Effect of dissemination of 2,4-dichlorophenoxyacetic acid (2,4-D) degradation plasmids on 2,4-D degradation and on bacterial community structure in two different soil horizons

Transfer of the 2,4-dichlorophenoxyacetic acid (2, I-D) degradation plasmid s pEMT1 and pJP4 from an introduced donor strain, Pseudomonas putida UWC3, to the indigenous bacteria of two different horizons (A horizon, depth of 0 to 30 cm; B horizon, depth of 30 to 60 cm) of a 2,4-D-contaminated soil wa s investigated as a means of bioaugmentation. When the soil was amended wit h nutrients, plasmid transfer and enhanced degradation of 2,4-D were observ ed, These findings were most striking in the B horizon, where the indigenou s bacteria were unable to degrade any of the 2,4-D (100 mg/kg of soil) duri ng at least 22 days but where inoculation with either of the two plasmid do nors resulted in complete 2,4-D degradation within 14 days. In contrast, in soils not amended with nutrients, inoculation of donors in the A horizon a nd subsequent formation of transconjugants (10(5) CFU/g of soil) could not increase the 2,4-D degradation rate compared to that of the noninoculated s oil. However, donor inoculation in the nonamended El-horizon soil resulted in complete degradation of 2,4-D within 19 days, while no degradation at al l was observed in noninoculated soil during 89 days. With plasmid pEMT1, th is enhanced degradation seemed to be due only to transconjugants (105 CFU/g of soil), since the donor was already undetectable when degradation starte d. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes showed that inoculation of the donors was followed by a shift in the microbial com munity structure of the nonamended B-horizon soils. The new 16S rRNA gene f ragments in the DGGE profile corresponded with the 16S rRNA genes of 2,4-D- degrading transconjugant colonies isolated on agar plates. This result indi cates that the observed change in the community was due to proliferation of transconjugants formed in soil. Overall, this work clearly demonstrates th at bioaugmentation can constitute an effective strategy for cleanup of soil s which are poor in nutrients and microbial activity, such as those of the B horizon.