Coupling pathway prediction and fluorescence spectroscopy to assess the impact of auxiliary substrates on micropollutant biodegradation.

Some bacteria can degrade organic micropollutants (OMPs) as primary carbon sources. Due to typically low OMP concentrations, these bacteria may benefit from supplemental assimilation of natural substrates present in the pool of dissolved organic matter (DOM). The biodegradability of such auxiliary substrates and the impacts on OMP removal are tightly linked to biotransformation pathways. Here, we aimed to elucidate the biodegradability and effect of different DOM constituents for the carbofuran degrader Novosphingobium sp. KN65.2, using a novel approach that combines pathway prediction, laboratory experiments, and fluorescence spectroscopy. Pathway prediction suggested that ring hydroxylation reactions catalysed by Rieske-type dioxygenases and flavin-dependent monooxygenases determine the transformability of the 11 aromatic compounds used as model DOM constituents. Our approach further identified two groups with distinct transformation mechanisms amongst the four growth-supporting compounds selected for mixed substrate biodegradation experiments with the pesticide carbofuran (Group 1: 4-hydroxybenzoic acid, 4-hydroxybenzaldehyde; Group 2: p-coumaric acid, ferulic acid). Carbofuran biodegradation kinetics were stable in the presence of both Group 1 and Group 2 auxiliary substrates. However, Group 2 substrates would be preferable for bioremediation processes, as they showed constant biodegradation kinetics under different experimental conditions (pre-growing KN65.2 on carbofuran vs. DOM constituent). Furthermore, Group 2 substrates were utilisable by KN65.2 in the presence of a competitor (Pseudomonas fluorescens sp. P17). Our study thus presents a simple and cost-efficient approach that reveals mechanistic insights into OMP-DOM biodegradation.

Carbofuran pesticide toxicity to the eye.

Pesticide exposure to eyes is a major source of ocular morbidities in adults and children all over the world. Carbofuran (CF), N-methyl carbamate, pesticide is most widely used as an insecticide, nematicide, and acaricide in agriculture, forestry, and gardening. Contact or ingestion of carbofuran causes high morbidity and mortality in humans and pets. Pesticides are absorbed in the eye faster than other organs of the body and damage ocular tissues very quickly. Carbofuran exposure to eye causes blurred vision, pain, loss of coordination, anti-cholinesterase activities, weakness, sweating, nausea and vomiting, abdominal pain, endocrine, reproductive, and cytotoxic effects in humans depending on amount and duration of exposure. Pesticide exposure to eye injures cornea, conjunctiva, lens, retina, and optic nerve and leads to abnormal ocular movement and vision impairment. Additionally, anticholinesterase pesticides like carbofuran are known to cause salivation, lacrimation, urination, and defecation (SLUD). Carbofuran and its two major metabolites (3-hydroxycarbofuran and 3-ketocarbofuran) are reversible inhibitors of acetylcholinesterase (AChE) which regulates acetylcholine (ACh), a neurohumoral chemical that plays an important role in corneal wound healing. The corneal epithelium contains high levels of ACh whose accumulation by AChE inhibition after CF exposure overstimulates muscarinic ACh receptors (mAChRs) and nicotinic ACh receptors (nAChRs). Hyper stimulation of mAChRs in the eye causes miosis (excessive constriction of the pupil), dacryorrhea (excessive flow of tears), or chromodacryorrhea (red tears). Recent studies reported alteration of autophagy mechanism in human cornea in vitro and ex vivo post carbofuran exposure. This review describes carbofuran toxicity to the eye with special emphasis on corneal morbidities and blindness.

Heterogeneous distribution of carbofuran shaped by Pseudomonas stutzeri biofilms enhances biodegradation of agrochemicals.

