Microbial Succession during a Field Evaluation of Phenol and Toluene as the Primary Substrates for Trichloroethene Cometabolism

Abstract

Microbial community composition and succession were studied in an aquifer that was amended with phenol, toluene, and chlorinated aliphatic hydrocarbons to evaluate the effectiveness of these aromatic substrates for stimulating trichloroethene (TCE) bioremediation. Samples were taken after the previous year's field studies, which used phenol as the primary substrate, and after three successive monthly treatments of phenol plus 1,1-dichloroethene (1,1-DCE) plus TCE, phenol plus TCE, and toluene plus TCE. Dominant eubacteria in the community were assessed after each of the four treatments by characterizing isolates from the most dilute most-probable-number tubes and by extracting DNA from aquifer samples. The succession of dominant phenol- and toluene-degrading strains was evaluated by genomic fingerprinting, cellular fatty acid methyl ester (FAME) analysis, and amplified ribosomal DNA restriction analysis (ARDRA). 1,1-DCE was found to drastically reduce microbial growth and species richness, which corresponded to the reduction in bioremediation effectiveness noted previously for this treatment (G. D. Hopkins and P. L. McCarty, Environ. Sci. Technol. 29:1628-1637, 1995). Only a few gram-positive isolates could be obtained after treatment with 1,1-DCE, and these were not seen after any other treatments. Microbial densities returned to their original levels following the subsequent phenol-TCE treatment, but the original species richness was not restored until after the subsequent toluene-TCE treatment. Genomic fingerprinting and FAME analysis indicated that six of the seven originally dominant microbial groups were still dominant after the last treatment, indicating that the community is quite resilient to toxic disturbance by 1,1-DCE. FAME analysis indicated that six microbial taxa were dominant: three members of the (beta) subclass of the class Proteobacteria (Comamonas-Variovorax, Azoarcus, and Burkholderia) and three gram-positive groups (Bacillus, Nocardia, and an unidentified group). ARDRA revealed that the dominant community members were stable during the three nontoxic treatments and that virtually all of the bands could be accounted for by isolates from five of the dominant taxa, indicating that the isolation protocol used likely recovered most of the dominant members of this community.