Phylogenetic and metabolic diversity of bacteria degrading aromatic compounds under denitrifying conditions,and description of Thauera phenylacetica sp. nov., Thauera aminoaromaticasp. nov., and Azoarcus buckelii sp. nov

Six strains of denitrifying bacteria isolated from various oxic and anoxic habitats on different monocyclic aromatic substrates were characterized by sequencing 16S rRNA genes, determining physiological and morphological traits, and DNA-DNA hybridization. According to these criteria, strains S100, SP and LG356 were identified as members of Thauera aromatica. Strains B5-1 and B5-2 were tentatively affiliated to the species Azoarcus tolulyticus. Strains B4P and S2 were only distantly related to each other and to other described Thauera species. These two strains are proposed as the type strains of two new species, Thauera phenylacetica sp. nov. and Thauera aminoaromaticasp. nov., respectively. By 16S rRNA gene analysis, strain U120 was highly related to the type strains of Azoarcus evansii and Azoarcus anaerobius, whereas corresponding DNA-DNA reassociation values indicated only a low degree of genomic relatedness. Based upon a low DNA similarity value and the presence of distinguishing physiological properties, strain U120 is proposed as the type strain of a new species, Azoarcus buckelii sp. nov. Almost all of the new isolates were obtained with different substrates. The highly varied substrate spectra of the isolates indicates that an even higher diversity of denitrifying bacteria degrading aromatic compounds would be discovered in the different habitats by using a larger spectrum of aromatic substrates for enrichment and isolation.     

Lithoautotrophic growth of sulfate-reducing bacteria,and description of Desulfobacterium autotrophicum gen. nov., sp. nov.

The capacity of mesophilic sulfate-reducing bacteria to grow lithoautotrophically with H₂, sulfate and CO₂ was investigated with enrichment cultures and isolated species. (a) Enrichments in liquid mineral media with H₂, sulfate and CO₂ consistently yielded mixed cultures of nonautotrophic, acetate-requiring Desulfovibrio species and autotrophic, acetate-producing Acetobacterium species (cell ratio approx. 20:1). (b) By direct dilution of mud samples in agar, various non-sporing sulfate reducers were isolated in pure cultures that did grow autotrophically. Two oval cell types (strains HRM2, HRM4) and one curved cell type (strain HRM6) from marine sediment were studied in detail. The strains grew in mineral medium supplemented only with vitamins (biotin, p-aminobenzoate, nicotinate). Carbon autotrophy was evident (i) from comparative growth experiments with non-autotrophic, acetate-requiring species, (ii) from high cell densities ruling out a cell synthesis from organic impurities in the mineral media, and (iii) by demonstrating that 96–99% of the cell carbon was derived from ¹⁴C-labelled CO₂. Autotrophic growth occurred with a doubling time of 16–20 h at 24–28°C. Formate, fatty acids up to palmitate, ethanol, lactate, succinate, fumarate, malate and other organic acids were also used and completely oxidized. The three strains possessed cytochromes of the b-and c-type, but no desulfoviridin. Strain HRM2 is described as a new species of a new genus, Desulfobacterium autotrophicum. (c) The capacity for autotrophic growth was also tested with sulfate-reducing bacteria that originally had been isolated on organic substrates. The incompletely oxidizing, non-sporing types such as Desulfovibrio and Desulfobulbus species and Desulfomonas pigra were confirmed to be obligate heterotrophs that required acetate for growth with H₂ and sulfate. In contrast, several of the completely oxidizing sulfate reducers were facultative autotrophs, such as Desulfosarcina variabilis, Desulfonema limicola, Desulfococcus niacini, and the newly isolated Desulfobacterium vacuolatum and Desulfobacter hydrogenophilus. The only incompletely oxidizing sulfate reducer that could grow autotrophically was the sporing Desulfotomaculum orientis, which obtained 96% of its cell carbon from ¹⁴C-labelled CO₂. Desulfovibrio baarsii and Desulfococcus multivorans may also be regarded as types of facultative autotrophs; they could not oxidize H₂, but grew on sulfate with formate as the only organic substrate.     

