The selection of mixed microbial inocula in environmental biotechnology: Example using petroleum contaminated tropical soils

The impact of inorganic N and P additions on a tropical soil contaminated with petroleum hydrocarbons was investigated using molecular and culture techniques. Microcosms were incubated for 42 days and sampled at 0, 1, 7, 28 and 42 days. Changes in bacterial community structure were determined using denaturing gradient gel electrophoresis (DGGE) of the rRNA following reverse transcription PCR using primers specific to the V3 region of the 16S rRNA gene. To identify which components of the microbial community were changing during incubation, PCR amplicons were resolved using DGGE and the banding patterns analyzed using stepwise discriminant function analysis (SDA). SDA showed that the number of bands needed to recover the differences between samples over time could be reduced from the initial 11 bands for the 16S rRNA transcript to 3 bands. Sequences originating from the rRNA gels (16S rRNA transcripts) were recovered in clades containing known cultured isolates of Bacillus marisflavi, Microbacterium oxydans and Pseudomonas oleovorans.Isolation studies on these soils using lubricant oil as a carbon source yielded 317 bacterial isolates, 3 of which showed high sequence similarity (> 96%) with the 16S rRNA transcripts identified using SDA as being important in differentiating between bacterial communities over time. These isolates were then tested singly and in combination for their ability to degrade lubricant oil. These analyses demonstrated that the consortium selected using the combined molecular-SDA approach was more effective at degrading the lubricant in both liquid media and in contaminated sand than the single isolates.