Biodegradation, harnessing the metabolic versatility of microorganisms to reduce agrochemical contaminations, is commonly studied with enriched planktonic cells but overlooking the dominant lifestyle of microorganisms is to form biofilms, which compromises the efficiency of biodegradation in natural environment. Here, we employed a carbofuran-degrading bacterium Pseudomonas stutzeri PS21 to investigate how the bacterial biofilms formed and responded to agrochemicals. First, the PS21 biofilms formed with a core of bacterial cells enclosing with extracellular polymeric substances (EPSs), and the biofilms were active and resilient when exposed to carbofuran (up to 50 mg L-1). The formation was regulated by the second messenger bis-(3'-5')-cyclic di-guanosine monophosphate signaling, which strengthened the structural resistance and metabolic basis of biofilms to remain the degrading efficiency as comparable as the planktonic cells. Second, carbofuran distributed heterogeneously in the near-biofilm microenvironment via the covalent adsorption of biofilms, which provided a spontaneous force that enhanced the combination of carbofuran with biofilms to maintain high degrading activity. Additionally, we elucidated the biodegradation was driven by the integrated metabolic system of biofilms involving the extracellular enzymes located in the EPSs. This study exhibited the structural and metabolic advantages of microbial biofilms, highlighting the attractive potentials of exploring biofilm-based strategies to facilitate the in-situ bioremediation of organic contaminations.

Comparative Genomic Analysis of Carbofuran-Degrading Sphingomonads Reveals the Carbofuran Catabolism Mechanism in Sphingobium sp. Strain CFD-1.

The worldwide use of the carbamate insecticide carbofuran has caused considerable concern about its environmental fate. Degradation of carbofuran by Sphingobium sp. strain CFD-1 is initiated via the hydrolysis of its ester bond by carbamate hydrolase CehA to form carbofuran phenol. In this study, another carbofuran-degrading strain, Sphingobium sp. CFD-2, was isolated. Subsequently, a cfd gene cluster responsible for the catabolism of carbofuran phenol was predicted by comparing the genomes of strains CFD-1, CFD-2, and Novosphingobium sp. strain KN65.2. The key genes verified to be involved in the catabolism of carbofuran phenol within the cfd cluster include the hydroxylase gene cfdC, epoxide hydrolase gene cfdF, and ring cleavage dioxygenase gene cfdE and are responsible for the successive conversion of carbofuran phenol, resulting in complete ring cleavage. These carbofuran-catabolic genes (cehA and the cfd cluster) are distributed on two plasmids in strain CFD-1 and are highly conserved among the carbofuran-degrading sphingomonad strains. The mobile genetic element IS6100 flanks cehA and the cfd gene cluster, indicating the importance of horizontal gene transfer in the formation of carbofuran degradation gene clusters. The elucidation of the molecular mechanism of carbofuran catabolism provides insights into the evolutionary scenario of the conserved carbofuran catabolic pathway. IMPORTANCE Owing to the extensive use of carbofuran over the past 50 years, bacteria have evolved catabolic pathways to mineralize this insecticide, which plays an important role in eliminating carbofuran residue in the environment. In this study, the cfd gene cluster, responsible for the catabolism of carbofuran phenol, was predicted by comparing sphingomonad genomes. The function of key enzymatic genes in this gene cluster was identified. Furthermore, the carbamate hydrolase gene cehA and the cfd gene cluster are highly conserved in different carbofuran-degrading strains. Additionally, the horizontal gene transfer elements flanking the cfd gene cluster were investigated. These findings help elucidate the molecular mechanism of microbial carbofuran degradation and enhance our understanding of the evolutionary mechanism of the carbofuran catabolic pathway.

Carbofuran self-poisoning: forensic and analytic investigations in twins and literature review.