Anaerobic degradation of benzene, toluene, ethylbenzene, and o-xylene in sediment-free iron-reducing enrichment cultures

Monoaromatic hydrocarbons such as benzene, toluene, ethylbenzene, and xylene (BTEX) are widespread contaminants in groundwater. We examined the anaerobic degradation of BTEX compounds with amorphous ferric oxide as electron acceptor. Successful enrichment cultures were obtained for all BTEX substrates both in the presence and absence of AQDS (9,10-anthraquinone-2,6-disulfonic acid). The electron balances showed a complete anaerobic oxidation of the aromatic compounds to CO2. This is the first report on the anaerobic degradation of o-xylene and ethylbenzene in sediment-free iron-reducing enrichment cultures.     

Culturable diversity of lithotrophic haloalkaliphilic sulfate-reducing bacteria in soda lakes and the description of Desulfonatronum thioautotrophicum sp. nov., Desulfonatronum thiosulfatophilum sp. nov., Desulfonatronovibrio thiodismutans sp. nov., and Desulfonatronovibrio magnus sp. nov

Soda lake sediments usually contain high concentrations of sulfide indicating active sulfate reduction. Monitoring of sulfate-reducing bacteria (SRB) in soda lakes demonstrated a dominance of two groups of culturable SRB belonging to the order Desulfovibrionales specialized in utilization of inorganic electron donors, such as formate, H₂ and thiosulfate. The most interesting physiological trait of the novel haloalkaliphilic SRB isolates was their ability to grow lithotrophically by dismutation of thiosulfate and sulfite. All isolates were obligately alkaliphilic with a pH optimum at 9.5–10 and moderately salt tolerant. Among the fifteen newly isolated strains, four belonged to the genus Desulfonatronum and the others to the genus Desulfonatronovibrio. None of the isolates were closely related to previously described species of these genera. On the basis of phylogenetic, genotypic and phenotypic characterization of the novel soda lake SRB isolates, two novel species each in the genera Desulfonatronum and Desulfonatronovibrio are proposed.     

Characterization of Porphyrobacter sanguineus sp. nov., an aerobic bacteriochlorophyll-containing bacterium capable of degrading biphenyl and dibenzofuran

Three strains of " Agrobacterium sanguineum", an aerobic marine bacterial species described previously, were re-characterized from phylogenetic and taxonomic viewpoints. 16S rDNA sequence comparisons showed that the " A. sanguineum" strains belong to the alpha-4 subgroup of alpha-Proteobacteria, with members of the genera Erythromicrobium and Porphyrobacter as their closest relatives. DNA-DNA hybridization studies indicated that the " A. sanguineum" strains were distinguishable from any previously known species of these genera. Bacteriochlorophyll a, monosaccharide-type glycosphingolipids, 2-OH fatty acids of C14:0, C15:0, C16:0, and C16:1, and ubiquinone-10 were detected in the " A. sanguineum" strains. The G+C of the DNA was 63.8-64.0 mol%. Two of the " A. sanguineum" strains, IAM 12620 (=ATCC 25659) and ATCC 25661, were able to grow with biphenyl and dibenzofuran as sole carbon source in the presence of 0.05% yeast extract. The medium in these cultures turned yellowish-orange at the exponential phase of growth due to the release of soluble chromogenic metabolites. The remaining " A. sanguineum" strain, ATCC 25660, and all test strains of Erythromicrobium and Porphyrobacter neither grew nor produced yellow-orange pigment with biphenyl or dibenzofuran. In PCR experiments, bphA1 gene, coding for the large subunit protein of biphenyl dioxygenase, was detected in " A. sanguineum" IAM 12620 and ATCC 25661. Based on these results, we propose classifying " A. sanguineum" IAM 12620 and ATCC 25661 as a new species of the genus Porphyrobacter with the name Porphyrobacter sanguineus sp. nov.     

Anaerobic degradation of indolic compounds by sulfate-reducing enrichment cultures,and description of Desulfobacterium indolicum gen. nov., sp. nov.

