Cholesterol degradation by Gordonia cholesterolivorans.

This paper reports physiological and genetic data about the type strain Gordonia cholesterolivorans, a strain that is able to degrade steroid compounds containing a long carbon side chain such as cholesterol (C(27)), cholestenone (C(27)), ergosterol (C(28)), and stigmasterol (C(29)). The length of the carbon side chain appears to be of great importance for this bacterium, as the strain is unable to grow using steroids with a shorter or nonaliphatic carbon side chain such as cholic acid (C(24)), progesterone (C(21)), testosterone, androsterone, 4-androstene-3,17-dione (all C(19)), and further steroids. This study also demonstrates that the degradation of cholesterol is a quite common feature of the genus Gordonia by comparing Gordonia cholesterolivorans with some other species of this genus (e.g., G. sihwensis, G. hydrophobica, G. australis, and G. neofelifaecis). Pyrosequencing of the genome of G. cholesterolivorans led to the identification of two conventional cholesterol oxidase genes on an 8-kb and a 12.8-kb genomic fragment with genetic organizations that are quite unique as compared to the genomes of other cholesterol-degrading bacteria sequenced so far. The identified two putative cholesterol oxidases of G. cholesterolivorans are both intracellularly acting enzymes of the class I type. Whereas one of these two cholesterol oxidases (ChoOx-1) shows high identity with an oxidoreductase of the opportunistic pathogen G. bronchialis and is not transcribed during growth with cholesterol, the other one (ChoOx-2) appears phylogenetically closer to cholesterol oxidases from members of the genus Rhodococcus and is transcribed constitutively. By using targeted gene disruption, a G. cholesterolivorans ChoOx-2 gene mutant strain that was unable to grow with steroids was obtained.

Coexistence of a sulphate-reducing Desulfovibrio species and the dehalorespiring Desulfitobacterium frappieri TCE1 in defined chemostat cultures grown with various combinations of sulfate and tetrachloroethene.

A two-member co-culture consisting of the dehalorespiring Desulfitobacterium frappieri TCE1 and the sulphate-reducing Desulfovibrio sp. strain SULF1 was obtained via anaerobic enrichment from soil contaminated with tetrachloroethene (PCE). In this co-culture, PCE dechlorination to cis-dichloroethene was due to the activity of the dehalorespiring bacterium only. Chemostat experiments with lactate as the primary electron donor for both strains along with varying sulphate and PCE concentrations showed that the sulphate-reducing strain outnumbered the dehalogenating strain at relatively high ratios of sulphate/PCE. Stable co-cultures with both organisms present at similar cell densities were observed when both electron acceptors were supplied in the reservoir medium in nearly equimolar amounts. In the presence of low sulphate/PCE ratios, the Desulfitobacterium sp. became the numerically dominant strain within the chemostat co-culture. Surprisingly, in the absence of sulphate, strain SULF1 did not disappear completely from the co-culture despite the fact that there was no electron acceptor provided with the medium to be used by this sulphate reducer. Therefore, we propose a syntrophic association between the sulphate-reducing and the dehalorespiring bacteria via interspecies hydrogen transfer. The sulphate reducer was able to sustain growth in the chemostat co-culture by fermenting lactate and using the dehalogenating bacterium as a 'biological electron acceptor'. This is the first report describing growth of a sulphate-reducing bacterium in a defined two-member continuous culture by syntrophically coupling the electron and hydrogen transfer to a dehalorespiring bacterium.

Reclassification of Desulfobacterium phenolicum as Desulfobacula phenolica comb. nov. and description of strain SaxT as Desulfotignum balticum gen. nov., sp. nov.

A mesophilic, sulfate-reducing bacterium (strain SaxT) was isolated from marine coastal sediment in the Baltic Sea and originally described as a 'Desulfoarculus' sp. It used a large variety of substrates, ranging from simple organic compounds and fatty acids to aromatic compounds as electron donors. Autotrophic growth was possible with H2, CO2 and formate in the presence of sulfate. Sulfate, thiosulfate and sulfite were used as electron acceptors. Sulfur and nitrate were not reduced. Fermentative growth was obtained with pyruvate, but not with fumarate or malate. Substrate oxidation was usually complete leading to CO2, but at high substrate concentrations acetate accumulated. CO dehydrogenase activity was observed, indicating the operation of the CO dehydrogenase pathway (reverse Wood pathway) for CO2 fixation and complete oxidation of acetyl-CoA. The rod-shaped cells were 0.8-1.0 microm wide and 1.5-2.5 microm long. Spores were not produced and cells stained Gram-negative. The temperature limits for growth were between 10 and 42 degrees C (optimum growth at 28-32 degrees C). Growth was observed at salinities ranging from 5 to 110 g NaCl l(-1), with an optimum at 10-25 g NaCl l(-1). The G+C content of the DNA was 62.4 mol%. Vitamins were required for growth. Based on the 16S rRNA gene sequence, strain SaxT represents a new genus within the delta-subclass of the Proteobacteria. The name Desulfotignum balticum gen. nov., sp. nov. is proposed. After the 16S rDNA sequences of all members of the genus Desulfobacterium were published (GenBank accession nos. AJ237601-AJ237604, AJ237606, AJ237607), the need to reclassify most members of the genus Desulfobacterium became obvious due to their strong phylogenetic affiliation to other genera. Here, we propose to reclassify Desulfobacterium phenolicum as Desulfobacula phenolica comb. nov. Desulfotignum balticum, Desulfobacterium phenolicum and Desulfobacula toluolica contain cellular fatty acids which have so far only been found in members of the genus Desulfobacter.

