Pyrosequencing study of a bacterial PAH-degrading consortium and the shifts produced in its composition by inoculation with a competitive strain

FESTA SABRINA; MACCHI MARIANELA; MORELLI IRMA S; COPPOTELLI BIBIANA M

Mar del Plata

Congreso; X Congreso Argentino de Microbiología General; 2014

SAMIGE

Bioaugmentation requires different species of introduced degrading microorganisms, which can compete with the indigenous microbial community in PAH-contaminated soil, in order to participate in the main carbon and energy flux processes and enhance PAH removal. PAH-degrading bacterial consortia, usually obtained by enrichment techniques from of environmental samples, have been proven to be diverse and complex providing a unique system to challenge the new molecular tools allowing the characterization of molecular interactions and microbial activities on a global level never before achieved. A consortium (CON) was obtained from a chronically contaminated soil with polycyclic aromatic hydrocarbons (PAH), five strains were isolated and identified as Sphingobium sp. (AM), Enterobacter sp. (B and B1) and Pseudomonas sp. (T and Bc). The CON was inoculated with Sphingomonas paucimobilis 20006FA, a strain able to degrade a wide range of PAH, generating a new consortium (CON+I). Both consortia were characterized in terms of structure, diversity and functionality. The CON and CON+I consortia were cultivated in LMM with 200 mg/l of phenanthrene for 15 days. The CON showed a degradation of 59 % with the accumulation of 1-hydroxy 2-naphtoic acid, the CON+I showed a degradation of 78% without accumulation of this intermediary. Population dynamic between the two consortia was observed at cultivable (counts) and non cultivable level (PCR-DGGE). This work aims to study shifts in diversity and composition of CON, by pyrosequencing technique, when is inoculated with a competent bacterial inoculant. The phylogenic composition of the consortia was profiled using pyrosequencing of PCR-amplified bacterial 16S rRNA gene fragments with the posterior analysis with EstimateS (Version 9). All representative sequences of each OTU were classified into the domain Bacteria, class Proteobacteria (Alpha, Beta and Gamma with 89.3%, 2.93% and 7.73% of the reads respectively). Relative phylotype frequency at the genus level revealed the presence of Inquilinus 1.1%, Achromobacter 2.9%, Sphingobium 87,7%, Pseudomonas 0.5%, Raoultella 2.4%, Luteibacter 0.6%., Kluyvera 4.2% and Sphingomonas 0.5%. A correlation of some of the sequences with the isolated strains was observed. Surprisingly in the CON+I the genera belonging to the family Sphingomonadaceae remained constant, while a marked increase at the level of Achromobacter 12.2% and Pseudomonas 1.7% was observed, suggesting a syntrophic interaction between the inoculated strain and these two genera. Additionally it could be observed a decrease in diversity with inoculation, being the Shannon index (H) 1.51 for CON and 1.29 for CON+I. Using a high throughput technique it was possible to observe that the relative composition of bacterial genera within the consortium are strongly affected by the introduction of an inoculant, giving clues that complex interactions between bacterial species could influence the biodegrading potential.