Characterization of a novel amidohydrolase with promiscuous esterase activity from a soil metagenomic library and its application in degradation of amide herbicides.

Amide herbicides have been extensively used worldwide and have received substantial attention due to their adverse environmental effects. Here, a novel amidohydrolase gene was identified from a soil metagenomic library using diethyl terephthalate (DET) as a screening substrate. The recombinant enzyme, AmiH52, was heterologously expressed in Escherichia coli and later purified and characterized, with the highest activity occurring at 40 ℃ and pH 8.0. AmiH52 was demonstrated to have both esterase and amidohydrolase activities, which exhibited highly specific activity for p-nitrophenyl butyrate (2669 U/mg) and degrading activity against several amide herbicides. In particular, it displayed the strongest activity against propanil, with a high degradation rate of 84% at 8 h. A GC-MS analysis revealed that propanil was transformed into 3,4-dichloroaniline (3,4-DCA) during this degradation. The molecular interactions and binding stability were then analyzed by molecular docking and molecular dynamics simulation, which revealed that several key amino acid residues, including Tyr164, Trp66, Ala59, Val283, Arg58, His33, His191, and His226, are involved in the specific interactions with propanil. This study provides a function-driven screening method for amide herbicide hydrolase from the metagenomic libraries and a promising propanil-degrading enzyme (AmiH52) for potential applications in environmental remediation.

Degradation of butachlor and propanil by Pseudomonas sp. strain But2 and Acinetobacter baumannii strain DT.

Herbicides have been extensively used globally, resulting in severe environmental pollution. Novel butachlor-degrading Pseudomonas sp. strain But2 isolated from soil can degrade butachlor regardless of the concentration and grows without a lag phase. Specific degradation was increased at 0.01-0.1 mM, and did not change significantly at higher concentrations. During degradation, 2-chloro-N-(2,6-diethylphenyl) acetamide, 2,6-diethylaniline, and 1,3-diethylbenzene were formed, which indicated that deamination occurred. Moreover, Pseudomonas sp. strains could tolerate propanil at up to 0.8 mM. The mixed bacterial culture of Pseudomonas sp. But2 and Acinetobacter baumannii DT (a propanil-degrading bacterial strain) showed highly effective biodegradation of both butachlor and propanil in liquid media and soil. For example, under treatment with the mixed culture, the half-lives of propanil and butachlor were 1 and 5 days, respectively, whereas those for the control were 3 and 15 days. The adjuvants present in herbicides reduced degradation in liquid media, but did not influence herbicide removal from the soil. The results showed that the mixed bacteria culture is a good candidate for the removal of butachlor and propanil from contaminated soils.

Biodegradation of propanil by Acinetobacter baumannii DT in a biofilm-batch reactor and effects of butachlor on the degradation process.

The herbicide, propanil, has been extensively applied in weed control, which causes serious environmental pollution. Acinetobacter baumannii DT isolated from soil has been used to determine the degradation rates of propanil and 3,4-dichloroaniline by freely suspended and biofilm cells. The results showed that the bacterial isolate could utilize both compounds as sole carbon and nitrogen sources. Edwards's model could be fitted well to the degradation kinetics of propanil, with the maximum degradation of 0.027 ± 0.003 mM h-1. The investigation of the degradation pathway showed that A. baumannii DT transformed propanil to 3,4-dichloroaniline before being completely degraded via the ortho-cleavage pathway. In addition, A. baumannii DT showed high tolerance to butachlor, a herbicide usually mixed with propanil to enhance weed control. The presence of propanil and butachlor in the liquid media increased the cell surface hydrophobicity and biofilm formation. Moreover, the biofilm reactor showed increased degradation rates of propanil and butachlor and high tolerance of bacteria to these chemicals. The obtained results showed that A. baumannii DT has a high potential in the degradation of propanil.

Mineralization of the herbicide swep by a two-strain consortium and characterization of a new amidase for hydrolyzing swep.

