Biodegradation of methyl tert-butyl ether by cold-adapted mixed and pure bacterial cultures.

An aerobic mixed bacterial culture (CL-EMC-1) capable of utilizing methyl tert-butyl ether (MTBE) as the sole source of carbon and energy with a growth temperature range of 3 to 30 degrees C and optimum of 18 to 22 degrees C was enriched from activated sludge. Transient accumulation of tert-butanol (TBA) occurred during utilization of MTBE at temperatures from 3 degrees C to 14 degrees C, but TBA did not accumulate above 18 degrees C. The culture utilized MTBE at a concentration of up to 1.5 g l(-1) and TBA of up to 7 g l(-1). The culture grew on MTBE at a pH range of 5 to 9, with an optimum pH of 6.5 to 7.1. The specific growth rate of the CL-EMC-1 culture on 0.1 g l(-1) of MTBE at 22 degrees C and pH 7.1 was 0.012 h(-1), and the growth yield was 0.64 g (dry weight) g(-1). A new MTBE-utilizing bacterium, Variovorax paradoxus strain CL-8, isolated from the mixed culture utilized MTBE, TBA, 2-hydroxy isobutyrate, lactate, methacrylate, and acetate as sole sources of carbon and energy but not 2-propanol, acetone, methanol, formaldehyde, or formate. Two other isolates, Hyphomicrobium facilis strain CL-2 and Methylobacterium extorquens strain CL-4, isolated from the mixed culture were able to grow on C(1) compounds. The combined consortium could thus utilize all of the carbon of MTBE.

[Growth of Rhodococcus opacus on mixtures of monohalogenated benzenes and phenols]. / Rost Rhodococcus opacus na smesiakh monogalogenirovannykh benzolov i fenolov.

The growth of Rhodococcus opacus GM-14 on mixtures of 2-chloro- and 2-bromophenol, of 4-chloro, 4-bromo-, and 4-iodophenol, and of chloro-, bromo-, and iodobenzenes was accompanied by consumption of the substrates and excretion of halogen ions to the medium. During the growth on monochlorophenols, the substrates were consumed sequentially in the following order: 4-chloro-, 3-chloro-, and then 2-chlorophenol. Chlorine ions were excreted in a two-phase manner in amounts comprising 79% of the theoretical yield. The diauxic growth of R. opacus GM-14 can be explained by the existence in this bacterium of two modified metabolic pathways for the ortho-cleavage of halogenated pyrocatechols. The first pathway included 4-halogeno- or dihalogenopyrocatechols as intermediates, whereas the second pathway included 3-halogenopyrocatechols.

Effect of aromatic compounds on cellular fatty acid composition of Rhodococcus opacus.

In cells of Rhodococcus opacus GM-14, GM-29, and 1CP, the contents of branched (10-methyl) fatty acids increased from 3% to 15 to 34% of the total fatty acids when the cells were grown on benzene, phenol, 4-chlorophenol, chlorobenzene, or toluene as the sole source of carbon and energy, in comparison with cells grown on fructose. In addition, the content of trans-hexadecenoic acid increased from 5% to 8 to 18% with phenol or chlorophenol as the carbon source. The 10-methyl branched fatty acid content of R. opacus GM-14 cells increased in a dose-related manner following exposure to phenol or toluene when toluene was not utilized as the growth substrate. The results suggest that 10-methyl branched fatty acids may participate in the adaptation of R. opacus to lipophilic aromatic compounds.

New aerobic ammonium-dependent obligately oxalotrophic bacteria: description of Ammoniphilus oxalaticus gen. nov., sp. nov. and Ammoniphilus oxalivorans gen. nov., sp. nov.

The genus Ammoniphilus is proposed for aerobic endospore-forming Gram-variable rod-shaped bacteria, which are ammonium-dependent, obligately oxalotrophic and haloalkalitolerant, oxidase- and catalase-positive, mesophilic and motile by peritrichous flagella. Cell wall contained two electron-dense layers. The external layer consists of a chain of electron-dense granules morphologically resembling the cellulosomes of Clostridium thermocellum. Two species are described, Ammoniphilus oxalaticus gen. nov., sp. nov. and Ammoniphilus oxalivorans gen. nov., sp. nov. The type strains of these species are strains RAOx-1 (= DSM 11538) and RAOx-FS (= DSM 11537), respectively. Ammoniphilus strains were isolated from the rhizosphere of sorrel (Rumex acetosa) and from decaying wood. The strains require a high concentration of ammonium ions and use oxalate as the sole organic source of carbon and energy for growth; no growth factors were required. Growth occurred at pH 6.8-9.5. The optimum temperature and pH for growth were 28-30 degrees C and 8.0-8.5. All strains grew in a saturated solution of ammonium oxalate, and tolerated 3% NaCl. Whole-cell hydrolysates contain meso-diaminopimelic acid and glucose. The menaquinone of the strains was MK 7, and the major cellular fatty acids were 12-methyl tetradecanoic, cis-hexadec-9-enoic and hexadecanoic acids. The G + C content of the DNA was 45-46 mol% for A. oxalaticus and 42 mol% for A. oxalivorans. The almost complete 16S rDNA sequence of three strains of the two species of Ammoniphilus shows that the genus falls into the radiation of the Clostridium-Bacillus subphylum of Gram-positive bacteria. The closest phylogenetic neighbour of Ammoniphilus is Oxalophagus oxalicus. The DNA-DNA hybridization value between strains RAOx-1 and RAOx-FS was 39.7%.

