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Four bacterial strains were isolated from enrichment cultures inoculated with soil from Bien Hoa military base in Vietnam contaminated with the herbicides 2,4-dichlorophenoxyacetate (2,4-D) and 2,4,5-trichlorophenoxyacetate (2,4,5-T). They were classified as Pseudomonas aeruginosa BT1 2.2, Sphingomonas histidinilytica BT1 5.2, Bordetella petrii BT1 9.2, and Achromobacter xylosoxidans BT1 10.2. All four were able to degrade 2,4-D and 2,4,5-T, but only the last three species used them as the sole sources of carbon and energy. Mass balance analyses suggest that between 33 and 46% of the carbon in the herbicides is incorporated into dry weight (DW). We obtained insight into their degradation pathways by the genomic analysis of these strains. A tfdCDEF gene cluster was found in A. xylosoxidans BT1 10.2 with amino acid sequences of their gene products showing high identity to those in B. petrii DSM12804. Bordetella petrii BT1 9.2 has a full complement of the tfdABCDEF genes. Surprisingly, the gene organization along with the amino acid sequences of the gene products are virtually identical to those of Cupriavidus pinatubonensis JMP134, referred to as type I tfd genes, and different from those of A. xylosoxidans BT1 10.2 and B. petrii DSM12804. We hypothesize that some of the genetic potential to degrade the herbicides has been recruited in recent mating events between these species and other members of the proteobacteria. This is the first report showing that B. petrii BT1 9.2 emerges as a key player in the degradation of 2,4-D.

We determined the degradation rates of the herbicides 2,4-D and 2,4,5-T by two different bacterial communities. One of these originated from soil heavily contaminated with herbicides from Bien Hoa airbase, the other from the same soil but amended with additional carbon and Gibbs energy sources. The community from the contaminated, but untreated, soil degraded both 2,4-D and 2,4,5-T within 5 days of cultivation. The one from the amended soil, however, hardly showed any degradation of the compounds throughout 23 days of cultivation. After refreshment of the medium and prolonged culturing, however, this community degraded both herbicides within 25 days with progressively increasing rates. nMDS analysis revealed a highly significant differentiation pattern of the two communities. Cultures inoculated with amended soil showed a significant increase of Bacillus and Paenibacillus upon prolonged exposure to the herbicides. The succession in the culture from untreated soil, on the other hand, was dominated by species from the Proteobacteria. We were able to isolate two of them and they were shown to be related to Bordetella petrii and Sphingomonas histidinilytica, successively. Subsequent PCR analyses of their DNA revealed the presence of key genes involved in the degradation of the herbicides. This study provides a more fundamental understanding of the biodegradation of 2,4-D and 2,4,5-T by displaying part of the bacterial community succession during their breakdown allowing a comprehensive view on potential key degraders.

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