Kinetics and synthesis of poly(3-hydroxybutyrate) by a putative-mutant of Bacillus licheniformis

Abstract

The present study deals with the kinetics of improved poly(3-hydroxybutyrate) (PHB) production by an L-cysteine HCl-depressed mutant of Bacillus licheniformis. Production of biodegradable polymers is to eliminate use of materials derived from petrochemicals and also because of their environmental impact. For the current study, mutant strain (NA-21) & wild-type (IIB-isl19) were used for PHB production. Submerged culture with two-stage fermentation technique was used for PHB production. Results indicated that PHB production was improved with 300 mM of –HNO₂. The superior mutant strain (NA-21) resulted in 2-fold more PHB as compared to the wild-type (IIB-isl9). It was selected, and resistance against L-cysteine HCl was developed. At 4 ppm concentration of L-cysteine HCl, PHB production by mutant strain (NA-cys4) was higher than its wild counterpart by 5.7-fold. Kinetic study of parameters including specific growth rate (µ h^− 1), growth (Y_x/s,Y_s/x), product yield coefficients (Y_p/s,Y_p/x), volumetric rate constants (Q_p, Q_s, Q_x) and specific rate constants (q_p, q_s, q_x), were also accomplished. Moreover, Y_p/x, Q_p and q_p = µ × Y_p/x were found to be very significant as 1.254 ± 0.06 (g/g biomass), 0.134 ± 0.01 (g/l/h) and 0.168 ± 0.01 (g/g/h), respectively. The effect of fatty acids on PHB production highlighted the improvement in PHB production by 1.94-fold. The highest PHB production during the study was 16.35 ± 3.12 g/l which highlighted its significance (p ≤ 0.05) and impact on the overall process. The variation in PBH yield between wild-type and mutant B. licheniformis is possibly because of induced DNA interstrand thus making unstable thymidine-thymidine dymers. From the results, it was concluded that improved PHB production on industrial scale is fairly possible and it holds the potential to contribute significantly to plastic circularity in the future.

Keywords

Kinetics / Bacillus licheniformis / Submerged culture / Poly(3-hydroxybutyrate) / Fermentation optimizations / Industrial biotechnology

References

Ali S, Nawaz W. Optimization of nutritional requirements for dopamine synthesis by calcium alginate-entrapped mutant strain of aspergillus oryzae EMS-6. Nat Prod Res, 2017, 31(3): 281-288.

Ansari S, Fatima T. Cyanobacterial polyhydroxybutyrate (PHB): screening, optimization and characterization. PLoS ONE, 2016, 11(6): e0158168.

Azin M, Noroozi E. Random mutagenesis and use of 2-deoxy-D-glucose as an antimetabolite for selection of α-amylase-overproducing mutants of aspergillus oryzae. World J Microbiol Biotechnol, 2001, 17: 747-750.

Bailey MJ. A note on the use of dinitrosalicylic acid for determining the products of enzymatic reactions. Appl Microbiol Biotechnol, 1988, 29: 494-496.

Chavan S, Yadav B, Tyagi RD, Drogui P. A review on production of polyhydroxyalkanoate (PHA) biopolyesters by thermophilic microbes using waste feedstocks. Bioresourc Technol, 2021, 341: 125900.

Engels HW, Pirkl HG, Albers R, Albach RW, Krause J, Hoffmann A, Dormish J. Polyurethanes: versatile materials and sustainable problem solvers for today’s challenges. Angewandte Chemie Inter, 2013, 52(36): 9422-9441.

Flora GD, Bhatt K, Tuteja U. Optimization of culture conditions for poly α-hydroxybutyrate production from isolated Bacillus species. J Cell Tissue Res, 2010, 10(2): 2235.

Getachew A, Woldesenbet F (2016) Production of biodegradable plastic by polyhydroxybutyrate (PHB) accumulating bacteria using low cost agricultural waste material. BMC Res Notes 9:509. https://doi.org/10.1186/s13104-016-2321-y

Gray N, Calvin M, Bhatia SC. Enzymes Biotechnology, 2010, New Delhi, India: CBS Publishers Incorp.

Hu S, Zhao C, Zhang Y, Wang X, He P, Chen S. Construct a synthetic Entner–doudoroff pathway in Bacillus licheniformis for enhancing lichenysin production. World J Microbiol Biotechnol, 2023, 39(7): 168.

Jose AA, Hazeena SH, Lakshmi NM, Madhavan A, Sirohi R, Tarafdar A, Binod P. Bacterial biopolymers: from production to applications in biomedicine. Sustainable Chemd Pharm, 2022, 25: 100582.

Kashiwaya Y, Takeshima T, Mori N, Nakashima K, Clarke K, Veech RL. d-β-Hydroxybutyrate protects neurons in models of Alzheimer’s and Parkinson’s disease. Proc Nat Acad Sci, 2000, 97(10): 5440-5444.

