Molecular Characterization, Optimization and Production of PHA by Indigenous Bacteria Using Alternative Nutrient Sources as Substrate
B. N. Dienye *
Department of Microbiology, University of Port Harcourt, Rivers State, Nigeria.
O. K. Agwa
Department of Microbiology, University of Port Harcourt, Rivers State, Nigeria.
G. O. Abu
Department of Microbiology, University of Port Harcourt, Rivers State, Nigeria.
*Author to whom correspondence should be addressed.
Abstract
Polyhydroxyalkanoates (PHA) are renewable, biodegradable biopolymer intracellularly accumulated by wide array of microorganisms as carbon reserve. This study investigates the influence of various cultural conditions on PHA production by a recently isolated local species under submerged fermentation. Six PHA producing strains were identified by 16S rDNA gene sequencing and strain Priestia flexa OWO1 showed satisfactory PHA productivity. The effects of production parameters were investigated and extraction of PHA was carried out using sodium hypochlorite method and maximum amount was detected after 72h. Maximum PHA production was obtained at incubation period of 48h, pH of 7.0 and temperature of 30oC. Amongst the hydrolysate of agro waste used, brewers spent grain (BSG) gave maximum yield of 3.01g/L while beans bran powder gave the highest PHA yields of 3.9 g/L amongst the organic nitrogen sources tested. Analysis of the crude PHA by Fourier Transform Infrared Spectroscopy (FT-IR) showed the presence of methyl, methylene as well as carbonyl functional groups. PHA production was higher after optimizing the production conditions as compared to basal medium therefore the utilization of these cheap renewable resources as alternative substrates for production of PHA make the process cost effective and sustainable.
Keywords: Polyhydroxyalkanoates, Priestia flexa, FTIR, submerged fermentation, hydrolysate
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Koller M, Rodr´ıguez- Contreras A. Techniques for tracing PHA-producing organisms and for qualitative and quantitative analysis of intra- and extracellular PHA. Engineering in Life Sciences. 2015;15:558–581.
Shamsuddin IM, Jafar AJ, Abubakar SAS, Yusuf S, Lateefah M, Aminu I. Bioplastics as Better Alternative to Petroplastics and Their Role in National Sustainability:A Review. Advances in Bioscience and Bioengineering. 2017;5(4):63-70.
Jain R, Tiwari A. Biosynthesis of planet friendly bioplastics using renewable carbon source Journal of Environmental Health Science and Engineering. 2015;13(11):2-5.
Bejagam KK, Gupta NS, Le K-S, Iverson CN, Marrone BL, Pilania G. Predicting the Mechanical Response of Polyhydroxyalkanoate Biopolymers Using Molecular Dynamics Simulations. Polymers. 2022;14 :345.
El-Abd MA, El-Sheikh HH, Desouky S, Shehab A. Identification, biodegradation and bio-evaluation of biopolymer produced from Bacillus thuringenesis. Journal of Applied Pharmaceutical Science. 2017;7 (04):103-110.
Al-Battashi H, Annamalai N, Al-Kindi S, Nair AS, Al-Bahry S, Verma JP, Sivakumar N. Production of bioplastic (poly-3-hydroxybutyrate) using waste paper as a feedstock:Optimization of enzymatic hydrolysis and fermentation employing Burkholderia sacchari. Journal of Cleaner Production. 2019;1-46
Muhammadi S, Muhammad A, Shafqat H. Bacterial polyhydroxyalkanoates eco-friendly next-generation plastic:Production, biocompatibility, biodegradation, physical properties, and application. Green Chemistry Letters and Reviews. 2015;8:3-4.
Satchasataporn K, Duangsri C, Charunchaipipat W, Laloknam S, Burut-Archanai S, Powtongsook S, Akrimajirachoote N, Raksajit W. Enhanced production of poly-3-hydroxybutyrate and carotenoids by Arthrospira platensis under combined glycerol and phosphorus supplementation. Science Asia. 2022;48: 1-9.
