ISOLATION, IDENTIFICATION AND SCREENING OF HALOPHILIC BACTERIA FOR POLYHYDROXYALKANOATE (PHA) IN HYPERSALINE LAGOS WATER BODY, NIGERIA

Fayemi, Scott O 1 , Boboye Bolatito 2 and Olukunle Oluwatoyin F 3 . 1. Redeemer’s University, Department of Biological Sciences, Faculty of Natural Sciences, PMB 230, Ede, Osun State, Nigeria. 2. Federal University of Technology, Akure (FUTA), Department of Biotechnology, PMB 704, Akure, Ondo State, Nigeria. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History Received: 10 September 2019 Final Accepted: 12 October 2019 Published: November 2019

The biomolecules such as polyhydroxyalkanoate (PHA) produced by halophilic microbes are stable due to their ability to live under extreme conditions (Oren, 2013;Parthasarathi et al., 2013;Santini and Warren, 2015). Polyhydroxyalkanoates (PHAs) are biopolymers and may be produced by bacteria in order to overcome environmental stresses that are characteristic of hypersalinity. The vast attention towards PHAs is mainly attributed to its properties that resemble some petrochemical plastics, as well as its biocompatibility and complete biodegradability. Microbial activity in the natural environment breaks down such bioplastics to release carbon dioxide and water or methane under aerobic and anaerobic conditions (Naik et al., 2008). PHAs are biodegradable and of immense importance in the applications of medicine, engineering, agriculture, telecommunications, transportation and household utilities (Aditi et al., 2015;Zhang et al., 2015;Manikandan and Senthilkumar, 2017). They are inevitably important as components of production for razors, utensils, diapers, feminine hygiene products, cosmetics containers, shampoo bottles and so on. PHAs can be incorporated into packaging components such as coatings, laminations and biodegradable printing inks (Reddy et al., 2003;Mojaveryazdia and Rezaniac, 2013). PHA structures include rigid thermoplastics, thermoplastic elastomers and grades useful in waxes, adhesives and binders. Properties range from elastomeric to resins as stiff as nylon 6 or polycarbonate (Hall, 1981 Saharan et al., 2014) revealed that little or no information has been found on the water bodies of Nigeria. It is therefore imperative to investigate the types of halophilic bacteria and their PHA producing potentials in Nigerian water bodies.

Collection of water samples
Marine water samples of three (3) replicates from each location of Lagos State, Nigeria Southern Atlantic Ocean water body were collected aseptically with the aid of a water sampler using the modified methods of water sampling by Bugnicourt et al., (2014). The water samples were collected at three different depths (surface: 0-0.07m, middle: 50 meters and bottom: 100 meters below water surface) into a 1 litre separate sterile plastic container. The locations considered for the depths of the water collection were recorded from the Geographic Positioning System (GPS) as (1) latitude 6.35ᴼ N longitude 3.28ᴼ E (ST1); 2) latitude 6.35ᴼ N longitude 3.40ᴼ E (ST2); and 3) latitude 6.36ᴼ N longitude 3.47ᴼ E (ST3).The temperatures of the collected water samples were recorded in situ.

Enumeration, isolation and identification of bacterial isolates
The sampled water was serially diluted from 10 -1 to 10 -7 , cultured by spread plate method on plate count agar (PCA) and incubated at 35°C for 24 to 48 hours. The colonies were counted.
Bacteria were characterized based on cultural characteristics which are; margins, elevation, growth shape. The methods of Holt (1994) were employed for the morphological characteristics of the cells and these includes gram stain reaction, cells spore, acid fast staining reaction, cell shape and cellular arrangements. The biochemical assays in order to determine the absence/presence of catalase, oxidase; citrate utilization, nitrate reduction, the probe 472 of the bacteria to determine if it is a strict anaerobe or not, the ability to hydrolyze starch. Also, included are assays to determine sugar fermentation ability, Vogues Prokauer (VP) and ability to grow in 6.5% sodium chloride. At the end of the assays on each of the isolates, each organism was identified with the Bergey's Manual of Systematic Bacteriology as utilized by Tissari, et al. (2010).

Determination of the effects of 3% (w/v) NaCl salt concentration on bacterial isolates
Minimal salt medium (MSM) was prepared with the following constituents: K 2 HPO 4 (0.5g/L), KH 2 PO 4 (0.04g/L), MgSO 4 (0.05g/L), Ammonium dihydrogen phosphate (0.04g/L), Ferrous sulphate (0.001g/L), glucose (8% w/v) and NaCl (3% w/v). The medium was sterilized in the autoclave at 121°C for 15 minutes at 15 psi, allowed to cool in the respective distributed test tubes, after which inoculation of pure isolates at 1 10 4 cell per mL was carried out aseptically, then subsequently incubated at 37°C for 2 to 72 hours before absorbance was taken at 520nm with the aid of Spectrophotometer.

Physicochemical analysis of water samples
Physical, chemical and trace/heavy metal analyses were conducted on the marine water samples according to Bugnicourt, et al. (2014). The physical parameters temperature, pH, turbidity, conductivity and total suspended solids were studied according to Galinha, et al., (2018). The chemical parameter total acidity, total alkalinity, chloride by titrimetric method, nitrate, sulphate were also determined with the aid of spectrophotometry methods (Germer et al., 2014). Dissolved oxygen and chemical oxygen demand were determined by Ibtisam and Karim, (2012) methods, while biological oxygen demand were determined according to Kosseva and Web, (2013) methods.

