Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Umar Mustapha M

PROVIDER: S-EPMC7355768 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Enhanced Carbofuran Degradation Using Immobilized and Free Cells of <i>Enterobacter</i> sp. Isolated from Soil.

Umar Mustapha Mohammed M Halimoon Normala N Wan Johari Wan Lutfi WL Abd Shukor Mohamed Yunus MY

Molecules (Basel, Switzerland) 20200616 12

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 ...[more]

PMID: 32560037

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Project description: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.

Project description:Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can be generated using a transposome. The commercially available, Tn5-derived transposome used in this protocol consists of a linear segment of DNA containing an R6Kγ replication origin, a gene for kanamycin resistance and two mosaic sequence ends, which serve as transposase binding sites. The transposome, provided as a DNA/transposase protein complex, is introduced by electroporation into the prototrophic strain, Enterobacter sp. YSU, and randomly incorporates itself into this host's genome. Transformants are replica plated onto Luria-Bertani agar plates containing kanamycin, (LB-kan) and onto M-9 medium agar plates containing kanamycin (M-9-kan). The transformants that grow on LB-kan plates but not on M-9-kan plates are considered to be auxotrophs. Purified genomic DNA from an auxotroph is partially digested, ligated and transformed into a pir+ Escherichia coli (E. coli) strain. The R6Kγ replication origin allows the plasmid to replicate in pir+ E. coli strains, and the kanamycin resistance marker allows for plasmid selection. Each transformant possesses a new plasmid containing the transposon flanked by the interrupted chromosomal region. Sanger sequencing and the Basic Local Alignment Search Tool (BLAST) suggest a putative identity of the interrupted gene. There are three advantages to using this transposome mutagenesis strategy. First, it does not rely on the expression of a transposase gene by the host. Second, the transposome is introduced into the target host by electroporation, rather than by conjugation or by transduction and therefore is more efficient. Third, the R6Kγ replication origin makes it easy to identify the mutated gene which is partially recovered in a recombinant plasmid. This technique can be used to investigate the genes involved in other characteristics of Enterobacter sp. YSU or of a wider variety of bacterial strains.

Dataset Information

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

Publications

Enhanced Carbofuran Degradation Using Immobilized and Free Cells of <i>Enterobacter</i> sp. Isolated from Soil.

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