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J C Spain

Publications and source records attributed to J C Spain.

At least 55 records · Page 3Linked to original sources
PubMed1994

Environmental biotechnology research: an overview.

Cleanup and treatment of hazardous wastes incur major operational costs for the U.S. Air Force. Bioremediation can provide a cost-effective alternative to traditional technologies for a wide range of natural organic compounds such as jet fuel. Bioventing and natural attenuation are emerging as treatments of choice in many instances. Synthetic organic chemicals are much more resistant to biodegradation. However, recent advances in biotechnology allow the development of strains able to use nitro- and chloro-substituted organic compounds as their sole source of carbon and energy. Current basic research is focused on expanding the range of synthetic chemicals amenable to biodegradation. At the same time, development of appropriate bioreactors and models for scale up are essential for practical application of the technology.

Biotechnology↗
PubMed1993

Biodegradation of 4-nitrotoluene by Pseudomonas sp. strain 4NT.

A strain of Pseudomonas spp. was isolated from nitrobenzene-contaminated soil on 4-nitrotoluene as the sole source of carbon, nitrogen, and energy. The organism also grew on 4-nitrobenzaldehyde, and 4-nitrobenzoate. 4-Nitrobenzoate and ammonia were detected in the culture fluid of glucose-grown cells after induction with 4-nitrotoluene. Washed suspensions of 4-nitrotoluene- or 4-nitrobenzoate-grown cells oxidized 4-nitrotoluene, 4-nitrobenzaldehyde, 4-nitrobenzyl alcohol, and protocatechuate. Extracts from induced cells contained 4-nitrobenzaldehyde dehydrogenase, 4-nitrobenzyl alcohol dehydrogenase, and protocatechuate 4,5-dioxygenase activities. Under anaerobic conditions, cell extracts converted 4-nitrobenzoate or 4-hydroxylaminobenzoate to protocatechuate. Conversion of 4-nitrobenzoate to protocatechuate required NADPH. These results indicate that 4-nitrotoluene was degraded by an initial oxidation of the methyl group to form 4-nitrobenzyl alcohol, which was converted to 4-nitrobenzoate via 4-nitrobenzaldehyde. The 4-nitrobenzoate was reduced to 4-hydroxylaminobenzoate, which was converted to protocatechuate. A protocatechuate 4,5-dioxygenase catalyzed meta-ring fission of the protocatechuate. The detection of 4-nitrobenzaldehyde and 4-nitrobenzyl alcohol dehydrogenase and 4-nitrotoluene oxygenase activities in 4-nitrobenzoate-grown cells suggests that 4-nitrobenzoate is an inducer of the 4-nitrotoluene degradative pathway.

Biodegradation, Environmental↗
PubMed1993

Degradation of nitrobenzene by a Pseudomonas pseudoalcaligenes.

A Pseudomonas pseudoalcaligenes able to use nitrobenzene as the sole source of carbon, nitrogen, and energy was isolated from soil and groundwater contaminated with nitrobenzene. The range of aromatic substrates able to support growth was limited to nitrobenzene, hydroxylaminobenzene, and 2-aminophenol. Washed suspensions of nitrobenzene-grown cells removed nitrobenzene from culture fluids with the concomitant release of ammonia. Nitrobenzene, nitrosobenzene, hydroxylaminobenzene, and 2-aminophenol stimulated oxygen uptake in resting cells and in extracts of nitrobenzene-grown cells. Under aerobic and anaerobic conditions, crude extracts converted nitrobenzene to 2-aminophenol with oxidation of 2 mol of NADPH. Ring cleavage, which required ferrous iron, produced a transient yellow product with a maximum A380. In the presence of NAD, the product disappeared and NADH was produced. In the absence of NAD, the ring fission product was spontaneously converted to picolinic acid, which was not further metabolized. These results indicate that the catabolic pathway involves the reduction of nitrobenzene to nitrosobenzene and then to hydroxylaminobenzene; each of these steps requires 1 mol of NADPH. An enzyme-mediated Bamberger-like rearrangement converts hydroxylaminobenzene to 2-aminophenol, which then undergoes meta ring cleavage to 2-aminomuconic semialdehyde. The mechanism for release of ammonia and subsequent metabolism are under investigation.

