Meiying Hu

Biodegradation of beta-cypermethrin and 3-phenoxybenzoic acid by a novel Ochrobactrum lupini DG-S-01

A newly isolated bacterium DG-S-01 from activated sludge utilized beta-cypermethrin (beta-CP) and its major metabolite 3-phenoxybenzoic acid (3-PBA) as sole carbon and energy source for growth in mineral salt medium (MSM). Based on the morphology, physio-biochemical characteristics, and 16S rDNA sequence analysis, DG-S-01 was identified as Ochrobactrum lupini. DG-S-01 effectively degraded beta-CP with total inocula biomass A(590 nm) = 0.1-0.8, at 20-40 °C, pH 5-9, initial beta-CP 50-400 mg L(-1) and metabolized to yield 3-PBA leading to complete degradation. Andrews equation was used to describe the special degradation rate at different initial concentrations. Degradation rate parameters q(max), K(s) and K(i) were determined to be 1.14 d(-1), 52.06 mg L(-1) and 142.80 mg L(-1), respectively. Maximum degradation was observed at 30 °C and pH 7.0. Degradation of beta-CP was accelerated when MSM was supplemented with glucose, beef extract and yeast extract. Studies on biodegradation in liquid medium showed that over 90% of the initial dose of beta-CP (50 mg L(-1)) was degraded under the optimal conditions within 5d. Moreover, the strain also degraded beta-cyfluthrin, fenpropathrin, cyhalothrin and deltamethrin. These results reveal that DG-S-01 may possess potential to be used in bioremediation of pyrethroid-contaminated 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.

Characterization of a novel cyfluthrin-degrading bacterial strain Brevibacterium aureum and its biochemical degradation pathway

Brevibacterium aureum DG-12, a new bacterial strain isolated from active sludge, was able to degrade and utilize cyfluthrin as a growth substrate in the mineral medium. Response surface methodology using central composite rotatable design of cultural conditions was successfully employed for optimization resulting in 88.6% degradation of cyfluthrin (50mgL(-1)) within 5days. The bacterium degraded cyfluthrin by cleavage of both the carboxylester linkage and diaryl bond to form 2,2,3,3-tetramethyl-cyclopropanemethanol, 4-fluoro-3-phenexy-benzoic acid, 3,5-dimethoxy phenol, and phenol, and subsequently transformed these compounds with a maximum specific degradation rate, half-saturation constant and inhibition constant of 1.0384day(-1), 20.4967mgL(-1), and 141.9013mgL(-1), respectively. A novel degradation pathway for cyfluthrin was proposed based on analysis of these metabolites. In addition, this strain was found capable of degrading a wide range of synthetic pyrethroid insecticides. Our results suggest that B. aureum DG-12 may be an ideal microorganism for bioremediation of the pyrethroid-contaminated environments.

Enhancement of cypermethrin degradation by a coculture of Bacillus cereus ZH-3 and Streptomyces aureus HP-S-01

Degradation of cypermethrin was significantly enhanced in a coculture of Bacillus cereus ZH-3 and Streptomyces aureus HP-S-01. In the pure culture, longer half-lives (t(1/2)=32.6-43.0h) of cypermethrin were observed, as compared to the mixed cocultures (t(1/2)=13.0h). The optimal degradation conditions were determined to be 28.2°C and pH 7.5 based on response surface methodology (RSM). Under these conditions, the mixed cultures completely metabolized cypermethrin (50mgL(-1)) within 72h. Analysis of degradation products of cypermethrin indicated that the microbial consortium converted cypermethrin to α-hydroxy-3-phenoxy-benzeneacetonitrile, 3-phenoxybenzaldehyde and 4-phenoxyphenyl-2,2-dimethyl-propiophenone, and subsequently transformed these compounds with a maximum specific degradation rate (q(max)), half-saturation constant (K(s)) and inhibition constant (K(i)) of 0.1051h(-1), 31.2289mgL(-1) and 220.5752mgL(-1), respectively. This is the first report of a proposed pathway of degradation of cypermethrin by hydrolysis of ester linkage and oxidization of 3-phenoxybenzyl in a coculture. Finally, this coculture is the first described mixed microbial consortium capable of metabolizing cypermethrin.