Indole (1.5 mmol/l) added to suflate-rich marine mud or sulfate-free sewage digestor sludge was anaerobically degraded within one week. Enrichments from sludge samples in defined indole-containing media with or without sulfate were selective for sulfate-reducing bacteria or mixed methanogenic associations, respectively. Other enrichments of sulfate-reducing bacteria were obtained with skatole, indoleacetate, indolepropionate, quinoline, and pyridine. From a marine enrichment with indole as sole electron donor and carbon source, an oval to rod-shaped, Gram-negative, nonsporing sulfate-reducing bacterium (strain In04) was isolated. Growth occurred in defined bicarbonate-buffered, sulfide-reduced media supplemented with vitamin B₁₂. Furthen aromatic compounds utilized as electron donors and carbon sources were anthranilic acid and quinoline. Nonaromatic compounds used as substrates were formate, acetate, propionate, ethanol, propanol, butanol, pyruvate, malate, fumarate, and succinate. However, growth with substrates other than indole was rather slow. Thiosulfate served as an alternative electron acceptor. Complete oxidation of indole to CO₂ was shown by stoichiometric measurements in batch culture with sulfate as electron acceptor. An average growth yield of 31.3 g cell dry weight was obtained per mol of indole oxidized. Pigment analysis revealed that cytochromes and menaquinone MK-7 (H₂) were present. Desulfoviridin could not be detected. Strain In04 is described as new species of the new genus Desulfobacterium indolicum.     

Holophaga foetida gen. nov., sp. nov., a new,homoacetogenic bacterium degrading methoxylated aromatic compounds

A polyphasic approach was used in which genotypic and phenotypic properties of a gram-negative, obligately anaerobic, rod-shaped bacterium isolated from a black anoxic freshwater mud sample were determined. Based on these results, the name Holophaga foetida gen. nov., sp. nov. is proposed. This microorganism produced dimethylsulfide and methanethiol during growth on trimethoxybenzoate or syringate. The only other compounds utilized were pyruvate and trihydroxybenzenes such as gallate, phloroglucinol, or pyrogallol. The aromatic compounds were degraded to acetate. Although comparison of the signature nucleotide pattern of the five established subclasses of Proteobacteria with the 16S rDNA sequence of Holophaga foetida revealed a relationship to members of the -subclass, the phylogenetic position within the radiation of this class is so deep and dependent upon the number and selection of reference sequences that its affiliation to the Proteobacteria must be considered tentative. The type strain is H. foetida strain TMBS4 (DSM 6591).F. Bak died on 27 December 1992. A very promising and productive career thus ended much too early     

Isolation, characterization, and polyaromatic hydrocarbon degradation potential of aerobic bacteria from marine macrofaunal burrow sediments and description of Lutibacterium anuloederans gen. nov., sp. nov., and Cycloclasticus spirillensus sp. nov.

Two new polyaromatic hydrocarbon-degrading marine bacteria have been isolated from burrow wall sediments of benthic macrofauna by using enrichments on phenanthrene. Strain LC8 (from a polychaete) and strain M4-6 (from a mollusc) are aerobic and gram negative and require sodium chloride (>1%) for growth. Both strains can use 2- and 3-ring polycyclic aromatic hydrocarbons as their sole carbon and energy sources, but they are nutritionally versatile. Physiological and phylogenetic analyses based on 16S ribosomal DNA sequences suggest that strain M4-6 belongs to the genus Cycloclasticus and represents a new species, Cycloclasticus spirillensus sp. nov. Strain LC8 appears to represent a new genus and species, Lutibacterium anuloederans gen. nov., sp. nov., within the Sphingomonadaceae. However, when inoculated into sediment slurries with or without exogenous phenanthrene, only L. anuloederans appeared to sustain a significant phenanthrene uptake potential throughout a 35-day incubation. In addition, only L. anuloederans appeared to enhance phenanthrene degradation in heavily contaminated sediment from Little Mystic Cove, Boston Harbor, Boston, Mass.     