BACKGROUND: Swep is an excellent carbamate herbicide that kills weeds by interfering with metabolic processes and inhibiting cell division at the growth point. Due to the large amount of use, swep residues in soil and water not only cause environmental pollution but also accumulate through the food chain, ultimately pose a threat to human health. This herbicide is degraded in soil mainly by microbial activity, but no studies on the biotransformation of swep have been reported. RESULTS: In this study, a consortium consisting of two bacterial strains, Comamonas sp. SWP-3 and Alicycliphilus sp. PH-34, was enriched from a contaminated soil sample and shown to be capable of mineralizing swep. Swep was first transformed by Comamonas sp. SWP-3 to the intermediate 3,4-dichloroaniline (3,4-DCA), after which 3,4-DCA was mineralized by Alicycliphilus sp. PH-34. An amidase gene, designated as ppa, responsible for the transformation of swep into 3,4-DCA was cloned from strain SWP-3. The expressed Ppa protein efficiently hydrolyzed swep and a number of other structural analogues, such as propanil, chlorpropham and propham. Ppa shared less than 50% identity with previously reported arylamidases and displayed maximal activity at 30 °C and pH 8.6. Gly449 and Val266 were confirmed by sequential error prone PCR to be the key catalytic sites for Ppa in the conversion of swep. CONCLUSIONS: These results provide additional microbial resources for the potential remediation of swep-contaminated sites and add new insights into the catalytic mechanism of amidase in the hydrolysis of swep.

Spirosoma sordidisoli sp. nov., a propanil-degrading bacterium isolated from a herbicide-contaminated soil.

A novel Gram-stain negative, aerobic, rod-shaped, asporogenous, propanil-degrading bacterial strain, TY50T, was isolated from a herbicide-contaminated soil in Nanjing, China. Strain TY50T was found to grow optimally at pH 9.0, 30 °C and in the absence of NaCl. The G + C content of the total DNA was determined to be 55.5 mol%. The 16S rRNA gene sequence of strain TY50T shows high identity to that of Spirosoma lacussanchae CPCC 100624T (99.3%), Spirosoma metallicum PR1014kT (94.8%) and Spirosoma soli MIMBbqt12T (94.6%). DNA-DNA hybridization indicated that the isolate had relatively low levels of DNA-DNA relatedness with S. lacussanchae CPCC 100624T (48.3%). Average nucleotide identity and the digital DNA-DNA hybridizations for draft genomes between strain TY50T and S. lacussanchae CPCC 100624T were 93.2% and 51.0%, respectively. The major cellular fatty acids of strain TY50T were identified as C16:1ω5c (24.5%) and summed feature 3 (C16:1ω6c/C16:1ω7c, 40.7%). MK-7 was found to be the predominant respiratory quinone. The major polar lipid profile includes phosphatidylethanolamine, an unidentified lipid and an unidentified aminolipid. These chemotaxonomic data support the affiliation of strain TY50T with the members of the genus Spirosoma. Strain TY50T can be distinguished from its close phylogenetic neighbours based on its phenotypic, genotypic, and chemotaxonomic features. Therefore, strain TY50T represents a novel member of the genus Spirosoma, for which the name Spirosoma sordidisoli sp. nov. is proposed. The type strain is TY50T (= KCTC 62494T = CCTCC AB 2018041T).

Characterization of an arylamidase from a newly isolated propanil-transforming strain of Ochrobactrum sp. PP-2.

Propanil, one of the most extensively used post-emergent contact herbicides, has also been reported to have adverse effect on environmental safety. A bacterial strain of Ochrobactrum sp. PP-2, which was capable of transforming propanil, was isolated from a propanil-contaminated soil collected from a chemical factory. An arylamidase gene mah responsible for transforming propanil to 3,4-dichloroaniline (3,4-DCA) was cloned from strain PP-2 by shotgun method and subsequently confirmed by function expression. The arylamidase Mah shares low amino acid sequence identity (27-50%) with other biochemically characterized amidases and shows less than 30% identities to other reported propanil hydrolytic enzymes. Mah was most active at pH 8 and 35 °C. Mah had a remarkable activity toward propanil (Km = 6.3 ±â€¯1.2 µM), showing the highest affinity efficiency for propanil as compared with other reported propanil hydrolytic enzymes. Our study also provides a new arylamidase for the hydrolysis of propanil.

Rhizobium album sp. nov., isolated from a propanil-contaminated soil.

