Optimization of Polyhydroxybutyrate Production by Bacillus Species Isolated from Dump Site Soil Using Multiple Linear Regression Analysis
DOI:
https://doi.org/10.56919/usci.2434.030Keywords:
Biodegradable plastics, polyhydroxybutyrate (PHB), Bacillus velezensis, sugarcane bagasse, multiple linear regression (MLR)Abstract
Petrochemical-based plastics cause considerable environmental degradation due to their non-biodegradable properties; however, biodegradable plastics, such as polyhydroxybutyrates (PHBs), provide a sustainable relief to the environment. This study used sugarcane bagasse as a carbon source to synthesize PHB by Bacillus species isolated from dumping site soil. Bacillus velezensis, which displays a high affinity to generate PHB based on the intensity of coloration upon radiation, was selected from 53 isolates. At 30°C, pH 7.5, and 48 hours of incubation, under the ideal conditions resulted in attaining the maximum PHB yield of 50.23% (w/w). Similarly, a predicted PHB yield of 66.05% (w/w) was estimated by a multiple linear regression (MLR) model utilizing ordinary least squares (OLS) regression, which identified temperature, pH, substrate concentration, and incubation time as critical variables. Scanning electron microscopy displayed intracellular PHB granules, and Fourier-transform infrared (FT-IR) analysis indicated the existence of unique PHB functional groups (C–H, CH₂, C=O, and C–O). Tests on biodegradability indicated that soil microorganisms can break down the produced PHB, highlighting the environmental benefits of this research. This study also demonstrates that sugarcane bagasse, an agricultural byproduct, is a cost-effective raw material for PHB synthesis. Additionally, the OLS model provided useful insights for optimizing yield, indicating promise for industrial applications. Future studies should examine other optimization statistical techniques like Response Surface Methodology (RSM) to boost PHB production in complex biological systems. These techniques could also address scalability, economic feasibility, and environmental benefits through cooperation with agro-waste companies and pilot-scale manufacturing activities.
References
Ajuzie, C. U., & Atuanya, E. I. (2014). Identification of the bacterial community responsible for decontaminating ELEME petrochemical industrial effluent using 16S rDNA, PCR-denaturing gradient gel electrophoresis. Nigerian Journal of Technology (NIJOTECH), 33(4), 1-7. https://doi.org/10.4314/njt.v33i4.6 DOI: https://doi.org/10.4314/njt.v33i4.6
Anon, T., Sudarat, S., & Sutticha, N. T. (2017). Efficient polyhydroxybutyrate production from Bacillus thuringiensis using sugarcane juice substrate. Turkish Journal of Biology. https://doi.org/103906/biy-1704-13
Bala, K. M. D., Santhi, R., & Jagadeeswari, S. (2016). Exploitation of agro-industrial residues as a substrate for biodegradable polymer production using polyhydroxybutyrate accumulating bacteria. International Journal of Current Microbiology and Applied Sciences, 5(1), 10-22. https://doi.org/10.20546/ijcmas.2016.501.002 DOI: https://doi.org/10.20546/ijcmas.2016.501.002
Bengio, Y., Courville, A., & Vincent, P. (2016). Representation learning: A review and new perspectives. IEEE Transactions on Pattern Analysis and Machine Intelligence. https://doi.org/10.1109/TPAMI.2013.50 DOI: https://doi.org/10.1109/TPAMI.2013.50
Bengio, Y., Goodfellow, I., & Courville, A. (2016). Deep learning. MIT Press.
Bharathi, B., Gowdhaman, D., & Ponnusami, V. (2016). Isolation and identification of polyhydroxybutyrate (PHB) producing Bacillus cereus BB613—a novel isolate. International Journal of Chemical Technology Research, 9(1), 1-7.
