Biodegradation of chlorpyrifos by enterobacter strain B-14 and its use in bioremediation of contaminated soils

Six chlorpyrifos-degrading bacteria were isolated from an Australian soil and compared by biochemical and molecular methods. The isolates were indistinguishable, and one (strain B-14) was selected for further analysis. This strain showed greatest similarity to members of the order Enterobacteriales and was closest to members of the Enterobacter asburiae group. The ability of the strain to mineralize chlorpyrifos was investigated under different culture conditions, and the strain utilized chlorpyrifos as the sole source of carbon and phosphorus. Studies with ring or uniformly labeled [(14)C]chlorpyrifos in liquid culture demonstrated that the isolate hydrolyzed chlorpyrifos to diethylthiophospshate (DETP) and 3, 5, 6-trichloro-2-pyridinol, and utilized DETP for growth and energy. The isolate was found to possess mono- and diphosphatase activities along with a phosphotriesterase activity. Addition of other sources of carbon (glucose and succinate) resulted in slowing down of the initial rate of degradation of chlorpyrifos. The isolate degraded the DETP-containing organophosphates parathion, diazinon, coumaphos, and isazofos when provided as the sole source of carbon and phosphorus, but not fenamiphos, fonofos, ethoprop, and cadusafos, which have different side chains. Studies of the molecular basis of degradation suggested that the degrading ability could be polygenic and chromosome based. Further studies revealed that the strain possessed a novel phosphotriesterase enzyme system, as the gene coding for this enzyme had a different sequence from the widely studied organophosphate-degrading gene (opd). The addition of strain B-14 (10(6) cells g(-1)) to soil with a low indigenous population of chlorpyrifos-degrading bacteria treated with 35 mg of chlorpyrifos kg(-1) resulted in a higher degradation rate than was observed in noninoculated soils. These results highlight the potential of this bacterium to be used in the cleanup of contaminated pesticide waste in the environment.     

Cloning of mpd gene from a chlorpyrifos-degrading bacterium and use of this strain in bioremediation of contaminated soil

An effective chlorpyrifos-degrading bacterium (named strain YC-1) was isolated from the sludge of the wastewater treating system of an organophosphorus pesticides manufacturer. Based on the results of phenotypic features, phylogenetic similarity of 16S rRNA gene sequences and BIOLOG test, strain YC-1 was identified as the genus Stenotrophomonas. The isolate utilized chlorpyrifos as the sole source of carbon and phosphorus for its growth and hydrolyzed chlorpyrifos to 3,5,6-trichloro-2-pyridinol. Parathion, methyl parathion, and fenitrothion also could be degraded by strain YC-1 when provided as the sole source of carbon and phosphorus. The gene encoding the organophosphorus hydrolase was cloned using a PCR cloning strategy based on the known methyl parathion degrading (mpd) gene of Plesiomonas sp. M6. Sequence blast result indicated this gene has 99% similar to mpd. The inoculation of strain YC-1 (10(6) cells g(-1)) to soil treated with 100 mg kg(-1) chlorpyrifos resulted in a higher degradation rate than in noninoculated soils. Theses results highlight the potential of this bacterium to be used in the cleanup of contaminated pesticide waste in the environment.     

Biodegradation of Chlorpyrifos and Its Hydrolysis Product 3,5,6-Trichloro-2-Pyridinol by a New Fungal Strain Cladosporium cladosporioides Hu-01

Intensive use of chlorpyrifos has resulted in its ubiquitous presence as a contaminant in surface streams and soils. It is thus critically essential to develop bioremediation methods to degrade and eliminate this pollutant from environments. We present here that a new fungal strain Hu-01 with high chlorpyrifos-degradation activity was isolated and identified as Cladosporium cladosporioides based on the morphology and 5.8S rDNA gene analysis. Strain Hu-01 utilized 50 mg·L⁻¹ of chlorpyrifos as the sole carbon of source, and tolerated high concentration of chlorpyrifos up to 500 mg·L⁻¹. The optimum degradation conditions were determined to be 26.8°C and pH 6.5 based on the response surface methodology (RSM). Under these conditions, strain Hu-01 completely metabolized the supplemented chlorpyrifos (50 mg·L⁻¹) within 5 d. During the biodegradation process, transient accumulation of 3,5,6-trichloro-2-pyridinol (TCP) was observed. However, this intermediate product did not accumulate in the medium and disappeared quickly. No persistent accumulative metabolite was detected by gas chromatopraphy-mass spectrometry (GC-MS) analysis at the end of experiment. Furthermore, degradation kinetics of chlorpyrifos and TCP followed the first-order model. Compared to the non-inoculated controls, the half-lives (t _1/2) of chlorpyrifos and TCP significantly reduced by 688.0 and 986.9 h with the inoculum, respectively. The isolate harbors the metabolic pathway for the complete detoxification of chlorpyrifos and its hydrolysis product TCP, thus suggesting the fungus may be a promising candidate for bioremediation of chlorpyrifos-contaminated water, soil or crop.     