Law JH, Slepecky RA. Assay of poly-β-hydroxybutyric acid. J Bacteriol, 1961, 82(1): 33-36.

Lawford HG, Roseau JD. Mannose fermentation by ethanologenic recombinants of E. Coli and kinetical aspects. Biotechnol Lett, 1993, 15: 615-620.

Lee SY, Lee YK, Chang HN. Stimulatory effects of amino acids and oleic acid on poly (3-hydroxybutyric acid) synthesis by recombinant Escherichia coli. J Ferment Bioengin, 1995, 79(2): 177-180.

Mantele W, Deniz E. UV–VIS absorption spectroscopy: Lambert-Beer reloaded. Spectrochimica Acta Part A: Mol Biomol Spectro, 2017, 173: 965-968.

Mohd A, Arif JS, Syed AA, Mejdi S, Riadh B, Mousa A, Abdelbaset ME, Waleed AA, Salem HA, Manojkumar S, Mitesh P. Polyhydroxybutyrate (PHB)-based biodegradable polymer from Agromyces indicus: enhanced production, characterization, and optimization. Polym (Basel), 2022, 14(19): 3982.

Nair IC, Pradeep S, Ajayan MS, Jayachandran K, Shashidhar S. Accumulation of intracellular polyhydroxybutyrate in Alcaligenes sp d 2 under phenol stress. Appl Biochem Biotechnl, 2009, 159: 545-552.

Pal A, Prabhu A, Kumar AA, Rajagopal B, Dadhe K, Ponnamma V, Shivakumar S. Optimization of process parameters for maximum poly(-β-)hydroxybutyrate (PHB) production by Bacillus thuringiensis IAM 12077. Pol J Microbiol, 2009, 58(2): 149-154.

Patwardhan PR, Srivastava AK. Model-based fed-batch cultivation of R. eutropha for enhanced biopolymer production. Biochem Engin J, 2004, 20(1): 21-28.

Pirt SJ, Callow DS. Continuous-flow culture of the filamentous mould Penicillium Chrysogenum and the control of its morphology. Nature, 1959, 184: 307-310.

Ramadas NV, Soccol CR, Pandey A. A statistical approach for optimization of polyhydroxybutyrate production by Bacillus sphaericus NCIM 5149 under submerged fermentation using central composite design. Appl Biochem Biotechnol, 2010, 162: 996-1007.

Rey MW, Ramaiya P, Nelson BA, Brody-Karpin SD, Zaretsky EJ, Tang M, Berka RM. Complete genome sequence of the industrial bacterium Bacillus licheniformis and comparisons with closely related Bacillus species. Genome Biol, 2004, 5(10): 1-12.

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

Shrivastav A, Kim HY, Kim YR (2013) Advances in the applications of polyhydroxyalkanoate nanoparticles for novel drug delivery system. BioMed Res Int 2013:581684

Siddiqi OH. Mutagenic action of nitrous acid on aspergillus nidulans. Genet Res, 1962, 3(2): 303-314.

Snedecor GW, Cochran WG. Statistical methods, 1980, Iowa, USA: National Publishing House.

Stam H, van Verseveld HW, de Vries W, Stouthamer AH. Utilization of poly-β-hydroxybutyrate in free-living cultures of Rhizobium ORS571. FEMS Microbiol Lett, 1986, 35(2–3): 215-220.

Thammasittirong A, Saechow S, Thammasittirong SNR. Efficient polyhydroxybutyrate production from Bacillus thuringiensis using sugarcane juice substrate. Turkish J Biol, 2017, 41(6): 992-1002.

Tripathi AD, Srivastava SK, Singh RP. Statistical optimization of physical process variables for bio-plastic (PHB) production by Alcaligenes Sp. Biomass Bioenergy, 2013, 55: 243-250.

Wang Q, Yu H, Xia Y, Kang Z, Qi Q (2009) Complete PHB mobilization in Escherichia coli enhances the stress tolerance: a potential biotechnological application. Microb Cell Fact 8(1):1–9

Vijayalakshmi S, Ranjitha J, Rajeswari VD. Enzyme production ability by Bacillus subtilis and Bacillus licheniformis-A comparative study. Asian J Pharm Clin Res, 2013, 6(4): 29-32.

Yeo JCC, Muiruri JK, Thitsartarn W, Li Z, He C. Recent advances in the development of biodegradable PHB-based toughening materials: approaches, advantages and applications. Mat Sci Engin C, 2018, 92: 1092-1116.

Zhou W, Bergsma S, Colpa DI, Euverink GW, Krooneman J. Polyhydroxyalkanoates (PHAs) synthesis and degradation by microbes and applications towards a circular economy. J Env Mang, 2023, 341: 118033.