Bharathi, D., Nandhini Devi, R., & Tholappan, P. (2016). Optimization and characterization of polyhydroxybutyrate (PHB) by Bacillus megaterium using response surface methodology. International Journal of Biological Macromolecules, 91, 175–184. https://doi.org/10.1016/j.ijbiomac.2016.09.036 DOI: https://doi.org/10.1016/j.ijbiomac.2016.09.036
Buchanan, R. E., & Gibbons, N. E. (Eds.). (1974). Bergey’s manual of determinative bacteriology (8th ed.).
Christina, T., Pallavi, S., Shrestha, R., & Prakash, M. (2018). Isolation of polyhydroxybutyrate (PHB) producing bacteria, optimization of culture conditions for PHB production, extraction, and characterization of PHB. Nepal Journal of Biotechnology, 6(1), 62-68. https://doi.org/10.3126/njb.v6i1.22339 DOI: https://doi.org/10.3126/njb.v6i1.22339
Cockerill, F. R., et al. (2012). Performance standards for antimicrobial disk susceptibility tests; approved standard—eleventh edition. Clinical Laboratory Standards Institute (CLSI), 32(1), 1-19.
Coppola, G., Gaudio, M. T., Lopresto, C. G., Calabrò, V., Curcio, S., & Chakraborty, S. (2021). Bioplastic from renewable biomass: A feasible solution for a greener environment. Earth Systems and Environment, 5, 231-251. https://doi.org/10.1007/s41748-021-00208-7 DOI: https://doi.org/10.1007/s41748-021-00208-7
Darshen, R., et al. (2014). "Quantitative estimation of reducing sugar (Glucose) by DNS method." Sciencevivid. Retrieved from Sciencevivid.
Donya, K., Stefan, P., Winfried, N., et al. (2017). Photosynthetic poly-β-hydroxybutyrate accumulation in unicellular Cyanobacterium Synechocystis sp. PCC 6714. AMB Express, 7, 143. https://doi.org/10.1186/s13568-017-0443-9 DOI: https://doi.org/10.1186/s13568-017-0443-9
El-Hamshary, O. I. M., Kadi, H. A., & Al-Twaty, N. H. (2018). Molecular characterization and UV improvement of some bioplastic-producing bacteria isolated from plants in Taif city, Saudi Arabia. Pharmacophore, 9, 7-18. https://doi.org/10.1007/s10924-018-1273-6
El-Hamshary, O. I., Abdel-Mawgoud, A. M., & Abou-Zeid, D. M. (2018). Screening of polyhydroxybutyrate-producing bacteria from rhizosphere soil and optimization of PHB production using agro-waste. Journal of Applied Polymer Science. https://doi.org/10.1007/s10924-018-1273-6
El-Kadi, S. (2010). Bioplastic production from inexpensive sources. ResearchGate. https://www.researchgate.net/publication/260480411
Gasser, E., Ballman, N. P., Drogem, S., Bohn, J., & Konig, H. (2014). Microbial production of biopolymers from the renewable resource wheat straw. Journal of Applied Microbiology, 117(4), 1035-1044. https://doi.org/10.1111/jam.12581 DOI: https://doi.org/10.1111/jam.12581
George, N., Debroy, A., Bhat, S., Singh, S., & Bindal, S. (2021). Biowaste to bioplastics: An eco-friendly approach for a sustainable future. Journal of Applied Biotechnology Reports, 8(3), 221–233.
Getachew, A., & Woldesenbet, F. (2016). Production of biodegradable plastic by polyhydroxybutyrate (PHB) accumulating bacteria using low-cost agricultural waste material. BMC Research Notes, 9, 509. https://doi.org/10.1186/s13104-016-2321-y DOI: https://doi.org/10.1186/s13104-016-2321-y
Giin-Yu, A. T., Chia-Lung, C., Ling, L., Liya, G., & Lin, W. (2014). Start a research on biopolymer polyhydroxybutyrate (PHB). Polymers, 6(2), 706-754. https://doi.org/10.3390/polym6030706 DOI: https://doi.org/10.3390/polym6030706
Graham, P.H., and Parker, C.A. (1964). Diagnostic features in the characterization of the root- nodule bacteria of legumes. Plant Soil 20:383-396. https://doi.org/10.1007/BF01373828 DOI: https://doi.org/10.1007/BF01373828
Gram, C. (1884). The differential staining of schizomycetes in tissue sections and in dried preparations. Fortschritte der Medicin, 2, 185-189.
