Shu Cai

Biodegradation of chloroacetamide herbicides by Paracoccus sp. FLY-8 in vitro.

A butachlor-degrading strain, designated FLY-8, was isolated from rice field soil and was identified as Paracoccus sp. Strain FLY-8 could degrade and utilize six chloroacetamide herbicides as carbon sources for growth, and the degradation rates followed the order alachlor > acetochlor > propisochlor > butachlor > pretilachlor > metolachlor. The influence of molecular structure of the chloroacetamide herbicides on the microbial degradation rate was first analyzed; the results indicated that the substitutions of alkoxymethyl side chain with alkoxyethyl side chain greatly reduced the degradation efficiencies; the length of amide nitrogens alkoxymethyl significantly affected the biodegradability of these herbicides: the longer the alkyl was, the slower the degradation efficiencies occurred. The phenyl alkyl substituents have no obvious influence on the degradation efficiency. The pathway of butachlor complete mineralization was elucidated on the basis of the results of metabolite identification and enzyme assays. Butachlor was degraded to alachlor by partial C-dealkylation and then converted to 2-chloro-N-(2,6-dimethylphenyl)acetamide by N-dealkylation, which subsequently transformed to 2,6-diethylaniline, which was further degraded via the metabolites aniline and catechol, and catechol was oxidized through an ortho-cleavage pathway. This study highlights an important potential use of strain FLY-8 for the in situ bioremediation of chloroacetamide herbicides and their metabolite-contaminated environment.

The biodegradation pathway of triethylamine and its biodegradation by immobilized Arthrobacter protophormiae cells

A bacterial strain named R4 was isolated from a wastewater treatment pool containing triethylamine (TEA) as the sole source of carbon and nitrogen. Strain R4 was identified as Arthrobacter protophormiae based on 16S rRNA gene sequence analysis and morphological and physiological properties. The optimal pH, temperature and concentration of NaCl for TEA degradation by strain R4 were 7.0, 30°C and 0.5%, respectively. Strain R4 could completely degrade 100 mg l(-1) TEA to ammonia in 32 h, and could also effectively degrade diethylamine (DEA) and ethylamine (EA) to ammonia. The degradation of TEA was strongly inhibited by some metal ions (Cu(2+), Mn(2+), Zn(2+), Co(2+), Ni(2+) and Ag(+)) (1.0mM). Addition of either SO(4)(2-) or NH(4)(+) reduced the degradation efficiency of TEA by strain R4 to a certain extent. The inhibition became significant when the concentration of SO(4)(2-) and NH(4)(+) reached to 11 mM and 30 mM, respectively. Cell-free extracts prepared from cells grown in TEA exhibited TEA monooxygenase, DEA monooxygenase and EA monooxygenase activity. Here, we propose the metabolic pathway of TEA degradation in strain R4. The efficiency of TEA removal by immobilized cells of strain R4 was found to be equivalent to that of free cells. In addition, the immobilized cells could be reused without reduction in their ability to degrade TEA.

Degradation of cyhalofop-butyl (CyB) by Pseudomonas azotoformans strain QDZ-1 and cloning of a novel gene encoding CyB-hydrolyzing esterase.

Cyhalofop-butyl (CyB) is a widely used aryloxyphenoxy propanoate (AOPP) herbicide for control of grasses in rice fields. Five CyB-degrading strains were isolated from rice field soil and identified as Agromyces sp., Stenotrophomonas sp., Aquamicrobium sp., Microbacterium sp., and Pseudomonas azotoformans; the results revealed high biodiversity of CyB-degrading bacteria in rice soil. One strain, P. azotoformans QDZ-1, degraded 84.5% of 100 mg L(-1) CyB in 5 days of incubation in a flask and utilized CyB as carbon source for growth. Strain QDZ-1 could also degrade a wide range of other AOPP herbicides. An esterase gene, chbH, which hydrolyzes CyB to cyhalofop acid (CyA), was cloned from strain QDZ-1 and functionally expressed. A chbH-disrupted mutant dchbH was constructed by insertion mutation. Mutant dchbH could not degrade and utilize CyB, suggesting that chbH was the only esterase gene responsible for CyB degradation in strain QDZ-1. ChbH hydrolyzed all AOPP herbicides tested as well as permethrin. The catalytic efficiency of ChbH toward different AOPP herbicides followed the order quizalofop-P-ethyl ≈ fenoxaprop-P-ethyl > CyB ≈ fluazifop-P-butyl > diclofop-methyl ≈ haloxyfop-P-methyl; the results indicated that the chain length of the alcohol moiety strongly affected the biodegradability of the AOPP herbicides, whereas the substitutions in the aromatic ring had only a slight influence.

Degradation of acetochlor by consortium of two bacterial strains and cloning of a novel amidase gene involved in acetochlor-degrading pathway.