Bacterial aerobic degradation of benzene,toluene, ethylbenzene and xylene

Several aerobic metabolic pathways for the degradation of benzene, toluene, ethylbenzene and xylene (BTEX), which are provided by two enzymic systems (dioxygenases and monooxygenases), have been identified. The monooxygenase attacks methyl or ethyl substituents of the aromatic ring, which are subsequently transformed by several oxidations to corresponding substituted pyrocatechols or phenylglyoxal, respectively. Alternatively, one oxygen atom may be first incorporated into aromatic ring while the second atom of the oxygen molecule is used for oxidation of either aromatic ring or a methyl group to corresponding pyrocatechols or protocatechuic acid, respectively. The dioxygenase attacks aromatic ring with the formation of 2-hydroxy-substituted compounds. Intermediates of the “upper” pathway are then mineralized by eitherortho-ormeta-ring cleavage (“lower” pathway). BTEX are relatively water-soluble and there-fore they are often mineralized by indigenous microflora. Therefore, natural attenuation may be considered as a suitable way for the clean-up of BTEX contaminants from gasoline-contaminated soil and groundwater.     

Isolation of microorganisms capable of degrading isoquinoline under aerobic conditions.

Isoquinoline-degrading microbial cultures were isolated from oil- and creosote-contaminated soils. The establishment of initial enrichment cultures required the use of emulsified isoquinoline. Once growth on isoquinoline was established, isoquinoline emulsification was no longer required for utilization of isoquinoline as the sole source of carbon and nitrogen by these cultures. An isoquinoline-degrading Acinetobacter strain was isolated from one of the enrichment cultures. The degradation of isoquinoline was accompanied by the accumulation of a red cell-associated pigment and of 1-hydroxyisoquinoline, which was further degraded to unknown intermediary ring-cleavage products and carbon dioxide.     

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.     

Description of the novel perchlorate-reducing bacteria Dechlorobacter hydrogenophilus gen. nov., sp. nov. and Propionivibrio militaris, sp. nov.

Novel dissimilatory perchlorate-reducing bacteria (DPRB) were isolated from enrichments conducted under conditions different from those of all previously described DPRB. Strain LT-1^T was enriched using medium buffered at pH 6.6 with 2-(N-morpholino)ethanesulfonic acid (MES) and had only 95% 16S rRNA gene identity with its closest relative, Azonexus caeni. Strain MP^T was enriched in the cathodic chamber of a perchlorate-reducing bioelectrical reactor (BER) and together with an additional strain, CR (99% 16S rRNA gene identity), had 97% 16S rRNA gene identity with Propionivibrio limicola. The use of perchlorate and other electron acceptors distinguished strains MP^T and CR from P. limicola physiologically. Strain LT-1^T had differences in electron donor utilization and optimum growth temperatures from A. caeni. Strains LT-1^T and MP^T are the first DPRB to be described in the Betaproteobacteria outside of the Dechloromonas and Azospira genera. On the basis of phylogenetic and physiological features, strain LT-1^T represents a novel genus in the Rhodocyclaceae; strain MP^T represents a novel species within the genus Propionivibrio. The names Dechlorobacter hydrogenophilus gen. nov., sp. nov and Propionivibrio militaris sp. nov. are proposed.     

Description of Oxalicibacterium horti sp. nov. and Oxalicibacterium faecigallinarum sp. nov., new aerobic, yellow-pigmented, oxalotrophic bacteria

Three strains of aerobic, Gram-negative, rod-shaped, non-spore-forming, yellow-pigmented bacteria (OD1^T, YOx^T and NS13), which were isolated in previous studies by enrichment in a mineral medium with potassium oxalate as the sole carbon source, were characterized. On the basis of 16S rRNA gene sequence similarity, strains OD1^T, YOx^T and NS13 belong to the Betaproteobacteria , most closely related to Oxalicibacterium flavum TA17^T (97.2–99.7% sequence similarity). The major whole-cell fatty acids were C_16:0, C_16:1ω7c and C_17:0 cyclo. The results of DNA–DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strains OD1^T and YOx^T from O. flavum TA17^T and from each other. Therefore, it is concluded that the strains OD1^T and YOx^T represent novel species within the genus Oxalicibacterium , for which the names Oxalicibacterium horti sp. nov. (type strain OD1^T=DSM 21640^T=NBRC 13594^T) and Oxalicibacterium faecigallinarum sp. nov. (type strain YOx^T=DSM 21641^T=CCM 2767^T) are proposed.     

