Isolation and Characterisation of Polyhydroxybutyrate (PHB) Producing Bacteria for Sustainable Bioplastic Development

Abstract

Polyhydroxybutyrate (PHB), a biodegradable biopolymer produced by certain bacteria, is one of the sustainable alternatives being sought after due to the growing environmental burden posed by traditional plastics. In this study, environmental samples were taken from Dumas Beach and Kholwad College Garden in South Gujarat in order to isolate, identify, and optimize PHB-producing bacterial strains. Using the Carbol Fuchsin, Sudan Black B, and Nile Blue A staining methods, a total of 25 bacterial isolates were acquired and tested. Based on morphological, biochemical, and staining investigations, two strains—Bacillus spp. and Pseudomonas spp.—were determined to be effective PHB producers. Molasses, leftover cooking oil, orange and potato peels, and other low-cost carbon sources were assessed for PHB production. The largest PHB concentration was found in potato peels, where Pseudomonas spp. could reach 89%. PHB yield was considerably increased by a pH of 7, a temperature of 37°C, and shaking conditions, according to the results of production parameter optimization. Fourier Transform Infrared Spectroscopy (FTIR), which showed distinctive peaks of ester carbonyl and C–H functional groups, verified that the isolated polymer was PHB. This research highlights the potential of using regionally isolated bacteria and agro-waste materials for low-cost, eco-friendly PHB production. These findings support sustainable bioplastic development and offer promising avenues for industrial-scale biopolymer synthesis using locally sourced resources.

Introduction

Global plastic production is rapidly increasing and is projected to nearly triple by 2050, reaching 590 million tonnes. Despite this growth, less than 10% of plastic is recycled, with much ending up polluting natural environments and aquatic systems, causing serious ecological and health risks, particularly from microplastics.

Polyhydroxybutyrate (PHB) is a biodegradable, biocompatible polymer produced by bacteria that offers an environmentally friendly alternative to petroleum-based plastics. Research, such as that by M. Adnan et al. (2022), highlights certain bacterial strains like Agromyces indicus as promising PHB producers, comparable to common synthetic polymers. However, the cost of production remains a barrier.

The study addresses gaps in identifying and optimizing native bacterial strains from organic-rich environments (soil, compost, agricultural waste) capable of producing PHB efficiently, especially using low-cost substrates. Current research largely focuses on known model bacteria under controlled lab conditions, limiting industrial scalability.

This research aims to isolate and characterize PHB-producing bacteria from environmental samples in South Gujarat, India, using staining techniques, morphological and biochemical tests, and optimize growth conditions to enhance PHB yield. Organic wastes were tested as alternative carbon sources, and PHB was extracted and quantified. Characterization was done using FTIR spectroscopy to confirm polymer identity.

The results showed that among 25 isolated bacterial colonies, 8 strains produced PHB, with two (including a Bacillus species) selected for detailed analysis. This work contributes to sustainable bioplastic production by exploring underutilized local bacteria and cost-effective substrates, helping reduce reliance on synthetic plastics and environmental pollution.

Conclusion

The goal of the current study was to identify and isolate native bacterial strains that might produce polyhydroxybutyrate (PHB), a biodegradable biopolymer that could eventually take the place of traditional plastics. Twenty-five bacterial isolates were found in the various environmental samples collected in South Gujarat\'s Dumas Beach and Kholwad College Garden. Two promising organisms, Bacillus spp. (Isolate 3) and Pseudomonas spp. (Isolate 4), were found by primary screening utilising Carbol Fuchsin, Sudan Black B, and Nile Blue A staining procedures. These strains demonstrated a notable intracellular buildup of PHB. The characterisation of these isolates was concomitantly done by biochemicals, and more on their PHB production was done using specific low cost organic waste sources. The tested four carbon sources that include potato peels, orange peels, molasses, and waste cooking oil showed that potato peels produced greater PHB accumulation in both isolates; 89% PHB accumulation in Pseudomonas spp. and 77% in Bacillus spp. Optimisation trial experiments showed that optimal PHB production was obtained at pH7, a temperature of 37o C and shaking (aerobic) conditions. Also, the second factor contributed to a small-scale biomass and PHB improvement due to the presence of nitrogen. These results present the idea that the nutritional and environmental conditions have a strong effect on the PHB synthesis. FTIR analysis of the extracted polymer confirmed the structural integrity of PHB through characteristic peaks corresponding to ester carbonyl and alkyl groups. This validates the biopolymer identity and supports the effectiveness of the extraction and purification method. Overall, this study demonstrates that native bacterial strains can be harnessed for cost-effective PHB production using agro-industrial waste. The use of regionally available organic materials not only reduces production costs but also supports circular waste management. Future work should explore scale-up strategies, bioreactor development, and molecular-level strain optimization to advance commercial applications. This research contributes meaningfully to sustainable plastic alternatives and localised bioeconomy development.

