Quantitative assessment of phenol hydroxylase diversity in bioreactors using a full cycle functional gene analysis

BASILE, L.A.; ERIJMAN, LEONARDO

Buenos Aires, Argentina

Congreso; Tercer Congreso Argentino de Microbiología General; 2007

Sociedad Argentina de Microbiología General

Biodiversity has allegedly a significant influence upon the dynamics, stability and functioning of ecosystems. One of the practical consequences of diversity within microbial assemblages is the occurrence of functional redundancy, i.e. the ability of a number of different species to carry out similar functions. In this work, an approach to assess quantitatively the diversity of functional genes in complex microbial environments was developed, and applied to the analysis of the genetic diversity of phenol-degrading potential of communities from laboratory-scale activated sludge. A 620 bp fragment of the gene coding for the major subunit of phenol hydroxylase (LmPH) was amplified with conserved primers from activated sludge extracted from two reactors fed with synthetic sewage plus phenol, and used to generate clone libraries. Following phylogenetic analysis, 59 sequences were clustered into six distinct subgroups with a genetic distance of 0.08, which likely reflects functionally relevant differences in enzyme function. Seven sets of primers were designed to target the six groups and used to obtain quantitative information on the dynamics of LmPH gene diversity, using real time PCR assays throughout nine months of bioreactors operation. Total LmPH gene copy number remained approximately steady in phenol-amended and control reactors. However, the increase in phenol-degrading activity observed in phenol-amended sludge was accompanied by a parallel increase in LmPH gene diversity. This effect was reversed upon deletion of phenol addition. Simultaneous analysis of PCR–DGGE banding patterns of 16S rRNA gene displayed variations over the time scale, but no significant differences were observed between phenol-amended and control reactors at each time point. In conclusion, our results indicate that a high genetic diversity for the LmPH gene emerges in sludge acclimatized to phenol, and the diversity is sustained as long as the supply of phenol is continued. The use of a full cycle functional gene approach appears a convenient strategy to get insight into functional redundancy in complex environments.