Effects of Soil pH on the Biodegradation of Chlorpyrifos and Isolation of a Chlorpyrifos-Degrading Bacterium

We examined the role of microorganisms in the degradation of the organophosphate insecticide chlorpyrifos in soils from the United Kingdom and Australia. The kinetics of degradation in five United Kingdom soils varying in pH from 4.7 to 8.4 suggested that dissipation of chlorpyrifos was mediated by the cometabolic activities of the soil microorganisms. Repeated application of chlorpyrifos to these soils did not result in the development of a microbial population with an enhanced ability to degrade the pesticide. A robust bacterial population that utilized chlorpyrifos as a source of carbon was detected in an Australian soil. The enhanced ability to degrade chlorpyrifos in the Australian soil was successfully transferred to the five United Kingdom soils. Only soils with a pH of ≥6.7 were able to maintain this degrading ability 90 days after inoculation. Transfer and proliferation of degrading microorganisms from the Australian soil to the United Kingdom soils was monitored by molecular fingerprinting of bacterial 16S rRNA genes by PCR-denaturing gradient gel electrophoresis (DGGE). Two bands were found to be associated with enhanced degradation of chlorpyrifos. Band 1 had sequence similarity to enterics and their relatives, while band 2 had sequence similarity to strains of Pseudomonas. Liquid enrichment culture using the Australian soil as the source of the inoculum led to the isolation of a chlorpyrifos-degrading bacterium. This strain had a 16S rRNA gene with a sequence identical to that of band 1 in the DGGE profile of the Australian soil. DNA probing indicated that genes similar to known organophosphate-degrading (opd) genes were present in the United Kingdom soils. However, no DNA hybridization signal was detected for the Australian soil or the isolated degrader. This indicates that unrelated genes were present in both the Australian soil and the chlorpyrifos-degrading isolate. These results are consistent with our observations that degradation of chlorpyrifos in these systems was unusual, as it was growth linked and involved complete mineralization. As the 16S rRNA gene of the isolate matched a visible DGGE band from the Australian soil, the isolate is likely to be both prominent and involved in the degradation of chlorpyrifos in this soil.     

Engineering chlorpyrifos-degrading Stenotrophomonas sp. YC-1 for heavy metal accumulation and enhanced chlorpyrifos degradation

Many ecosystems are currently co-contaminated with pesticides and heavy metals, such as chlorpyrifos and cadmium. A promising strategy to remediate mixed chlorpyrifos-cadmium-contaminated sites is the use of chlorpyrifos-degrading bacteria endowed with cadmium removal capabilities. In this work, a gene coding for synthetic phytochelatins (EC20) with high cadmium-binding capacity was introduced into a chlorpyrifos-degrading bacterium, Stenotrophomonas sp. YC-1, resulting in an engineered strain with both cadmium accumulation and chlorpyrifos degradation capabilities. To improve the cadmium-binding efficiency of whole cells, EC20 was displayed on the cell surface of Stenotrophomonas sp. YC-1 using the truncated ice nucleation protein (INPNC) anchor. The surface localization of the INPNC-EC20 fusion protein was demonstrated by cell fractionation, Western blot analysis, and immunofluorescence microscopy. Expression of EC20 on the cell surface not only improved cadmium binding, but also alleviated the cellular toxicity of cadmium. As expected, the chlorpyrifos degradation rate was reduced in the presence of cadmium for cells without EC20 expression. However, expression of EC20 (higher cadmium accumulation) completely restored the level of chlorpyrifos degradation. These results demonstrated that EC20 expression not only enhanced cadmium accumulation, but also reduced the toxic effect of cadmium on chlorpyrifos degradation.     