Isolation and characterization of a fungus able to degrade pyrethroids and 3-phenoxybenzaldehyde

Fungal strain HU, isolated from activated sludge and identified as a member of the genus Cladosporium based on morphology and sequencing of 28S rRNA, was shown to degrade 90% of fenvalerate, fenpropathrin, β-cypermethrin, deltamethrin, bifenthrin, and permethrin (100 mgL(-1)) within 5 days. Fenvalerate was utilized as sole carbon and energy source and co-metabolized in the presence of sucrose. Degradation of fenvalerate occurred at pH 5-10 at 18-38°C. The fungus first hydrolyzed the carboxylester linkage to produce α-hydroxy-3-phenoxy-benzeneacetonitrile and 3-phenoxybenzaldehyde, and subsequently degraded these two compounds with a q(max), K(s) and K(i) of 1.73 d(-1), 99.20 mgL(-1) and 449.75 mgL(-1), respectively. Degradation followed first-order kinetics. These results show that the fungal strain may possess potential to be used in bioremediation of pyrethroid-contaminated environments.

Purification and Characterization of a Novel Chlorpyrifos Hydrolase from Cladosporium cladosporioides Hu-01

Chlorpyrifos is of great environmental concern due to its widespread use in the past several decades and its potential toxic effects on human health. Thus, the degradation study of chlorpyrifos has become increasing important in recent years. A fungus capable of using chlorpyrifos as the sole carbon source was isolated from organophosphate-contaminated soil and characterized as Cladosporium cladosporioides Hu-01 (collection number: CCTCC M 20711). A novel chlorpyrifos hydrolase from cell extract was purified 35.6-fold to apparent homogeneity with 38.5% overall recovery by ammoniumsulfate precipitation, gel filtration chromatography and anion-exchange chromatography. It is a monomeric structure with a molecular mass of 38.3 kDa. The pI value was estimated to be 5.2. The optimal pH and temperature of the purified enzyme were 6.5 and 40°C, respectively. No cofactors were required for the chlorpyrifos-hydrolysis activity. The enzyme was strongly inhibited by Hg2+, Fe3+, DTT, β-mercaptoethanol and SDS, whereas slight inhibitory effects (5–10% inhibition) were observed in the presence of Mn2+, Zn2+, Cu2+, Mg2+, and EDTA. The purified enzyme hydrolyzed various organophosphorus insecticides with P-O and P-S bond. Chlorpyrifos was the preferred substrate. The Km and Vmax values of the enzyme for chlorpyrifos were 6.7974 μM and 2.6473 μmol·min−1, respectively. Both NH2-terminal sequencing and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometer (MALDI-TOF-MS) identified an amino acid sequence MEPDGELSALTQGANS, which shared no similarity with any reported organophosphate-hydrolyzing enzymes. These results suggested that the purified enzyme was a novel hydrolase and might conceivably be developed to fulfill the practical requirements to enable its use in situ for detoxification of chlorpyrifos. Finally, this is the first described chlorpyrifos hydrolase from fungus.

Silencing of Rieske iron–sulfur protein using chemically synthesised siRNA as a potential biopesticide against Plutella xylostella

BACKGROUND Extensive applications and frequent long-term use of pesticides can affect behavioural mechanisms and physiological and biochemical aspects of insects, leading to resistance. However, insect control strategies involving a different mode of action would be valuable for managing the emergence of insect resistance. In this context, the development of RNA interference technology has brought a turning point in the creation of new biopesticides. RESULTS Full-length cDNA of Rieske iron-sulfur protein (RISP) was cloned and characterised from Plutella xylostella L. Three siRNAs specific to RISP sequences were designed and chemically synthesised, and fed to P. xylostella larvae by coating cabbage leaves. This resulted in specific gene silencing of RISP, and consequently brought significant mortality of P. xylostella larvae compared with the control treatment. Silencing of RISP leads to significantly lower transcript levels of RISP compared with the control. In addition, the amount of ATP in the surviving larvae was lower than in the control. However, surviving larvae gradually recovered to normal transcript and protein levels. CONCLUSION This is the first demonstration of the potential use of chemically synthesised siRNA in the development of new biopesticides as a mitochondrial electron transport inhibitor.

The mitochondria-mediate apoptosis of Lepidopteran cells induced by azadirachtin.

Mitochondria have been shown to play an important role in apoptosis using mammalian cell lines. However, this seems not to be the case in Drosophila, an insect model organism; thus more in-depth studies of insect cell apoptosis are necessary. In the present study, mitochondrial involvement during azadirachtin- and camptothecin-induced apoptosis in Spodoptera frugiperda Sf9 cells (isolated from Spodoptera frugiperda pupal ovarian tissue) was investigated. The results showed that both azadirachtin and camptothecin could induce apoptosis in Sf9 cells. Reactive oxygen species (ROS) generation, activation of mitochondrial permeability transition pores (MPTPs) and loss of mitochondrial membrane potential (MMP) were observed very early during apoptosis and were followed subsequently by the release of cytochrome-c from the mitochondria. Furthermore, the results also revealed that the opening of MPTPs and the loss of MMP induced by azadirachtin could be significantly inhibited by the permeability transition pore (PTP) inhibitor cyclosporin A (CsA), which was used to identify the key role of mitochondria in the apoptosis of Sf9 cells. However, in camptothecin-treated Sf9 cells, CsA could not suppress the opening of MPTPs and the loss of MMP when apoptosis was induced. The data from caspase-3 and caspase-9 activity assays and detection of apoptosis by morphological observation and flow cytometry also uncovered the different effect of CsA on the two botanical apoptosis inducers. Although different mechanisms of apoptosis induction exist, our study revealed that mitochondria play a crucial role in insect cell line apoptosis.

