Abstract
Polyhydroxyalkanoates (PHAs) are biodegradable polyesters accumulated in a wide variety of microorganisms as intracellular carbon and energy storage compounds. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is one of the most valuable biopolymers because of its superior mechanical properties. Here, we developed a bioprocess utilizing recombinant Bacillus megaterium strain for PHBV over-production from glucose, without any precursor addition. PHA production was performed in a controlled bioreactor by batch and fed-batch modes using wild-type B. megaterium and rec-B. megaterium cells overexpressing the native phaC gene. The effect of oxygen transfer rate on biomass formation and PHA accumulation was also investigated, under different dissolved oxygen levels. Structural and thermal properties of PHA were characterized by GC–FID, 1H‐NMR, TGA and DSC analyses. Significantly, the copolymer produced from glucose as the carbon source in rec-B. megaterium was composed of 58 mol% of 3‐hydroxyvalerate monomers. After 66 h, rec-B. megaterium cells in fed-batch fermentation with a pre-determined growth rate µ0 = 0.1 h−1 produced the highest CDW (7.7 g L−1) and PHA concentration (6.1 g L−1). Moreover, an exponential glucose feeding profile resulted in 2.2-fold increase in PHA yield compared to batch cultivation. Overall, this study paves the way to an enhanced biopolymer production process in B. megaterium cells, where the highest product yield on cell was obtained as YP/X = 0.8 g g−1.
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References
Krishnan S, Chinnadurai GS, Perumal P (2017) Polyhydroxybutyrate by Streptomyces sp.: production and characterization. Int J Biol Macromol 104:1165–1171
Kumar M, Singhal A, Verma PK, Thakur IS (2017) Production and characterization of polyhydroxyalkanoate from lignin derivatives by Pandoraea sp. ISTKB ACS Omega 2:9156–9163
Ferre-Guell A, Winterburn J (2018) Biosynthesis and characterization of polyhydroxyalkanoates with controlled composition and microstructure. Biomacromol 19:996–1005
Koller M, Hesse P, Bona R, Kutschera C, Atlic A, Braunegg G (2007) Potential of various archae- and eubacterial strains as industrial polyhydroxyalkanoate producers from whey. Macromol Biosci 7:218–226
Urtuvia V, Maturana N, Peña C, Díaz-Barrera A (2020) Accumulation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Azotobactervinelandii with different 3HV fraction in shake flasks and bioreactor. Bioprocess Biosyst Eng 43:1469
Marangoni C, Furigo A, de Aragão GMF (2002) Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Ralstoniaeutropha in whey and inverted sugar with propionic acid feeding. Process Biochem 38:137–141
Suhazsini P, Keshav R, Narayanan S, Chaudhuri A, Radha P (2020) A study on the synthesis of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) by Bacillusmegaterium utilizing cheese whey permeate. J Polym Environ 28:1390–1405
Güngörmedi G, Demirbilek M, Mutlu MB, Denkbaş EB, Çabuk A (2014) Polyhydroxybutyrate and hydroxyvalerate production by Bacillusmegaterium strain A1 isolated from hydrocarbon-contaminated soil. J Appl Polym Sci 131:40530
Haywood GW, Anderson AJ, Roger Williams D, Dawes EA, Ewing DF (1991) Accumulation of a poly(hydroxyalkanoate) copolymer containing primarily 3-hydroxyvalerate from simple carbohydrate substrates by Rhodococcus sp. NCIMB 40126. Int J Biol Macromol 13:83–88
Porras MA, Ramos FD, Diaz MS, Cubitto MA, Villar MA (2019) Modeling the bioconversion of starch to P(HB-co-HV) optimized by experimental design using Bacillusmegaterium BBST4 strain. Environ Technol 40:1185–1202
Valappil SP, Rai R, Bucke C, Roy I (2008) Polyhydroxyalkanoate biosynthesis in Bacillus cereus SPV under varied limiting conditions and an insight into the biosynthetic genes involved. J Appl Microbiol 104:1624–1635
Aldor IS, Kim S-W, Prather KLJ, Keasling JD (2002) Metabolic engineering of a novel propionate-independent pathway for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in recombinant Salmonellaenterica serovar typhimurium. Appl Environ Microbiol 68:3848–3854