Grigore, M. E., Grigorescu, R. M., Iancu, L., Ion, R. M., Zaharia, C., & Andrei, E. R. (2019). Methods of synthesis, properties, and biomedical applications of polyhydroxyalkanoates: A review. Journal of Biomaterials Science, Polymer Edition, 30, 695–712. https://doi.org/10.1080/09205063.2019.1605866 DOI: https://doi.org/10.1080/09205063.2019.1605866
Hong, K., Sun, S., Tian, W., Chen, G. Q., & Huang, W. (1999). A rapid method for detecting bacterial polyhydroxyalkanoates in intact cells by Fourier transform infrared spectroscopy. Applied Microbiology and Biotechnology, 51(5), 523-526. https://doi.org/10.1007/s002530051427 DOI: https://doi.org/10.1007/s002530051427
Kumar, M., Rathour, R., Singh, R., et al. (2020). Bacterial polyhydroxyalkanoates: Opportunities, challenges, and prospects. Journal of Cleaner Production, 263, 121500. https://doi.org/10.1016/j.jclepro.2020.121500 DOI: https://doi.org/10.1016/j.jclepro.2020.121500
Lee, H.-J., Kim, S.-G., Cho, D.-H., et al. (2022). Finding of novel lactate-utilizing Bacillus sp. YHY22 and its evaluation for polyhydroxybutyrate (PHB) production. International Journal of Biological Macromolecules, 201, 653–661. https://doi.org/10.1016/j.ijbiomac.2022.01.025 DOI: https://doi.org/10.1016/j.ijbiomac.2022.01.025
Magar, S. P., Ingle, A. B., & Ganorkar, R. N. (2015). Production of bioplastic (PHA) from emulsified cotton seed oil medium by Ralstonia spp. International Journal of Engineering Research and General Science, 3(1), 436–441.
Markl, E. (2018). PHB—bio-based and biodegradable replacement for PP: A review. Novel Techniques in Nutrition and Food Science, 2, 000546. https://doi.org/10.31031/ntnf.2018.02.000546 DOI: https://doi.org/10.31031/NTNF.2018.02.000546
Michael, P., Loganayagi, R., Nancy, D., Ranandkumar, S. G., & Arulselvi, P. (2012). Isolation and characterization of indigenous Ralstonia strain YRF1 for high polyhydroxybutyrate (PHB) production. Journal of Applied Biology, 48, 9424–9427.
Mostafa, F. A., Galal, M. A., & Elkady, M. F. (2015). Biodegradable polyhydroxybutyrate production by Bacillus cereus using sugarcane bagasse. Biotechnology Research International. https://doi.org/10.1080/09168451.2015.1008733
Mostafa, Y. S., Alrumman, S. M., Alamri, S. A., Otaif, K. A., Mostafa, M. S., & Alfaify, A. M. (2020). Production by the marine bacterium Pseudodonghicola xiamenensis through date syrup valorization and structural assessment of the biopolymer bioplastic (poly-3-hydroxybutyrate). Scientific Reports, 10, 8815. https://doi.org/10.1038/s41598-020-65858-5 DOI: https://doi.org/10.1038/s41598-020-65858-5
Ng, A. (2018). Machine Learning Course (CS229). Stanford University.
Ng, A. (2018). Machine learning yearning: Technical strategy for AI engineers in the era of deep learning.
