Aerobic Degradation of N-Methyl-4-Nitroaniline (MNA) by Pseudomonas sp. Strain FK357 Isolated from Soil

N-Methyl-4-nitroaniline (MNA) is used as an additive to lower the melting temperature of energetic materials in the synthesis of insensitive explosives. Although the biotransformation of MNA under anaerobic condition has been reported, its aerobic microbial degradation has not been documented yet. A soil microcosms study showed the efficient aerobic degradation of MNA by the inhabitant soil microorganisms. An aerobic bacterium, Pseudomonas sp. strain FK357, able to utilize MNA as the sole carbon, nitrogen, and energy source, was isolated from soil microcosms. HPLC and GC-MS analysis of the samples obtained from growth and resting cell studies showed the formation of 4-nitroaniline (4-NA), 4-aminophenol (4-AP), and 1, 2, 4-benzenetriol (BT) as major metabolic intermediates in the MNA degradation pathway. Enzymatic assay carried out on cell-free lysates of MNA grown cells confirmed N-demethylation reaction is the first step of MNA degradation with the formation of 4-NA and formaldehyde products. Flavin-dependent transformation of 4-NA to 4-AP in cell extracts demonstrated that the second step of MNA degradation is a monooxygenation. Furthermore, conversion of 4-AP to BT by MNA grown cells indicates the involvement of oxidative deamination (release of NH₂ substituent) reaction in third step of MNA degradation. Subsequent degradation of BT occurs by the action of benzenetriol 1, 2-dioxygenase as reported for the degradation of 4-nitrophenol. This is the first report on aerobic degradation of MNA by a single bacterium along with elucidation of metabolic pathway.     

Metabolism of 2-Chloro-4-Nitroaniline via Novel Aerobic Degradation Pathway by Rhodococcus sp. Strain MB-P1

2-chloro-4-nitroaniline (2-C-4-NA) is used as an intermediate in the manufacture of dyes, pharmaceuticals, corrosion inhibitor and also used in the synthesis of niclosamide, a molluscicide. It is marked as a black-listed substance due to its poor biodegradability. We report biodegradation of 2-C-4-NA and its pathway characterization by Rhodococcus sp. strain MB-P1 under aerobic conditions. The strain MB-P1 utilizes 2-C-4-NA as the sole carbon, nitrogen, and energy source. In the growth medium, the degradation of 2-C-4-NA occurs with the release of nitrite ions, chloride ions, and ammonia. During the resting cell studies, the 2-C-4-NA-induced cells of strain MB-P1 transformed 2-C-4-NA stoichiometrically to 4-amino-3-chlorophenol (4-A-3-CP), which subsequently gets transformed to 6-chlorohydroxyquinol (6-CHQ) metabolite. Enzyme assays by cell-free lysates prepared from 2-C-4-NA-induced MB-P1 cells, demonstrated that the first enzyme in the 2-C-4-NA degradation pathway is a flavin-dependent monooxygenase that catalyzes the stoichiometric removal of nitro group and production of 4-A-3-CP. Oxygen uptake studies on 4-A-3-CP and related anilines by 2-C-4-NA-induced MB-P1 cells demonstrated the involvement of aniline dioxygenase in the second step of 2-C-4-NA degradation. This is the first report showing 2-C-4-NA degradation and elucidation of corresponding metabolic pathway by an aerobic bacterium.     

Degradation of Bromoxynil Octanoate by Strain Acinetobacter sp. XB2 Isolated from Contaminated Soil

Bromoxynil octanoate (BOO), the most widespread herbicide applied to maize, is potentially toxic to both animals and humans. In this article, a highly effective BOO-degrading bacterial strain, XB2, was isolated from the soil of a herbicide factory. The strain was identified as an Acinetobacter sp. based on its 16S rRNA gene sequence analysis, morphological, physiological, and biochemical properties. This strain could use BOO as its sole carbon source and could degrade 100?mg?l(-1) BOO to non-detectable levels in 72?h (h). The optimal pH and temperature for strain XB2's growth and degradation of BOO in MSM are 7.0 and 30°C, respectively. We propose the following pathway of BOO degradation by strain XB2: the first step is the scission of the ester bond to form bromoxynil, bromoxynil then transformed to 3,5-dibromo-4-hydroxybenzoic acid?due to the hydrolysis of nitriles, and debromination finally results in the formation of 3-bromo-4-hydroxybenzoic acid. Inoculating BOO-treated soil samples with strain XB2 resulted in a higher rate of BOO degradation than in non-inoculated soil, regardless of whether the soil had previously been sterilized.     