Two bacterial strains Sphingobium quisquiliarum DC-2 and Sphingobium baderi DE-13 were isolated from activated sludge. Acetochlor was transformed by S. quisquiliarum DC-2 to a transitory intermediate 2-chloro-N-(2-methyl-6-ethylphenyl)acetamide (CMEPA), which was further transformed to 2-methyl-6-ethylaniline (MEA), and MEA could not be degraded by strain DC-2. S. baderi DE-13, incapable of degrading acetochlor, showed capability of degrading MEA to an intermediate 2-methyl-6-ethylaminophenol (MEAOH). MEAOH was further transformed to 2-methyl-6-ethylbenzoquinoneimine (MEBQI), which was mineralized by strain DE-13. A gene, cmeH, encoding an amidase that catalyzed the amide bond cleavage of CMEPA was cloned from strain DC-2. CmeH was expressed in Escherichia coli BL21 and homogenously purified using Ni-nitrilotriacetic acid affinity. CmeH efficiently hydrolyzed CMEPA and other important herbicide, such as propanil, fenoxaprop-p-ethyl and clodinafop-propargyl.

Cloning of a Novel Arylamidase Gene from Paracoccus sp. Strain FLN-7 That Hydrolyzes Amide Pesticides

ABSTRACT The bacterial isolate Paracoccus sp. strain FLN-7 hydrolyzes amide pesticides such as diflubenzuron, propanil, chlorpropham, and dimethoate through amide bond cleavage. A gene, ampA, encoding a novel arylamidase that catalyzes the amide bond cleavage in the amide pesticides was cloned from the strain. ampA contains a 1,395-bp open reading frame that encodes a 465-amino-acid protein. AmpA was expressed in Escherichia coli BL21 and homogenously purified using Ni-nitrilotriacetic acid affinity chromatography. AmpA is a homodimer with an isoelectric point of 5.4. AmpA displays maximum enzymatic activity at 40°C and a pH of between 7.5 and 8.0, and it is very stable at pHs ranging from 5.5 to 10.0 and at temperatures up to 50°C. AmpA efficiently hydrolyzes a variety of secondary amine compounds such as propanil, 4-acetaminophenol, propham, chlorpropham, dimethoate, and omethoate. The most suitable substrate is propanil, with Km and k cat values of 29.5 μM and 49.2 s−1, respectively. The benzoylurea insecticides (diflubenzuron and hexaflumuron) are also hydrolyzed but at low efficiencies. No cofactor is needed for the hydrolysis activity. AmpA shares low identities with reported arylamidases (less than 23%), forms a distinct lineage from closely related arylamidases in the phylogenetic tree, and has different biochemical characteristics and catalytic kinetics with related arylamidases. The results in the present study suggest that AmpA is a good candidate for the study of the mechanism for amide pesticide hydrolysis, genetic engineering of amide herbicide-resistant crops, and bioremediation of amide pesticide-contaminated environments.

Degradation of piperazine by Paracoccus sp. TOH isolated from activated sludge

Piperazine is widely used as an intermediate in the manufacture of insecticides, rubber chemicals, corrosion inhibitors, and urethane. In this study, a highly effective piperazine-degrading bacteria strain, TOH, was isolated from the acclimated activated sludge of a pharmaceutical plant. This strain, identified as Paracoccus sp., utilises piperazine as the sole source of carbon, nitrogen and energy for growth. The optimal pH and temperature for the growth of TOH were 8.0 and 30°C, respectively. The effects of co-substrates and heavy metals on the degradation efficiency of piperazine were investigated. The results indicated that exogenously supplied glucose promoted the degradation of piperazine, while the addition of ammonium chloride slightly inhibited piperazine degradation. Metal ions such as Ni(2+) and Cd(2+) inhibited the degradation of piperazine, whereas Mg(2+) increased it. In addition, metabolic intermediates were identified by mass spectrometry, allowing a degradation pathway for piperazine to be proposed for the first time.

Catellibacterium nanjingense sp. nov., a propanil-degrading bacterium isolated from activated sludge, and emended description of the genus Catellibacterium

A novel facultatively anaerobic, non-spore-forming, non-motile, catalase- and oxidase-positive, Gram-negative and rod-shaped bacterial strain, designated Y12(T), was isolated from activated sludge of a wastewater bio-treatment facility. The strain was able to degrade about 90% of added propanil (100 mg l(-1)) within 3 days of incubation. Growth occurred in the presence of 0-4.5% (w/v) NaCl (optimum 0.5%), at 10-40 °C (optimum 28 °C) and at pH 5.5-10.0 (optimum pH 7.0). Vesicular internal membrane structures and photoheterotrophic growth were not observed. The major respiratory quinone was ubiquinone-10 and the major cellular fatty acid was summed feature 8 (C(18:1)ω6c and/or C(18:1)ω7c). The genomic DNA G+C content of strain Y12(T) was 63.7 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparison revealed that strain Y12(T) was a member of the genus Catellibacterium, as it showed highest sequence similarities to Catellibacterium caeni DCA-1(T) (99.1%) and <96.0% similarities with other species of the genus Catellibacterium. Strain Y12(T) showed low DNA-DNA relatedness values with C. caeni DCA-1(T). Based on phenotypic, genotypic and phylogenetic properties, strain Y12(T) represents a novel species of the genus Catellibacterium, for which the name Catellibacterium nanjingense sp. nov. is proposed. The type strain is Y12(T) (=CCTCC AB 2010218(T) =KCTC 23298(T)). An emended description of the genus Catellibacterium is also presented.