Thermosediminibacter oceani gen. nov., sp. nov. and Thermosediminibacter litoriperuensis sp. nov., new anaerobic thermophilic bacteria isolated from Peru Margin

A new group of anaerobic thermophilic bacteria was isolated from enrichment cultures obtained from deep sea sediments of Peru Margin collected during Leg 201 of the Ocean Drilling Program. A total of ten isolates were obtained from cores of 1–2 m below seafloor (mbsf) incubated at 60°C: three isolates came from the sediment 426 m below sea level with a surface temperature of 9°C (Site 1227), one from 252 m below sea level with a temperature of 12°C (Site 1228), and six isolates under sulfate-reducing condition from the lower slope of the Peru Trench (Site 1230). Strain JW/IW-1228P from the Site 1228 and strain JW/YJL-1230-7/2 from the Site 1230 were chosen as representatives of the two identified clades. Based on the 16S rDNA sequence analysis, these isolates represent a novel group with Thermovenabulum and Caldanaerobacter as their closest relatives. The temperature range for growth was 52–76°C with an optimum at around 68°C for JW/IW-1228P and 43–76°C with an optimum at around 64°C for JW/YJL-1230-7/2. The pH^25C range for growth was from 6.3 to 9.3 with an optimum at 7.5 for JW/IW-1228P and from 5 to 9.5 with an optimum at 7.9–8.4 for JW/YJL-1230-7/2. The salinity range for growth was from 0% to 6% (w/v) for JW/IW-1228P and from 0% to 4.5% (w/v) for JW/YJL-1230-7/2. The G+C content of the DNA was 50 mol% for both JW/IW-1228P and JW/YJL-1230-7/2. DNA–DNA hybridization yielded 52% similarity between the two strains. According to 16S rRNA gene sequence analysis, the isolates are located within the family, Thermoanaerobacteriaceae. Based on their morphological and physiological properties and phylogenetic analysis, it is proposed that strain JW/IW-1228P^T is placed into a novel taxa, Thermosediminibacter oceani, gen. nov., sp. nov. (DSM 16646^T=ATCC BAA-1034^T), and JW/YJL-1230-7/2^T into Thermosediminibacter litoriperuensis sp. nov. (DSM 16647^T =ATCC BAA-1035^T).An erratum to this article can be found at     

Characterization and description of Anaeromyxobacter dehalogenans gen. nov., sp. nov., an aryl-halorespiring facultative anaerobic myxobacterium

Five strains were isolated which form a physiologically and phylogenetically coherent group of chlororespiring microorganisms and represent the first taxon in the Myxobacteria capable of anaerobic growth. The strains were enriched and isolated from various soils and sediments based on their ability to grow using acetate as an electron donor and 2-chlorophenol (2-CPh) as an electron acceptor. They are slender gram-negative rods with a bright red pigmentation that exhibit gliding motility and form spore-like structures. These unique chlororespiring myxobacteria also grow with 2,6-dichlorophenol, 2,5-dichlorophenol, 2-bromophenol, nitrate, fumarate, and oxygen as terminal electron acceptors, with optimal growth occurring at low concentrations (<1 mM) of electron acceptor. 2-CPh is reduced by all strains as an electron acceptor in preference to nitrate, which is reduced to ammonium. Acetate, H(2), succinate, pyruvate, formate, and lactate were used as electron donors. None of the strains grew by fermentation. The 16S ribosomal DNA (rDNA) sequences of the five strains form a coherent cluster deeply branching within the family Myxococcaceae within the class Myxobacteria and are mostly closely associated with the Myxococcus subgroup. With the exception of anaerobic growth and lack of a characteristic fruiting body, these strains closely resemble previously characterized myxobacteria and therefore should be considered part of the Myxococcus subgroup. The anaerobic growth and 9.0% difference in 16S rDNA sequence from those of other myxobacterial genera are sufficient to place these strains in a new genus and species designated Anaeromyxobacter dehalogenans. The type strain is 2CP-1 (ATCC BAA-258).