A novel Gram-stain negative, facultatively anaerobic, non-spore-forming, motile and rod-shaped bacterium (NS-104T) was isolated from a propanil-contaminated soil in Nanjing, China. Growth occurred at pH 5.0-9.0 (optimum 6.0), 16-37 °C (optimum 30 °C) and in the presence of 0-2.0% (w/v) NaCl (optimum, without NaCl). Strain NS-104T showed high 16S rRNA gene sequence identity to Rhizobium azooxidifex DSM 100211T (96.7%). The phylogenetic analysis of the 16S rRNA gene as well as the housekeeping genes recA, atpD and glnA demonstrated that strain NS-104T belongs to the genus Rhizobium. Strain NS-104T did not form nodules on six different legumes, and the nodD, nodC and nifH genes were neither amplified by PCR nor found in the draft genome of strain NS-104T. The sole respiratory quinone was ubiquinone Q-10. The polar lipid profile included the major amounts phosphatidylmonomethylethanolamine, phosphatidylglycerol and moderate amounts of phosphatidylethanolamine, phosphatidylcholine, diphosphatidylglycerol and unidentified aminolipids. The major cellular fatty acids were C18:1ω7c (39.6%), C19:0 cyclo ω8c (29.8%) and C16:0 (11.5%). The G + C content of strain NS-104T was 61.9 mol%. Strain NS-104T therefore represents a new species, for which the name Rhizobium album sp. nov. is proposed, with the type strain NS-104T (= KCTC 62327T = CCTCC AB 2017250T).

Metabolic Hydrolysis of Aromatic Amides in Selected Rat, Minipig, and Human In Vitro Systems.

The release of aromatic amines from drugs and other xenobiotics resulting from the hydrolysis of metabolically labile amide bonds presents a safety risk through several mechanisms, including geno-, hepato- and nephrotoxicity. Whilst multiple in vitro systems used for studying metabolic stability display serine hydrolase activity, responsible for the hydrolysis of amide bonds, they vary in their efficiency and selectivity. Using a range of amide-containing probe compounds (0.5-10 µM), we have investigated the hydrolytic activity of several rat, minipig and human-derived in vitro systems - including Supersomes, microsomes, S9 fractions and hepatocytes - with respect to their previously observed human in vivo metabolism. In our hands, human carboxylesterase Supersomes and rat S9 fractions systems showed relatively poor prediction of human in vivo metabolism. Rat S9 fractions, which are commonly utilised in the Ames test to assess mutagenicity, may be limited in the detection of genotoxic metabolites from aromatic amides due to their poor concordance with human in vivo amide hydrolysis. In this study, human liver microsomes and minipig subcellular fractions provided more representative models of human in vivo hydrolytic metabolism of the aromatic amide compounds tested.

Oryzisolibacter propanilivorax gen. nov., sp. nov., a propanil-degrading bacterium.

Strain EPL6T, a Gram-negative, motile, short rod was isolated from a propanil and 3,4-dichloroaniline enrichment culture produced from rice paddy soil. Based on the analyses of the 16S rRNA gene sequence, strain EPL6T was observed to be a member of the family Comamonadaceae, sharing the highest pairwise identity with type strains of the species Alicycliphilus denitrificans K601T (96.8 %) and Melaminivora alkalimesophila CY1T (96.8 %). Strain EPL6T was able to grow in a temperature range of 15-37 °C, pH 6-9 and in the presence of up to 4 % (w/v) NaCl and tested positive for catalase and oxidase reactions. The major respiratory quinone was Q8. The genomic DNA had a G+C content of 69.4±0.9 mol%. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol, and the major fatty acid methyl esters comprised C16 : 0, C18 : 1ω7c and summed feature 3 (C16 : 1ω7c/iso-C15 : 0 2-OH). Comparison of the genome sequence of strain EPL6T and of its closest neighbours, Melaminivora alkalimesophila CY1T and Alicycliphilus denitrificans K601T, yielded values of ANI ≤84.1 % and of AAI ≤80.3 %. Therefore, the genetic, phylogenetic, phenotypic and chemotaxonomic characteristics support the classification of this organism into a new taxon. Considering the genetic divergence of strain EPL6T from the type strains of the closest species, which belong to distinct genera, we propose a new genus within the family Comamonadaceae, named Oryzisolibacter propanilivorax gen. nov., sp. nov., represented by the isolate EPL6T as the type strain of the species (=LMG 28427T=CECT 8927T).