Aminophenols↗
PubMed1993

Cloning and characterization of Pseudomonas sp. strain DNT genes for 2,4-dinitrotoluene degradation.

The degradation of 2,4-dinitrotoluene (DNT) by Pseudomonas sp. strain DNT is initiated by a dioxygenase attack to yield 4-methyl-5-nitrocatechol (MNC) and nitrite. Subsequent oxidation of MNC by a monooxygenase results in the removal of the second molecule of nitrite, and further enzymatic reactions lead to ring fission. Initial studies on the molecular basis of DNT degradation in this strain revealed the presence of three plasmids. Mitomycin-derived mutants deficient in either DNT dioxygenase only or DNT dioxygenase and MNC monooxygenase were isolated. Plasmid profiles of mutant strains suggested that the mutations resulted from deletions in the largest plasmid. Total plasmid DNA partially digested by EcoRI was cloned into a broad-host-range cosmid vector, pCP13. Recombinant clones containing genes encoding DNT dioxygenase, MNC monooxygenase, and 2,4,5-trihydroxytoluene oxygenase were characterized by identification of reaction products and the ability to complement mutants. Subcloning analysis suggests that the DNT dioxygenase is a multicomponent enzyme system and that the genes for the DNT pathway are organized in at least three different operons.

Cloning, Molecular↗
PubMed1992

Chlorobenzene degradation by bacteria isolated from contaminated groundwater.

Bacterial isolates were obtained from groundwater and soils contaminated with chlorobenzene (CB). The isolates were tested to determine whether the natural community could remove the groundwater contaminants. These isolates were identified and characterized as to their ability to grow on CB and related aromatic compounds. The complete consortium could mineralize approximately 54% of the CB within 7 days, with no accumulation of 3-chlorocatechol. Metabolic pathways were evaluated for several isolates. One phenotype was characterized by the ability to degrade CB by the modified ortho pathway. One strain also degraded p-dichlorobenzene by using the same pathway. Isolates exhibiting a second phenotype degraded p-cresol, benzene, and phenol by the classical ortho pathway and accumulated 3-chlorocatechol when grown in the presence of CB. Strains of the third phenotype grew on complex media in the presence of CB but did not transform any of the aromatic compounds tested. The results suggest that the indigenous microbial community at the contaminated site would be able to degrade CB if provided with the appropriate conditions.

Bacteria↗
PubMed1992

Biodegradation of mixtures of substituted benzenes by Pseudomonas sp. strain JS150.

Pseudomonas sp. strain JS150 was isolated as a nonencapsulated variant of Pseudomonas sp. strain JS1 that contains the genes for the degradative pathways of a wide range of substituted aromatic compounds. Pseudomonas sp. strain JS150 grew on phenol, ethylbenzene, toluene, benzene, naphthalene, benzoate, p-hydroxybenzoate, salicylate, chlorobenzene, and several 1,4-dihalogenated benzenes. We designed experiments to determine the conditions required for induction of the individual pathways and to determine whether multiple substrates could be biodegraded simultaneously. Oxygen consumption studies with whole cells and enzyme assays with cell extracts showed that the enzymes of the meta, ortho, and modified ortho cleavage pathways can be induced in strain JS150. Strain JS150 contains a nonspecific toluene dioxygenase with a substrate range similar to that found in strains of Pseudomonas putida. The presence of the dioxygenase along with multiple pathways for metabolism of substituted catechols allows facile extension of the growth range by spontaneous mutation and degradation of mixtures of substituted benzenes and phenols. Chlorobenzene-grown cells of strain JS150 degraded mixtures of chlorobenzene, benzene, toluene, naphthalene, trichloroethylene, and 1,2- and 1,4-dichlorobenzenes in continuous culture. Under similar conditions, phenol-grown cells degraded a mixture of phenol, 2-chloro-, 3-chloro, and 2,5-dichlorophenol and 2-methyl- and 3-methylphenol. These results indicate that induction of appropriate biodegradative pathways in strain JS150 permits the biodegradation of complex mixtures of aromatic compounds.

