Bioaugmentation of activated sludge with two Pseudomonas putida strains for the degradation of 4-chlorophenol

The augmentation of activated sludge with two strains of Pseudomonas putida, CP1 and A(a) was investigated. Both strains of bacteria degraded 1.56 mM 4-chlorophenol. P. putida CP1 degraded the chemical using a modified ortho-cleavage pathway while P. putida A(a) used the meta-cleavage pathway. When activated sludge incapable of 4-chlorophenol degradation was augmented with either strain, substrate degradation occurred and followed the same biochemical pathways as when the bacteria were grown in pure culture. Insertion, in tandem, of the genes for gentamycin resistance and green fluorescent protein into the chromosomes of the two strains, enabled the survival and spatial location of the bacteria in the mixed microbial population to be monitored. Labelling the bacteria did not alter their degradative capabilities. P. putida CP1::Tn7-gfp survived in higher numbers than P. putida A(a)::Tn7-gfp following addition to the activated sludge. This was attributed to the ability of this strain to flocculate and become integrated in the activated sludge floc. Addition of P. putida CP1::Tn7-gfp or A(a)::Tn7-gfp to activated sludge resulted in smaller decreases in total cell numbers indicating a protective effect of the introduced P. putida strains on the overall microbial population from the harmful effects of 4-chlorophenol. The non-flocculant strain A(a) did not survive as well as CP1 in the activated sludge system and required a higher inoculum size to effect substrate degradation.