Rocha CJL, Alvarez-Castillo E, Yanez MRE, Bengoechea C, Guerrero A, Ledesma MTO. Development of bioplastics from a microalgae consortium from wastewater Journal of Environmental Management. 2020;263 (110353):1-8.
Mitra R, Xu T, Xiang H. Ha, J. Current developments on polyhydroxyalkanoates synthesis by using halophiles as a promising cell factory. Microbial Cell Factories. 2020;19 (86):1-30.
Samrot AV, Samanvitha KS, Shobana N, Renitta RE, Senthilkumar P, Kumar SS, et al. The synthesis, characterization and applications of polyhydroxyalkanoates (PHA) and PHA-Based nanoparticles. Polymers. 2021;13(3302):1-29.
McAdam B, Fournet MB, McDonald P, Mojicevic M. Production of polyhydroxybutyrate (PHB) and factors impacting its chemical and mechanical characteristics. Polymers. 2020;12:2- 20.
Amadu AA, Qiu S, Ge S, Addico GND, Ameka G K, Yu Z, et al. A review of biopolymer (Poly-β-hydroxybutyrate) synthesis in microbes cultivated on wastewater. Science of the Total Environment. 2021;756 (143729).
Mascarenhas J, Aruna K. Production and characterization of polyhydroxyalkanoates (Pha) By Bacillus Megaterium strain jha using inexpensive agro-industrial wastes. International Journal of Recent Scientific Research. 2019;10(7):33359-33374.
Tufail S, Munir S, Jamil N. Variation analysis of bacterial polyhydroxyalkanoates production using saturated and unsaturated hydrocarbons. Brazilian Journal of Microbiology. 2017;48 (1):629-636.
Sayyed RZ, Shaikh SS, Wani SJ, Rehman MT, Al Ajmi MF, Haque S, El Enshasy HA. Production of biodegradable polymer from agro-wastes in Alcaligenes sp. and Pseudomonas sp. Molecules. 2021;26: 2443.
Choi T-R, Park Y-L, Song H-S, Lee SM, Park SL, Lee HS et al. Fructose-based production of short-chain-length and medium-chain-length polyhydroxyalkanoate copolymer by Arctic Pseudomonas sp. B14-6. Polymers. 2021;13 (1398):1-14
Hamdy SM, Danial AW, Sanaa MF, El‑Ra G, Shoreit AAM, Hesham A EL. Production and optimization of bioplastic (Polyhydroxybutyrate) from Bacillus cereus strain SH‑02 using response surface methodology. BMC Microbiology. 2022;22:183.
Ahmed OB, Dablool AS. Quality improvement of the DNA extracted by boiling method in gram negative bacteria.International Journal of Bioassays 6.04. 2017;5347-5349.
Srinvivasan R, Karaoz U, Volegova M, Macichan J, Kato-Maeda M, Miller S, Nadarajan R, Brodie EL, Lynch SV. Use of 16SrRNA gene for identification of a broad range of clinically relevant bacterial pathogens. PLoS One. 2015;10(2): 0117617.
Mohapatra S, Samantaray DP, Samantaray S. Phylogenetic heterogeneity of the rhizospheric soil bacterial isolates producing PHAs revealed by comparative analysis of 16s-rRNA. International Journal of Current Microbiology and Applied Science. 2014;3(5):680-690.
Chandani N, Mazumder PB, Bhattacharjee A. Potential of dumpsite bacterial isolate in producing polyhydroxybutyrate under stress prone environment. Indian Journal of Experimental Biology. 2020;58:643-650.
Naheed N, Jamil N. Optimization of biodegradable plastic production on sugar cane molasses in Enterobacter sp. SEL2. Brazilian Journal of Microbiology. 2014;45 (2):417-426.
Bharathi B, Gowdhaman D, Ponnusami V. Isolation and identification of polyhydroxybutyrate (PHB) producing Bacillus cereus BB613-A Novel Isolate. International Journal of ChemTech Research. 2016;9(11):224-228.
