-sulfur; in contrast, when growing by aerobic respiration, the range of sulfonates able to serve as sulfur source was greater. Both bacteria displayed a preferential utilization of sulfate-sulfur to that of the sulfonates tested. Thus, bacterial assimilation of sulfonate-sulfur during anaerobic growth has direct parallels with features until now recognized only for aerobic assimilatory processes.     

Desulfonation of Linear Alkylbenzenesulfonate Surfactants and Related Compounds by Bacteria

Pseudomonas putida S-313 (= DSM 6884) grew in sulfate-free medium when the sole sulfur source supplied was one of several arylsulfonates involved in the synthesis, application, or biodegradation of linear alkyl-benzenesulfonate (LAS) surfactants. 2-(4-Sulfophenyl)butyric acid, 4-n-butyl-1-methyl-6-sulfotetralin, and 4-toluenesulfonic acid were each completely utilized during growth, as were the model LAS 1-(4-sulfophenyl) octane and the arylsulfonate dyestuff Orange II. The product in each case was the corresponding phenol, which was identified by gas chromatography-mass spectrometry or ¹H nuclear magnetic resonance. Stoichiometric conversion of 4-toluenesulfonic acid to 4-cresol was observed. The molar growth yields observed were 2.4 to 2.8 kg of protein per mol of S, which were comparable to the yield for sulfate. Commercial LAS disappeared from growth medium inoculated with strain S-313, but negligible growth occurred; digestion of cells in alkali led to recovery of the LAS mixture, which seemingly sorbed to the cells. However, mixed culture L6 was readily obtained from batch enrichment cultures containing commercial LAS as a sole sulfur source and an inoculum from domestic sewage. Culture L6 desulfonated components of the LAS surfactant to the corresponding phenols, which were identified by gas chromatography-mass spectrometry. Compounds with shorter alkyl chains were desulfonated preferentially, as were the centrally substituted isomers. In the presence of 200 μM sulfate, culture L6 grew well and LAS disappeared, although this was due purely to sorption, as shown by digestion of the cells in alkali. Thus, under sulfate-limited conditions, LAS can be desulfonated directly.     

Taurine-Sulfur Assimilation and Taurine-Pyruvate Aminotransferase Activity in Anaerobic Bacteria

We demonstrated the ability of strictly fermentative, as well as facultatively fermentative, bacteria to assimilate sulfonate sulfur for growth. Taurine (2-aminoethanesulfonate) can be utilized by Clostridium pasteurianum C1 but does not support fermentative growth of two Klebsiella spp. and two different Clostridium spp. However, the latter are able to assimilate the sulfur of a variety of other sulfonates (e.g., cysteate, 3-sulfopyruvate, and 3-sulfolactate) anaerobically. A novel taurine-pyruvate aminotransferase activity was detected in cell extracts of C. pasteurianum C1 grown with taurine as the sole sulfur source. This activity was not detected in extracts of other bacteria examined, in C. pasteurianum C1 grown with sulfate or sulfite as the sulfur source, or in a Klebsiella isolate assimilating taurine-sulfur by aerobic respiration. More common aminotransferase activities (e.g., with aspartate or glutamate as the amino donor and pyruvate, oxalacetate, or (alpha)-ketoglutarate as the amino acceptor) were present, no matter what sulfur source was used for growth. Partial characterization of the taurine-pyruvate aminotransferase revealed an optimal temperature of 37(deg)C and a broad optimal pH range of 7.5 to 9.5.     

Assimilation of sulfur from alkyl- and arylsulfonates by Clostridium spp.

Organisms able to utilize one of several alkyl- and arylsulfonates as sole source of sulfur under anoxic conditions were enriched. Three fermenting bacteria, all putative Clostridium spp., were isolated in pure culture. All three organisms had wide substrate ranges for alkylsulfonates, taurine and arylsulfonates, presumably due to three different enzyme systems. One organism, strain KNNDS (DSM 10612) was selected for further characterization. The organism was possibly a new Clostridium sp., with Clostidium intestinalis as its nearest neighbor (97.6% similarity of rDNA). Strain KNNDS catalyzed complete sulfonate utilization concomitant with growth. Growth yields of approximtely 3 kg protein/mol sulfur were observed, independent of the sulfur source [e.g. sulfate, sulfide, 4-(phenyl)butyl-1-sulfonate, 2,6-naphthyldisulfonate or 4-nitrocatechol sulfate]. We failed to detect significant amounts of either an arylsulfonatase or an arylsulfatase, and we hypothesize different arylsulfatases [EC 3.1.6.1] in aerobes and in Clostridium spp. Received: 15 October / Accepted: 29 November 1996     

Analysis of the Anaerobic Microbial Community Capable of Degrading p-Toluene Sulfonate