Benzene Derivatives↗
PubMed1992

Oxidation of nitrotoluenes by toluene dioxygenase: evidence for a monooxygenase reaction.

Pseudomonas putida F1 and Pseudomonas sp. strain JS150 initiate toluene degradation by incorporating molecular oxygen into the aromatic nucleus to form cis-1,2-dihydroxy-3-methylcyclohexa-3,5-diene. When toluene-grown cells were incubated with 2- and 3-nitrotoluene, the major products identified were 2- and 3-nitrobenzyl alcohol, respectively. The same cells oxidized 4-nitrotoluene to 2-methyl-5-nitrophenol and 3-methyl-6-nitrocatechol. Escherichia coli JM109(pDTG601), which contains the toluene dioxygenase genes from P. putida F1 under the control of the tac promoter, oxidized the isomeric nitrotoluenes to the same metabolites as those formed by P. putida F1 and Pseudomonas sp. strain JS150. These results extend the range of substrates known to be oxidized by this versatile enzyme and demonstrate for the first time that toluene dioxygenase can oxidize an aromatic methyl substituent.

Biodegradation, Environmental↗
PubMed1991

Simultaneous biodegradation of chlorobenzene and toluene by a Pseudomonas strain.

Pseudomonas sp. strain JS6 grows on a wide range of chloro- and methylaromatic substrates. The simultaneous degradation of these compounds is prevented in most previously studied isolates because the catabolic pathways are incompatible. The purpose of this study was to determine whether strain JS6 could degrade mixtures of chloro- and methyl-substituted aromatic compounds. Strain JS6 was maintained in a chemostat on a minimal medium with toluene or chlorobenzene as the sole carbon source, supplied via a syringe pump. Strain JS6 contained an active catechol 2,3-dioxygenase when grown in the presence of chloroaromatic compounds; however, in cell extracts, this enzyme was strongly inhibited by 3-chlorocatechol. When cells grown to steady state on toluene were exposed to 50% toluene-50% chlorobenzene, 3-chlorocatechol and 3-methylcatechol accumulated in the medium and the cell density decreased. After 3 h, the enzyme activities of the modified ortho ring fission pathway were induced, the metabolites disappeared, and the cell density returned to previous levels. In cell extracts, 3-methylcatechol was degraded by both catechol 1,2- and catechol 2,3-dioxygenase. Strain JS62, a catechol 2,3-dioxygenase mutant of JS6, grew on toluene, and ring cleavage of 3-methylcatechol was catalyzed by catechol 1,2-dioxygenase. The transient metabolite 2-methyllactone was identified in chlorobenzene-grown JS6 cultures exposed to toluene. These results indicate that strain JS6 can degrade mixtures of chloro- and methylaromatic compounds by means of a modified ortho ring fission pathway.

Biodegradation, Environmental↗
PubMed1991

Biotransformation of nitrobenzene by bacteria containing toluene degradative pathways.

