AbstractPolyhydroxyalkanoates (PHAs) are biopolymers that are of interest because they can be used to produce bio-plastics. They are produced by many types of bacteria when environmental conditions are rich in carbon but poor in nitrogen. In this study environmental samples were collected from a waste water treatment plant at Petersfield and three different garden compost. Bacterial isolates were recovered after growth on a range of media, including minimal salt medium (MSM) and Lysogeny broth medium (LB). The minimal media contained a low nitrogen, high carbon ratio. PHA production was initially tested with staining methods Sudan Black B, Nile Blue, Nile Red. The Sudan Black B staining gave positive result for 48 out of the 73 isolates producing a bluish black colour when screened. While the Nile Blue A and Nile red tested positive for 18 out of 73 isolates which showed bright orange. From the isolates recovered from the waste water and compost, 40 % and 60% respectively were capable of producing PHAs. The identified bacteria came from the following genera identified with 16S rRNA sequencing are: Alcaligenes, Aeromonas, Arthrobacter, Bacillus, Pseudomonas, Citrobacter, Acinetobacter, Klebsiella and Raoultella. The presence of a PHA synthase gene (phaC) was confirmed by PCR amplification using different primers (I-179R and I-179L) (phaCF1BO and phaCR2BO), however some strains did not amplify for the phaC gene although they gave a positive staining reaction with Sudan Black B. Therefore, plasmids isolated from the environmental strains were used for the transformation of E. coli XL-1 Blue (negative strain for PHA producer) and the result showed that only plasmids from four environmental strains were transferred and gave positive result with PHA testing methods that included PCR and staining with Sudan Black and Nile Red.
PHA were extracted from the strains and characterized by FTIR spectroscopy and NMR for the production of PHA. The result of the FTIR showed peaks at 1630 - 1650 cm-1 corresponding to C=O carbonyl stretching groups which indicated the presence of an ester, amide or amino acid rather than PHA. This observation was confirmed by NMR analysis, which was unsuccessful in detecting the backbone of PHA Nevertheless, strains did successfully amplify the phaC gene and one Pseudomonas strain showed the presence of the phaC1, phaC2 and phaZ genes.
A biochip was designed and tested for the detection of phaC gene sequences in different strains residing in strains and environmental samples. The biochip contained 15 probes for class 1, 2 and 3 PHA synthases. Hybridization of the probes to test cassettes showed that the probes function well. The designed and fine-tuned probes can detect phaC gene sequences in bacterial consortium from environmental sources.
|Date of Award||18 Apr 2018|
|Supervisor||Vitaly Zinkevich (Supervisor)|