Spatial distribution and physiological state of bacteria in a sand column experiment during the biodegradation of toluene

Toxic organic contaminants frequently serve as growth substrates for bacteria. However, long-term exposure to the organic contaminants can result in significant stress or “injury” to bacterial cells such that bacteria may lose, either temporarily or permanently, their capacity to degrade a specific toxic organic contaminant.In order to understand the relationship between biodegradability and physiological conditions of bacteria after a prolonged exposure to a contaminant, biomass samples collected from a sand column experiment, with toluene as the carbon source, were analyzed for bacterial physiology and spatial population distribution in the porous media. The column was seeded with three bacterial isolates that perform aerobic (Pseudomonas putida F1), denitrifying (Thauera aromatica T1), and facultative (Ralstonia pickettii PKO1) degradation of toluene were analyzed. Total, viable but not culturable with toluene, and toluene-culturable cells were enumerated using 4′6-diamidino-2-phenylindole (DAPI) staining and plate counting methods. Comparison of three types of cell counts showed that toluene-culturable cells were less than 40% of the total cell numbers. However, viable colonies transferred to a toluene media after cultivation on rich media regained their ability to degrade toluene. This implies that the temporary loss of their toluene degradation capacity is either due to an intracellular accumulation of degradation by-products, which have to be consumed in order for the cells to degrade toluene, or it is possible that cells have shifted to degrade other substrates such as toluene degradation intermediates or organic materials resulting from cell turnover. Comparison of cell counts with toluene concentration showed no exponential increase in total and viable cell numbers, as reported for flat bed biofilm reactor experiments. The overall fraction of toluene-culturable cells was highest at the highest toluene concentration near the column inlet, which indicates that the observed temporary loss of toluene culturability was not solely caused by a direct toxic effect from the long-term exposure to toluene.