CHARACTERIZATION OF POLYHYDROXYLALKANOATE (PHA) PRODUCING HALOPHILIC BACTERIA FROM HYPERSALINE WATER BODY IN LAGOS, NIGERIA

Abstract

The need to reduce the environmental pollution and destruction of ozone layer as well as the understanding that synthetic plastic disposal contributes to these problems makes this study necessary. This study was aimed at charactering polyhydroxylalkanoate (PHA) producing halophilic bacteria from hypersaline water body in Lagos, Southern Nigeria. Bacteria were isolated from water samples obtained at three locations of coordinates 6.35ᴼ N 3.28ᴼ E; 6.35ᴼ N 3.40ᴼ E; and 6.36ᴼ N 3.47ᴼ E and from depths of 0.07 m (top), 50 m (middle) and 100 m (bottom) below water surface using standard water sampling methods. Conventional (cultural, morphological, biochemical) and molecular (DNA extraction, amplification of 16S rRNA gene sequencing, blast on NCBI website) methods were exploited for bacterial identification. Physicochemical analysis were carried out on water samples using standard methods of AOAC for assays on physical, chemical and trace/heavy metals. Various conditions of pHs, temperatures and salt concentrations were carried out on bacterial isolates for their growth effects. Screening and synthesis of PHA was performed by Sudan black B staining techniques on isolates; while, the best PHA yielding bacteria was investigated in minimal salt medium (MSM) supplemented with 8% (w/v) glucose. The growth indexes of the bacterial isolates were determined by optical density (OD) at 520 nm from UV spectrophotometer. The genetic relatedness was studied by constituting the phylogenetic trees of yield groups with the aid of computer software (Eugene version 1.32.0) application. The probe for the presence of PHA types was also carried out with the aid of multiplex PCR using phaC, phaC1 (scl) and phaC2 (mcl-) primers. PHA yield identification was carried out by subjecting the products to Fourier-transform Infrared (FTIR) and Gas-Chromatography Mass-Spectrometry (GC-MS). All data obtained were subjected to statistical analysis. The highest bacterial load of 20.66 105 CFU/mL was obtained at 50m below water surface from latitude 6.35ᴼ N longitude 3.40ᴼ while the physicochemical results obtained for the sampled water revealed that of a typical hypersaline environment (Dissolved oxygen of 4.08 ± 0.067 – 4.42 ± 0.05 and pH of 7.70 ± 0.067 – 7.96 ± 0.05). Hence, the conventional identification revealed 18 different identical bacterial isolates with the highest occurrence of Corynebacterium kutscheri at all sites and depths. But, from the molecular identification methods, PHA screened bacterial isolates showed 49 novel (97% and less resemblance to sequence data found deposited with NCB) out of 50 bacterial isolates obtained, but Bacillus subtilis strain ASKAMI3 showed 100% resemblance with other bacteria at repository NCBI gene bank. The general optimal growth conditions obtained for the bacterial isolates were pH 6 and 7 across all periods except, Bacillus badius and Lactobacillus delbrueckii viii strains that had their optimal growth at pH 2. For temperature optimal growth, 60 °C for 24 hours incubation was obtained for halophiles. Optimal growth of bacterial isolates revealed 18 hours for 6–12% salt concentration for halotolerant bacteria. Out of the 62.6% (n=190) PHA producing potential bacterial isolates, 44.5% actually produced PHA. The highest PHA weight yield of 1.2630 ± 0.0170 g/L was obtained from Alcaligenes faecalis strain N1-4 (94.83% resemblance). This bacterium was positive for short chain length–scl (phaC1) PHA production potential. However, the analysis from FTIR and GC-MS detected the different building compounds for PHA production. This research work has established the fact that bacterial isolates obtained from the hypersaline water body of Lagos, Nigeria were able to produce PHA that could be employed in the various applications in medicine, engineering, agriculture, entertainment and other household utilities. Furthermore, the proper integration of this research outcome into bioplastics production will definitely be a major contributor to the solution to global warming.