Utilization of Halogenated Benzenes, Phenols, and Benzoates by Rhodococcus opacus GM-14.

Strain GM-14 was isolated by selective enrichment from contaminated soil with chlorobenzene as the sole source of carbon and energy. It utilizes an exceptionally wide spectrum of haloaromatic substrates. It is a gram-positive, weakly acid-fast actinomycete, with a morphological cycle from cocci and short rods to long rods and branched filaments; it grew optimally at 28(deg)C; and it tolerated 5% NaCl in rich medium. The chemotaxonomic characteristics, the diagnostic biochemical tests, the whole-cell fatty acid composition, and 16S rDNA analysis were consistent with Rhodococcus opacus. R. opacus GM-14 grew on 48 of 117 different aromatic and haloaromatic compounds. It utilized phenol at concentrations up to 1.2 g/liter, 3- and 4-methylphenols up to 0.5 g/liter, 2- and 4-chlorophenols up to 0.25 g/liter, and 3-chlorophenol up to 0.1 g/liter. It grew in saturated aqueous solutions of benzene, chlorobenzene, and 1,3- and 1,4-dichlorobenzene (up to 13, 3, 0.5, and 0.5 g/liter, respectively). The specific growth rate of strain GM-14 on phenol and 3- and 4-chlorophenols in batch culture was 0.27 to 0.29 h(sup-1), and that on benzene and chlorobenzene was similar to the rate on fructose, i.e., 0.2 h(sup-1). The growth yield on benzene and on chlorobenzene (<=0.4 g liter(sup-1)) was 40 to 50 g (dry weight) per mol of substrate consumed, equalling 8 g of dry weight biomass per mol of substrate carbon, similar to that obtained on acetate. During growth of strain GM-14 on chlorobenzene, 1,3-dichlorobenzene, and all isomers of monochlorophenol, stoichiometric amounts of chloride were released, and 50% of the stoichiometric amount was released from 1,4-dichlorobenzene.

Degradation of 2-chlorobenzoic and 2,5-dichlorobenzoic acids in pure culture by Pseudomonas stutzeri.

A strain of Pseudomonas stutzeri KS25 utilizing 2-chlorobenzoic and 2,5-dichlorobenzoic acids as the sole carbon and energy source was isolated from polychlorophenol-contaminated soil and sewage, using the method of enrichment cultures. This strain was also able to grow on 2-fluoro-, 2-iodo-, 2-bromo- and 2,5-dihydroxybenzoate, but did not utilize 3-, 4-chloro-, 2,4- and 2,6-dichlorobenzoates as the sole carbon and energy source, however, it cometabolized 3-chloro-, 2,4- and 2,6-dichlorobenzoates, but not 4-chlorobenzoate. The yield of released chlorine during utilization of 2-chloro- and 2,5-dichlorobenzoates amounted to 100% of the theoretical. The concentration of 2-chloro- and 2,5-dichlorobenzoates, not substantially inhibiting the isolated microorganism, was within the range 0.25-0.5 and 2.5-3.0 g/L, respectively.

Degradation of 2-chlorobenzoic and 2,5-dichlorobenzoic acids in soil columns by Pseudomonas stutzeri.

The heterocontinuous flow cultivation technique was used for the study of 2-chlorobenzoic and 2,5-dichlorobenzoic acid degradation in soil columns inoculated with Pseudomonas stutzeri. 2-Chlorobenzoic and 2,5-dichlorobenzoic acids disappeared from the soil columns within 8 and 12 d, respectively. The presence of the haloaromatics increased the survival of strain KS25 in soil. Viable cell numbers in the soil columns flushed with 2-chlorobenzoic and 2,5-dichlorobenzoic acids were 1.3 and 2 times higher, respectively, than those without the chlorobenzoic acids after 30 d of incubation.

Cloning and expression of the Arthrobacter globiformis KZT1 fcbA gene encoding dehalogenase (4-chlorobenzoate-4-hydroxylase) in Escherichia coli.

The fsbA gene controlling the first step of 4-chlorobenzoic acid (4CBA) metabolism in the Gram-positive soil bacterium Arthrobacter globiformis KZT1 has been cloned and analysed in Escherichia coli. The E. coli minicells analysis showed that a polypeptide(s) with Mr = 58 kDa (and/or Mr = 32 kDa) can be the fcbA product(s). Despite the gene dose amplification and control of the E. coli inducible Plac promoter, the level of functional expression of the fcbA gene in E. coli cells seems comparable only with that in the parental KZT1 strain. Effective 4CBA dechlorination by recombinant cells during growth in the presence of substrate within a range of concentrations 0.1 g/l to 0.7 g/l as well as a sudden reduction in the reaction efficiency at higher substrate concentrations were observed.

