Response of the soil bacterial community to the addition of toluene and toluene-degrading bacteria

Changes in soil bacterial communities after toluene and/or toluene degrading bacteria were added were monitored by growth on various media, and by the culture-independent method of Reverse Sample Genome Probing (RSGP). A total of 397 isolates that were cultured from toluene-amended and non-amended soil were identified using fatty acid methyl ester (FAME) analysis. In 75% of the soil samples, gram-positive bacteria dominated, primarily Bacillus sp. and Cellulomonas sp. In contrast, RSGP revealed Proteobacteria (α, β, and γ subgroups) to be the dominant species, with Bacillus sp. dominant in only one soil sample. In toluene-treated soil, FAME and RSGP analyses indicated that by day 5 the major bacterial populations were gram-negative bacteria, in particular, Pseudomonas sp., Acinetobacter sp., and Alcaligenes sp. When toluene and Pseudomonas putida D8 were coincidentally introduced, the proportion of Pseudomonas sp. in the bacterial population recovered using non-selective medium increased from 16 to 62% and then rapidly decreased to about 9%. When we used selective medium to monitor the population of P. putida D8, a rapid initial increase followed by a gradual decline was also observed. In samples analyzed by RSGP, D8 was one of the major species of the bacterial community at day 2, but its signal intensity dropped to 9.5% by day 5. The influence of D8 addition on the bacterial profile was monitored in growth-based examinations. Bacillus sp. and Pseudomonas sp. were initially dominant. By day 5, Bacillus sp. decreased while the Proteobacteria, (including Acinetobacter sp., Agrobacterium sp., Alcaligenes sp., Burkholderia sp., Erwinia sp., and Pseudomonas sp.) increased. At the same time, D8 decreased to a level indistinguishable from background. Conversely, RSGP analysis revealed the population dominance of P. putida (including D8) and Rhizobium fredii at day 2. Populations shifted toward Agrobacterium tumefaciens, Bacillus subtilis, R. fredii, and D8 by day 5. P. putida D8 levels could be monitored using RSGP when cultivation failed. However, cultivation of Bacillus sp. was always successful, while the organism was only occasionally detected by RSGP. While cultivation and RSGP methods comparably detected the same major bacterial populations, the overall bacterial diversity was greater with RSGP than with growth-based testing.