Nonpolar nitroaromatic compounds have been considered resistant to attack by oxygenases because of the electron withdrawing properties of the nitro group. We have investigated the ability of seven bacterial strains containing toluene degradative pathways to oxidize nitrobenzene. Cultures were induced with toluene vapor prior to incubation with nitrobenzene, and products were identified by high-performance liquid chromatography and gas chromatography-mass spectrometry. Pseudomonas cepacia G4 and a strain of Pseudomonas harboring the TOL plasmid (pTN2) did not transform nitrobenzene. Cells of Pseudomonas putida F1 and Pseudomonas sp. strain JS150 converted nitrobenzene to 3-nitrocatechol. Transformation of nitrobenzene in the presence of 18O2 indicated that the reaction in JS150 involved the incorporation of both atoms of oxygen in the 3-nitrocatechol, which suggests a dioxygenase mechanism. P. putida 39/D, a mutant strain of P. putida F1, converted nitrobenzene to a compound tentatively identified as cis-1,2-dihydroxy-3-nitrocyclohexa-3,5-diene. This compound was rapidly converted to 3-nitrocatechol by cells of strain JS150. Cultures of Pseudomonas mendocina KR-1 converted nitrobenzene to a mixture of 3- and 4-nitrophenol (10 and 63%, respectively). Pseudomonas pickettii PKO1 converted nitrobenzene to 3- and 4-nitrocatechol via 3- and 4-nitrophenol. The nitrocatechols were slowly degraded to unidentified metabolites. Nitrobenzene did not serve as an inducer for the enzymes that catalyzed its oxidation. These results indicate that the nitrobenzene ring is subject to initial attack by both mono- and dioxygenase enzymes.

Biodegradation, Environmental↗
PubMed1991

Biodegradation of 2,4-dinitrotoluene by a Pseudomonas sp.

Previous studies of the biodegradation of nonpolar nitroaromatic compounds have suggested that microorganisms can reduce the nitro groups but cannot cleave the aromatic ring. We report here the initial steps in a pathway for complete biodegradation of 2,4-dinitrotoluene (DNT) by a Pseudomonas sp. isolated from a four-member consortium enriched with DNT. The Pseudomonas sp. degraded DNT as the sole source of carbon and energy under aerobic conditions with stoichiometric release of nitrite. During induction of the enzymes required for growth on DNT, 4-methyl-5-nitrocatechol (MNC) accumulated transiently in the culture fluid when cells grown on acetate were transferred to medium containing DNT as the sole carbon and energy source. Conversion of DNT to MNC in the presence of 18O2 revealed the simultaneous incorporation of two atoms of molecular oxygen, which demonstrated that the reaction was catalyzed by a dioxygenase. Fully induced cells degraded MNC rapidly with stoichiometric release of nitrite. The results indicate an initial dioxygenase attack at the 4,5 position of DNT with the concomitant release of nitrite. Subsequent reactions lead to complete biodegradation and removal of the second nitro group as nitrite.

Biodegradation, Environmental↗
PubMed1991

Pathway for Biodegradation of p-Nitrophenol in a Moraxella sp.

A Moraxella strain grew on p-nitrophenol with stoichiometric release of nitrite. During induction of the enzymes for growth on p-nitrophenol, traces of hydroquinone accumulated in the medium. In the presence of 2,2'-dipyridyl, p-nitrophenol was converted stoichiometrically to hydroquinone. Particulate enzymes catalyzed the conversion of p-nitrophenol to hydroquinone in the presence of NADPH and oxygen. Soluble enzymes catalyzed the conversion of hydroquinone to gamma-hydroxymuconic semialdehyde, which was identified by high-performance liquid chromatography (HPLC)-mass spectroscopy. Upon addition of catalytic amounts of NAD, gamma-hydroxymuconic semialdehyde was converted to beta-ketoadipic acid. In the presence of pyruvate and lactic dehydrogenase, substrate amounts of NAD were required and gamma-hydroxymuconic semialdehyde was converted to maleylacetic acid, which was identified by HPLC-mass spectroscopy. Similar results were obtained when the reaction was carried out in the presence of potassium ferricyanide. Extracts prepared from p-nitrophenol-growth cells also contained an enzyme that catalyzed the oxidation of 1,2,4-benzenetriol to maleylacetic acid. The enzyme responsible for the oxidation of 1,2,4-benzenetriol was separated from the enzyme responsible for hydroquinone oxidation by DEAE-cellulose chromatography. The results indicate that the pathway for biodegradation of p-nitrophenol involves the initial removal of the nitro group as nitrite and formation of hydroquinone. 1,4-Benzoquinone, a likely intermediate in the initial reaction, was not detected. Hydroquinone is converted to beta-ketoadipic acid via gamma-hydroxymuconic semialdehyde and maleylacetic acid.