Influence of different electron donors and acceptors on dehalorespiration of tetrachloroethene by Desulfitobacterium frappieri TCE1.

Strain TCE1, a strictly anaerobic bacterium that can grow by reductive dechlorination of tetrachloroethene (PCE) and trichloroethene (TCE), was isolated by selective enrichment from a PCE-dechlorinating chemostat mixed culture. Strain TCE1 is a gram-positive, motile, curved rod-shaped organism that is 2 to 4 by 0.6 to 0.8 microm and has approximately six lateral flagella. The pH and temperature optima for growth are 7.2 and 35 degrees C, respectively. On the basis of a comparative 16S rRNA sequence analysis, this bacterium was identified as a new strain of Desulfitobacterium frappieri, because it exhibited 99.7% relatedness to the D. frappieri type strain, strain PCP-1. Growth with H(2), formate, L-lactate, butyrate, crotonate, or ethanol as the electron donor depends on the availability of an external electron acceptor. Pyruvate and serine can also be used fermentatively. Electron donors (except formate and H(2)) are oxidized to acetate and CO(2). When L-lactate is the growth substrate, strain TCE1 can use the following electron acceptors: PCE and TCE (to produce cis-1,2-dichloroethene), sulfite and thiosulfate (to produce sulfide), nitrate (to produce nitrite), and fumarate (to produce succinate). Strain TCE1 is not able to reductively dechlorinate 3-chloro-4-hydroxyphenylacetate. The growth yields of the newly isolated bacterium when PCE is the electron acceptor are similar to those obtained for other dehalorespiring anaerobes (e.g., Desulfitobacterium sp. strain PCE1 and Desulfitobacterium hafniense) and the maximum specific reductive dechlorination rates are 4 to 16 times higher (up to 1.4 micromol of chloride released. min(-1). mg of protein(-1)). Dechlorination of PCE and TCE is an inducible process. In PCE-limited chemostat cultures of strain TCE1, dechlorination is strongly inhibited by sulfite but not by other alternative electron acceptors, such as fumarate or nitrate.

Anaerobic incorporation of the radiolabeled explosive TNT and metabolites into the organic soil matrix of contaminated soil after different treatment procedures.

Four bioreactor designs were performed to evaluate the level of incorporation of 14C-labeled 2,4,6-trinitrotoluene (TNT) and metabolites into the organic soil matrix of different anaerobically treated contaminated soils. The contaminated soils were amended with molasses slivers (80:20% per weight) as auxiliary substrate to enhance microbial activity. After 5 weeks (bioreactors 1 and 2), 8 weeks (bioreactor 3) and 12 weeks (bioreactor 4) of anaerobic incubation, we determined 41%, 58%, 72%, and 54%, respectively, of the initially applied radioactivity immobilized in various soil fractions. After alkaline hydrolyses of the solvent-extracted soils, low quantities of radiolabel were found in the humic and fulvic acid fractions, whereas the bulk of 14C activity was found to be strongly bound to the humin fraction (solid soil residues). The amounts of solvent extractable radioactivity were 53%, 40%, 16%, and 29% for bioreactors 1, 2, 3, and 4, respectively. The level of TNT transformation at the end of the experiments was within 90-94%. Regarding the results presented in this study, we can assume that there is the possibility of high incorporation levels of TNT metabolites into the soil organic matrix mediated by microbial cometabolism under strictly anoxic conditions.

Identification of oxidized TNT metabolites in soil samples of a former ammunition plant.

