Optimization of cultivation medium and cyclic fed-batch fermentation strategy for enhanced polyhydroxyalkanoate production by Bacillus thuringiensis using a glucose-rich hydrolyzate

Abstract

The accumulation of petrochemical plastic waste is detrimental to the environment. Polyhydroxyalkanoates (PHAs) are bacterial-derived polymers utilized for the production of bioplastics. PHA-plastics exhibit mechanical and thermal properties similar to conventional plastics. However, high production cost and obtaining high PHA yield and productivity impedes the widespread use of bioplastics. This study demonstrates the concept of cyclic fed-batch fermentation (CFBF) for enhanced PHA productivity by Bacillus thuringiensis using a glucose-rich hydrolyzate as the sole carbon source. The statistically optimized fermentation conditions used to obtain high cell density biomass (OD₆₀₀ of 2.4175) were: 8.77 g L⁻¹ yeast extract; 66.63% hydrolyzate (v/v); a fermentation pH of 7.18; and an incubation time of 27.22 h. The CFBF comprised three cycles of 29 h, 52 h, and 65 h, respectively. After the third cyclic event, cell biomass of 20.99 g L⁻¹, PHA concentration of 14.28 g L⁻¹, PHA yield of 68.03%, and PHA productivity of 0.219 g L⁻¹ h⁻¹ was achieved. This cyclic strategy yielded an almost threefold increase in biomass concentration and a fourfold increase in PHA concentration compared with batch fermentation. FTIR spectra of the extracted PHAs display prominent peaks at the wavelengths unique to PHAs. A copolymer was elucidated after the first cyclic event, whereas, after cycles CFBF 2–4, a terpolymer was noted. The PHAs obtained after CFBF cycle 3 have a slightly higher thermal stability compared with commercial PHB. The cyclic events decreased the melting temperature and degree of crystallinity of the PHAs. The approach used in this study demonstrates the possibility of coupling fermentation strategies with hydrolyzate derived from lignocellulosic waste as an alternative feedstock to obtain high cell density biomass and enhanced PHA productivity.

Keywords

Polyhydroxyalkanoate / Glucose-rich hydrolyzate / Response surface methodology / Cyclic fed-batch fermentation / PHA productivity / Pulp and paper mill sludge

References

[1] Andersen RKI, Jayaraman K. Influence of carbon and nitrogen sources on the growth and sporulation of Bacillus thuringiensis var galleriae for biopesticide production. Chem Biochem Eng Q, 2003, 17: 225-231.

[2] Annamalai N, Sivakumar N. Production of polyhydroxybutyrate from wheat bran hydrolysate using Ralstonia eutropha through microbial fermentation. J Biotechnol, 2016, 237: 13-17.

[3] Annamalai N, Al-battashi H, Al-bahry S, Sivakumar N. Biorefinery production of poly-3-hydroxybutyrate using waste office paper hydrolysate as feedstock for microbial fermentation. J Biotechnol, 2017, 265: 25-30.

[4] Apparao U, Krishnaswamy VG. Production of polyhydroxyalkanoate (PHA) by a moderately halotolerant bacterium Klebsiella pneumoniae U1 isolated from rubber plantation area. Int J Environ Bioremediat Biodegrad, 2015, 3: 54-61.

[5] Aramvash A, Moazzeni Zavareh F, Gholami Banadkuki N. Comparison of different solvents for extraction of polyhydroxybutyrate from Cupriavidus necator. Eng Life Sci, 2018, 18: 20-28.

[6] Argyropoulos D, Lynch HC. Recombinant β-glucanase production and plasmid stability of Bacillus subtilis in cyclic fed batch culture. Biotechnol Tech, 1997, 11: 187-190.

[7] Baidurah S, Kubo Y, Kuno M, . Rapid and direct compositional analysis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in whole bacterial cells by thermally assisted hydrolysis and methylation-gas chromatography. Anal Sci, 2015, 31: 79-83.

[8] Beber VC, de Barros S, Banea MD, . Effect of Babassu natural filler on PBAT/PHB biodegradable blends: an investigation of thermal, mechanical, and morphological behavior. Materials, 2018, 11: 1-16.

[9] Buzarovska A, Grozdanov A. Crystallization kinetics of poly(hydroxybutyrate-co-hydroxyvalerate) and poly(dicyclohexylitaconate) PHBV/PDCHI blends: thermal properties and hydrolytic degradation. J Mater Sci, 2009, 44: 1844-1850.

[10] Cao Y, Meng DJ, Lu J, Long J. Statistical optimization of xylanase production by Aspergillus niger AN-13 under submerged fermentation using response surface methodology. African J Biotechnol, 2008, 7: 631-638.

