P63: Comparison of TNT degradation rate among different clostridium strains reveals hydrogenase plays major role in TNT degradation

2,4,6-trinitrotoluene (TNT) is a widely used nitroaromatic compound, which is recalcitrant to degradation and has been of environmental concern. Among different microorganisms that act in TNT biodegradation, clostridium species are distinguished by their fast degradation rate. Here we report a comparison of different C. acetobutylicum strains for degradation of TNT in vivo. Using the stable colorimetric protocol, we adapted the method in actively growing cultures to assay the TNT degradation rate. C. acetobutylicum ATCC 824 degraded TNT at a rate of 11215 ± 342 µM/Hour/A₆₀₀ during early exponential phase, while the rate decreased to 749 ± 792 µM/Hour/A₆₀₀ when the cells were in stationary phase (24 hour cultures). A series of mutant strains derived from C. acetobutylicum ATCC 824, such as mutantB, 824(pta) 824(buk) and M5 strains were tested for TNT degradation and only minor differences were observed.  Similar to that observed in a cell lysate assay, 824(pSOS-as-hydA), the antisense downregulated hydrogenase mutant, showed significantly reduced TNT degradation compared to the wild type. These results indicate that in C. acetobutylicum hydA plays the major role in TNT degradation. The degradation rate of TNT by some environmentally abundant clostridia e.g. Clostridium phytofermentans was also measured and showed strong TNT degradation upon growth on other feedstocks, such as cellobiose, indicating a wide role for various clostridia in environmental degradation of nitroaromatics.