Carbofuran is a pesticide widely used in agricultural context to kill insects, mites, and flies by ingestion or contact. Along with literature review, we aimed to (i) present the clinical, autopsy, and toxicological findings of carbofuran self-poisonings in two 69-year-old twins, resulting in the death of one of them and (ii) assess carbofuran metabolite distribution using molecular networking. Quantitative analysis of carbofuran and its main metabolites (3-hydroxycarbofuran and 3-ketocarbofuran) was carried out using an original liquid chromatography-tandem mass spectrometry method on biological samples (cardiac or peripheral blood, urine, bile, and gastric contents). Toxicological analysis of post-mortem samples (twin 1) highlighted high concentrations of carbofuran and its metabolites in cardiac blood, bile, and gastric contents. These compounds were also quantified in blood and/or urine samples of the living brother (twin 2), confirming poisoning. Using molecular networking approach to facilitate visualization of mass spectrometry datasets and sample-to-sample comparisons, we detected two more metabolites (7-phenol-carbofuran and 3-hydroxycarbofuran glucuronide) in bile (twin 1) and urine (twin 2). These results highlight the value of (i) these compounds as carbofuran consumption markers and (ii) bile samples in post-mortem analysis to confirm poisoning. From an analytical point of view, molecular networking allowed the detection and interpretation of carbofuran metabolite ammonium adducts which helped to confirm their identification annotations, as well as their structural data. From a clinical point of view, the different outcomes between the two brothers are discussed. Overall, these cases provide novel information regarding the distribution of carbofuran and its metabolites in poisoning context.

Identification of the key amino acid sites of the carbofuran hydrolase CehA from a newly isolated carbofuran-degrading strain Sphingbium sp. CFD-1.

A novel carbofuran-degrading strain CFD-1 was isolated and preliminarily identified as Sphingbium sp. This strain was able to utilize carbofuran as the sole carbon source for growth. The carbofuran hydrolase gene cehA was cloned from strain CFD-1 and expressed in Escherichia coli. CehA could hydrolyze carbamate pesticides including carbofuran and carbaryl efficiently, while it showed poor hydrolysis ability against isoprocarb, propoxur, oxamyl and aldicarb. CehA displayed maximal enzymatic activity at 40 °C and pH 7.0. The apparent Km and Kcat values of CehA for carbofuran were 133.22 ±â€¯5.70 µM and 9.48 ±â€¯0.89 s-1, respectively. The site-directed mutation experiment showed that His313, His315, His453 and His495 played important roles in the hydrolysis of carbofuran by CehA. Furthermore, the sequence of cehA is highly conserved among different carbofuran-degrading strains, and there are mobile elements around cehA, indicating that it may be transferred horizontally between different strains.

Mechanism insights into the transformation of carbosulfan during apple drying processes.

The transformation of carbosulfan (CSN) in apples was investigated during oven-drying, microwave drying, and sun-drying. CSN transformed primarily into carbofuran (COA) during these drying processes. The conversion kinetics of CSN and COA was fitted by curve regression and mainly conformed to quadratic models (R2 = 0.70-0.97). Oven-drying promoted the transformation of CSN into COA. Microwave drying resulted in the highest scavenging capacity against CSN and COA (41%-100%). Moreover, a transformation mechanism was proposed on the basis of density functional theory (DFT) calculation. The COA originated from a series of chemical reactions involving hydroxyl substitution, cleavage, and oxidation; this result was further confirmed on the basis of molecular electrostatic potential (MEP) and molecular orbital theory. Furthermore, the toxicity and stability of CSN and COA were evaluated with the T.E.S.T. program. COA was less toxic than CSN to aquatic organisms but more toxic than CSN to rats. Therefore, COA production should be avoided during drying. Microwave drying was found to be the optimum choice for drying apples.

Pesticide bioremediation in liquid media using a microbial consortium and bacteria-pure strains isolated from a biomixture used in agricultural areas.