Biochemical characterization of a key step involved in 2H4MB production in Decalepis hamiltonii.

Decalepis hamiltonii is widely known for its flavour molecule 2-Hydroxy-4-Methoxy Benzaldehyde (2H4MB), a structural isomer of vanillin. As the biosynthetic pathway of 2H4MB is not known, we hypothesised 2H4MB origins could be from phenylpropanoid pathway (PPP). Accordingly, a study was conducted using PPP inhibitors (viz. piperonylic acid, MDCA and propanil) against in vitro root cultures of D. hamiltonii to find the branch of PPP which catalyses the 2H4MB formation. HPLC analysis was carried out to quantify 2H4MB levels in control and respective inhibitor treated root cultures in vitro. The results obtained revealed that piperonylic acid did not inhibit 2H4MB biosynthesis in the given period, whereas MDCA and propanil had the marked inhibitory effect. The inhibitory effect was evident with 13.2, 33.6 and 37.9% decrease in 2H4MB levels at 50, 100 and 150mM concentration of MDCA respectively in comparison with control roots. Similarly, the inhibitory effect of propanil on 2H4MB biosynthesis was obvious with 23.7, 49.5 and 57.9% decrease in 2H4MB levels at 50, 100 and 150µM concentration of inhibitor respectively when compared with control roots. Propanil showed a greater slow down effect on 2H4MB biosynthesis compared to MDCA. Incorporation of 0.1, 0.5 and 1.0mM ferulic acid as a precursor to in vitro root cultures of D. hamiltonii showed an increase in 2H4MB levels at the rate of 3.1, 107 and 94.1% respectively as quantified by HPLC analysis. However, ferulic acid in conjunction with propanil did not show any increase in 2H4MB levels. This clearly explains that ferulic acid is channelled through the 4-CL (4-coumarate CoA ligase) enzyme, where it would be converted to feruloyl-CoA and could be further converted to 2H4MB in D. hamiltonii.

Investigation of the binding and simultaneous quantifications of propanil and bromoxynil herbicide concentrations in human serum albumin.

This study reported the use of UV-visible and fluorescence spectroscopy and partial-least-square (PLS) multivariate regression for accurate and simultaneous quantifications of two widely used herbicides, propanil, 3',4'-dichloropropionanilide (PPL) and bromoxynil, 3,5-dibromo-4-hydroxybenzonitrile (BXL) in human serum albumin (HSA) at physiological conditions. The binding affinity and thermodynamic properties of PPL-HSA and BXL-HSA complexes were also investigated. Partial-least-square (PLS) regression was used to collate the variability in the absorption or emission spectra of PPL-HSA and BXL-HSA complexes with PPL and/or BXL concentrations in HSA samples. The binding constants of 7.66× 108 M-1 for PPL-HSA and 4.88× 106 M-1 for BXL-HSA complexes were calculated at physiological conditions (temperature, 310 K; pH 7.4). Thermodynamic parameter values: enthalpy (ΔH) (13.99 kJ mol-1), entropy (ΔS) (0.078 kJ mol-1 K-1), and Gibbs free energy (ΔG) (-10.19 kJ mol-1) were determined for PPL-HSA complexation at physiological conditions. However, differences in thermodynamic property values of: ΔH (-214.3 kJ mol-1), ΔS (-0.563 kJ mol-1 K-1), and ΔG (-39.70 kJ mol-1) were observed for BXL-HSA complexes. The binding constants and negative ΔG values indicated strong binding affinity and thermodynamically favorability of PPL-HSA and BXL-HSA complex formation. Results of the PLS regression calibration showed good linearity (R2 ≥ 0.998289), high sensitivity, and impressive low limit-of-detections (LODs) of 1.38× 10-8 M for PPL and 1.68× 10-8 M for BXL that are comparable and/or lower than many previously reported LODs for herbicide and pesticide analyses. Most importantly, PLS regression is capable of simultaneous quantifications of PPL and BXL concentrations in HSA samples with good accuracy and low errors of 3.66%. UV-visible spectrophotometers and spectrofluorometers are fairly inexpensive, easy to use, and are readily available in almost every laboratory, making this protocol excellent and affordable for routine analysis of weed/pest control chemical residues in humans. The results of this study are significant and remarkable that will provide critical insight into the binding mechanism of herbicide toxicity in humans and non-target organisms, which are of special interest in the area of biomedical study, environmental risk assessment, and ecotoxicology.