Aerobic Degradation of 1,1,1-Trichloroethane by Mycobacterium spp. Isolated from Soil

Two strains of 1,1,1-trichloroethane (TCA)-degrading bacteria, TA5 and TA27, were isolated from soil and identified as Mycobacterium spp. Strains TA5 and TA27 could degrade 25 and 75 mg · liter of TCA⁻¹ cometabolically in the presence of ethane as a carbon source, respectively. The compound 2,2,2-trichloroethanol was produced as a metabolite of the degradation process.     

Degradation and Detoxification of Nicosulfuron by a Pseudomonas Strain Isolated from a Contaminated Cornfield Soil

ABSTRACT

The dissipation and detoxification of nicosulfuron (NS) by Pseudomonas aeruginosa B9 isolated from a cornfield soil was investigated. The fastest decline of NS occurred at 40 µg ml^?1 in liquid media with 0.25% glucose plus 0.05% yeast extract (DT₅₀ = 4 days) with a notable pH reduction (pH ? 5). Bioassay tests showed considerable phytotoxicity of NS for Cress (Lepidium sativum L.) with 50% shoot growth inhibition (SGI) at 40 µg ml^?1. The dissipation of NS (40 µg ml^?1) by the B9 isolate reduced the SGI significantly (SGI: up to 45 ± 3%) compared to the non-inoculated media (SGI: up to 58 ± 4%). In soils with the B9 isolate, NS dissipation, especially at 0.3 µg g^?1, was faster with a more significant SGI reduction (k = 0.08 ± 0.00 day^?1; SGI = 2 ± 1%) compared to non-inoculated samples (k = 0.03 ± 0.00 day^?1; SGI = 8 ± 1%). NS initially inhibited soil respiration, microbial biomass carbon, and dehydrogenase activity. The effect was however transient, and these parameters recovered within 10 days, especially in the presence of the isolate. Overall, this study proves Pseudomonas aeruginosa B9 as a suitable candidate for bioremediation of NS in contaminated sites.     

Aerobic TNT Reduction via 2-Hydroxylamino-4,6-Dinitrotoluene by Pseudomonas aeruginosa Strain MX Isolated from Munitions-Contaminated Soil

Bioremediation of munitions-contaminated soil requires effective transformation and detoxification of high concentrations of 2,4,6-trinitrotoluene (TNT). Pseudomonas aeruginosa strain MX, isolated from munitions-contaminated soil, aerobically transformed TNT (100 mg/L) in culture medium within 15 h, causing transient accumulation of hydroxylaminodinitrotoluenes (HADNTs). The predominance of 2-hydroxylamino-4,6-dinitrotoluene (2HADNT), as well as 2-amino-4,6-dinitrotoluene (2ADNT) and 4,4' ,6,6' -tetranitro-2,2' -azoxytoluene (2,2'AZT), indicated preferential reduction of the TNT ortho nitro group. While only 12% of the TNT was transformed to 2ADNT, up to 65% was transformed to tetranitroazoxytoluenes (AZTs), which accumulated as a precipitate. The precipitate was formed by microscopic particles adhering to bacterial cells, which subsequently formed clusters containing lysed cells. Toxicity toward bacteria was primarily attributed to 2ADNT, because pure AZTs preincubated with sterile medium had little effect on the strain. While the culture medium containing TNT exhibited toxicity toward corn (Zea mays L.) and witchgrass (Panicum capillare L.), little phytotoxicity was observed after incubating with P. aeruginosa strain MX for 4 d. Strong binding of HADNTs to soil and low AZT bioavailability may further promote the detoxification of TNT in soil.     