Journal Article↗
PubMed1989

Monohydroxylation of phenol and 2,5-dichlorophenol by toluene dioxygenase in Pseudomonas putida F1.

Pseudomonas putida F1 contains a multicomponent enzyme system, toluene dioxygenase, that converts toluene and a variety of substituted benzenes to cis-dihydrodiols by the addition of one molecule of molecular oxygen. Toluene-grown cells of P. putida F1 also catalyze the monohydroxylation of phenols to the corresponding catechols by an unknown mechanism. Respirometric studies with washed cells revealed similar enzyme induction patterns in cells grown on toluene or phenol. Induction of toluene dioxygenase and subsequent enzymes for catechol oxidation allowed growth on phenol. Tests with specific mutants of P. putida F1 indicated that the ability to hydroxylate phenols was only expressed in cells that contained an active toluene dioxygenase enzyme system. 18O2 experiments indicated that the overall reaction involved the incorporation of only one atom of oxygen in the catechol, which suggests either a monooxygenase mechanism or a dioxygenase reaction with subsequent specific elimination of water.

Catechols↗
PubMed1989

Degradation of p-chlorotoluene by a mutant of Pseudomonas sp. strain JS6.

Pseudomonas sp. strain JS6 grows on chlorobenzene, p-dichlorobenzene, or toluene as a sole source of carbon and energy. It does not grow on p-chlorotoluene (p-CT). Growth on glucose in the presence of p-CT resulted in the accumulation of 4-chloro-2,3-dihydroxy-1-methylbenzene (3-chloro-6-methylcatechol), 4-chloro-2,3-dihydroxy-1-methylcyclohexa-4,6-diene (p-CT dihydrodiol), and 2-methyl-4-carboxymethylenebut-2-en-4-olide (2-methyl dienelactone). Strain JS21, a spontaneous mutant capable of growth on p-CT, was isolated from cultures of strain JS6 after extended exposure to p-CT. In addition to growing on p-CT, JS21 grew on all of the substrates that supported growth of the parent strain, including p-dichlorobenzene, chlorobenzene, benzene, toluene, benzoate, p-hydroxybenzoate, phenol, and ethylbenzene. The pathway for degradation of p-CT by JS21 was investigated by respirometry, isolation of intermediates, and assay of enzymes in cell extracts. p-CT was converted to 3-chloro-6-methylcatechol by dioxygenase and dihydrodiol dehydrogenase enzymes. 3-Chloro-6-methylcatechol underwent ortho ring cleavage catalyzed by a catechol 1,2-dioxygenase to form 2-chloro-5-methyl-cis,cis-muconate, which was converted to 2-methyl dienelactone. A dienelactone hydrolase converted 2-methyl dienelactone to 2-methylmaleylacetic acid. Preliminary results indicate that a change in wild-type induction patterns allows JS21 to grow on p-CT.

Biotransformation↗
PubMed1988

Degradation of 1,2-dichlorobenzene by a Pseudomonas sp.

A Pseudomonas sp. that was capable of growth on 1,2-dichlorobenzene (o-DCB) or chlorobenzene as a sole source of carbon and energy was isolated by selective enrichment from activated sludge. The initial steps involved in the degradation of o-DCB were investigated by isolation of metabolites, respirometry, and assay of enzymes in cell extracts. Extracts of o-DCB-grown cells converted radiolabeled o-DCB to 3,4-dichloro-cis-1,2-dihydroxycyclohexa-3,5-diene (o-DCB dihydrodiol). 3,4-Dichlorocatechol and o-DCB dihydrodiol accumulated in culture fluids of cells exposed to o-DCB. The results suggest that o-DCB is initially converted by a dioxygenase to a dihydrodiol, which is converted to 3,4-dichlorocatechol by an NAD+-dependent dehydrogenase. Ring cleavage of 3,4-dichlorocatechol is by a catechol 1,2-oxygenase to form 2,3-dichloro-cis,cis-muconate. Preliminary results indicate that chloride is eliminated during subsequent lactonization of the 2,3-dichloro-cis,cis-muconate, followed by hydrolysis to form 5-chloromaleylacetic acid.