Assay for PHA Production
Each bacterial isolate was grown in 100 mL minimal salt medium supplemented with 8% (w/v) glucose in a 250 mL conical flask and incubated for 37 °C for 72 hours 100 rpm in an incubator. Thereafter, PHA production was investigated using Sudan Black B staining methods (Khanna and Srivastava, 2005). Data were recorded based on the positive observation of black colouration of the cells and negative observation of pink coloration of the cells. All numerical data were subjected to statistical analysis using versions of IBM SPSS 2.0 and Microsoft Excel 2010 software. St1= latitude 6.35ᴼ N longitude 3.28ᴼ E; St2= latitude 6.35ᴼ N longitude 3.40ᴼ E; St3= latitude6.36ᴼ N longitude 3.47ᴼ E; Top= 0.07m water surface; Middle= 50m below water surface; Bottom= 100m below water surface       Legend: a=Yersinia pestis; b=Bacillus badius; c=Corynebacterium kutsceri; d=Neiserra veilloneilla; e=Bacillus megaterium; f=Enterobacter intermedius; g=Klebsiella pneumonia subsp pneumoniae; h=Enterobacter amnigenus; i=Staphylococcus epidermidis; j=Micrococcus luteus; k=Staphylococcus aureus; l=Micrococcus varians; m=Mycobacterium smegmatids; n=Staphylococcus saprophyticus; o=Mycobacterium delbrueckii; p=Bacillus megaterium; q=Lactobacillus delbrueckii; r=Serratia marcescens;

Results:-
Figure shows the effect of 3% (w/v) salt on bacteria isolated from Lagos, Nigerian hypersaline water body. There was no significant (p<0.05) increase in the isolated bacteria growth before 41 hours of incubation. However, Lactobacillus delbrueckii among the other bacteria showed a notable decline in growth (OD -0.77) after 2 hours of incubation. The highest growth (OD of 2.87) was recorded for Neisseria veillonella at 53 hours of incubation but, declined at 72 hours to OD 2.68. The graph generally showed that the highest growth for many of the bacterial was obtained at 53 hours of incubation before.    Legend. St1= latitude 6.35ᴼ N longitude 3.28ᴼ E; St2= latitude 6.35ᴼ N longitude 3.40ᴼ E; St3= latitude 6.36ᴼ N longitude 3.47ᴼE. Table 4.5 shows the physical characteristics of the hypersaline water body of Lagos, Nigeria, where pH ranged between (7.70±0.067 and 7.96±0.05) for st2 at 100 m below water surface and st2 at 0-7 cm water surface; Turbidity between (0.166±0.07 and 37.4±0.04) NTU (Nephelometric unit) for 0-7 cm water surface in St2 and 50 m below water surface at St1 respectively; conductivity between (37300±462 and 97 43100±731.20) µS/cm for 100 m below water surface and 50 m below water surface in St1 and St3 respectively; and TDS between (20500±530 and 0723900±94.07) mg/L for 100 m below water surface and 50 m below water surface in St1 and St3 respectively.  3 reveals that Corynebacterium kutscheri is widely distributed organism found across all sites and depths sampled in this research. It also shows that there is no significant difference (p < 0.05) in the occurrence of this same bacterial isolates between 0-0.07m and 50 m below surface of the water but reveals tremendous difference between the aforementioned depths with that of 100 m below water surface (p < 0.05). This may be as a result of the lower dissolved oxygen recorded at 100 m below water surface compared to the values that of 0-0.07 m and 50 m below surface water which has no significant values (p < 0.05  3 shows the initiation of exponential period at 53 hours from the effects of 3% salt on the growth of bacteria isolates from Lagos, Nigeria hypersaline water body. Hence, apart from the adjustment of bacterial isolates to temperature, osmotic pressure, atmospheric pressure, pH, and moisture availability among other environmental factors (Kaye and Baross, 2004), the bacterial isolates displayed the characteristic of accumulating enough salt concentration ( known as lag period of which may also be term period of growth adjustment) in order to initiate the proliferation (cellular multiplication), and this may be because their genetic makeup may require sodium chloride or salt for growth (Elabed et al., 2019). Figure 4.3 also displayed the 'salt shock' experienced of Lactobacillus delbrueckii where the initial inoculum size was seen to be significantly decreased (p ≥ 0.05) before 53 hour after culture when it fully recovered. Thus, the acclimatizing period of over 52 hours of accumulation may be required for 3 % of salts (NaCl) concentration for Lactobacillus delbrueckii for the initiation of proliferation as observed in this research. Figure 4.3 clearly shows that Lactobacillus delbrueckii experienced 'salt shock' at 2 hours of incubation before progressive recovery at 41 hours after incubation.

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All eighteen (18) bacterial isolated conventionally are identified as Bacillus species 4 (22.22%) species, Corynebacterium specie, 1 (0.06%), Enterobacter 2 (0.11%), Klebsiella 1 (0.06%), and Micrococcus 2 (0.11%) species. Mycobacterium also recorded 2 (0.06%) species, Neisseria veillonella 1 (0.06%), Staphylococcus species 3 (0.17%) and Yersinia 1 (0.06%). Hence from these bacterial isolates, Bacillus species have been predominantly known to produce PHA under different environmental conditions and carbon source (Halami, 2009). Also, Enterobacter species do accumulates PHA production (Ceyhan and Guven, 2011) even when isolated from other source but may be dependent upon the conditions as well as genetic inclination for PHA production. Corynebacterium species is another PHA producer when circumstance triggers the production. These bacteria are members of the taxonomically related genera Rhodococcus, Nocardia and Corynebacterium, and it has been discovered that 3HV and 3HB monomers present in PHA is dependent on the carbon source and Laos noted that 3HV is generally the major 3-hydroxyacid produced (Anderson et al, 1990).

Conclusion:-
The data available from this research hereby suggests the presence of Corynebacterium kutsceri proliferation at all the three depths and sites researched in this work, it also pointed that their population is reduced as the available concentration of dissolved oxygen decreases. C. kutsceri is a PHA producer among others viz: