Screening and Characterization of Polyhydroxyalkanoates-Producing Bacteria Isolated from Soda Lakes in Kenya for Biopolymer Production with Fruit Peels Residues as Feedstock

Abstract

Plastics derived from fossil fuels are associated with major post-use challenges and environmental pollution. Polyhydroxyalkanoates (PHAs) are bio-based polymers synthesized by microorganisms as storage products under constrained nutrition and excess carbon sources. Bacterial-based PHAs are ideal for replacing synthetic plastics because they possess desirable physical and mechanical properties and are readily biodegradable. Extremophiles such as those isolated from high salt and pH environments are ideal microorganisms for PHA production because of their ability to grow in growth media that minimize contaminations. Hence, this study aimed to (1) assess the diversity of polyhydroxyalkanoate (PHA)-producing bacteria from the Kenyan soda lakes Simbi and Magadi, (2) optimize the fermentation conditions for PHA production by Halomonas alkalicola and Bacillus sp. JSM, (3) evaluate the impact of co-feeding volatile fatty acids (VFAs) on PHA accumulation, and (4) investigate the potential of using fruit waste residues as substrates for microbial PHA production by Halomonas alkalicola. Water, sediment, and soil samples were collected from eight locations of Lake Simbi and nine locations of Lake Magadi for isolation of PHA-producing bacteria using serial dilution and spread plating techniques. Isolates were screened for PHA production, and their identities were confirmed using morphological, biochemical, and molecular methods. Growth conditions were optimized for isolates with the highest PHA accumulation abilities for enhanced production. PHA production was also assessed using the short-chain fatty acids (SFCAs) acetate, butyrate, and propionate as co-substrates. The fermentation conditions (pH, temperature, NaCl concentration, incubation time, and carbon and nitrogen sources) were optimized and co-substrate utilization assessed by adding volatile fatty acids (acetate, propionate, and butyrate) to the fermentation medium. Lastly, fruit waste residues (banana, mango, orange, and pineapple peels) were acid-pretreated and tested as alternative carbon sources for PHA production by Halomonas alkalicola. The findings revealed that lakes Simbi and Magadi were potent sources of PHA producing bacteria. Of the 218 isolates screened, 31 were positive for PHA production. The isolates belonged to six genera namely Arthrobacter spp. (1), Bacillus spp. (23), Exiguobacterium spp. (2), Halomonas spp. (1), Paracoccus spp. (2), and Rhodobaca spp (2). Gram negative bacterium Halomonas alkalicola isolated from Lake Simbi had the highest PHA concentration of 0.397 g/L of PHAs, which was significantly higher than that of the other 30 isolates (p < 0.01), on preliminary assessment. Initial assessment further revealed that Bacillus sp. JSM had the highest PHA content of 19.14%, which was significantly higher than that of other isolates (p < 0.05). The two were selected for further optimization. Through optimization of culture conditions, the PHA titers for H. alkalicola and Bacillus sp. were improved to 1.419 g/L and 0.514 g/L, respectively. For H. alkalicola, co-feeding the growth substrates with 0.1% (w/v) acetate, butyrate, and propionate resulted in PHA production of 1.47±0.03 g/L, 1.41±0.07 g/L, and 1.28±0.06 g/L. Notably, the polymer synthesized in the presence of propionate precursors contained 18.36±0.77 3-hydroxyvalerate (3HV) monomer fraction, which was significantly higher than that without the precursors (control) or while using acetate or butyrate (p < 0.001). Further investigations revealed that Halomonas alkalicola was capable of utilizing fruit waste residues as sole carbon sources for PHA accumulation. The bacterium was capable of accumulating 0.41 g/L, 0.39 g/L, 0.45 g/L, and 0.28 g/L of PHAs from banana peels, mango peels, orange peels, and pineapple peels, respectively under optimized conditions of 3.0% (w/v) H2SO4 pretreatment, 4%(w/v) substrate loading, and 20:1 to 30:1 carbon/nitrogen ratios. Although the bacterium showed preference for commercially available simple sugars over fruit waste residues as carbon sources, it demonstrated potential for beneficial use of waste streams for production of useful biomaterials. Thus, soda lakes bacteria isolated in this study were not only capable of producing biodegradable polymers but could also utilize fruit waste streams. In conclusion, this study shows the presence of diverse halophilic bacteria from Kenyan soda lakes, of which a high-yielding strain Halomonas alkalicola could be harnessed through optimal culture conditions for PHA production. Consequently, this study recommends the use of H. alkalicola for PHA production with orange peels hydrolysates as a carbon source. In addition, further capacity development is recommended through large-scale production, genetic improvement, and identification of high-potent strains for commercialization of polyhydroxyalkanoates production in Kenya. Future studies should prioritize the exploration of new soda lakes and other extreme environments for bioprospection of PHA-producing bacteria and the use of new feedstock sources. This study highlights the potential of soda lake bacteria as a sustainable solution for bioplastic production, contributing to environmental conservation and promoting waste management in Kenya.