Biodegradation of chlorpyrifos and its hydrolyzing metabolite 3,5,6-trichloro-2-pyridinol by Sphingobacterium sp. JAS3

Biodegradation of chlorpyrifos and its metabolite 3,5,6-trichloro-2-pyridinol (TCP) were studied in aqueous medium and in soil with a novel bacterial strain JAS3. The molecular characterization based on 16S rRNA sequence analysis revealed the strain JAS3 as Sphingobacterium sp. The strain JAS3 was able to grow in minimal salt medium (MSM) supplemented with 300 mg l^?1 of chlorpyrifos as sole carbon source. The degradation of chlorpyrifos and its primary metabolite TCP were examined by HPLC. After 5 d, Sphingobacterium sp. JAS3 degraded chlorpyrifos and its metabolite TCP to benzene, 1,3-bis(1,1-dimethylethyl) was analyzed by GCMS. Degradation of chlorpyrifos and TCP in soil with and without addition of nutrients was also studied. The ability to degrade chlorpyrifos makes this strain a useful candidate for remediation of pesticide contaminated sites.     

Biodegradation of methyl parathion and p-nitrophenol by a newly isolated Agrobacterium sp. strain Yw12

Strain Yw12, isolated from activated sludge, could completely degrade and utilize methyl parathion as the sole carbon, phosphorus and energy sources for growth in the basic salt media. It could also completely degrade and utilize p-nitrophenol as the sole carbon and energy sources for growth in the minimal salt media. Phenotypic features, physiological and biochemical characteristics, and phylogenetic analysis of 16S rRNA sequence showed that this strain belongs to the genus of Agrobacterium sp. Response surface methodology was used to optimize degradation conditions. Under its optimal degradation conditions, 50 mg l⁻¹ MP was completely degraded within 2 h by strain Yw12 and the degradation product PNP was also completely degraded within 6 h. Furthermore, strain Yw12 could also degrade phoxim, methamidophos, chlorpyrifos, carbofuran, deltamethrin and atrazine when provided as the sole carbon and energy sources. Enzymatic analysis revealed that the MP degrading enzyme of strain Yw12 is an intracellular enzyme and is expressed constitutively. These results indicated that strain Yw12 might be used as a potential and effective organophosphate pesticides degrader for bioremediation of contaminated sites.     

Biodegradation of chlorpyrifos and 3,5,6-trichloro-2-pyridinol by a newly isolated Paracoccus sp. strain TRP

A bacterium, isolated from activated sludge and named strain TRP, could biodegrade chlorpyrifos and 3,5,6-trichloro-2-pyridinol. Phenotypic features, physiological and chemotaxonomic characteristics, and phylogenetic analysis of 16S rRNA sequence revealed that the isolate belongs to the genus of Paracoccus. Strain TRP could also degrade pyridine, methyl parathion and carbonfuran when provided as sole carbon and energy sources. Native-PAGE and enzymatic degradation assay of the cell-free extracts indicated that an alternative degradation mechanism might involve an inducible enzyme. Degradation study of chlorpyrifos by strain TRP was examined by GC–MS and HPLC; no persistent accumulated metabolite was observed. To the best of our knowledge, this is the first report of a bacterium that could completely mineralize chlorpyrifos. This isolate will be potentially useful in biotreatment of wastewaters and bioremediation of contaminated soils.     

Secretory production of biologically active rat interleukin-2 by Clostridium acetobutylicum DSM792 as a tool for anti-tumor treatment

A bacterium, isolated from contaminated soils around a chemical factory and named strain DSP3 was capable of biodegrading both chlorpyrifos and 3,5,6-trichloro-2-pyridinol. Based on the results of phenotypic features, phylogenetic similarity of 16S rRNA gene sequences, DNA G+C content, and DNA homology between strain DSP3 and reference strains, strain DSP3 was identified as Alcaligenes faecalis. Chlorpyrifos was utilized as the sole source of carbon and phosphorus by strain DSP3. We examined the role of strain DSP3 in the degradation of chlorpyrifos and 3,5,6-trichloro-2-pyridinol under different culture conditions. Parathion and diazinon could also be degraded by strain DSP3 when provided as the sole sources of carbon and phosphorus. An addition of strain DSP3 (10(8)cells g(-1)) to soil with chlorpyrifos (100 mg kg(-1)) resulted in a higher degradation rate than the one obtained from non-inoculated soils. Different degradation rates of chlorpyrifos in six types of treated soils suggested that soils used for cabbage growing in combination with inoculation of strain DSP3 showed enhanced microbial degradation of chlorpyrifos.     

