An investigation of mono-chlorophenol degradation and the bioaugmentation of activated sludge by two pseudomonas putida species

The degradation of all three mono-chlorophenols by two Pseudomonas putida species, A(a) and CPI, was investigated Pseudomonas putida A(a) metabolised 4-chlorophenol to completion via the meta-cleavage pathway but the degradation of 2- and 3- chlorophenol was incomplete Pseudomonas putida CPI degraded all three chlorophenol isomers to completion via the modified ortho-cleavage pathway. The genes for the key enzymes of the chlorophenol degradative pathways, catechol 2,3- dioxygenase of the meta-cleavage pathway from P putida A(a) and chlorocatechol 1,2- dioxygenase of the modified or^o-cleavage pathway from P putida CPI were amplified by PCR and identified by nucleotide sequencing. Large plasmids were detected in both strains Southern hybridization to DIG-labelled probes confirmed the presence of the genes for chlorocatechol 2,3-dioxygenase on the 1 17 kb plasmid in A(a) and chlorocatechol 1,2-dioxygenase on the 110 kb plasmid in CPI. The bacteria were tagged by inserting the gene for green fluourescent protein (GFP), into the chromosomes of P putida A(a) and P putida CPI using Tn7 and Tn5 based delivery vectors Tn7 inserted specifically at a neutral chromosomal site m P putida A(a) and an unknown site in P putida CPI. Bioaugmentation of activated sludge resulted in the complete removal of 4-chlorophenol GFP was used as a visual marker to observe the location of the introduced strains, revealing that CPI Tn7-gfp was located almost entirely withm the sludge floes while P putida A(a) Tn 7-gfp was evenly distributed throughout the mixed liquor P putida CPI Tn 7-gfp survived in greater numbers than P putida A(a) Tn 7-gfp. The addition of the bacteria had a protective effect on the indigenous batenal population however this effect was more pronounced with the addition of P putida CPI Tn 7-gfp. The floc-forming ability of P putida CPI Tn 7-gfp may result in its enhanced survival leading to better degradation of 4-chlorophenol