Uptake and decomposition of the herbicide propanil in the plant Bidens pilosa L. dominating in the Yangtze Three Gorges Reservoir (TGR), China.

Propanil (3',4'-dichloropropionanilide) is a selective post emergence herbicide for controlling broad leaf and grass weeds in rice (Oryza sativa L.). After being taken up by plants, the fate of propanil in decomposing plant material is of particular importance to the phytoremediation of the environment. Therefore, we investigated the biotransformation of propanil in the plant Bidens pilosa under conditions close to those present in the Three Gorges Reservoir (TGR), China. Plants pre-treated with 14C-ring-labeled propanil were either (treatment a) directly submerged in TGR water for 90 days or (treatment b) pre-extracted with organic solvents, and subsequently only insoluble materials and non-extractable residues (NER) of the pesticide fractions were similarly incubated. After incubation in TGR water (treatment a), 30 % of applied radioactivity was released into water and simultaneously, amounts of NER in the plant debris appeared to increase with time finally amounting to 40 % of applied 14C. The radioactivity contained in the extractable fractions were identified as propanil, 3,4-dichloroaniline (DCA), and N-ß-D-glucopyranosyl-3,4-dichloroaniline (DCA-Glu). In treatment b, significant 14C amounts were released to the water (6 % of applied 14C) and the solubilized radioactivity fractions were demonstrated to agree with those found in the extractable fractions. Therefore, if residues of the pesticide propanil are taken up by plants, it may enter again the aquatic environment after plant death and submergence. This phenomenon may have a potential impact on aquatic organisms, which to our knowledge has not been reported before. As plant uptake and degradation of xenobiotics are recognized as detoxification, we consider B. pilosa with its high uptake potential, at least for propanil, as suitable species for phytoremediation.

Novel insights into the metabolic pathway of iprodione by soil bacteria.

Microbial degradation constitutes the key soil dissipation process for iprodione. We recently isolated a consortium, composed of an Arthrobacter sp. strain C1 and an Achromobacter sp. strain C2, that was able to convert iprodione to 3,5-dichloroaniline (3,5-DCA). However, the formation of metabolic intermediates and the role of the strains on iprodione metabolism remain unknown. We examined the degradation of iprodione and its suspected metabolic intermediates, 3,5-dichlorophenyl-carboxamide (metabolite I) and 3,5-dichlorophenylurea-acetate (metabolite II), by strains C1 and C2 and their combination under selective (MSM) and nutrient-rich conditions (LB). Bacterial growth during degradation of the tested compounds was determined by qPCR. Strain C1 rapidly degraded iprodione (DT50 = 2.3 h) and metabolite II (DT50 = 2.9 h) in MSM suggesting utilization of isopropylamine, transiently formed by hydrolysis of iprodione, and glycine liberated during hydrolysis of metabolite II, as C and N sources. In contrast, strain C1 degraded metabolite I only in LB and growth kinetics suggested the involvement of a detoxification process. Strain C2 was able to transform iprodione and its metabolites only in LB. Strain C1 degraded vinclozolin, a structural analog of iprodione, and partially propanil, but not procymidone and phenylureas indicating a structure-dependent specificity related to the substituents of the carboxamide moiety.

Modelling the biodegradation kinetics of the herbicide propanil and its metabolite 3,4-dichloroaniline.