Growth of Escherichia coli Coexpressing Phosphotriesterase and Glycerophosphodiester Phosphodiesterase, Using Paraoxon as the Sole Phosphorus Source

Phosphotriesterases catalyze the hydrolytic detoxification of phosphotriester pesticides and chemical warfare nerve agents with various efficiencies. The directed evolution of phosphotriesterases to enhance the breakdown of poor substrates is desirable for the purposes of bioremediation. A limiting factor in the identification of phosphotriesterase mutants with increased activity is the ability to effectively screen large mutant libraries. To this end, we have investigated the possibility of coupling phosphotriesterase activity to cell growth by using methyl paraoxon as the sole phosphorus source. The catabolism of paraoxon to phosphate would occur via the stepwise enzymatic hydrolysis of paraoxon to dimethyl phosphate, methyl phosphate, and then phosphate. The Escherichia coli strain DH10B expressing the phosphotriesterase from Agrobacterium radiobacter P230 (OpdA) is unable to grow when paraoxon is used as the sole phosphorus source. Enterobacter aerogenes is an organism capable of growing when dimethyl phosphate is the sole phosphorus source. The enzyme responsible for hydrolyzing dimethyl phosphate has been previously characterized as a nonspecific phosphohydrolase. We isolated and characterized the genes encoding the phosphohydrolase operon. The operon was identified from a shotgun clone that enabled E. coli to grow when dimethyl phosphate is the sole phosphorus source. E. coli coexpressing the phosphohydrolase and OpdA grew when paraoxon was the sole phosphorus source. By constructing a short degradative pathway, we have enabled E. coli to use phosphotriesters as a sole source of phosphorus.     

Effects of soil pH on the biodegradation of chlorpyrifos and isolation of a chlorpyrifos-degrading bacterium

We examined the role of microorganisms in the degradation of the organophosphate insecticide chlorpyrifos in soils from the United Kingdom and Australia. The kinetics of degradation in five United Kingdom soils varying in pH from 4.7 to 8.4 suggested that dissipation of chlorpyrifos was mediated by the cometabolic activities of the soil microorganisms. Repeated application of chlorpyrifos to these soils did not result in the development of a microbial population with an enhanced ability to degrade the pesticide. A robust bacterial population that utilized chlorpyrifos as a source of carbon was detected in an Australian soil. The enhanced ability to degrade chlorpyrifos in the Australian soil was successfully transferred to the five United Kingdom soils. Only soils with a pH of >/=6.7 were able to maintain this degrading ability 90 days after inoculation. Transfer and proliferation of degrading microorganisms from the Australian soil to the United Kingdom soils was monitored by molecular fingerprinting of bacterial 16S rRNA genes by PCR-denaturing gradient gel electrophoresis (DGGE). Two bands were found to be associated with enhanced degradation of chlorpyrifos. Band 1 had sequence similarity to enterics and their relatives, while band 2 had sequence similarity to strains of Pseudomonas. Liquid enrichment culture using the Australian soil as the source of the inoculum led to the isolation of a chlorpyrifos-degrading bacterium. This strain had a 16S rRNA gene with a sequence identical to that of band 1 in the DGGE profile of the Australian soil. DNA probing indicated that genes similar to known organophosphate-degrading (opd) genes were present in the United Kingdom soils. However, no DNA hybridization signal was detected for the Australian soil or the isolated degrader. This indicates that unrelated genes were present in both the Australian soil and the chlorpyrifos-degrading isolate. These results are consistent with our observations that degradation of chlorpyrifos in these systems was unusual, as it was growth linked and involved complete mineralization. As the 16S rRNA gene of the isolate matched a visible DGGE band from the Australian soil, the isolate is likely to be both prominent and involved in the degradation of chlorpyrifos in this soil.     