Alcohol Oxidoreductases↗
PubMed1988

Oxidation of substituted phenols by Pseudomonas putida F1 and Pseudomonas sp. strain JS6.

The biodegradation of benzene, toluene, and chlorobenzenes by Pseudomonas putida involves the initial conversion of the parent molecules to cis-dihydrodiols by dioxygenase enzyme systems. The cis-dihydrodiols are then converted to the corresponding catechols by dihydrodiol dehydrogenase enzymes. Pseudomonas sp. strain JS6 uses a similar system for growth on toluene or dichlorobenzenes. We tested the wild-type organisms and a series of mutants for their ability to transform substituted phenols after induction with toluene. When grown on toluene, both wild-type organisms converted methyl-, chloro-, and nitro-substituted phenols to the corresponding catechols. Mutant strains deficient in dihydrodiol dehydrogenase or catechol oxygenase activities also transformed the phenols. Oxidation of phenols was closely correlated with the induction and activity of the toluene dioxygenase enzyme system.

Biodegradation, Environmental↗
PubMed1987

Degradation of 1,4-dichlorobenzene by a Pseudomonas sp.

A Pseudomonas species able to degrade p-dichlorobenzene as the sole source of carbon and energy was isolated by selective enrichment from activated sludge. The organism also grew well on chlorobenzene and benzene. Washed cells released chloride in stoichiometric amounts from o-, m-, and p-dichlorobenzene, 2,5-dichlorophenol, 4-chlorophenol, 3-chlorocatechol, 4-chlorocatechol, and 3,6-dichlorocatechol. Initial steps in the pathway for p-dichlorobenzene degradation were determined by isolation of metabolites, simultaneous adaptation studies, and assay of enzymes in cell extracts. Results indicate that p-dichlorobenzene was initially converted by a dioxygenase to 3,6-dichloro-cis-1,2-dihydroxycyclohexa-3,5-diene, which was converted to 3,6-dichlorocatechol by an NAD+-dependent dehydrogenase. Ring cleavage of 3,6-dichlorocatechol was by a 1,2-oxygenase to form 2,5-dichloro-cis, cis-muconate. Enzymes for degradation of haloaromatic compounds were induced in cells grown on chlorobenzene or p-dichlorobenzene, but not in cells grown on benzene, succinate, or yeast extract. Enzymes of the ortho pathway induced in cells grown on benzene did not attack chlorobenzenes or chlorocatechols.

Biodegradation, Environmental↗
PubMed1984

Comparison of p-Nitrophenol Biodegradation in Field and Laboratory Test Systems.

Acclimation of microbial communities exposed to p-nitrophenol (PNP) was measured in laboratory test systems and in a freshwater pond. Laboratory tests were conducted in shake flasks with water, shake flasks with water and sediment, eco-cores, and two sizes of microcosm. The sediment and water samples used in the laboratory experiments were obtained from the pond. After a 6-day acclimation period, PNP was biodegraded rapidly in the pond. When the pond was treated with PNP a second time, biodegradation began immediately. The acclimation periods in laboratory test systems that contained sediment were similar to that in the pond. The acclimation period was threefold longer in shake flasks without sediment. PNP was biodegraded more slowly by microbial communities acclimated in the laboratory than it was in the pond, and the rate of biodegradation varied with the type of test. The number of bacteria able to mineralize PNP increased by 3 orders of magnitude in the pond during the acclimation period. Similar increases accompanied acclimation in the laboratory systems.

Journal Article↗