Isolation, Identification and Genetics of Nylon6 Degrading Pseudomonas putida

Sixty-four soil samples (contaminatedwith nylon waste for many years) were collected from different places in Baghdad. From these samples, fortysevenisolates were obtained as a degrader for nylon6 film.These isolates were repeatedly tested in mineral salt medium supplemented withnylon6 film to ensure their degradation ability; only 27 isolates appeared tohave degradation ability fornylon6 filmas a sole source of carbon and nitrogen. These isolates (27 isolates) were screened for their ability to degrade nylon6 film according to growth density;it was found that these isolates were varied in their growth density. Themost efficient isolates were T1, M3, S3A, S12 andS17; however, S3A isolate is the best among them. According to growth density, 20 isolates were selected for identification; they were identified depending on morphological, cultural, and biochemical characteristics.Results showed that 16 isolates belonged to Pseudomonas spp.(Pseudomonas putida (14) isolates, Pseudomonas stutzeri (1) isolate and Pseudomonas sp. (1) isolate, while the other four belonged toMoraxella spp. The plasmid profile for Pseudomonas putidaS3A was studied. Results showed that this isolate harbored small plasmid DNA bands.In order to study the role of its plasmid in degradation of nylon6 film, curing experiment was performed by using sodium dodecyl sulfate (SDS) and showed that two colonies had lost their ability to degrade nylon6 film as a sole source of carbon and nitrogen. Plasmid DNA extraction from one of these colonies indicated the loss of plasmid DNA bands, and this referred that the plasmid DNA bands could be responsible for degrading
nylon6 film in P. putida S3A. Optimum conditions for degradation of nylon6 film by P. putida S3A were
investigated. It was found that these conditions are growing this bacterium in mineral salt medium (pH 6.5) containing 0.1% of nylon6 film and incubated with shaking (180rpm) at 37 °C for seven days. It was found also that this bacterium was able to survive up to 0.7%of nylon6 film. In an attempt to investigate the ability of bacteria to degrade crude nylon, the efficient five isolates(T1, M3, S3A, S12 and S17) were grown on crude nylon6 and nylon66 as a sole source of carbon and nitrogen. Results showed that nylon66 is
more susceptible to degrade than nylon6 by these isolates, and maximum growth was recorded byP. putida S3A. To insure isolation of bacteria capable to degrade crude nylon6, nylon6 was processed by UV irradiation and transferred to film, and this film was used to isolate bacteria.From the Fourier Transformer Infrared Red Spectroscopy (FTIR) results, it can be concluded that the irradiated nylon6 became less difficult to degrade because U.V light photodegradenylon6 into small pieces, and reduced its molecular weight. It can be concluded also that nylon6 film was degraded byP. putida S3A, which used the (N-H, C=O and C-H)groups as carbon and nitrogen source.