Metabolism of the Aliphatic Nitramine 4-Nitro-2,4-Diazabutanal by Methylobacterium sp. Strain JS178

The aliphatic nitramine 4-nitro-2,4-diazabutanal (NDAB; C₂H₅N₃O₃) is a ring cleavage metabolite that accumulates during the aerobic degradation of the energetic compound hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by various Rhodococcus spp. NDAB is also produced during the alkaline hydrolysis of either RDX or octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) and during the photolysis of RDX. Traces of NDAB were observed in a soil sampled from an ammunition-manufacturing facility contaminated with both HMX and RDX, suggesting natural attenuation. In this study, we report the isolation of a soil bacterium that is able to degrade NDAB under aerobic conditions. The isolate is a pink-pigmented facultative methylotroph affiliated with the genus Methylobacterium. The strain, named Methylobacterium sp. strain JS178, degrades NDAB as a sole nitrogen source, with concomitant growth and formation of 1 molar equivalent of nitrous oxide (N₂O). Comparison of the growth yield of strain JS178 grown on NDAB, nitrite (NO₂⁻), or ammonium (NH₄⁺) as a nitrogen source revealed that 1 N equivalent is assimilated from each mole of NDAB, which completes the nitrogen mass balance. In radiotracer experiments, strain JS178 mineralized 1 C of the [¹⁴C]NDAB produced in situ from [¹⁴C]RDX by Rhodococcus sp. strain DN22. Studies on the regulation of NDAB degradation indicated that allantoin, an intermediate in the purine catabolic pathway and a central molecule in the storage and transport of nitrogen in plants, up-regulated the enzyme(s) involved in the degradation of the nitramine. The results reveal the potential for the sequential participation of rhodococci and methylobacteria to effect the complete degradation of RDX.     

Chlorpyrifos-induced stress response in the chlorpyrifos-degrader Klebsiella sp. CPK

A chlorpyrifos-degrading bacterium, Klebsiella sp. CPK, which can biodegrade chlorpyrifos and transform it into 3,5,6-trichloro-2-pyridinol, was isolated by the enrichment culture technique. A classic stringent response triggered by chlorpyrifos stress was identified through the detection of (p)ppGpp accumulation in this strain. Sequence analysis of the (p)ppGpp synthetase RelA in Klebsiella sp. CPK showed that it only had (p)ppGpp synthetase activity. Compared to its parent, the △relA strain was more sensitive to several stress conditions, such as high salt, low pH values and a high concentration of chlorpyrifos. In addition, growth curves and semi-quantitative RT-PCR indicated that chlorpyrifos stress affected the growth and relA expression. Together, these results indicated that chlorpyrifos could mount a stringent response in the Klebsiella sp. CPK strain, and relA expression modulated the response of the Klebsiella sp. CPK strain to chlorpyrifos stress.     

Biodegradation of Methyl tert-Butyl Ether by a Bacterial Pure Culture

A bacterial strain, PM1, which is able to utilize methyl tert-butyl ether (MTBE) as its sole carbon and energy source, was isolated from a mixed microbial consortium in a compost biofilter capable of degrading MTBE. Initial linear rates of MTBE degradation by 2 × 10⁶ cells ml⁻¹ were 0.07, 1.17, and 3.56 μg ml⁻¹ h⁻¹ for initial concentrations of 5, 50, and 500 μg MTBE ml⁻¹, respectively. When incubated with 20 μg of uniformly labeled [¹⁴C]MTBE ml⁻¹, strain PM1 converted 46% to ¹⁴CO₂ and 19% to ¹⁴C-labeled cells within 120 h. This yield is consistent with the measurement of protein accumulation at different MTBE concentrations from which was estimated a biomass yield of 0.18 mg of cells mg MTBE⁻¹. Strain PM1 was inoculated into sediment core material collected from a contaminated groundwater plume at Port Hueneme, California, in which there was no evidence of MTBE degradation. Strain PM1 readily degraded 20 μg of MTBE ml⁻¹ added to the core material. The rate of MTBE removal increased with additional inputs of 20 μg of MTBE ml⁻¹. These results suggest that PM1 has potential for use in the remediation of MTBE-contaminated environments.     