Chen Q, Wang Q, Wei G, Liang Q, Qi Q (2011) Production in Escherichiacoli of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with differing monomer compositions from unrelated carbon sources. Appl Environ Microbiol 77:4886–4893
Yang JE, Choi YJ, Lee SJ, Kang K-H, Lee H, Oh YH, Lee SH, Park SJ, Lee SY (2014) Metabolic engineering of Escherichiacoli for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from glucose. Appl Microbiol Biotechnol 98:95–104
Han J, Hou J, Zhang F, Ai GM, Li M, Cai SF, Liu HL, Wang L, Wang ZJ, Zhang SL, Cai L, Zhao DH, Zhou J, Xiang H (2013) Multiple propionyl coenzyme a-supplying pathways for production of the bioplastic poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in Haloferaxmediterranei. Appl Environ Microbiol 79:2922–2931
Heinrich D, Raberg M, Steinbuchel A (2015) Synthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from unrelated carbon sources in engineered Rhodospirillumrubrum. FEMS Microbiol Lett 362:fnv038
Zhang YZ, Liu GM, Weng WQ, Ding JY, Liu SJ (2015) Engineering of Ralstoniaeutropha for the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from glucose. J Biotechnol 195:82–88
Reddy SV, Thirumala M, Mahmood S (2009) Production of PHB and P (3HB-co-3HV) biopolymers by Bacillusmegaterium strain OU303A isolated from municipal sewage sludge. World J Microbiol Biotechnol 25:391–397
Porras MA, Vitale C, Villar MA, Cubitto MA (2017) Bioconversion of glycerol to poly(HB-co-HV) copolymer in an inexpensive medium by a Bacillusmegaterium strain isolated from marine sediments. J Environ Chem Eng 5:1–9
Akdoğan M, Çelik E (2018) Purification and characterization of polyhydroxyalkanoate (PHA) from a Bacillusmegaterium strain using various dehydration techniques. J Chem Technol Biotechnol 93:2292–2298
Ray S, Kalia VC (2017) Co-metabolism of substrates by Bacillusthuringiensis regulates polyhydroxyalkanoate co-polymer composition. Bioresour Technol 224:743–747
Luzier WD (1992) Materials derived from biomass/biodegradable materials. Proc Natl Acad Sci USA 89:839–842
Sim SJ, Snell KD, Hogan SA, Stubbe J, Rha C, Sinskey AJ (1997) PHA synthase activity controls the molecular weight and polydispersity of polyhydroxybutyrate in vivo. Nat Biotechnol 15:63–67
Chek MF, Hiroe A, Hakoshima T, Sudesh K, Taguchi S (2019) PHA synthase (PhaC): interpreting the functions of bioplastic-producing enzyme from a structural perspective. Appl Microbiol Biotechnol 103:1131–1141
Raza ZA, Tariq MR, Majeed MI, Banat IM (2019) Recent developments in bioreactor scale production of bacterial polyhydroxyalkanoates. Bioprocess Biosyst Eng 42:901–919
Huschner F, Grousseau E, Brigham CJ, Plassmeier J, Popovic M, Rha C, Sinskey AJ (2015) Development of a feeding strategy for high cell and PHA density fed-batch fermentation of Ralstonia eutropha H16 from organic acids and their salts. Process Biochem 50:165–172
Suwannasing W, Imai T, Kaewkannetra P (2015) Cost-effective defined medium for the production of polyhydroxyalkanoates using agricultural raw materials. Bioresour Technol 194:67–74
Choi SY, Rhie MN, Kim HT, Joo JC, Cho IJ, Son J, Jo SY, Sohn YJ, Baritugo K-A, Pyo J, Lee Y, Lee SY, Park SJ (2020) Metabolic engineering for the synthesis of polyesters: a 100-year journey from polyhydroxyalkanoates to non-natural microbial polyesters. Metab Eng 58:47–81
Choi SY, Cho IJ, Lee Y, Kim Y-J, Kim K-J, Lee SY (2020) Microbial polyhydroxyalkanoates and nonnatural polyesters. Adv Mater 32:1907138
Kanjanachumpol P, Kulpreecha S, Tolieng V, Thongchul N (2013) Enhancing polyhydroxybutyrate production from high cell density fed-batch fermentation of Bacillusmegaterium BA-019. Bioprocess Biosyst Eng 36:1463–1474
RamKumar Pandian S, Deepak V, Kalishwaralal K, Rameshkumar N, Jeyaraj M, Gurunathan S (2010) Optimization and fed-batch production of PHB utilizing dairy waste and sea water as nutrient sources by Bacillusmegaterium SRKP-3. Bioresour Technol 101:705–711
Blunt W, Sparling R, Gapes DJ, Levin DB, Cicek N (2018) The role of dissolved oxygen content as a modulator of microbial polyhydroxyalkanoate synthesis. World J Microbiol Biotechnol 34:106
de Almeida A, Giordano AM, Nikel PI, Pettinari MJ (2010) Effects of aeration on the synthesis of poly(3-hydroxybutyrate) from glycerol and glucose in recombinant Escherichiacoli. Appl Environ Microbiol 76:2036