Noel, R.K. and Penelope, J.P. (2011). Methods in Microbiology. Journal of biotechnology. 86 (2), 87-95, 2011. https://doi.org/10.1016/B978-0-12-387730-7.00003-6 DOI: https://doi.org/10.1016/B978-0-12-387730-7.00003-6
Padovani, G., Carlozzi, P., Seggiani, M., et al. (2016). PHB-rich biomass and BioH2 production by means of photosynthetic microorganisms. Chemical Engineering Transactions, 49, 55-60. https://doi.org/10.3303/CET1649010
Park, D. H., & Kim, B. S. (2011). Production of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by Ralstonia eutropha from soybean oil. New Biotechnology, 28(6), 719–724. https://doi.org/10.1016/j.nbt.2011.01.007 DOI: https://doi.org/10.1016/j.nbt.2011.01.007
Pohare, M. S., Singha, M. D., & Sharma, P. A. (2015). Optimization of polyhydroxybutyrate production using local isolates. Research Journal of Chemical Sciences, 5(9), 42-47.Polyhydroxybutyrate (PHB) – A review of microbial synthesis and applications by M. E. Grigore, R. M. Grigorescu, L. Iancu, & V. Constantin (2019). https://doi.org/10.9734/IJBCRR/2019/v26i430082.
Poszytek, K., Ciezkowska, M., Sklodowska, A., & Drewniak, L. (2016). Microbial consortium with high cellulolytic activity (MCHCA) for enhanced biogas production. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2016.00324 DOI: https://doi.org/10.3389/fmicb.2016.00324
Poszytek, K., Galazka, A., & Karczewska, K. (2016). Microbial degradation of lignocellulosic biomass: Enzymes, pathways, and products. Applied Microbiology and Biotechnology, 100, 7857-7875. https://doi.org/10.1007/s00253-016-7857-5
Ramadas, N. V., Singh, S. K., Soccol, C. R., & Pandey, A. (2009). Polyhydroxybutyrate production using agro-industrial residue as substrate by Bacillus sphaericus NCIM 5149. Brazilian Archives of Biology and Technology, 52(1), 17-23. https://doi.org/10.1590/S1516-89132009000100003 DOI: https://doi.org/10.1590/S1516-89132009000100003
Sagar Aryal (2019). "Indole Test- Principle, Reagents, Procedure, Result Interpretation and Limitations." Available at Microbiology Info.
Samrot, A. V., Samanvitha, S. K., Shobana, N., et al. (2021). The synthesis, characterization, and applications of polyhydroxyalkanoates (PHAs) and PHA-based nanoparticles. Polymers (Basel), 13(1933), 1-15. https://doi.org/10.3390/polym13193302 DOI: https://doi.org/10.3390/polym13193302
Shreema, M. (2014). Characterization of Physical, Spectroscopic and Thermal Properties of Polyhydroxybutyrate (PHB) using FT-IR Spectroscopy. Journal of Polymer Science, 26(4), 1-15.
Singh, G., Mittal, A., Kumari, A., Goel, A., Aggarwal, N.K., and Yadav, A. (2011). Optimization of Poly-B-Hydroxybutyrate Production from Bacillus species. European Journal of Biological Sciences, 3 (4): 112-116.
Souvik, B., Bhargavi, S., Rithanya, T., & Lilly, M. S. (2024). PHB production by Bacillus megaterium LSRB 0103 using cornstarch and urea. Current Microbiology, 81, 139. https://doi.org/10.1007/s00284-024-03667-z DOI: https://doi.org/10.1007/s00284-024-03667-z
Sunday, A., Adeyinka, E. A., & Florence, O. A. (2023). Screening of microorganisms producing polymer (PHB) from dumping site soil in Ilorin metropolis, Kwara State, Nigeria. Journal of Biochemistry, Microbiology, and Biotechnology, 11(1), 26-30. https://doi.org/10.54987/jobimb.v11i1.802 DOI: https://doi.org/10.54987/jobimb.v11i1.802
Sunitha, P., & Ujwala, B. (2013). Screening, isolation, and quantification of PHB-producing soil bacteria. International Journal of Engineering Science and Invention, 2(9), 1-6.