Microorganisms' role in pesticide degradation has been studied widely. Insitu treatments of effluents containing pesticides such as biological beds (biobeds) are efficient biological systems where biomixture (mixture of substrates) and microorganisms are the keys in pesticide treatment; however, microbial activity has been studied poorly, and its potential beyond biobeds has not been widely explored. In this study, the capacity of microbial consortium and bacteria-pure strains isolated from a biomixture (soil-straw; 1:1, v/v) used to treat agricultural effluents under real conditions were evaluated during a bioremediation process of five pesticides commonly used Yucatan Mexico. Atrazine, carbofuran, and glyphosate had the highest degradations (>90%) using the microbial consortium; 2,4-D and diazinon were the most persistent (DT50 = 8.64 and 6.63 days). From the 21 identified bacteria species in the microbial consortium, Pseudomonas nitroreducens was the most abundant (52%) according to identified sequences. For the pure strains evaluation 2,4-D (DT50 = 9.87 days), carbofuran (DT50 = 8.27 days), diazinon (DT50 = 8.80 days) and glyphosate (DT50 = 8.59 days) were less persistent in the presence of the mixed consortium (Ochrobactrum sp. DGG-1-3, Ochrobactrum sp. Ge-14, Ochrobactrum sp. B18 and Pseudomonas citronellolis strain ADA-23B). Time, pesticide, and strain type were significant (P < 0.05) in pesticide degradation, so this process is multifactorial. Microbial consortium and pure strains can be used to increase the biobed efficiency by inoculation, even in the remediation of soil contaminated by pesticides in agricultural areas.

Efficacy of soil-borne Enterobacter sp. for carbofuran degradation: HPLC quantitation of degradation rate.

Excessive use of pesticides in agricultural fields is a matter of great concern for living beings as well as the environment across the world, in particular, the third world countries. Therefore, there is an urgent need to find out an effective way to degrade these hazardous chemicals from the soil in an environment-friendly way. In the current project, a bacterial species were isolated through enrichment culture from carbofuran-supplemented rice-field soil and identified as a carbofuran degrader. The rate of carbofuran degradation by this bacterial species was evaluated using reverse-phase high-performance liquid chromatography (RP-HPLC), which confirmed the ability to utilize as a carbon source up to 4 µg/ml of 99% technical grade carbofuran. The morphological, physiological, biochemical characteristics and phylogenetic analysis of the 16S rRNA sequence showed that this strain belongs to the genus of Enterobacter sp. (sequence accession number LC368285 in DDBJ), and the optimum growth condition for the isolated strain was 37°C at pH 7.0. Moreover, an antibiotic sensitivity test showed that it was susceptible to azithromycin, penicillin, ceftazidime, ciprofloxacin, and gentamycin, and the minimal inhibitory concentration value of gentamycin was 400 µg/ml against the bacteria. It shows beyond doubt from the RP-HPLC quantification that the isolated bacterium has the ability to detoxify carbofuran (99% pure). Finally, the obtained results imply that the isolated strain of Enterobacter can be used as a potential and effective carbofuran degrader for bioremediation of contaminated sites through bioaugmentation.

Enhanced Carbofuran Degradation Using Immobilized and Free Cells of Enterobacter sp. Isolated from Soil.

Extensive use of carbofuran insecticide harms the environment and human health. Carbofuran is an endocrine disruptor and has the highest acute toxicity to humans than all groups of carbamate pesticides used. Carbofuran is highly mobile in soil and soluble in water with a lengthy half-life (50 days). Therefore, it has the potential to contaminate groundwater and nearby water bodies after rainfall events. A bacterial strain BRC05 was isolated from agricultural soil characterized and presumptively identified as Enterobacter sp. The strain was immobilized using gellan gum as an entrapment material. The effect of different heavy metals and the ability of the immobilized cells to degrade carbofuran were compared with their free cell counterparts. The results showed a significant increase in the degradation of carbofuran by immobilized cells compared with freely suspended cells. Carbofuran was completely degraded within 9 h by immobilized cells at 50 mg/L, while it took 12 h for free cells to degrade carbofuran at the same concentration. Besides, the immobilized cells completely degraded carbofuran within 38 h at 100 mg/L. On the other hand, free cells degraded the compound in 68 h. The viability of the freely suspended cell and degradation efficiency was inhibited at a concentration greater than 100 mg/L. Whereas, the immobilized cells almost completely degraded carbofuran at 100 mg/L. At 250 mg/L concentration, the rate of degradation decreased significantly in free cells. The immobilized cells could also be reused for about nine cycles without losing their degradation activity. Hence, the gellan gum-immobilized cells of Enterobacter sp. could be potentially used in the bioremediation of carbofuran in contaminated soil.