Utilization of DNA as a Sole Source of Phosphorus, Carbon, and Energy by Shewanella spp.: Ecological and Physiological Implications for Dissimilatory Metal Reduction

The solubility of orthophosphate (PO₄³⁻) in iron-rich sediments can be exceedingly low, limiting the bioavailability of this essential nutrient to microbial populations that catalyze critical biogeochemical reactions. Here we demonstrate that dissolved extracellular DNA can serve as a sole source of phosphorus, as well as carbon and energy, for metal-reducing bacteria of the genus Shewanella. Shewanella oneidensis MR-1, Shewanella putrefaciens CN32, and Shewanella sp. strain W3-18-1 all grew with DNA but displayed different growth rates. W3-18-1 exhibited the highest growth rate with DNA. While strain W3-18-1 displayed Ca²⁺-independent DNA utilization, both CN32 and MR-1 required millimolar concentrations of Ca²⁺ for growth with DNA. For S. oneidensis MR-1, the utilization of DNA as a sole source of phosphorus is linked to the activities of extracellular phosphatase(s) and a Ca²⁺-dependent nuclease(s), which are regulated by phosphorus availability. Mass spectrometry analysis of the extracellular proteome of MR-1 identified one putative endonuclease (SO1844), a predicted UshA (bifunctional UDP-sugar hydrolase/5′ nucleotidase), a predicted PhoX (calcium-activated alkaline phosphatase), and a predicted CpdB (bifunctional 2′,3′ cyclic nucleotide 2′ phosphodiesterase/3′ nucleotidase), all of which could play important roles in the extracellular degradation of DNA under phosphorus-limiting conditions. Overall, the results of this study suggest that the ability to use exogenous DNA as the sole source of phosphorus is widespread among the shewanellae, and perhaps among all prokaryotes, and may be especially important for nutrient cycling in metal-reducing environments.     

Development of a Freeze-Dried Fungal Wettable Powder Preparation Able to Biodegrade Chlorpyrifos on Vegetables

Continuous use of the pesticide chlorpyrifos has resulted in harmful contaminations in environment and species. Based on a chlorpyrifos-degrading fungus Cladosporium cladosporioides strain Hu-01 (collection number: CCTCC M 20711), a fungal wettable powder preparation was developed aiming to efficiently remove chlorpyrifos residues from vegetables. The formula was determined to be 11.0% of carboxymethyl cellulose-Na, 9.0% of polyethylene glycol 6000, 5.0% of primary alcohol ethoxylate, 2.5% of glycine, 5.0% of fucose, 27.5% of kaolin and 40% of freeze dried fungi by response surface methodology (RSM). The results of quality inspection indicated that the fungal preparation could reach manufacturing standards. Finally, the degradation of chlorpyrifos by this fungal preparation was determined on pre-harvest cabbage. Compared to the controls without fungal preparation, the degradation of chlorpyrifos on cabbages, which was sprayed with the fungal preparation, was up to 91% after 7 d. These results suggested this freeze-dried fungal wettable powder may possess potential for biodegradation of chlorpyrifos residues on vegetables and provide a potential strategy for food and environment safety against pesticide residues.     

Mineralization of the Cyclic Nitramine Explosive Hexahydro-1,3,5-Trinitro-1,3,5-Triazine by Gordonia and Williamsia spp.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a cyclic nitroamine explosive that is a major component in many military high-explosive formulations. In this study, two aerobic bacteria that are capable of using RDX as the sole source of carbon and nitrogen to support their growth were isolated from surface soil. These bacterial strains were identified by their fatty acid profiles and 16S ribosomal gene sequences as Williamsia sp. KTR4 and Gordonia sp. KTR9. The physiology of each strain was characterized with respect to the rates of RDX degradation and [U-¹⁴C]RDX mineralization when RDX was supplied as a sole carbon and nitrogen source in the presence and absence of competing carbon and nitrogen sources. Strains KTR4 and KTR9 degraded 180 μM RDX within 72 h when RDX served as the only added carbon and nitrogen source while growing to total protein concentrations of 18.6 and 16.5 μg/ml, respectively. Mineralization of [U-¹⁴C]RDX to ¹⁴CO₂ was 30% by strain KTR4 and 27% by KTR9 when RDX was the only added source of carbon and nitrogen. The addition of (NH₄)₂SO₄ greatly inhibited KTR9's degradation of RDX but had little effect on that of KTR4. These are the first two pure bacterial cultures isolated that are able to use RDX as a sole carbon and nitrogen source. These two genera possess different physiologies with respect to RDX mineralization, and each can serve as a useful microbiological model for the study of RDX biodegradation with regard to physiology, biochemistry, and genetics.     