Aerobic Degradation of 3-Methylindole by Pseudomonas aeruginosa Gs Isolated from Mangrove Sediment

3-Methylindole (3MI), an N-heterocyclic aromatic compound also called skatole, is associated with animal waste and industrial processing. A pure culture of bacterium capable of using 3MI as the sole source of carbon and energy was isolated from mangrove sediment using an enrichment technique and identified as Pseudomonas aeruginosa Gs based on 16S rDNA sequence. Microbial degradation of 3MI was studied in batch culture experiments for several factors, including initial substrate concentrations, pH, and salinity. The optimum pH and salinity was 7.0 and 5‰, respectively. Degradation of 3MI by P. aeruginosa Gs was quantified by reversed-phase high-performance liquid chromatography. Two metabolites of 3MI degradation were detected and proposed to be indoline-3-carboxylic acid and indoline-3-ol based on data obtained from HPLC/MS. Our results suggest that 3MI can be rapidly degraded by indigenous microorganisms found in mangrove sediment.     

Degradation of 2-Methylnaphthalene by Pseudomonas sp. Strain NGK1

Pseudomonas sp. strain NGK1, a soil bacterium isolated by naphthalene enrichment from biological waste effluent treatment, capable of utilizing 2-methylnaphthalene as sole source of carbon and energy. To deduce the pathway for biodegradation of 2-methylnaphthalene, metabolites were isolated from the spent medium and identified by thin-layer chromatography and high-performance liquid chromatography. The characterization of purified metabolites, oxygen uptake studies, and enzyme activities revealed that the strain degrades 2-methylnaphthalene through more than one pathway. The growth of the bacterium, utilization of 2-methylnaphthalene, and 4-methylsalicylate accumulation by Pseudomonas sp. strain NGK1 were studied at various incubation periods. Received: 20 March 2001 / Accepted: 25 April 2001     

Degradation of Mono-chlorinated Dibenzo-p-Dioxins by Janibacter sp. Strain YA Isolated from River Sediment

Strain YA was newly isolated from an enrichment culture of river sediment and was identified as Janibacter sp. It was able to utilize dibenzofuran as the sole source of carbon and energy. Strain YA degraded > 90% of 1-chloro-dibenzo-p-dioxin (1-CDD) and > 80% of 2-chloro-dibenzo-p-dioxin in 18 hours with each initial concentration at 40 mg/L. A novel metabolite, 2-chloro-2′,6-dihydroxydiphenylether, was observed in 1-CDD degradation. From the metabolites detected by gas chromatography–mass spectrometry, strain YA was supposed to have at least two types of oxidation pathways in 1-CDD degradation.     

Monooxygenase-Mediated 1,2-Dichloroethane Degradation by Pseudomonas sp. Strain DCA1

A bacterial strain, designated Pseudomonas sp. strain DCA1, was isolated from a 1,2-dichloroethane (DCA)-degrading biofilm. Strain DCA1 utilizes DCA as the sole carbon and energy source and does not require additional organic nutrients, such as vitamins, for optimal growth. The affinity of strain DCA1 for DCA is very high, with a K_m value below the detection limit of 0.5 μM. Instead of a hydrolytic dehalogenation, as in other DCA utilizers, the first step in DCA degradation in strain DCA1 is an oxidation reaction. Oxygen and NAD(P)H are required for this initial step. Propene was converted to 1,2-epoxypropane by DCA-grown cells and competitively inhibited DCA degradation. We concluded that a monooxygenase is responsible for the first step in DCA degradation in strain DCA1. Oxidation of DCA probably results in the formation of the unstable intermediate 1,2-dichloroethanol, which spontaneously releases chloride, yielding chloroacetaldehyde. The DCA degradation pathway in strain DCA1 proceeds from chloroacetaldehyde via chloroacetic acid and presumably glycolic acid, which is similar to degradation routes observed in other DCA-utilizing bacteria.     