Interactions of Paraoxonase-1 with Pharmacologically Relevant Carbamates.

Mammalian paraoxonase-1 hydrolyses a very broad spectrum of esters such as certain drugs and xenobiotics. The aim of this study was to determine whether carbamates influence the activity of recombinant PON1 (rePON1). Carbamates were selected having a variety of applications: bambuterol and physostigmine are drugs, carbofuran is used as a pesticide, while Ro 02-0683 is diagnostic reagent. All the selected carbamates reduced the arylesterase activity of rePON1 towards the substrate S-phenyl thioacetate (PTA). Inhibition dissociation constants (Ki), evaluated by both discontinuous and continuous inhibition measurements (progress curves), were similar and in the mM range. The rePON1 displayed almost the same values of Ki constants for Ro 02-0683 and physostigmine while, for carbofuran and bambuterol, the values were approximately ten times lower and two times higher, respectively. The affinity of rePON1 towards the tested carbamates was about 3-40 times lower than that of PTA. Molecular modelling of rePON1-carbamate complexes suggested non-covalent interactions with residues of the rePON1 active site that could lead to competitive inhibition of its arylesterase activity. In conclusion, carbamates can reduce the level of PON1 activity, which should be kept in mind, especially in medical conditions characterized by reduced PON1 levels.

Hydrolase CehA and Monooxygenase CfdC Are Responsible for Carbofuran Degradation in Sphingomonas sp. Strain CDS-1.

Carbofuran, a broad-spectrum systemic insecticide, has been extensively used for approximately 50 years. Diverse carbofuran-degrading bacteria have been described, among which sphingomonads have exhibited an extraordinary ability to catabolize carbofuran; other bacteria can only convert carbofuran to carbofuran phenol, while all carbofuran-degrading sphingomonads can degrade both carbofuran and carbofuran phenol. However, the genetic basis of carbofuran catabolism in sphingomonads has not been well elucidated. In this work, we sequenced the draft genome of Sphingomonas sp. strain CDS-1 that can transform both carbofuran and carbofuran phenol but fails to grow on them. On the basis of the hypothesis that the genes involved in carbofuran catabolism are highly conserved among carbofuran-degrading sphingomonads, two such genes, cehACDS-1 and cfdCCDS-1, were predicted from the 84 open reading frames (ORFs) that share ≥95% nucleic acid similarities between strain CDS-1 and another sphingomonad Novosphingobium sp. strain KN65.2 that is able to mineralize the benzene ring of carbofuran. The results of the gene knockout, genetic complementation, heterologous expression, and enzymatic experiments reveal that cehACDS-1 and cfdCCDS-1 are responsible for the conversion of carbofuran and carbofuran phenol, respectively, in strain CDS-1. CehACDS-1 hydrolyzes carbofuran to carbofuran phenol. CfdCCDS-1, a reduced flavin mononucleotide (FMNH2)- or reduced flavin adenine dinucleotide (FADH2)-dependent monooxygenase, hydroxylates carbofuran phenol at the benzene ring in the presence of NADH, FMN/FAD, and the reductase CfdX. It is worth noting that we found that carbaryl hydrolase CehAAC100, which was previously demonstrated to have no activity toward carbofuran, can actually convert carbofuran to carbofuran phenol, albeit with very low activity.IMPORTANCE Due to the extensive use of carbofuran over the past 50 years, bacteria have evolved catabolic pathways to mineralize this insecticide, which plays an important role in eliminating carbofuran residue in the environment. This study revealed the genetic determinants of carbofuran degradation in Sphingomonas sp. strain CDS-1. We speculate that the close homologues cehA and cfdC are highly conserved among other carbofuran-degrading sphingomonads and play the same roles as those described here. These findings deepen our understanding of the microbial degradation mechanism of carbofuran and lay a foundation for the better use of microbes to remediate carbofuran contamination.