This study models the biodegradation kinetics of two toxic xenobiotic compounds in enriched mixed cultures: a commonly applied herbicide (3,4-dichloropropionanilide or propanil) and its metabolite (3,4-dichloroaniline or DCA). The dependence of the metabolite degradation kinetics on the presence of the parent compound was investigated, as well as the influence of the feeding operation strategy. Model equations were proposed incorporating substrate inhibition of the parent compound and the metabolite during dump feed operation of a sequencing batch reactor (SBR). The kinetic parameters of the biomass were compared to step feed degradation of the SBR. The relationship between propanil and DCA degradation rates with the concentration of each compound was studied. A statistical comparison was carried out between the model predictions and experimental results. Substrate inhibition by both propanil and DCA was prominent during dump feed operation but insignificant during step feed. With both feeding strategies, the metabolite degradation was found to be dependent on the concentration of both the parent compound and the metabolite, suggesting that the DCA degrading enzymatic activity was independent of the detachment of the propionate moiety from the propanil molecule. After incorporating this finding into the model equations, the model was able to describe well the propanil and DCA degradation profiles, with an r (2) correlation >0.95 for each case. A kinetic model was developed for the degradation of the herbicide propanil and its metabolite DCA. An exponential inhibition term was incorporated to describe the substrate inhibition during dump feeding. The kinetics of metabolite degradation was dependent of the sum of the concentrations of metabolite and parent compound, which could also be of relevance to future xenobiotic modelling applications from wastewater.

Biodegradation of the herbicide propanil, and its 3,4-dichloroaniline by-product in a continuously operated biofilm reactor.

The persistence of propanil in soil and aquatic environments along with the possible accumulation of toxic degradation products, such as chloroanilines, is of environmental concern. In this work, a continuous small-scale bioprocess to degrade the herbicide propanil, its main catabolic by-product, 3,4-dichloroaniline (3,4-DCA), and the herbicide adjuvants is carried out. A microbial consortium, constituted by nine bacterial genera, was selected. The isolated strains, identified by amplification and sequencing of their 16S rDNA, were: Acidovorax sp., Luteibacter (rhizovicinus), Xanthomonas sp., Flavobacterium sp., Variovorax sp., Acinetobacter (calcoaceticus), Pseudomonas sp., Rhodococcus sp., and Kocuria sp. The ability of the microbial consortium to degrade the herbicide was evaluated in a biofilm reactor at propanil loading rates ranging from 1.9 to 36.8 mg L(-1) h(-1). Complete removal of propanil, 3,4-DCA, chemical oxygen demand and total organic carbon was obtained at propanil loading rates up to 24.9 mg L(-1) h(-1). At higher loading rates, the removal efficiencies decayed. Four of the identified strains could grow individually in propanil, and 3,4-DCA: Pseudomonas sp., Acinetobacter calcoaceticus, Rhodococcus sp., and Xanthomonas sp. The Kokuria strain grew on 3,4-DCA, but not on propanil. The first three bacteria have been related to biodegradation of phenyl urea herbicides or chlorinated anilines. Although some strains of the genera Xanthomonas and Kocuria have a role in the biodegradation of several xenobiotic compounds, as far as we know, there are no reports about degradation of propanil by Xanthomonas or 3,4-DCA by Kocuria species.

Propionate addition enhances the biodegradation of the xenobiotic herbicide propanil and its metabolite.

This study investigated ways of stimulating the biodegradation rates of the commonly applied herbicide, 3,4-dichloropropionanilide (propanil), and its metabolite, 3,4-dichloroaniline (DCA), as well as the growth rate of propanil- and DCA-degrading organisms in a mixed culture. Propionate, the other metabolite of propanil, stimulated the specific degradation rates of both propanil and DCA after a brief acclimation period. A metabolic model developed to characterise the metabolism of propanil and DCA biodegradation showed that the efficiency of oxidative phosphorylation (i.e. P/O ratio), which measures the metabolic efficiency, increased over time by 6- to 10-fold. This increase was accompanied by a 5- to 10-fold increase in the propanil and DCA biodegradation degradation rates. The biodegradation rates of the culture were unaffected when using an irrigation water matrix (Tejo river, Portugal), highlighting the utility of the culture for bioaugmentation purposes.

Diversity of propanil-degrading bacteria isolated from rice rhizosphere and their potential for plant growth promotion.