Degradation of Fumonisin B1 by a Bacterial Strain Isolated from Soil

A mixed microbial culture degrading fumonisin B l was obtained from soil samples using an enrichment culture procedure. A bacterial isolate from the enrichment culture (strain NCB 1492) degraded fumonisin B1 after incubation for 3 h, as indicated by TLC and HPLC analysis. On the basis of the sequence analysis of 16S rDNA, strain NCB 1492 was related to the Delftia/Comamonas group. Thin-layer chromatographic analysis indicated the presence of metabolites in the NCB 1492 culture filtrates after degradation of fumonisin B1 supplied as sole carbon and nitrogen source in phosphate buffer. Four metabolites were identified by mass spectrometry analysis.     

Microbial Degradation of Acetamiprid by Ochrobactrum sp. D-12 Isolated from Contaminated Soil

Neonicotinoid insecticides are one of the most important commercial insecticides used worldwide. The potential toxicity of the residues present in environment to humans has received considerable attention. In this study, a novel Ochrobactrum sp. strain D-12 capable of using acetamiprid as the sole carbon source as well as energy, nitrogen source for growth was isolated and identified from polluted agricultural soil. Strain D-12 was able to completely degrade acetamiprid with initial concentrations of 0–3000 mg·L⁻¹ within 48 h. Haldane inhibition model was used to fit the special degradation rate at different initial concentrations, and the parameters q _max, K _s and K _i were determined to be 0.6394 (6 h)⁻¹, 50.96 mg·L⁻¹ and 1879 mg·L⁻¹, respectively. The strain was found highly effective in degrading acetamiprid over a wide range of temperatures (25–35°C) and pH (6–8). The effects of co-substrates on the degradation efficiency of acetamiprid were investigated. The results indicated that exogenously supplied glucose and ammonium chloride could slightly enhance the biodegradation efficiency, but even more addition of glucose or ammonium chloride delayed the biodegradation. In addition, one metabolic intermediate identified as N-methyl-(6-chloro-3-pyridyl)methylamine formed during the degradation of acetamiprid mediated by strain D-12 was captured by LC-MS, allowing a degradation pathway for acetamiprid to be proposed. This study suggests the bacterium could be a promising candidate for remediation of environments affected by acetamiprid.     

Aerobic Mineralization of Hexachlorobenzene by Newly Isolated Pentachloronitrobenzene-Degrading Nocardioides sp. Strain PD653