Persistence and metabolism of carbofuran in the soil and sugarcane plant.

Persistence and metabolism of carbofuran in the soil and sugarcane plant were studied under tropical sugarcane ecosystem. Residues of carbofuran and its metabolites in the soil, sugarcane leaf, and juice were determined by employing matrix-specific sample preparation methods and gas chromatography equipped with mass spectrometry. The recoveries of carbofuran, 3-keto carbofuran, and 3-hydroxy carbofuran were in the range of 88.75 ± 2.58-100.25 ± 2.38, 90.38 ± 2.61-98.24 ± 4.78, and 89.25 ± 3.11-98.10 ± 3.19%, respectively, at three levels of fortification across the three matrices involved in the study. At recommended dose (carbofuran 3% CG at 2 kg a.i./ha), the initial deposit of carbofuran in the soil was 14.390 ± 1.727 µg/g. The total residues comprising both carbofuran and 3-hydroxy carbofuran were detected up to 105 days after treatment with the half-life of 10.83 days. The parent compound and its metabolite were detected and quantified in the sugarcane plant (leaves and juice) from 14 days after application of carbofuran in the soil. The total residues (carbofuran and 3-hydroxy carbofuran) were detected in the leaves and cane juice up to 75 and 30 days after treatment, respectively.

Removal of carbamates and detoxification potential in a biomixture: Fungal bioaugmentation versus traditional use.

The use of fungal bioaugmentation represents a promising way to improve the performance of biomixtures for the elimination of pesticides. The ligninolyitc fungus Trametes versicolor was employed for the removal of three carbamates (aldicarb, ALD; methomyl, MTM; and methiocarb, MTC) in defined liquid medium; in this matrix ALD and MTM showed similar half-lives (14d), nonetheless MTC exhibited a faster removal, with a half-life of 6.5d. Then the fungus was employed in the bioaugmentation of an optimized biomixture to remove the aforementioned carbamates plus carbofuran (CFN). Bioaugmented and non-bioaugmented systems removed over 99% ALD and MTM after 8d of treatment, nonetheless a slight initial delay in the removal was observed in the bioaugmented biomixtures (removal after 3d: ALD 87%/97%; MTM 86%/99%, in bioaugmented/non-bioaugmented systems). The elimination of the other carbamates was slower, but independent of the presence of the fungus: >98% for MTM after 35d and >99.5% for CFN after 22d. Though the bioaugmentation did not improve the removal capacity of the biomixture, it favored a lower production of transformation products at the first stages of the treatment, and in both cases, a marked decrease in the toxicity of the matrix was swiftly achieved along the process (from 435 to 448 TU to values <1TU in 16d).

Carbofuran removal in continuous-photocatalytic reactor: Reactor optimization, rate-constant determination and carbofuran degradation pathway analysis.

Carbofuran (CBF) removal in a continuous-flow photocatalytic reactor with granular activated carbon supported titanium dioxide (GAC-TiO2) catalyst was investigated. The effects of feed flow rate, TiO2 concentration and addition of supplementary oxidants on CBF removal were investigated. The central composite design (CCD) was used to design the experiments and to estimate the effects of feed flow rate and TiO2 concentration on CBF removal. The outcome of CCD experiments demonstrated that reactor performance was influenced mainly by feed flow rate compared to TiO2 concentration. A second-order polynomial model developed based on CCD experiments fitted the experimental data with good correlation (R2 ∼ 0.964). The addition of 1 mL min-1 hydrogen peroxide has shown complete CBF degradation and 76% chemical oxygen demand removal under the following operating conditions of CBF ∼50 mg L-1, TiO2 ∼5 mg L-1 and feed flow rate ∼82.5 mL min-1. Rate constant of the photodegradation process was also calculated by applying the kinetic data in pseudo-first-order kinetics. Four major degradation intermediates of CBF were identified using GC-MS analysis. As a whole, the reactor system and GAC-TiO2 catalyst used could be constructive in cost-effective CBF removal with no impact to receiving environment through getaway of photocatalyst.