Three strains of Clostridium sp., 14 (VKM B-2201), 42 (VKM B-2202), and 21 (VKM B-2279), two methanogens, Methanobacterium formicicum MH (VKM B-2198) and Methanosarcina mazei MM (VKM B-2199), and one sulfate-reducing bacterium, Desulfovibrio sp. SR1 (VKM B-2200), were isolated in pure cultures from an anaerobic microbial community capable of degrading p-toluene sulfonate. Strain 14 was able to degrade p-toluene sulfonate in the presence of yeast extract and bactotryptone and, like strain 42, to utilize p-toluene sulfonate as the sole sulfur source with the production of toluene. p-Toluene sulfonate stimulated the growth of Ms. mazei MM on acetate. The sulfate-reducing strain Desulfovibrio sp. SR1 utilized p-toluene sulfonate as an electron acceptor. The putative scheme of p-toluene sulfonate degradation by the anaerobic microbial community is discussed.     

Taurine serves as sole source of nitrogen for aerobic and anaerobic growth by <Emphasis Type="Italic">Klebsiella</Emphasis> sp.

The sulfur of taurine can be assimilated by Klebsiella sp. during aerobic growth, but not fermentative growth. However, taurine’s N can be utilized by this bacterium as sole nitrogen source for both aerobic and anaerobic growth. Two other amino-containing sulfonates (3-aminopropanesulfonate and cysteate) were also examined for their abilities to serve as nitrogen sources for Klebsiella sp. during the different growth conditions. The result shows that 3-aminopropanesulfonate only supports aerobic growth while cysteate does not under either condition.     

2-Chloro-4-amino-1,3,5-triazine-6(5H)-one: a new intermediate in the biodegradation of chlorinated s-triazines

Pseudomonas sp. strain A grew with 2-chloro-1,3,5-triazine-4,6-diamine as the sole and growth-limiting source of nitrogen. The substrate was utilized quantitatively and concomitantly with growth and with excretion of a product which was identified as 2-chloro-4-amino-1,3,5-triazine-6(5H)-one. The reaction yielded 1 mol of organic product and 1 mol of NH4+ per mol of substrate.     

2-Chloro-4-amino-1,3,5-triazine-6(5H)-one: a new intermediate in the biodegradation of chlorinated s-triazines.

Pseudomonas sp. strain A grew with 2-chloro-1,3,5-triazine-4,6-diamine as the sole and growth-limiting source of nitrogen. The substrate was utilized quantitatively and concomitantly with growth and with excretion of a product which was identified as 2-chloro-4-amino-1,3,5-triazine-6(5H)-one. The reaction yielded 1 mol of organic product and 1 mol of NH4+ per mol of substrate.     

Mineralization of 4-aminobenzenesulfonate (4-ABS) by Agrobacterium sp. strain PNS-1

A bacterial strain, PNS-1, isolated from activated sludge, could utilize sulphanilic acid (4-ABS) as the sole organic carbon and energy source under aerobic conditions. Determination and comparison of 16S r DNA sequences showed that the strain PNS-1 is closely related to the species of Agrobacterium genus. Growth on 4-ABS was accompanied with ammonia and sulfate release. TOC results showed complete mineralization of sulphanilic acid. This strain was highly specific for 4-ABS as none of the sulphonated aromatics used in the present study including other ABS isomers were utilized. Strain PNS-1 could, however, utilize all the tested monocyclic aromatic compounds devoid of a sulfonate group. No intermediates could be detected either in the growth phase or with dense cell suspensions. Presence of chloramphenicol completely inhibited 4-ABS degradation by cells pregrown on succinate, indicating that degradation enzymes are inducible. No plasmid could be detected in the Agrobacterium sp. Strain PNS-1 suggesting that 4-ABS degradative genes may be chromosomal encoded.     

Sulfonates: novel electron acceptors in anaerobic respiration

The enrichment and isolation in pure culture of a bacterium, identified as a strain of Desulfovibrio, able to release and reduce the sulfur of isethionate (2-hydroxyethanesulfonate) and other sulfonates to support anaerobic respiratory growth, is described. The sulfonate moiety was the source of sulfur that served as the terminal electron acceptor, while the carbon skeleton of isethionate functioned as an accessory electron donor for the reduction of sulfite. Cysteate (alanine-3-sulfonate) and sulfoacetaldehyde (acetaldehyde-2-sulfonate) could also be used for anaerobic respiration, but many other sulfonates could not. A survey of known sulfate-reducing bacteria revealed that some, but not all, strains tested could utilize the sulfur of some sulfonates as terminal electron acceptor. Isethionate-grown cells of Desulfovibrio strain IC1 reduced sulfonate-sulfur in preference to that of sulfate; however, sulfate-grown cells reduced sulfate-sulfur in preference to that of sulfonate. Received: 2 May 1996 / Accepted: 8 June 1996     