A novel aerobic pentachloronitrobenzene-degrading bacterium, Nocardioides sp. strain PD653, was isolated from an enrichment culture in a soil-charcoal perfusion system. The bacterium also degraded hexachlorobenzene, a highly recalcitrant environmental pollutant, accompanying the generation of chloride ions. Liberation of ¹⁴CO₂ from [U-ring-¹⁴C]hexachlorobenzene was detected in a culture of the bacterium and indicates that strain PD653 is able to mineralize hexachlorobenzene under aerobic conditions. The metabolic pathway of hexachlorobenzene is initiated by oxidative dechlorination to produce pentachlorophenol. As further intermediate metabolites, tetrachlorohydroquinone and 2,6-dichlorohydroquinone have been detected. Strain PD653 is the first naturally occurring aerobic bacteria capable of mineralizing hexachlorobenzene.Hexachlorobenzene (C₆Cl₆; HCB) is one of the most persistent environmental pollutants. Its average half-life in soil is approximately 9 years (2). When HCB is liberated in environment, it is bioaccumulated in plants, zooplankton, and shellfish. Finally, HCB is accumulated in the human body via the food chain, whereupon its possible toxicity adversely affects human health as a result of long-term exposure and accumulation. Therefore, HCB was listed as one of the 12 persistent organic pollutants in the Stockholm Convention.A number of studies have been attempted to develop cleanup technology for environmental pollutants. Microbial degradation is a promising effective way to remediate environmental pollutants, including persistent organic pollutants. However, heavily chlorinated benzenes, especially HCB, are resistant to microbial degradation. Several studies have been reported on the reductive dechlorination of HCB. Reductive dechlorination of HCB to pentachlorobenzene by cytochrome P-450 was found in rat hepatic microsomes (22). Microbial transformation of HCB to trichlorobenzene and dichlorobenzene by reductive dechlorination was observed in anaerobic sewage sludge and a mixed culture (5, 7). Yeh and Pavlostathis maintained such an HCB-dechlorinating mixed culture for more than 1 year by adding surfactants as carbon sources (30). One of the microorganisms that reductively dechlorinates HCB is “Dehalococcoides” sp. strain CBDB1 (12). Dehalococcoides sp. strain CBDB1 dechlorinated HCB and pentachlorobenzene via dehalorespiration and gave a final end product mixture comprised of 1,3,5-trichlorobenzene, 1,3-dichlorobenzene, and 1,4-dichlorobenzene. These reductive dechlorinating processes take a longer time and leave less-chlorinated compounds such as trichlorobenzene and dichlorobenzene as end products.Strictly aerobic, naturally occurring microorganisms that degrade and completely mineralize HCB have not been found. On the other hand, a microorganism capable of mineralizing pentachlorophenol (PCP), Sphingobium chlorophenolicum strain ATCC 39723, was isolated, and its gene organization involved in PCP metabolism was shown (4). Conversion of HCB to PCP was reported by using the genetically engineered mutant of cytochrome P-450_cam (CYP101) (13). Wild-type CYP101 from Pseudomonas putida had low degrading activity for dichlorobenzene and trichlorobenzene but did not decompose more highly chlorinated benzenes. The F87W/Y96F/V247L mutant showed improved di- and trichlorobenzene-degrading activity, but activity toward highly chlorinated benzenes including HCB was still low. The activity upon highly chlorinated benzenes was further improved in the mutant CYP101, F87W/Y96F/L244A/V247L (6). The rate of HCB degradation was increased 200-fold in the mutant. Yan et al. introduced the mutant CYP101 gene into S. chlorophenolicum strain ATCC 39723 by homologous recombination, to produce a complete HCB degrader (28). This genetically engineered bacterium degraded HCB almost completely within 12 h, together with formation of PCP as an intermediate. However, the application of genetically engineered microorganisms in natural areas is strictly restricted in many countries. HCB-degrading aerobes derived from natural sources are still required for remediation of HCB-contaminated areas.We describe here isolation and identification of a novel aerobic soil bacterial species capable of aerobically mineralizing HCB. The characterization of metabolites caused by oxidative removal of the chlorine groups from HCB is also described.     

Poly(Aspartic Acid) Degradation by a Sphingomonas sp. Isolated from Freshwater

A poly(aspartic acid) degrading bacterium (strain KT-1 [JCM10459]) was isolated from river water and identified as a member of the genus Sphingomonas. The isolate degraded only poly(aspartic acid)s of low molecular masses (<5 kDa), while the cell extract hydrolyzed high-molecular-mass poly(aspartic acid)s of 5 to 150 kDa to yield aspartic acid monomer.     

Survival in Sterile Soil and Atrazine Degradation of Pseudomonas sp. Strain ADP Immobilized on Zeolite

In laboratory settings, the ability of bacteria and fungi to degrade many environmental contaminants is well proven. However, the potential of microbial inoculants in soil remediation has not often been realized because catabolically competent strains rarely survive and proliferate in soil, and even if they do, they usually fail to express their desired catabolic potential. One method to address the survival problem is formulating the microorganisms with physical and chemical support systems. This study investigates the survival of Pseudomonas sp. strain ADP in sterile soil and its retention of atrazine-degrading functionality. Assessment was conducted with free and zeolite-immobilized bacteria incorporated into the soil. Pseudomonas sp. strain ADP remained viable for at least 10 weeks when stored at 15°C in sterile soil. Cell numbers increased for both free and zeolite-immobilized bacteria during this period, except for free cells when grown in Miller's Luria-Bertani medium, which exhibited constant cell numbers over the 10 weeks. Only the zeolite-immobilized cell retained full functionality to degrade atrazine after 10 weeks in sterile soil regardless of the medium used to culture Pseudomonas sp. strain ADP. Functionality was diminished in free-cell inoculations except when using an improved culture medium. Survival of zeolite-immobilized Pseudomonas sp. strain ADP separated from the soil matrix after 10 weeks’ incubation was significantly (p < .05) greater than in soil inoculated with free cells or in the soil fraction inoculated by release from zeolite-immobilized Pseudomonas sp. strain ADP.     