[11] Chaijamrus S, Udpuay N (2008) Production and characterization of polyhydroxybutyrate from molasses and corn steep liquor produced by Bacillus megaterium ATCC 6748. Agric Eng Int CIGR Ejournal 1–12

[12] Cheng L, Wang J, Fu Q, . Optimization of carbon and nitrogen sources and substrate feeding strategy to increase the cell density of Streptococcus suis. Biotechnol Biotechnol Equip, 2015, 29: 779-785.

[13] Czyrski A, Sznura J. The application of Box–Behnken-design in the optimization of HPLC separation of fluoroquinolones. Sci Rep, 2019, 9: 1-10.

[14] Duff SJB, Moritz JW, Andersen KL. Simultaneous hydrolysis and fermentation of pulp mill primary clarifier sludge. Can J Chem Eng, 1994, 72: 1013-1020.

[15] Ferreira BS, Schlottbom C (2016) Production of polyhydroxybutyrate from lignocellulosic hydrolysates—optimization of Bacillus sacchari fermentation and scale up from 2 L to 200 L. Eppendorf 1–6

[16] Gahlawat G, Srivastava AK. Enhancing the production of polyhydroxyalkanoate biopolymer by Azohydromonas australica using a simple empty and fill bioreactor cultivation strategy. Chem Biochem Eng Q, 2018, 31: 479-485.

[17] García YG, Carlos CJCM, . Procurement of fermentable sugars from cardboard waste for the cultivation of yeasts for biotechnological use. Rev Mex Ciencias For, 2015, 6: 88-105.

[18] Getachew A, Woldesenbet F. Production of biodegradable plastic by polyhydroxybutyrate (PHB) accumulating bacteria using low cost agricultural waste material. BMC Res Notes, 2016, 9: 1-9.

[19] Gholamveisi N, Azar SM, Moravej R. Bacillus thuringiensis strain NG, a novel isolated strain for production of various polyhydroxyalkanoates. Biol J Microorg, 2018, 6: 13-20.

[20] Govender L (2013) Seasonal variation of microflora and their effects on the quality of wood chips intended for pulping. PhD Dissertation, University of KwaZulu-Natal

[21] Gowda V, Shivakumar S. Agrowaste-based polyhydroxyalkanoate (PHA) production using hydrolytic potential of Bacillus thuringiensis IAM 12077. Braz Arch Biol Technol, 2014, 57: 55-61.

[22] Haas C, El-najjar T, Virgolini N, . High cell-density production of poly(3-hydroxybutyrate) in a membrane bioreactor. N Biotechnol, 2017, 37: 117-122.

[23] Hanrahan G, Garza C, Garcia E, Miller K. Experimental design and response surface modeling: A method development application for the determination of reduced inorganic species in environmental samples. J Environ Inform, 2007, 9: 71-79.

[24] Hassan MA, Bakhiet EK, Ali SG, Hussien HR. Production and characterization of polyhydroxybutyrate (PHB) produced by Bacillus sp. isolated from Egypt. J Appl Pharm Sci, 2016, 6: 46-51.

[25] Ibrahim MHA, Steinbüchel A. High-cell-density cyclic fed-batch fermentation of a poly(3-hydroxybutyrate)-accumulating thermophile, Chelatococcus sp. strain MW10. Appl Environ Microbiol, 2010, 76: 7890-7895.

[26] Kehail AA, Foshey M, Chalivendra V, Brigham CJ. Thermal and mechanical characterization of solvent-cast poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). J Polym Res, 2015, 22: 1-8.

[27] Kucera D, Benesova P, Ladicky P, . Production of polyhydroxyalkanoates using hydrolyzates of spruce sawdust: comparison of hydrolyzates detoxification by application of overliming, active carbon, and lignite. Bioengineering, 2017, 4: 1-9.

[28] Kunasundari B, Sudesh K. Isolation and recovery of microbial polyhydroxyalkanoates. Express Polym Lett, 2011, 5: 620-634.

[29] Łabuzek S, Radecka I. Biosynthesis of PHB tercopolymer by Bacillus cereus UW85. J Appl Microbiol, 2001, 90: 353-357.

[30] Larsson S, Palmqvist E, Hahn-Hägerdal B, . The generation of fermentation inhibitors during dilute acid hydrolysis of softwood. Enzyme Microb Technol, 1999, 24: 151-159.

[31] Limkar MB, Pawar SV, Rathod VK. Statistical optimization of xylanase and alkaline protease co-production by Bacillus spp using Box–Behnken Design under submerged fermentation using wheat bran as a substrate. Biocatal Agric Biotechnol, 2019, 17: 455-464.

[32] Liu J, Zhao Y, Diao M, . Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production by Rhodospirillum rubrum using a two-step culture strategy. J Chem, 2019, 2019: 1-8.