Non-diauxic fermentation of acid hydrolysed pine by a strain of Clostridium thermohydrosulfuricum isolated from a New Zealand hot spring

Abstract Fermentation of dilute acid hydrolysed pine could be achieved by all of 18 thermophilic glycolytic anaerobic bacteria tested, while only seven isolates representing the species Clostridium thermohydrosulfuricum, Thermoanaerobium brockii and Thermoanaerobacter ethanolicus were able to ferment undiluted hydrolysate. A strain of C. thermohydrosulfuricum , designated Rt8.B1, was notably better at fermenting the hydrolysate and was studied further. Studies showed that glucose and xylose present in the hydrolysate were utilized simultaneously, a phenomenon which also occurred in a medium containing glucose and xylose as the added carbohydrates.     

Syntrophic Effects in a Subsurface Clostridial Consortium on Fe(III)-(Oxyhydr)oxide Reduction and Secondary Mineralization

In this study, we cultivated from subsurface sediments an anaerobic clostridial consortium that was composed of a fermentative Fe-reducer Clostridium species (designated as strain FGH) and a novel sulfate-reducing bacterium belonging to the clostridia family Vellionellaceae (designated as strain RU4). In pure culture, Clostridium sp. strain FGH mediated the reductive dissolution/transformation of iron oxides during growth on peptone. When Clostridium sp. FGH was grown with strain RU4 on peptone, the rates of iron oxide reduction were significantly higher. Iron reduction by the consortium was mediated by multiple mechanisms, including biotic reduction by Clostridium sp. FGH and biotic/abiotic reactions involving biogenic sulfide formed by strain RU4. The Clostridium sp. FGH produced hydrogen during fermentation, and the presence of hydrogen inhibited growth and iron reduction activity. The sulfate-reducing partner strain RU4 was stimulated by the presence of H₂and generated reactive sulfide which promoted the chemical reduction of the iron oxides. Characterization of Fe(II) mineral products showed the formation of nanoparticulate magnetite during ferrihydrite reduction, and the precipitation of iron sulfides during goethite and hematite reduction. The results suggest an important pathway for iron reduction and secondary mineralization by fermentative sulfate-reducing microbial consortia through syntrophy-driven biotic/abiotic reactions with biogenic sulfide.

Supplemental materials are available for this article. Go to the publisher's online edition of Geomicrobiology Journal to view the supplemental file.     

Clostridium tunisiense sp. nov., a new proteolytic, sulfur-reducing bacterium isolated from an olive mill wastewater contaminated by phosphogypse

A new sporulated fermentative bacterium designated strain E1(T) (T=type strain), was isolated from an anaerobic mud of an olive mill wastewater basin contaminated by phosphogypse produced by a Tunisian factory. Strain E1(T) was a motile Gram-positive slightly curved rod with spherical terminal spore swelling the cell. It grew between 18 degrees C and 43 degrees C with an optimum at 37 degrees C and pH 7.8 (range 5.5-8.7), without NaCl (range 0-3%). Strain E1(T) was a chemoorganotrophic anaerobic bacterium fermenting only proteins and amino acids. Yeast extract was required for growth. Elemental sulfur was used as terminal electron acceptor. The G+C content of the DNA was 32.6 mol%. The closest phylogenetical relatives of strain E1(T) were Clostridium thiosulfatireducens and C. subterminale (97.3% similarity for partial rRNA gene sequences). DNA-DNA hybridization values between strain E1(T) and both species were 17% and 20.8%, respectively. On the basis of differences in genotypic and phenotypic characteristics, strain E1(T) (DSM 15206(T), CIP 107666(T)) is proposed as the type strain of a new species, C. tunisiense sp. nov. GenBank accession number for the 16S rRNA gene sequence of strain E1(T) is AY187622.     

Azoreductase activity of anaerobic bacteria isolated from human intestinal microflora

A plate assay was developed for the detection of anaerobic bacteria that produce azoreductases. With this plate assay, 10 strains of anaerobic bacteria capable of reducing azo dyes were isolated from human feces and identified as Eubacterium hadrum (2 strains), Eubacterium spp. (2 species), Clostridium clostridiiforme, a Butyrivibrio sp., a Bacteroides sp., Clostridium paraputrificum, Clostridium nexile, and a Clostridium sp. The average rate of reduction of Direct Blue 15 dye (a dimethoxybenzidine-based dye) in these strains ranged from 16 to 135 nmol of dye per min per mg of protein. The enzymes were inactivated by oxygen. In seven isolates, a flavin compound (riboflavin, flavin adenine dinucleotide, or flavin mononucleotide) was required for azoreductase activity. In the other three isolates and in Clostridium perfringens, no added flavin was required for activity. Nondenaturing polyacrylamide gel electrophoresis showed that each bacterium expressed only one azoreductase isozyme. At least three types of azoreductase enzyme were produced by the different isolates. All of the azoreductases were produced constitutively and released extracellularly.     