Molecular Mechanism of Nicotine Degradation by a Newly Isolated Strain,Ochrobactrum sp. Strain SJY1

A newly isolated strain, SJY1, identified as Ochrobactrum sp., utilizes nicotine as a sole source of carbon, nitrogen, and energy. Strain SJY1 could efficiently degrade nicotine via a variant of the pyridine and pyrrolidine pathways (the VPP pathway), which highlights bacterial metabolic diversity in relation to nicotine degradation. A 97-kbp DNA fragment containing six nicotine degradation-related genes was obtained by gap closing from the genome sequence of strain SJY1. Three genes, designated vppB, vppD, and vppE, in the VPP pathway were cloned and heterologously expressed, and the related proteins were characterized. The vppB gene encodes a flavin-containing amine oxidase converting 6-hydroxynicotine to 6-hydroxy-N-methylmyosmine. Although VppB specifically catalyzes the dehydrogenation of 6-hydroxynicotine rather than nicotine, it shares higher amino acid sequence identity with nicotine oxidase (38%) from the pyrrolidine pathway than with its isoenzyme (6-hydroxy-l-nicotine oxidase, 24%) from the pyridine pathway. The vppD gene encodes an NADH-dependent flavin-containing monooxygenase, which catalyzes the hydroxylation of 6-hydroxy-3-succinoylpyridine to 2,5-dihydroxypyridine. VppD shows 62% amino acid sequence identity with the hydroxylase (HspB) from Pseudomonas putida strain S16, whereas the specific activity of VppD is ∼10-fold higher than that of HspB. VppE is responsible for the transformation of 2,5-dihydroxypyridine. Sequence alignment and phylogenetic analysis suggested that the VPP pathway, which evolved independently from nicotinic acid degradation, might have a closer relationship with the pyrrolidine pathway. The proteins and functional pathway identified here provide a sound basis for future studies aimed at a better understanding of molecular principles of nicotine degradation.     

Degradation of 4-aminophenol by newly isolated Pseudomonas sp. strain ST-4

Aromatic compounds and their substituted forms are hazardous to the environment. Biodegradation by microorganisms can be used to remove these pollutants from soil and water. During the present investigations, Pseudomonas sp. strain ST-4 was used for the degradation of 4-aminophenol. The strain was able to use 4-aminophenol as growth substrate showing growth up to 400 ppm on mineral salt media plates. In broth, degradation up to 84% was observed. Induction with 4-aminophenol proved to be effective as it increased the degradation rate more than by the uninduced cell. Biodegradation was found to be more effective than autoxidation of 4-aminophenol, indicating bioremediation as main process to eliminate aromatic amines. In order to locate the responsible genes for degradation, curing and then isolation of plasmid showed the involvement of plasmid encoded genes in this mechanism since the cured strains do not grow with 4-aminophenol.     

Degradation of high concentrations of cresols by Pseudomonas sp. CP4

Pseudomonas sp. CP₄, a potent phenol-degrading laboratory isolate could mineralize all three isomers of cresol. This strain readily utilized up to 1.4, 1.1 and 2.2 g/l of o- m- and p-cresol, respectively as the sole sources of carbon and energy. These are the highest concentrations of cresols reported to be degraded by a bacterial strain. The rates of degradation of the three isomers were in the order: o- > p- > m-cresol. All the isomers of cresol were catabolized through a meta-cleavage pathway. Fairly high catechol 2,3-dioxygenase (C230) activity against catechol was observed in the cell-free extracts of the culture grown on these compounds and were in the order: m- > o- > p-cresol.