Expanded insecticide catabolic activity gained by a single nucleotide substitution in a bacterial carbamate hydrolase gene.

Carbofuran-mineralizing strain Novosphingobium sp. KN65.2 produces the CfdJ enzyme that converts the N-methylcarbamate insecticide to carbofuran phenol. Purified CfdJ shows a remarkably low KM towards carbofuran. Together with the carbaryl hydrolase CehA of Rhizobium sp. strain AC100, CfdJ represents a new protein family with several uncharacterized bacterial members outside the proteobacteria. Although both enzymes differ by only four amino acids, CehA does not recognize carbofuran as a substrate whereas CfdJ also hydrolyzes carbaryl. None of the CfdJ amino acids that differ from CehA were shown to be silent regarding carbofuran hydrolytic activity but one particular amino acid substitution, i.e., L152 to F152, proved crucial. CfdJ is more efficient in degrading methylcarbamate pesticides with an aromatic side chain whereas CehA is more efficient in degrading the oxime carbamate nematicide oxamyl. The presence of common flanking sequences suggest that the cfdJ gene is located on a remnant of the mobile genetic element Tnceh carrying cehA. Our results suggest that these enzymes can be acquired through horizontal gene transfer and can evolve to degrade new carbamate substrates by limited amino acid substitutions. We demonstrate that a carbaryl hydrolase can gain the additional capacity to degrade carbofuran by a single nucleotide transversion.

Biodegradation of carbofuran in soils within Nzoia River Basin, Kenya.

Carbofuran (2,3-dihydro-2,2-dimethylbenzofuran-7-yl methylcarbamate) has been used within the Nzoia River Basin (NRB), especially in Bunyala Rice Irrigation Schemes, in Kenya for the control of pests. In this study, the capacity of native bacteria to degrade carbofuran in soils from NRB was investigated. A gram positive, rod-shaped bacteria capable of degrading carbofuran was isolated through liquid cultures with carbofuran as the only carbon and nitrogen source. The isolate degraded 98% of 100-µg mL(-1) carbofuran within 10 days with the formation of carbofuran phenol as the only detectable metabolite. The degradation of carbofuran was followed by measuring its residues in liquid cultures using high performance liquid chromatography (HPLC). Physical and morphological characteristics as well as molecular characterization confirmed the bacterial isolate to be a member of Bacillus species. The results indicate that this strain of Bacillus sp. could be considered as Bacillus cereus or Bacillus thuringiensis with a bootstrap value of 100% similar to the 16S rRNA gene sequences. The biodegradation capability of the native strains in this study indicates that they have great potential for application in bioremediation of carbofuran-contaminated soil sites.

Genetic and metabolic analysis of the carbofuran catabolic pathway in Novosphingobium sp. KN65.2.