Azoreductase activity of anaerobic bacteria isolated from human intestinal microflora.

A plate assay was developed for the detection of anaerobic bacteria that produce azoreductases. With this plate assay, 10 strains of anaerobic bacteria capable of reducing azo dyes were isolated from human feces and identified as Eubacterium hadrum (2 strains), Eubacterium spp. (2 species), Clostridium clostridiiforme, a Butyrivibrio sp., a Bacteroides sp., Clostridium paraputrificum, Clostridium nexile, and a Clostridium sp. The average rate of reduction of Direct Blue 15 dye (a dimethoxybenzidine-based dye) in these strains ranged from 16 to 135 nmol of dye per min per mg of protein. The enzymes were inactivated by oxygen. In seven isolates, a flavin compound (riboflavin, flavin adenine dinucleotide, or flavin mononucleotide) was required for azoreductase activity. In the other three isolates and in Clostridium perfringens, no added flavin was required for activity. Nondenaturing polyacrylamide gel electrophoresis showed that each bacterium expressed only one azoreductase isozyme. At least three types of azoreductase enzyme were produced by the different isolates. All of the azoreductases were produced constitutively and released extracellularly.     

Bacterial dehalogenation of chlorobenzoates and coculture biodegradation of 4,4'-dichlorobiphenyl.

Acinetobacter sp. strain 4CB1 was isolated from a polychlorobiphenyl-contaminated soil sample by using 4-chlorobenzoate as a sole source of carbon and energy. Resting cells of Acinetobacter sp. strain 4CB1 hydrolytically dehalogenated 4-chlorobenzoate under aerobic and anaerobic conditions, but 4-hydroxybenzoate accumulated only under anaerobic conditions. Cell extracts of Acinetobacter sp. strain 4CB1 oxidized 4-hydroxybenzoate by an NADH-dependent monooxygenase to form protocatechuate, which was subsequently oxidized by both ortho- and meta-protocatechuate dioxygenase reactions. When grown on biphenyl, Acinetobacter sp. strain P6 cometabolized 4,4'-dichlorobiphenyl primarily to 4-chlorobenzoate; however, when this strain was grown in a coculture with Acinetobacter sp. strain 4CB1, 4-chlorobenzoate did not accumulate but was converted to inorganic chloride. When resting cells of Acinetobacter sp. strain 4CB1 were incubated anaerobically with 3,4-dichlorobenzoate, they accumulated 4-carboxy-1,2-benzoquinone as a final product. Since 3,4-dichlorobenzoate is a product that is formed from the cometabolism of 3,4-dichloro-substituted tetrachlorobiphenyls by Acinetobacter sp. strain P6, the coculture has a potential application for dehalogenation and mineralization of specific polychlorobiphenyl congeners.     

Bacterial dehalogenation of chlorobenzoates and coculture biodegradation of 4,4'-dichlorobiphenyl

Acinetobacter sp. strain 4CB1 was isolated from a polychlorobiphenyl-contaminated soil sample by using 4-chlorobenzoate as a sole source of carbon and energy. Resting cells of Acinetobacter sp. strain 4CB1 hydrolytically dehalogenated 4-chlorobenzoate under aerobic and anaerobic conditions, but 4-hydroxybenzoate accumulated only under anaerobic conditions. Cell extracts of Acinetobacter sp. strain 4CB1 oxidized 4-hydroxybenzoate by an NADH-dependent monooxygenase to form protocatechuate, which was subsequently oxidized by both ortho- and meta-protocatechuate dioxygenase reactions. When grown on biphenyl, Acinetobacter sp. strain P6 cometabolized 4,4'-dichlorobiphenyl primarily to 4-chlorobenzoate; however, when this strain was grown in a coculture with Acinetobacter sp. strain 4CB1, 4-chlorobenzoate did not accumulate but was converted to inorganic chloride. When resting cells of Acinetobacter sp. strain 4CB1 were incubated anaerobically with 3,4-dichlorobenzoate, they accumulated 4-carboxy-1,2-benzoquinone as a final product. Since 3,4-dichlorobenzoate is a product that is formed from the cometabolism of 3,4-dichloro-substituted tetrachlorobiphenyls by Acinetobacter sp. strain P6, the coculture has a potential application for dehalogenation and mineralization of specific polychlorobiphenyl congeners.