Getachew A, Woldesenbet F. Production of biodegradable plastic by polyhydroxybutyrate (PHB) accumulating bacteria using low cost agricultural waste material. BMC Research Notes. 2016;9:509.
Odeniyi OA, Adeola O J. Production and characterization of polyhydroxyalkanoic acid from Bacillus thuringiensis using different carbon substrates. International Journal of Biological Macromolecules. 2017;104:407–413.
Aburas MMA. Production of poly β-hydroxybutyrate from Pseudomonas putida MA102 isolated from waste water sample. Journal of American Science. 2016;12(5): 107-112.
Basnett P, Marcello E, Lukasiewicz B, Panchal B, Nigmatullin R, Knowles JC, Roy I. Biosynthesis and characterization of a novel, biocompatible medium chain length polyhydroxyalkanoate by Pseudomonas mendocina CH50 using coconut oil as the carbon source. Journal of Materials Science: Materials in Medicine. 2018;29:179.
Biradar G G, Shivasharana CT, Kaliwal BB. Isolation and characterization of polyhydroxybutyrate (PHB) producing Bacillus species from agricultural soil. European Journal of Experimental Biology. 2015;5(3):58-65.
Saratale RG, Cho SK, Saratale GD, Ghodake GS, Bharagava RN, Kim DS, Nair S, Shin HS. Efficient bioconversion of sugarcane bagasse into polyhydroxybutyrate (PHB) by Lysinibacillus sp. and its characterization. Bioresource Technology. 2021; 324:124673.
Ferreira EM, Procópio ARL, Pereira Junior RC, Zanotto SP, Procópio R EL. Polyhydroxyalkanoate (PHA) production by Lynisibacillus sp. strain UEA-20.171 African Journal of Biotechnology. 2016; 15(34):1827-1834.
Gupta RS, Patel S, Saini N, Chen S. Robust demarcation of 17 distinct Bacillus species clades proposed as novel Bacillaceae genera, by phylogenomic and comparative genomic analyses: description of Robertmurraya kyonggiensis sp. nov. and proposal for an emended genus Bacillus limiting it only to the members of the substilis and cereus clades of species. International Journal of Systematic and Evolutionary Microbiology. 70(11):5753-5798.
Jangra MR. Nehra KS. Isolation, screening and characterization of new strains with optimization studies to augment bacterial PHB production. Bulletin of Environment, Pharmacology and Life Sciences. 2017;6(8):34-44.
El-Sheekh MM, El-Abd M., El-Diwany AI, Ismail AS, Omar TH. Poly-3-hydroxybutyrate (PHB) production by Bacillus flexus ME-77 using some industrial wastes. Rendiconti Fisiche. Acc. Lincei. 2015;26:109–119.
Damle P, Vaidya VK. Isolation, identification and characterization of polyhydroxybutyrate producing Bacillus flexus. International Journal of Engineering Sciences & Research Technology. 2016; 5(11):106-114.
Gabr GA. Isolation and identification of bacterial strains able to biopolymer Polyhydroxybutyrate (Phb) production from soil of Al-Kharj probes, Saudi Arabia. Journal of Pharmaceutical Research International. 2018;21(6):1-11
Johri M, Rajaguru B, Pillai G. Optimization of cultural conditions for polyhydroxybutyrate (phb) production by Alcaligenes sp. from mangrove soil. Journal of Advanced Scientific Research 2021;HBIA:19-25.
Corchado- Lopo C, Martínez-Avila O, Marti E, Llim´os J, Busquets AM, Kucera D, Obruca S, Llenas, L, Ponsa S. Brewer’s spent grain as a no-cost substrate for polyhydroxyalkanoates production: Assessment of pretreatment strategies and different bacterial strains. New Biotechnolog. 2021;62:60–67.