The widespread agricultural application of carbofuran and concomitant contamination of surface and ground waters has raised health concerns due to the reported toxic effects of this insecticide and its degradation products. Most bacteria that degrade carbofuran only perform partial degradation involving carbamate hydrolysis without breakdown of the resulting phenolic metabolite. The capacity to mineralize carbofuran beyond the benzofuran ring has been reported for some bacterial strains, especially sphingomonads, and some common metabolites, including carbofuran phenol, were identified. In the current study, the catabolism of carbofuran by Novosphingobium sp. KN65.2 (LMG 28221), a strain isolated from a carbofuran-exposed Vietnamese soil and utilizing the compound as a sole carbon and nitrogen source, was studied. Several KN65.2 plasposon mutants with diminished or abolished capacity to degrade and mineralize carbofuran were generated and characterized. Metabolic profiling of representative mutants revealed new metabolic intermediates, in addition to the initial hydrolysis product carbofuran phenol. The promiscuous carbofuran-hydrolyzing enzyme Mcd, which is present in several bacteria lacking carbofuran ring mineralization capacity, is not encoded by the Novosphingobium sp. KN65.2 genome. An alternative hydrolase gene required for this step was not identified, but the constitutively expressed genes of the unique cfd operon, including the oxygenase genes cfdC and cfdE, could be linked to further degradation of the phenolic metabolite. A third involved oxygenase gene, cfdI, and the transporter gene cftA, encoding a TonB-dependent outer membrane receptor with potential regulatory function, are located outside the cfd cluster. This study has revealed the first dedicated carbofuran catabolic genes and provides insight in the early steps of benzofuran ring degradation.

Sublethal toxicity of carbofuran on the African catfish Clarias gariepinus: Hormonal, enzymatic and antioxidant responses.

The present study examined the impacts of carbofuran on endocrinology of the catfish, Clarias gariepinus, for the first time and evaluated cortisol (CRT), triiodothyronine (T3), thyroxin (T4), 17ß-estradiol (E2) and testosterone (TST) and the oxidative stress markers including SOD, CAT, GSTs, GSH. The toxic effects on the metabolic enzymes, G6PDH and LDH, in addition to lipid peroxidation (LPO) and DNA damage as biomarkers in Nile catfish, to sublethal exposures of carbofuran (0.16 and 0.49mg/L, for 35 days) were studied. Statistically significant differences between selected parameters between control and carbofuran-treated fish were recorded. Carbofuran caused a significant (p<0.05) increase in CRT and T3 levels; the mean levels of T4, TST, E2 exhibited significant decreases (p<0.05) in carbofuran-treated fish. Toxicity of carbofuran on liver, kidney, gills, gonads and muscles after 35 days of exposure was found. Glycogen levels showed a highly significant decrease in liver and gills (p< 0.001), a significant decrease (p< 0.05) in kidney and muscles, and insignificant changes (p>0.05) in gonads of treated fish. The two metabolic enzymes G6PDH and LDH in all tissues exhibited significant decreases (p<0.05) in treated fish. SOD, CAT, GSH and GST levels showed significant decreases (p<0.05) in all tissues of fish after exposure to carbofuran. LPO levels increased significantly (p<0.05) in all tissues except gonads after 5 weeks of exposure to carbofuran. There was a significant (p<0.05) increase in DNA fragmentation percentage in treated fish. Our results provide a clear evidence on the response of C. gariepinus to sublethal doses of carbofuran and allow us to consider catfish as a good bioindicator to reflect the endocrine disrupting impacts of carbofuran, and reflect the potential of this pesticide to cause disturbance in antioxidant defense system as well as metabolism and induction of lipid peroxidation (LPO) and DNA damage in contaminated ecosystems.

Selection of support materials for immobilization of Burkholderia cepacia PCL3 in treatment of carbofuran-contaminated water.

This study investigated the utilization of agricultural matrices as the support materials for cell immobilization to improve the technique of bioremediation. Coir, bulrush, banana stem and water hyacinth stem in both delignified and undelignified forms were used to immobilize Burkholderia cepacia PCL3 in bioremediation of carbofuran at 5 mg l(-1) in synthetic wastewater. Undelignified coir was found to be the most suitable support material for cell immobilization, giving the short half-life of carbofuran of 3.40 d (2.8 times shorter than the treatments with free cells). In addition, it could be reused three times without a loss in ability to degrade carbofuran. The growth and degradation ability of free cells were completely inhibited at the initial carbofuran concentrations of 250 mg l(-1), while there was no inhibitory effect of carbofuran on the immobilized cells. The results indicated a great potential for using the agricultural matrices as support material for cell immobilization to improve the overall efficiency of carbofuran bioremediation in contaminated water by B. cepacia PCL3.