Bacterial consortia utilizing volatile organic compounds: mapping genomic determinants of essential biodegradation pathways

• Autores: Vasiliy Khomenkov, A. B. Shevelev, Vitaliy Zhukov, N. A. Zagustina, Vladimir Popov
• Localización: Biotechniques for air pollution control: proceedings of the international congress Biotechniques for Air Pollution Control : A Coruña, Spain, October 5-7, 2005 / Christian Kennes (dir. congr.), María C. Veiga (dir. congr.), 2005, ISBN 84-9749-163-7, págs. 61-67
• Idioma: inglés
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  • Genotyping of a consortium of microorganisms used in biofiltration application is often required. A standard RT-PCR approach substantially improves qualitative resolution of the genotyping and makes it a really an express method. However, development of RT-PCR method for each type of consortium requires preliminary characterization of its polymorphism extent and identification of dominant and ubiquitous species in order to adjust RT-PCR primers to their genomic sequence tags e.g. 16S genes. The goal of the present study was mapping of genomic determinants of essential biodegradation pathways in microbial consortia developed under real experimental condition in laboratory trickling gas-phase biofilters and adapted to degrading a number of abundant VOC’s: ethyl benzene, m-xylene, styrene and o-xylene. Herewith we report 16S-rRNA-based geno-systematic study of four related mixed bacterial cultures isolated from laboratory trickling biofilters. 16S-rDNA random cloning and RFLPassay was applied to the total genomic DNA of the cultures. 16S-rDNA schizo-types belonging to the major species were identified with two restrictases. Sequencing of four dominant 16SrDNA plasmid clones elucidated that Pseudomonas fluorescens (AY730552) was the principal specie in the ethylbenzene-utilizing culture. Achromobacter xylosoxydans (AY753652),Pseudomonas veronii( AY748440) and Delftia acidovorans (AY753653) prevailed in o-xylene, styrene and m-xylene utilizing cultures respectively. Minor components in any of four mixed cultures were essentially the same and belonged to genera Mesorhizobium, Pedobacter and Paenibacillus. Therefore, random sequencing of 16S-rRNA cloned from total genomic DNA was demonstrated to be a suitable method for qualitative assay of VOC-utilizing mixed culture polymorphism under real experimental conditions. Species identification was accomplished by cloning and sequencing of key genes encoding major aromatic VOC biodegradation pathway enzymes such as cathechol-1,2-dioxygenase, cathechol-2,3-dioxygenase and xylenemonooxygenase. Certain alleles of these genes occurred in any population but their sequences were culture-specific. Results of cathechol-1,2- and cathechol-2,3-dioxygenase enzymatic activity assessment in crude extracts of the mixed cultures were in a good agreement with respective genotyping data.