Llimós J, Martínez-Avila O, Marti E, Corchado-Lopo C, Llenas L, Gea T, et al. Brewer’s spent grain biotransformation to produce lignocellulolytic enzymes and polyhydroxyalkanoates in a two- stage valorization scheme. Biomass Conversion and Biorefinery. 2020;12:3921-3932.
Danial AW, Hamdy SM, Alrumman, SA, Gad El-Rab SMF, Shoreit AA M, Hesham AE-L. Bioplastic Production by Bacillus wiedmannii AS-02 OK576278 Using Different Agricultural Wastes. Microorganisms. 2021;9 (2395):1-19.
El-Kadi SM, Elbagory M, EL-Zawawy HAH, EL-Shaer, HFA, Shoukry AA, El-Nahrawy S, Omara, AE-D, Ali DFI. Biosynthesis of Poly-ß-Hydroxybutyrate (PHB) from Different Bacterial Strains Grown on Alternative Cheap Carbon Sources. Polymers, 2021;13(3801):1-20.
Rathika R, Janaki V, Shanthi K, Kamala Kannan S. Bioconversion of agro-industrial effluents for polyhydroxyalkanoates production using Bacillus subtilis RS1. International Journal of Environmental Science and Technology. 2019;16(10): 5725-5734.
Sharma P, Bajaj B. K. Economical production of poly-3-hydroxybutyrate by Bacillus cereus under submerged and solid-state fermentation. Journal of Material and Environmental Science. 2016;7(4):1219-1228.
Bose SA, Raja S, Jeyaram K, Arockiasam S, Velmuruga S. Investigation of fermentation condition for production enhancement of polyhydroxyalkanoate from cheese whey by Pseudomonas Sp. Journal of Microbiology, Biotechnology and Food Science, 2020;9(5):890-989.
Guinhawa DJE, Arevalo AC, Cueto MGC, Lontok DJC, Sandro KAC, Magbojos-Magtibay CR. Isolation, biochemical characterization and optimization of polyhydroxyalkanoates (PHA) producing bacteria. The STETH, 2018;12:72-89.
Omkars J, Rohini S. Production of biodegradable plastic by polyhydroxybutyrate (Phb) accumulating bacteria using low cost agricultural waste material. Global Scientific Journal. 2018; 6(7):1091-1108.
Flora G, Bhatt K, Tuteja U. Optimization of culture conditions form poly Â-Hydroxybutyrate production from isolated bacillus species. Journal of Cell and Tissue Research. 2010;10:2235–2242.
Sadaf N, Kamthane DC. PHB production from bacteria isolated from oil contaminated soil. Bioscience Discovery. 2019;10(2):103-107.
Saratale RG, Cho SK, Saratale GD, Ghodake GS, Bharagava RN, Kim DS, et al. Efficient bioconversion of sugarcane bagasse into polyhydroxybutyrate (PHB) by Lysinibacillus sp. and its characterization. Bioresource Technology. 2021;324 (124673):1-6.
Belal EB, Farid MA. Production of Poly-β-hydroxybutyric acid (PHB) by Bacillus cereus. International Journal of Current Microbiology and Applied Sciences. 2016;5 (7):442-460.
Aly MM., Alharbi S, Bokhari F. Synthesis of biodegradable polymer (PHB) by Bacillus subtilis isolated from rhizosphere soil sample and Optimization of growth conditions. IOSR Journal of Pharmacy and Biological Sciences. 2019;14 (6):34-44.
Mahato RP, Kumar S, Singh P. Optimization of growth conditions to produce sustainable polyhydroxyalkanoate bioplastic by Pseudomonas aeruginosa EO1. Frontier in Microbiology. 2021;12 (711588):1-9.
Ahmad S, Amir A, Zafaryab M, Osama K, Faridi SA, Siddiqui MH, Rizvi MMA, Khan MA. Production and characterization of polyhydroxybutryrate biopolymer from Azohydromonas australica using sucrose as a sole carbon source. Journal of Microbial and Biochemical Technology. 2017;9(3):082-086.