Genetic control of degradation of chlorinated benzoic acids in Arthrobacter globiformis, Corynebacterium sepedonicum and Pseudomonas cepacia strains.

The strains of Arthrobacter globiformis KZT1, Corynebacterium sepedonicum KZ4 and Pseudomonas cepacia KZ2 capable of early dehalogenation and complete oxidation of 4-chloro-, 2,4-dichloro-and 2-chlorobenzoic acids, respectively, have been analyzed for the origin of the genetic control of degradation. The occurrence and molecular sizes of plasmids in all the strains have been established. Plasmid pBS1501 was shown to control 4-chlorobenzoate dehalogenation in the case of KZT1 strain. The same possibility is proposed for plasmid pBS1502 for dehalogenation of 2,4DCBA by KZ4 strain. The chromosome localization of the genes controlling oxidation of 4-hydroxybenzoate in strain KZT1 is shown. Localization of the whole set of genes responsible for 2CBA degradation in the strain KZ2 chromosome is suggested.

[Cloning of the Arthrobacter globiformis fcbA gene for dehalogenase and construction of a hybrid pathway of 4-chlorobenzoic acid degradation in Pseudomonas putida]. / Klonirovanie gena degalogenazy fcbA Arthrobacter globiformis i konstruirovanie gibridnogo puti degradatsii 4-khlorbenzoata v Pseudomonas putida.

The Artrobacter globiformis KZT1 fcbA gene responsible for dehalogenase (4-chlorobenzoate-4-hydroxylase) activity was cloned in Escherichia coli and Pseudomonas putida cells. The character of the fcbA gene expression was studied. Notwithstanding amplification of the gene dose and control of the inducible Plac promoter, the level of substrate dehalogenation by recombinant E. coli strains was lower, as compared with that in the original KZT1 strain. Cloning of the fcbA gene in P. putida KZ6R cells utilizing 4HBA resulted in a recombinant pathway of 4CBA degradation, which proved more effective for substrate consumption, in comparison with the original KZT1 strain.

[Utilization of 3-chlorobenzoic acid by a mixed culture of microorganisms]. / Utilizatsiia 3-khlorbenzoinoi kisloty smeshannoi kul'turoi mikroorganizmov.

A mixed microbial culture (Acinetobacter calcoaceticus INMI-KZ-3 and Alcaligenes faecalis INMI-KZ-5) completely utilises 3-chlorobenzoic acid (3-CBA), and the liberation of chlorine atoms is 100% of the theoretically possible one. A. faecalis growth in the mixed culture at the account of 2-chloro-cis,cis-muconic acid which is accumulated in the course of 3-CBA metabolism by A. calcoaceticus INMI-KZ-3.

[Utilization of 3-chlorobenzoic acid by Acinetobacter calcoaceticus]. / Utilizatsiia 3-khlorbenzoinoi kisloty Acinetobacter calcoaceticus.

A strain of Acinetobacter calcoaceticus assimilating 3-chlorobenzoic acid (3-CBA) as a sole source of carbon and energy was isolated from soil near Moscow. When 3-CBA is utilized, 2-chloro-cis, cis-muconic acid is accumulated in the medium. The liberation of chlorine atoms is 50--60% of the theoretically possible value. The oxidation of 3-CBA yields 3-chloropyrocatechol (3-CPC) and 4-chloropyrocatechol (4-CPC). 4-CPC serves as a source of nutrition for A. calcoaceticus. The oxidation of 3-CPC yields 2-chloro-cis, cis-muconic acid which is accumulated in the cultural broth without further utilization. The enzyme system of 3-CBA is inducible.

[Utilization of 4-chlorobenzoic acid by Arthrobacter globiformis]. / Utilizatsiia 4-khlorbenzoinoi kisloty Arthrobacter globiformis.

A strain of Arthrobacter globiformis utilizing 4-chlorobenzoic acid as a sole source of carbon and energy was isolated by the method of enrichment cultures from vegetable garden soil near Moscow. The yield of released chlorine upon the utilization of 4-chlorobenzoic acid exceeded 96% of the theoretically possible one. Biotin stimulated noticeably the utilization of the acid. The concentration of 4-chlorobenzoic acid that apparently did not inhibit the growth of the isolated organism was within the range of 0.5-0.6 g per litre. The strain utilized a number of mono- and dihydroxybenzoic acids but not benzoic acid. This observation make possible that dehalogenization occurs at the first step of preparative metabolism. The oxidation of 4-chlorobenzoic acid, 4-hydroxybenzoic acid, protocatechuic acid and galactose by the cells grown on these compounds has been studied and shown that the oxidation system for 4-chlorobenzoic acid is an inducible one whereas that for 4-hydroxybenzoic and protocatechuic acids is a constitutive or a semiconstitutive one.