Thammasittirong, A., & Attathom, T. (2010). Polyhydroxybutyrate production by Bacillus megaterium BA-019 and Alcaligenes eutrophus ATCC 17699 from cassava starch hydrolysate. ScienceAsia, 26(4), 165-170.
Tille P.M. (2014). Bailey and Scott’s diagnostic microbiology. Thirteen editions. Mosby, Inc., an affiliate of Elsevier Inc. 3251 Riverport Lane. St. Louis. Missouri 63043.
Wang, S. R., Ru, B., Dai, G. X., Sun, W. X., Qiu, K. Z., & Zhou, J. S. (2015). Pyrolysis mechanism study of minimally damaged hemicellulose polymers isolated from agricultural waste straw samples. Bioresource Technology, 190, 211–218. https://doi.org/10.1016/j.biortech.2015.04.098 DOI: https://doi.org/10.1016/j.biortech.2015.04.098
Wong, Y. M., Brigham, C. J., Rha, C. K., Sinkey, A. J., & Sudesh, K. (2012). Biosynthesis and characterization of polyhydroxyalkanoate containing high 3-hydroxyhexanoate monomer fraction from crude palm kernel oil by recombinant Cupriavidus necator. Bioresource Technology, 121, 320-327. https://doi.org/10.1016/j.biortech.2012.07.015 DOI: https://doi.org/10.1016/j.biortech.2012.07.015
Zakaria, M. R., Ariffin, H., Johar, N. A. M., et al. (2015). Biosynthesis and characterization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer from wild-type Comamonas sp. EB172. Polymers, 7(3), 599-618.
Zaman, Q., Grover, H., Mahato, N., Kumar, V., & Rajan, R. (2021). Enhanced production of polyhydroxybutyrate (PHB) in marine Bacillus sp. through genetic engineering. Journal of Biotechnology and Bioengineering, 10(7), 25-35.
Downloads
Published
Issue
Section
License

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
UMYU Scientifica recognizes the importance of protecting authors’ intellectual property while promoting the free exchange of scientific knowledge. The journal adopts a copyright-retention model that empowers authors to maintain ownership of their work while granting the journal rights necessary for publication and dissemination.
1. Copyright Ownership
Authors publishing with UMYU Scientifica retain full copyright and publishing rights to their work. By submitting a manuscript, authors agree to grant the journal a non-exclusive license to publish, reproduce, distribute, and archive the article in all forms and media for the purpose of scholarly communication.
2. Licensing Terms
All articles are published under the Creative Commons Attribution–NonCommercial (CC BY-NC) license.
This license permits others to:
- Share - copy and redistribute the material in any medium or format.
- Adapt - remix, transform, and build upon the material.
- For non-commercial purposes only, provided that proper credit is given to the original author(s) and UMYU Scientifica as the source, a link to the license is provided, and any modifications are clearly indicated.
Commercial reuse or distribution of the content requires written permission from both the author and the editorial office.
3. Author Rights
Authors are free to:
- Deposit all versions of their manuscript (preprint, accepted version, and published version) in institutional, disciplinary, or public repositories without embargo.
- Use and distribute their published article for non-commercial scholarly purposes, including teaching, conference presentations, and research sharing.
- Include their work in future books, theses, or compilations, provided proper citation to the journal is made.
4. Publisher’s Rights
Upon publication, UMYU Scientifica retains the right to:
- Host, index, and disseminate the article through the journal’s website and partner databases.
- Archive the content in long-term preservation systems such as the PKP Preservation Network (PKP-PN) and the Umaru Musa Yar’adua University Institutional Repository.
5. Attribution and Citation
Users must give appropriate credit to the author(s), include a link to the article’s DOI or the journal webpage, and indicate if changes were made. Proper citation is required whenever the work is reused or referenced.
6. License Reference
For detailed terms of use, please refer to the Creative Commons Attribution–NonCommercial 4.0 International License (CC BY-NC 4.0):
https://creativecommons.org/licenses/by-nc/4.0/