[33] Lynch HC, Bushell ME. The physiology of erythromycin biosynthesis in cyclic fed batch culture. Microbiology, 1995, 141: 3105-3111.

[34] Madrid LM, Díaz JCQ. Ethanol production from paper sludge using Kluyveromyces marxianus. Dyna, 2011, 78: 185-191.

[35] Marques S, Alves L, Roseiro JC, Gírio FM. Conversion of recycled paper sludge to ethanol by SHF and SSF using Pichia stipitis. Biomass Bioenergy, 2008, 32: 400-406.

[36] Martínez-Sanz M, Villano M, Oliveira C, . Characterization of polyhydroxyalkanoates synthesized from microbial mixed cultures and of their nanobiocomposites with bacterial cellulose nanowhiskers. N Biotechnol, 2014, 31: 364-376.

[37] Martin-Sampedro R, Eugenio ME, Revilla E, . Integration of kraft pulping on a forest biorefinery by the addition of a steam explosion pretreatment. BioResources, 2011, 6: 513-528.

[38] Melo JDD, Carvalho LFM, Medeiros AM, . A biodegradable composite material based on polyhydroxybutyrate (PHB) and carnauba fibers. Compos Part B Eng, 2012, 43: 2827-2835.

[39] Montanheiroa TLdA, Passadora FR, de Oliveiraa MP, . Preparation and characterization of maleic anhydride grafted poly(hydroxybutirate-co-hydroxyvalerate)—PHBV-g-MA. Mater Res, 2016, 19: 229-235.

[40] Moritz JW, Duff SJB. Simultaneous saccharification and extractive fermentation of cellulosic substrates. Biotechnol Bioeng, 1996, 49: 504-511.

[41] Możejko-Ciesielska J, Kiewisz R. Bacterial polyhydroxyalkanoates: still fabulous?. Microbiol Res, 2016, 192: 271-282.

[42] Muhammadi S, Afzal M, Hameed S. Bacterial polyhydroxyalkanoates-eco-friendly next generation plastic: production, biocompatibility, biodegradation, physical properties and applications. Green Chem Lett Rev, 2015, 8: 56-77.

[43] Munir S, Iqbal S, Jamil N. Polyhydroxyalkanoates (PHA) production using paper mill wastewater as carbon source in comparison with glucose. J Pure Appl Microbiol, 2015, 9: 453-460.

[44] Nguyen H-YT, Tran G-B. Optimization of fermentation conditions and media for production of glucose isomerase from Bacillus megaterium using response surface methodology. Scientifica, 2018, 2018: 1-11.

[45] Peters D. Carbohydrates for fermentation. Biotechnol J, 2006, 1: 806-814.

[46] Pillai AB, Kumar AJ, Thulasi K, Kumarapillai H. Evaluation of short-chain-length polyhydroxyalkanoate accumulation in Bacillus aryabhattai. Braz J Microbiol, 2017, 48: 451-460.

[47] Pirt SJ. The theory of fed batch culture with reference to the penicillin fermentation. J Appl Chem Biotechnol, 1974, 24: 415-424.

[48] Pradhan S, Dikshit PK, Moholkar VS. Production, ultrasonic extraction, and characterization of poly(3-hydroxybutyrate) (PHB) using Bacillus megaterium and Cupriavidus necator. Polym Adv Technol, 2018, 29: 2392-2400.

[49] Prasetyo J, Kato T, Park EY. Efficient cellulase-catalyzed saccharification of untreated paper sludge targeting for biorefinery. Biomass Bioenergy, 2010, 34: 1906-1913.

[50] Rai R, Keshavarz T, Roether JA, . Medium chain length polyhydroxyalkanoates, promising new biomedical materials for the future. Mater Sci Eng R Rep, 2011, 72: 29-47.

[51] Rodrigues PR, Nunes JMN, Lordelo LN, Druzian JI. Assessment of polyhydroxyalkanoate synthesis in submerged cultivation of Cupriavidus necator and Burkholderia cepacia strains using soybean as substrate. Braz J Chem Eng, 2019, 36: 73-83.

[52] Rohini D, Phadnis S, Rawal SK. Synthesis and characterization of poly-β-hydroxybutyrate from Bacillus thuringiensis R1. Indian J Biotechnol, 2006, 5: 276-283.

[53] Sandhya M, Aravind J, Kanmani P. Production of polyhydroxyalkanoates from Ralstonia eutropha using paddy straw as cheap substrate. Int J Environ Sci Technol, 2013, 10: 47-54.

[54] Saranya V, Shenbagarathai R. Production and characterization of PHA from recombinant E. coli harbouring PHAC1 gene of indigenous Pseudomonas sp. LDC-5 using molasses. Braz J Microbiol, 2011, 42: 1109-1118.