Senior PJ, Beech GA, Ritchie GA, Dawes EA (1972) The role of oxygen limitation in the formation of poly-β-hydroxybutyrate during batch and continuous culture of Azotobacterbeijerinckii. Biochem J 128:1193–1201
Sambrook JF, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Woodbury
Moore SJ, Lawrence AD, Biedendieck R, Deery E, Frank S, Howard MJ, Rigby SEJ, Warren MJ (2013) Elucidation of the anaerobic pathway for the corrin component of cobalamin (vitamin B12). Proc Natl Acad Sci 110:14906
Çelik E, Çalık P, Oliver SG (2009) Fed-batch methanol feeding strategy for recombinant protein production by Pichiapastoris in the presence of co-substrate sorbitol. Yeast 26:473–484
Taguchi H (1966) Dynamic measurement of the volumetric oxygen transfer coefficient in a fermentation system. J Ferment Technol 44:881–889
Yıldırım Z, Çelik E (2017) Periplasmic and extracellular production of cellulase from recombinant Escherichiacoli cells. J Chem Technol Biotechnol 92:319–324
Braunegg G, Sonnleitner B, Lafferty RM (1978) A rapid gas chromatographic method for the determination of poly-β-hydroxybutyric acid in microbial biomass. Eur J Appl Microbiol Biotechnol 6:29–37
Fei T, Cazeneuve S, Wen Z, Wu L, Wang T (2016) Effective recovery of poly-β-hydroxybutyrate (PHB) biopolymer from Cupriavidusnecator using a novel and environmentally friendly solvent system. Biotechnol Progress 32:678–685
Bunk B, Schulz A, Stammen S, Münch R, Warren MJ, Rohde M, Jahn D, Biedendieck R (2010) A short story about a big magic bug. Bioeng Bugs 1:85–91
Srirangan K, Liu X, Tran TT, Charles TC, Moo-Young M, Chou CP (2016) Engineering of Escherichiacoli for direct and modulated biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer using unrelated carbon sources. Sci Rep 6:36470
McCool GJ, Cannon MC (2001) PhaC and PhaR are required for polyhydroxyalkanoic acid synthase activity in Bacillusmegaterium. J Bacteriol 183:4235–4243
Chabes A, Domkin V, Larsson G, Liu A, Gräslund A, Wijmenga S, Thelander L (2000) Yeast ribonucleotide reductase has a heterodimeric iron-radical-containing subunit. Proc Natl Acad Sci 97:2474
Laszlo V, Hoda MA, Garay T, Pirker C, Ghanim B, Klikovits T, Dong YW, Rozsas A, Kenessey I, Szirtes I, Grusch M, Jakopovic M, Samarzija M, Brcic L, Kern I, Rozman A, Popper H, Zöchbauer-Müller S, Heller G, Altenberger C, Ziegler B, Klepetko W, Berger W, Dome B, Hegedus B (2015) Epigenetic down-regulation of integrin α7 increases migratory potential and confers poor prognosis in malignant pleural mesothelioma. J Pathol 237:203–214
Liberal V, Martinsson-Ahlzén H-S, Liberal J, Spruck CH, Widschwendter M, McGowan CH, Reed SI (2012) Cyclin-dependent kinase subunit (Cks) 1 or Cks2 overexpression overrides the DNA damage response barrier triggered by activated oncoproteins. Proc Natl Acad Sci 109:2754
Faccin DJL, Rech R, Secchi AR, Cardozo NSM, Ayub MAZ (2013) Influence of oxygen transfer rate on the accumulation of poly(3-hydroxybutyrate) by Bacillus megaterium. Process Biochem 48:420–425
Koller M, Braunegg G (2015) Potential and prospects of continuous polyhydroxyalkanoate (PHA) production. Bioengineering 2:94–121
Mohapatra S, Maity S, Dash HR, Das S, Pattnaik S, Rath CC, Samantaray D (2017) Bacillus and biopolymer: prospects and challenges. Biochem Biophys Rep 12:206–213
Lee SY (1996) Plastic bacteria? Progress and prospects for polyhydroxyalkanoate production in bacteria. Trends Biotechnol 14:431–438
Ryu HW, Hahn SK, Chang YK, Chang HN (1997) Production of poly(3-hydroxybutyrate) by high cell density fed-batch culture of Alcaligeneseutrophus with phosphate limitation. Biotechnol Bioeng 55:28–32
García C, Alcaraz W, Acosta-Cárdenas A, Ochoa S (2019) Application of process system engineering tools to the fed-batch production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from a vinasses–molasses mixture. Bioprocess Biosyst Eng 42:1023–1037
Sun ZY, Ramsay JA, Guay M, Ramsay BA (2006) Automated feeding strategies for high-cell-density fed-batch cultivation of Pseudomonasputida KT2440. Appl Microbiol Biotechnol 71:423–431