References

[1] M. Anwar, M. E. Konnova, and S. Dastgir , “Circular plastic economy for sustainable development: current advances and future perspectives,” 2025. https://doi.org/10.1039/D5SU00225G [2] U. Environment, “Plastic Pollution,” UNEP - UN Environment Programme, 2022. https://www.unep.org/plastic-pollution? [3] Agence France-Presse, “9 Percent of Plastic Worldwide is Recycled, OECD Says,” Voice of America, Feb. 23, 2022. https://www.voanews.com/a/percent-of-plastic-worldwide-is-recycled-oecd-says-/6455012.html? (accessed Jun. 30, 2025). [4] S. Price, U. Kuzhiumparambil, M. Pernice, and P. J. Ralph, “Cyanobacterial Polyhydroxybutyrate for Sustainable Bioplastic,” 2020. https://opus.lib.uts.edu.au/bitstream/10453/140834/2/Binder2.pdf [5] M. Adnan et al., “Polyhydroxybutyrate (PHB)-Based Biodegradable Polymer from Agromyces indicus: Enhanced Production, Characterization, and Optimization,” Polymers, vol. 14, no. 19, p. 3982, Sep. 2022, doi: https://doi.org/10.3390/polym14193982. [6] M. Adnan et al., “Characterization and Process Optimization for Enhanced Production of Polyhydroxybutyrate (PHB)-Based Biodegradable Polymer from Bacillus flexus Isolated from Municipal Solid Waste Landfill Site,” Polymers, vol. 15, no. 6, p. 1407, Jan. 2023, doi: https://doi.org/10.3390/polym15061407. [7] R. Ibrahim, Jesil Mathew Aranjani, Navya Prasanna, A. Biswas, and P. Kumar, “Production, isolation, optimization, and characterization of microbial PHA from Bacillus australimaris,” Scientific Reports, vol. 15, no. 1, Mar. 2025, doi: https://doi.org/10.1038/s41598-025-92146-x. [8] N. Chathalingath, J. S. Kingsly, and A. Gunasekar, “Biosynthesis and Biodegradation of Polyhydroxybutyrate from Priestia flexa; A Promising Mangrove Halophyte Towards the Development of Sustainable Eco-friendly Bioplastics,” Microbiological Research, p. 127270, Nov. 2022, doi: https://doi.org/10.1016/j.micres.2022.127270. [9] Y. S. Mostafa, S. A. Alrumman, K. A. Otaif, S. A. Alamri, M. S. Mostafa, and T. Sahlabji, “Production and Characterization of Bioplastic by Polyhydroxybutyrate Accumulating Erythrobacter aquimaris Isolated from Mangrove Rhizosphere,” Molecules, vol. 25, no. 1, p. 179, Jan. 2020, doi: https://doi.org/10.3390/molecules25010179. [10] Yannick Patrice Didion et al., “A novel strategy for extraction of intracellular poly(3-hydroxybutyrate) from engineered Pseudomonas putida using deep eutectic solvents: Comparison with traditional biobased organic solvents,” Separation and purification technology, vol. 338, pp. 126465–126465, Jun. 2024, doi: https://doi.org/10.1016/j.seppur.2024.126465. [11] G. S. Bhat, B. K. Deekshitha, V. Thivaharan, and M. S. Divyashree, “Physicochemical cell disruption of Bacillus sp. for recovery of polyhydroxyalkanoates: future bioplastic for sustainability,” 3 Biotech, vol. 14, no. 2, Feb. 2024, doi: https://doi.org/10.1007/s13205-024-03913-y.

Copyright

Copyright © 2025 Darshit Rameshbhai Goyani, Tankariwala Aneesha Aiyub, Dr. A. A. Raval. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.