Dokdonella kunshanensis sp. nov., isolated from activated sludge, and emended description of the genus Dokdonella

A gram-negative, aerobic, non-motile, non-spore-forming rod, designated DC-3(T), was isolated from activated sludge of a wastewater treatment plant in China. Comparative 16S rRNA gene sequence analysis showed that strain DC-3(T) belonged to the family Xanthomonadaceae and formed a lineage within the genus Dokdonella. Strain DC-3(T) shared the highest 16S rRNA gene sequence similarity with Dokdonella soli KIS28-6(T) (97.1 %) and Dokdonella fugitiva A3(T) (97.1 %). The G+C content of the genomic DNA was 71.5 mol%. The major respiratory quinone was Q-8 and the major fatty acids were iso-C17 : 1ω9c (31.6 %), iso-C15 : 0 (12.6 %), iso-C16 : 0 (21.3 %), iso-C17 : 0 (13.1 %) and iso-C11 : 0 3-OH (6.5 %), which supported the affiliation of strain DC-3(T) with the genus Dokdonella. DNA-DNA relatedness between strain DC-3(T) and its closest phylogenetic neighbours was <30 %. The results of physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain DC-3(T) from the recognized species of the genus Dokdonella. On the basis of its phenotypic properties and phylogenetic distinctiveness, strain DC-3(T) represents a novel species of the genus Dokdonella, for which the name Dokdonella kunshanensis sp. nov. is proposed. The type strain is DC-3(T) ( = CCTCC AB 2011179(T)  = KACC 16511(T)). The description of the genus Dokdonella is also emended.

Enhanced Biological Phosphorus Removal with P seudomonas putida GM6 from Activated Sludge

Abstract The enhanced biological phosphorus removal (EBPR) method is widely adopted for phosphorus removal from waste-water, yet little is known about its microbiological and molecular mechanisms. Therefore, it is difficult to predict and control the deterioration of the EBPR process in a large-scale municipal sewage treatment plant. This study used a novel strain isolated in the laboratory, Pseudomonas putida GM6, which had a high phosphate accumulating ability and could recover rapidly from the deteriorated system and enhance the capability of phosphorus removal in activated sludge. Strain GM6 marked with gfp gene, which was called GMTR, was delivered into a bench-scale sequencing batch reactor (SBR) of low efficiency, to investigate the colonization of GMTR and removal of phosphorus. After 21 days, the proportion of GMTR in the total bacteria of the sludge reached 9.2%, whereas the phosphorus removal rate was 96%, with an effluent concentration of about 0.2 mg L −1 . In the reactor with the addition of GMTR, phosphorus was removed quickly, in 1 h under anaerobic conditions, and in 2 h under aerobic conditions. These evidences were characteristic of EBPR processes. Field testing was conducted at a hospital sewage treatment facility with low phosphorus removal capability. Twenty- one days after Pseudomonas putida GM6 was added, effluent phosphorus concentration remained around 0.3 mg L −1 , corresponding to a removal rate of 96.8%. It was therefore demonstrated that Pseudomonas putida GM6couldbeused for a quick startup and enhancement of wastewater biological phosphorus removal, which provided a scientific basis for potential large-scale engineering application.

Expression, Characterization, and Site-Directed Mutation of a Multiple Herbicide-Resistant Acetohydroxyacid Synthase (rAHAS) from Pseudomonas sp. Lm10

A multiple herbicide-resistant acetohydroxyacid synthase (rAHAS) gene was cloned from Pseudomonas sp. Lm10. Sequence analysis showed that the rAHAS regulatory subunit was identical to that of Pseudomonasputida KT2440 (sensitive AHAS, sAHAS), whereas six different sites [H134→N (rAHAS→sAHAS), A135→P, S136→T, I210→V, F264→Y, and S486→W] were found in the catalytic subunit. The rAHAS and sAHAS were over expressed, purified and characterized. rAHAS showed higher resistance to four kinds of AHAS-inhibitor herbicides than sAHAS. The resistance factor of rAHAS was 56.0-fold, 12.6-fold, 6.5-fold, and 9.2-fold as compared with sAHAS when metsulfuron-methyl, imazethapyr, flumetsulam, and pyriminobac-methyl used as inhibitor, respectively. The specific activity of rAHAS was lower than that of sAHAS and the Km value of rAHAS for pyruvate was approximately onefold higher than the corresponding value for sAHAS. Data from site-directed mutagenesis demonstrated that alteration at A135, F264, and S486 resulted in resistance reduction, while the mutation at H134, S136, and I210 has little effect on the resistance. A135 was mainly responsible for resistance to imidazolinone; F264 conferred resistance to sulfonylurea and triazolopyrimidine sulfonamide; and S486 showed multiple herbicides resistance to the four herbicides.