Zhou Y, Han L-R, He H-W, Sang B, Yu D-L, Feng J, Zhang X (2018) Effects of agitation, aeration and temperature on production of a novel glycoprotein GP-1 by Streptomyceskanasenisi ZX01 and scale-up based on volumetric oxygen transfer coefficient. Molecules 23:125
Sinha J, Tae Bae J, Pil Park J, Hyun Song C, Won Yun J (2001) Effect of substrate concentration on broth rheology and fungal morphology during exo-biopolymer production by Paecilomyces japonica in a batch bioreactor. Enzyme Microb Technol 29:392–399
Garcia-Ochoa F, Gomez E, Santos VE, Merchuk JC (2010) Oxygen uptake rate in microbial processes: an overview. Biochem Eng J 49:289–307
Wang C, Zheng Y, Sun Y, Fan J, Qin Q, Zhao Z (2016) A novel biodegradable polyurethane based on poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and poly(ethylene glycol) as promising biomaterials with the improvement of mechanical properties and hemocompatibility. Polym Chem 7:6120–6132
Alsafadi D, Al-Mashaqbeh O (2017) A one-stage cultivation process for the production of poly-3-(hydroxybutyrate-co-hydroxyvalerate) from olive mill wastewater by Haloferax mediterranei. N Biotechnol 34:47–53
de Paula FC, de Paula CBC, Gomez JGC, Steinbüchel A, Contiero J (2017) Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production from biodiesel by-product and propionic acid by mutant strains of Pandoraea sp. Biotechnol Progress 33:1077–1084
Han J, Qiu Y-Z, Liu D-C, Chen G-Q (2004) Engineered Aeromonas hydrophila for enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with alterable monomers composition. FEMS Microbiol Lett 239:195–201
Fukui T, Kichise T, Iwata T, Doi Y (2001) Characterization of 13 kDa granule-associated protein in Aeromonascaviae and biosynthesis of polyhydroxyalkanoates with altered molar composition by recombinant bacteria. Biomacromol 2:148–153
Kuciel S, Mazur K, Jakubowska P (2019) Novel biorenewable composites based on poly (3-hydroxybutyrate-co-3-hydroxyvalerate) with natural fillers. J Polym Environ 27:803–815
Singh S, Mohanty AK, Sugie T, Takai Y, Hamada H (2008) Renewable resource based biocomposites from natural fiber and polyhydroxybutyrate-co-valerate (PHBV) bioplastic. Compos A 39:875–886
Hasan SK, Zainuddin S, Tanthongsack J, Hosur M, Allen L (2018) A study of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) biofilms’ thermal and biodegradable properties reinforced with halloysite nanotubes. J Compos Mater 52:3199–3207
Salgaonkar BB, Bragança JM (2015) Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Halogeometricumborinquense strain E3. Int J Biol Macromol 78:339–346
Luangthongkam P, Laycock B, Evans P, Lant P, Pratt S (2019) Thermophilic production of poly(3-hydroxybutyrate-co-3-hydrovalerate) by a mixed methane-utilizing culture. N Biotechnol 53:49–56
Don T-M, Chen CW, Chan T-H (2006) Preparation and characterization of poly(hydroxyalkanoate) from the fermentation of Haloferaxmediterranei. J Biomater Sci Polym Ed 17:1425–1438
Alkotaini B, Sathiyamoorthi E, Kim BS (2015) Potential of Bacillusmegaterium for production of polyhydroxyalkanoates using the red algae Gelidiumamansii. Biotechnol Bioprocess Eng 20:856–860
Alkotaini B, Koo H, Kim BS (2016) Production of polyhydroxyalkanoates by batch and fed-batch cultivations of Bacillusmegaterium from acid-treated red algae. Korean J Chem Eng 33:1669–1673
Rodriguez-Contreras A, Koller M, Dias MMD, Calafell-Monfort M, Braunegg G, Marques-Calvo MS (2013) High production of poly(3-hydroxybutyrate) from a wild Bacillusmegaterium Bolivian strain. J Appl Microbiol 114:1378–1387
Acknowledgements
This work was supported by Hacettepe University Scientific Research Projects Coordination Unit (Grant number FBB-2016-11851). We would like to thank Belgin Aslan for her help in TG and DSC analyses and Beray Temelli for her help in 1H-NMR analysis.
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Akdoğan, M., Çelik, E. Enhanced production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) biopolymer by recombinant Bacillus megaterium in fed-batch bioreactors. Bioprocess Biosyst Eng 44, 403–416 (2021). https://doi.org/10.1007/s00449-020-02452-z
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DOI: https://doi.org/10.1007/s00449-020-02452-z