[55] Schroeder BG, Zanoni PRS, Magalhães WLE, . Evaluation of biotechnological processes to obtain ethanol from recycled paper sludge. J Mater Cycles Waste Manage, 2015, 19: 463-472.

[56] Shang L, Fei Q, Zhang YH, . Thermal properties and biodegradability studies of poly(3-hydroxybutyrate-co-3-hydroxyvalerate). J Polym Environ, 2012, 20: 23-28.

[57] Sharma PK, Munir RI, de Kievit T, Levin DB. Synthesis of polyhydroxyalkanoates (PHAs) from vegetable oils and free fatty acids by wild-type and mutant strains of Pseudomonas chlororaphis. Can J Microbiol, 2017, 63: 1009-1024.

[58] Sheu D, Wang Y, Lee C. Rapid detection of polyhydroxyalkanoate-accumulating bacteria isolated from the environment by colony PCR. Microbiology, 2000, 146: 2019-2025.

[59] Sin MC, Tan IKP, Annuar MSM, Gan SN. Viscoelastic, spectroscopic, and microscopic characterization of novel bio-based plasticized poly(vinyl chloride) compound. Int J Polym Sci, 2014

[60] Sindhu R, Kuttiraja M, Prabisha TP, . Development of a combined pretreatment and hydrolysis strategy of rice straw for the production of bioethanol and biopolymer. Bioresour Technol, 2016, 215: 110-116.

[61] Singh M, Kumar P, Ray S, Kalia VC. Challenges and opportunities for customizing polyhydroxyalkanoates. Indian J Microbiol, 2015, 55: 235-249.

[62] Singh S, Sithole B, Lekha P, . Pretreatment and enzymatic saccharification of sludge from a prehydrolysis kraft and kraft pulping mill. J Wood Chem Technol, 2020

[63] Smitha KV, Pradeep BV. Application of Box-Behnken design for the optimization of culture conditions for novel fibrinolytic enzyme production by Bacillus altitudinis S-CSR 0020. J Pure Appl Microbiol, 2017, 11: 1447-1456.

[64] Surendran A, Lakshmanan M, Chee JY, . Can polyhydroxyalkanoates be produced efficiently from waste plant and animal oils?. Front Bioeng Biotechnol, 2020, 8: 1-15.

[65] Tesfaye T, Sithole B, Ramjugernath D, Ndlela L. Optimisation of surfactant decontamination and pre-treatment of waste chicken feathers by using response surface methodology. Waste Manage, 2018, 72: 371-388.

[66] Tesfaye T, Sithole B, Ramjugernath D, Ndlela L. Valorisation of waste chicken feathers: optimisation of decontamination and pre-treatment with bleaching agents using response surface methodology. Waste Manag, 2018, 72: 371-388.

[67] Thammasittirong A, Saechow S, Thammasittirong SN-R. Efficient polyhydroxybutyrate production from Bacillus thuringiensis using sugarcane juice substrate. Turk J Biol, 2017, 41: 992-1002.

[68] Torri C, Cordiani H, Samorì C, . Fast procedure for the analysis of poly(hydroxyalkanoates) in bacterial cells by off-line pyrolysis/gas-chromatography with flame ionization detector. J Chromatogr A, 2014, 1359: 230-236.

[69] Vahabi H, Michely L, Moradkhani G, . Thermal stability and flammability behavior of poly(3-hydroxybutyrate) (PHB) based composites. Materials (Basel), 2019, 12: 1-14.

[70] Volova TG, Zhila NO, Shishatskaya EI, . The physicochemical properties of polyhydroxyalkanoates with different chemical structures. Polym Sci Ser A, 2013, 55: 427-437.

[71] Wang Y, Liu S. Production of (R)-3-hydroxybutyric acid by Burkholderia cepacia from wood extract hydrolysates. AMB Express, 2014, 4: 1-10.

[72] Wellen RMR, Rabello MS, Júnior ICA, . Melting and crystallization of poly(3-hydroxybutyrate): effect of heating/cooling rates on phase transformation. Polímeros, 2015, 25: 296-304.

[73] Yang S, Franden MA, Yang Q, . Identification of inhibitors in lignocellulosic slurries and determination of their effect on hydrocarbon-producing microorganisms. Front Bioeng Biotechnol, 2018, 6: 1-14.

[74] Yu J, Stahl H. Microbial utilization and biopolyester synthesis of bagasse hydrolysates. Bioresour Technol, 2008, 99: 8042-8048.

[75] Zhang W, Alvarez-Gaitan JP, Dastyar W, . Value-added products derived from waste activated sludge: A biorefinery perspective. Water, 2018, 10: 1-20.

Funding

Department of Science and Technology, Republic of South Africa(Waste Roadmap project)