The herbicide propanil has long been used in rice production in southern Brazil. Bacteria isolated from contaminated soils in Massaranduba, Santa Catarina, Brazil, were found to be able to grow in the presence of propanil, using this compound as a carbon source. Thirty strains were identified as Pseudomonas (86.7%), Serratia (10.0%), and Acinetobacter (3.3%), based on phylogenetic analysis of 16S rDNA. Little genetic diversity was found within species, more than 95% homology, suggesting that there is selective pressure to metabolize propanil in the microbial community. Two strains of Pseudomonas (AF7 and AF1) were selected in bioreactor containing chemotactic growth medium, with the highest degradation activity of propanil exhibited by strain AF7, followed by AF1 (60 and 40%, respectively). These strains when encapsulated in alginate exhibited a high survival rate and were able to colonize the rice root surfaces. Inoculation with Pseudomonas strains AF7 and AF1 significantly improved the plant height of rice. Most of the Pseudomonas strains produced indoleacetic acid, soluble mineral phosphate, and fixed nitrogen. These bacterial strains could potentially be used for the bioremediation of propanil-contaminated soils and the promotion of plant growth.

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.

Degradation of propanil by bacterial isolates and mixed populations from a pristine lake.

The microbial transformation rates of propanil, a commonly used herbicide, were investigated using water from a pristine lake in northeast Georgia. Microbial degradation rates were measured using natural water microflora, the natural water microflora amended with five bacterial species (Aerobacter aerogenes, Aeromonas hydrophila, Acinetobacter calcoaceticus, Proteus mirabilis, and Aeromonas salmonicida) isolated from the same lake, and the five isolates individually. Transformation rate constants for propanil were compared for the mixed microbial assemblages and isolates at similar initial bacterial concentrations (approximately 5.0 x 10(-3) bacteria/mL). Degradation started within 60 hours and was completed by 160 hours in all experiments. The mean first-order rate constant for natural microflora was -(4.80 +/- 0.620) x 10(-3) h-1. Natural waters amended with the bacterial isolates yielded rate constants ranging from -(0.39 +/- 0.186) x 10(-3) h-1 to -(2.13 +/- 0.029) x 10(-3) h-1 with an overall mean of -(1.63 +/- 0.242) x 10(-3) h-1. After 660 hours following the first amendment of propanil, (i.e., 500 hours after propanil degradation was complete), each sample was again amended with propanil. Subsequent degradation rates ranged from -(21.3 +/- 0.186) x 10(-3) h-1 to -(64.2 +/- 0.786) x 10(-3) h-1 and the mean rate constant was -(37.5 +/- 0.922) x 10(-3) h-1. No significant differences were observed between first-order rate constants among isolates following the first or the second addition of propanil. After the second spike, however, the average of rate constants was approximately 20 times greater than that following the first spike. Rates for the individual isolates varied greatly from one isolate to another, ranging from virtually no degradation with A. calcoaceticus to -(21.6 +/- 0.332) x 10(-3) h-1 for the composite treatment of all isolates.

Role of metabolites in propanil-induced hemolytic anemia.

Hemolytic anemia and methemoglobinemia induced by exposure to certain arylamines, such as aniline and dapsone, are known to be mediated by their N-hydroxylamine metabolites. The arylamide propanil (3,4-dichloropropionanilide), a herbicide used extensively in rice fields, is also thought to induce methemoglobinemia through the action of metabolites. However, the hemolytic potential of this compound has not previously been reported. The present studies were undertaken to determine the hemolytic potential of propanil, and, if positive, the role of metabolites in this hemotoxicity. The survival of previously administered 51Cr-labeled erythrocytes in rats was reduced in a dose-dependent manner by ip administration of both propanil and its deacylated metabolite, 3,4-dichloroaniline (ED50 for both ca. 1.8 mmol/kg). When labeled erythrocytes were exposed in vitro to propanil or 3,4-dichloroaniline and then readministered to rats, no decrease in erythrocyte survival was observed, which indicated that these compounds were not direct-acting hemolytic agents. In contrast, erythrocyte survival was markedly reduced by ip administration or in vitro exposure to N-hydroxy-3,4-dichloroaniline. In addition, N-hydroxy-3,4-dichloroaniline was detected in the blood of propanil-treated rats in amounts sufficient to account for the hemolytic activity of the parent compound. These data indicate that N-hydroxy-3,4-dichloroaniline mediates propanil-induced hemolytic anemia, and that occupational exposure to propanil may result in an increased risk of hemolytic episodes.