Characterization of a fungal strain capable of degrading chlorpyrifos and its use in detoxification of the insecticide on vegetables

A fungal strain capable of utilizing chlorpyrifos as sole carbon and energy sources was isolated from soil by enrichment cultivation approach. The half-lives of degradation (DT₅₀) for chlorpyrifos at concentrations of 1, 10, and 100 mg l⁻¹ by the fungal strain DSP in mineral salt medium were measured to be 2.03, 2.93, and 3.49 days, respectively. Two cell-free extracts [E (1:10) and E (1:20)] from the fungal strain DSP in bran–glucose medium were prepared and used to enhance chlorpyrifos degradation on vegetables. Compared with the controls, the DT₅₀ of chlorpyrifos were reduced by 70.3%, 65.6%, 80.6%, 80.6%, and 86.1%, and by 53.8%, 43.2%, 66.0%, 54.3%, and 67.7% on E (1:20) and E (1:10) treated pakchoi, water spinach, Malabar spinach, haricot beans, and pepper, respectively. The 7-day residual values (R ₇) of chlorpyrifos on E (1:10) treated vegetables were all lower than the corresponding maximum residue levels of European Union (EU MRLs), except that the R ₇ value on haricot beans was slightly higher than the corresponding EU MRLs. The results indicate that cell-free extracts could rapidly degrade chlorpyrifos residues on vegetables.     

Formation of Hydride-Meisenheimer Complexes of Picric Acid (2,4,6-Trinitrophenol) and 2,4-Dinitrophenol during Mineralization of Picric Acid by Nocardioides sp. Strain CB 22-2

There are only a few examples of microbial conversion of picric acid (2,4,6-trinitrophenol). None of the organisms that have been described previously is able to use this compound as a sole source of carbon, nitrogen, and energy at high rates. In this study we isolated and characterized a strain, strain CB 22-2, that was able to use picric acid as a sole source of carbon and energy at concentrations up to 40 mM and at rates of 1.6 mmol · h⁻¹ · g (dry weight) of cells⁻¹ in continuous cultures and 920 μmol · h⁻¹ · g (dry weight) of cells⁻¹ in flasks. In addition, this strain was able to use picric acid as a sole source of nitrogen at comparable rates in a nitrogen-free medium. Biochemical characterization and 16S ribosomal DNA analysis revealed that strain CB 22-2 is a Nocardioides sp. strain. High-pressure liquid chromatography and UV-visible light data, the low residual chemical oxygen demand, and the stoichiometric release of 2.9 ± 0.1 mol of nitrite per mol of picric acid provided strong evidence that complete mineralization of picric acid occurred. During transformation, the metabolites detected in the culture supernatant were the [H⁻]-Meisenheimer complexes of picric acid and 2,4-dinitrophenol (H⁻-DNP), as well as 2,4-dinitrophenol. Experiments performed with crude extracts revealed that H⁻-DNP formation indeed is a physiologically relevant step in picric acid metabolism.     

Physiological and Biochemical Characterization of a Novel Nicotine-Degrading Bacterium Pseudomonas geniculata N1

Management of solid wastes with high nicotine content, such as those accumulated during tobacco manufacturing, poses a major challenge, which can be addressed by using bacteria such as Pseudomonas and Arthrobacter. In this study, a new species of Pseudomonas geniculata, namely strain N1, which is capable of efficiently degrading nicotine, was isolated and identified. The optimal growth conditions for strain N1 are a temperature of 30°C, and a pH 6.5, at a rotation rate of 120 rpm min⁻¹ with 1 g l⁻¹ nicotine as the sole source of carbon and nitrogen. Myosmine, cotinine, 6-hydroxynicotine, 6-hydroxy-N-methylmyosmine, and 6-hydroxy-pseudooxynicotine were detected as the five intermediates through gas chromatography-mass and liquid chromatography-mass analyses. The identified metabolites were different from those generated by Pseudomonas putida strains. The analysis also highlighted the bacterial metabolic diversity in relation to nicotine degradation by different Pseudomonas strains.