Microbial detoxification of bifenthrin by a novel yeast and its potential for contaminated soils treatment.

Bifenthrin is one the most widespread pollutants and has caused potential effect on aquatic life and human health, yet little is known about microbial degradation in contaminated regions. A novel yeast strain ZS-02, isolated from activated sludge and identified as Candida pelliculosa based on morphology, API test and 18S rDNA gene analysis, was found highly effective in degrading bifenthrin over a wide range of temperatures (20–40°C) and pH (5–9). On the basis of response surface methodology (RSM), the optimal degradation conditions were determined to be 32.3°C and pH 7.2. Under these conditions, the yeast completely metabolized bifenthrin (50 mg·L−1) within 8 days. This strain utilized bifenthrin as the sole carbon source for growth as well as co-metabolized it in the presence of glucose, and tolerated concentrations as high as 600 mg·L−1 with a q max, K s and K i of 1.7015 day−1, 86.2259 mg·L−1 and 187.2340 mg·L−1, respectively. The yeast first degraded bifenthrin by hydrolysis of the carboxylester linkage to produce cyclopropanecarboxylic acid and 2-methyl-3-biphenylyl methanol. Subsequently, 2-methyl-3-biphenylyl methanol was further transformed by biphenyl cleavage to form 4-trifluoromethoxy phenol, 2-chloro-6-fluoro benzylalcohol, and 3,5-dimethoxy phenol, resulting in its detoxification. Eventually, no persistent accumulative product was detected by gas chromatopraphy-mass spectrometry (GC-MS) analysis. This is the first report of a novel pathway of degradation of bifenthrin by hydrolysis of ester linkage and cleavage of biphenyl in a microorganism. Furthermore, strain ZS-02 degraded a variety of pyrethroids including bifenthrin, cyfluthrin, deltamethrin, fenvalerate, cypermethrin, and fenpropathrin. In different contaminated soils introduced with strain ZS-02, 65–75% of the 50 mg·kg−1 bifenthrin was eliminated within 10 days, suggesting the yeast could be a promising candidate for remediation of environments affected by bifenthrin. Finally, this is the first described yeast capable of degrading bifenthrin.

Fenpropathrin Biodegradation Pathway in Bacillus sp. DG-02 and Its Potential for Bioremediation of Pyrethroid-Contaminated Soils

The widely used insecticide fenpropathrin in agriculture has become a public concern because of its heavy environmental contamination and toxic effects on mammals, yet little is known about the kinetic and metabolic behaviors of this pesticide. This study reports the degradation kinetics and metabolic pathway of fenpropathrin in Bacillus sp. DG-02, previously isolated from the pyrethroid-manufacturing wastewater treatment system. Up to 93.3% of 50 mg L(-1) fenpropathrin was degraded by Bacillus sp. DG-02 within 72 h, and the degradation rate parameters qmax, Ks, and Ki were determined to be 0.05 h(-1), 9.0 mg L(-1), and 694.8 mg L(-1), respectively. Analysis of the degradation products by gas chromatography-mass spectrometry led to identification of seven metabolites of fenpropathrin, which suggest that fenpropathrin could be degraded first by cleavage of its carboxylester linkage and diaryl bond, followed by degradation of the aromatic ring and subsequent metabolism. In addition to degradation of fenpropathrin, this strain was also found to be capable of degrading a wide range of synthetic pyrethroids including deltamethrin, λ-cyhalothrin, β-cypermethrin, β-cyfluthrin, bifenthrin, and permethrin, which are also widely used insecticides with environmental contamination problems with the degradation process following the first-order kinetic model. Bioaugmentation of fenpropathrin-contaminated soils with strain DG-02 significantly enhanced the disappearance rate of fenpropathrin, and its half-life was sharply reduced in the soils. Taken together, these results depict the biodegradation mechanisms of fenpropathrin and also highlight the promising potentials of Bacillus sp. DG-02 in bioremediation of pyrethroid-contaminated soils.