Water extracts of soil samples of the former ammunition plant "Tanne" near Clausthal-Zellerfeld, Lower Saxony, Germany, were investigated for highly polar oxidized 2,4,6-trinitrotoluene (TNT) metabolites. 0.4 to 9.0 mg/kg dry soil 2,4,6-trinitrobenzoic acid (TNBA) and 5.8 to 544 mg/kg dry soil 2-amino-4,6-dinitrobenzoic acid (2-ADNBA) were found. In addition to the oxidized metabolites, TNT, 4- and 2-aminodinitrotoluene (4- and 2-ADNT), and 2,4-dinitrotoluene (2,4-DNT) were extractable with water. Most interestingly, in one sample, 2-ADNBA represented the main contaminant. The origin of the oxidized nitroaromatics is unknown at this time. They might be generated chemically or photochemically. Furthermore, a biological synthesis seems possible.

Mass balance studies with 14C-labeled 2,4,6-trinitrotoluene (TNT) mediated by an Anaerobic desulfovibrio species and an Aerobic serratia species.

Investigations were carried out to evaluate the level of incorporation of radiolabeled 2,4,6-trinitrotoluene (TNT) and metabolites into the bacterial biomass of two different bacterial species after cometabolically mediated TNT transformation. Biotransformation experiments with 14C-TNT indicated that TNT was not mineralized; however, carbon derived from TNT became associated with the cells. It was found that more than 42% of the initially applied radiolabel was associated with the cell biomass after cometabolic 14C-TNT transformation with the strictly anerobic Desulfovibrio species strain SHV, whereas with the strictly aerobic Serratia plymuthica species strain B7, 32% of cell-associated 14C activity was measured. The remainder of the radiolabel was present in the supernatants of the liquid cultures in the form of different TNT metabolites. Under anoxic conditions with the Desulfovibrio species, TNT was ultimately transformed to 2,4,6-triaminotoluene (TAT) and both diaminonitrotoluene isomers, whereas under oxic conditions with the Serratia species, TNT was converted to hydroxylaminodinitrotoluenes and aminodinitrotoluenes, with 4-amino-2,6-dinitrotoluene (4ADNT) being the major end product. In both culture supernatants, small amounts of very polar, radiolabeled, but unidentified metabolites were detected. At the end of the experiments approximately 92% and 96% of the originally applied radioactivity was recovered in the studies with the Serratia and Desulfovibrio species, respectively.

Microbial Degradation of Diphenylamine Under Anoxic Conditions

Diphenylamine (DPA) was cometabolically degraded in anoxic sediment-water batch enrichments and in cultures of newly isolated sulfate-reducing bacteria. In gas chromatography-mass spectrometry (GC-MS) measurements, aniline was identified as a major breakdown product of the diphenylamine structure. After its identification, aniline was quantified by reversed phase high pressure liquid chromatography (HPLC). The fate of the other carbon ring system remained unclear, because benzene (as a product of reductive cleavage), phenol (as a product of hydrolytic cleavage), and/or other ring cleavage products of diphenylamine were not observed in our experiments with the methods employed.

Cometabolic transformation and cleavage of nitrodiphenylamines by three newly isolated sulfate-reducing bacterial strains.

Three sulfate-reducing bacterial strains (Desulfovibrio sp. strain SHV, Desulfococcus sp. strain WHC, and Desulfomicrobium sp. strain WHB) with the capacity to cometabolize 2-nitrodiphenylamine, 4-nitrodiphenylamine, and 2,4-dinitrodiphenylamine were newly isolated. Before breaking down the diphenylamine structure, these strains cometabolically reduce the nitrodiphenylamines to the corresponding aminodiphenylamines during anaerobic oxidation of the growth substrate lactate (Desulfovibrio strain SHV and Desulfomicrobium strain WHC) or benzoate (Desulfococcus strain WHB), leading to the formation of aniline and a smaller quantity of methylaniline. These compounds were not further metabolized by the sulfate reducers. The anaerobic metabolism of aminodiphenylamines also led to the formation of heterocyclic condensation products such as phenazine and acridine derivatives, provided that they contained an amino group in the ortho position of the diphenylamine (e.g., 2-aminodiphenylamine or 2,4-diaminodiphenylamine). In addition, low levels of indole and benzothiazole derivatives were identified, but these also were not further metabolized by the three sulfate-reducing strains.

Reduction of Nitrated Diphenylamine Derivatives under Anaerobic Conditions.

2-Nitrodiphenylamine, 4-nitrodiphenylamine, and 2,4-dinitrodiphenylamine were anaerobically metabolized in sediment-water batch enrichments inoculated with mud from the German North Sea coast. The first intermediate in 2,4-dinitrodiphenylamine degradation was 2-amino-4-nitrodiphenylamine, which appeared in large (nearly stoichiometric) amounts before being completely reduced to 2,4-diaminodiphenylamine. Of the second theoretically expected metabolite, 4-amino-2-nitrodiphenylamine, only traces were detected by gas chromatographic-mass spectrometric analysis in highly concentrated extracts. In addition, low levels of 4-nitrodiphenylamine, which may be the product of ortho deamination of intermediately produced 2-amino-4-nitrodiphenylamine, were observed. 2-Nitrodiphenylamine and 4-nitrodiphenylamine were primarily reduced to 2-aminodiphenylamine and 4-aminodiphenylamine, respectively. Diphenylamine was never detected in any experiment as a theoretically possible intermediate. Results from studies with dense cell suspensions of anaerobic, aromatic-compound-mineralizing bacteria confirmed the transformation reactions, which were carried out by microorganisms indigenous to the anaerobic coastal water sediment.

Toxicity of diphenylamine and some of its nitrated and aminated derivatives to the luminescent bacterium Vibrio fischeri.

Aqueous samples containing various nitrated and aminated diphenylamine derivatives were subjected to the luminescent bacterium Vibrio fischeri NRRL-B-11177 to determine their ecotoxicological potential. As the most important toxicological parameter, EC50, the concentration needed to reduce bacterial luminescence by 50%, was calculated. All compounds tested must be classified to the category "very toxic to aquatic organisms" using the widely accepted classification scheme of D. Strupp, H.P. Lühr, H. T. Grunder, J. Gerdesmann, and J. Ahlers (1990, UWSF--Z. Umweltchem. Okotox. 2, 151-156). Only 2, 4-diaminodiphenylamine can be classified as "less toxic to aquatic organisms". EC50 values after 30, 60, and 90 min of incubation of the test compounds are presented. For many of the compounds tested in this study there are no toxicological data in the literature.

Mineralization of monofluorobenzoate by a diculture under sulfate-reducing conditions.

A mesophilic, dehalogenating, sulfate-reducing diculture was isolated from an anaerobic lake sediment. One strain of the diculture is proposed to be an endospore-forming Desulfotomaculum species, the second strain was a vibrioid, motile and non-sporeforming species which is tentatively assigned to the genus Desulfovibrio. The diculture was able to mineralize 4- and 2-fluorobenzoate both isomers being incompletely oxidized with the release of acetate, which was subsequently used by both sulfate-reducing strains. Other electron donors used for growth included benzoate, 3- and 4-hydroxybenzoate, protocatechuate, catechol, phenol, 2,5-dimethoxyphenol, fatty acids up to C8, malate and pyruvate. The culture obtained from a freshwater habitat grew optimally at NaCl concentrations of 0.3-0.5 g l-1, 33-37 degrees C, and pH 7.4. Our experiments showed that certain fluorinated aromatic hydrocarbons could serve as sole sources of carbon and energy for sulfate-reducing bacteria.

Microbial degradation of explosives and related compounds.

The pollution of soil and water with explosives and related compounds caused by military activities has been known for a long time, but progress in understanding the environmental fate of such substances has only been made in the last few years. Microbial processes could be used for the remediation of explosives-contaminated soils and waste waters because it has been shown that a variety of different microorganisms are able to metabolize these chemical compounds. In some cases even a complete mineralization has been found, whereas in others only biotransformation reactions took place, producing more or less toxic and/or recalcitrant metabolites. Studies with pure cultures of bacteria and fungi have given detailed insights into the biodegradation pathways of at least some nitroorganic compounds. Additionally, some of the key enzymes have been isolated and purified or studied in crude extracts. This review summarizes information on the biodegradation and biotransformation pathways of several important explosives. This may be useful in developing microbiological methods for a safe and economic clean-up of soil and water contaminated with such compounds. It also shows the necessity of further investigations concerning the microbial metabolism of these substances.

Complete oxidation of benzoate and 4-hydroxybenzoate by a new sulfate-reducing bacterium resembling Desulfoarculus.

A new sulfate-reducer "strain SAX" was isolated from an anaerobic marine sediment [Saxild, Denmark]. The isolate was a gram-negative, motile and non-spore-forming rod which sometimes appeared as a curved rod. Strain SAX differed from all described Desulfovibrio-, Desulfobotulus- and Desulfoarculus-species by the ability to degrade aromatic compounds such as benzoate, 4-hydroxybenzoate and phenol completely to CO2. Electron donors used included lactate, pyruvate, malate, fumarate, crotonate and butyrate, while pyruvate was fermented in the absence of an external electron acceptor. Sulfate, thiosulfate or sulfite served as electron acceptors with benzoate as the donor, while nitrate and nitrite did not. The sulfate-reducing bacterium required vitamins and NaCl-concentrations of about 20 g/l. The optimum temperature for growth of strain SAX was 30 degrees C and the optimum pH value was 7.3. The DNA base composition was 62.4 mol% G+C. The strain possessed cytochrome c3, but no desulfoviridin. On the basis of these characteristics and because strain SAX could not be ascribed to any of the existing species therefore assignment as a new species to the genus Desulfoarculus was suggested.