Process optimisation and genomic analysis of poly(3-hydroxybutyrate) production by Mycolicibacterium smegmatis using sugarcane bagasse

Abstract

This study investigates the optimisation of poly(3-hydroxybutyrate) (PHB) production by Mycolicibacterium smegmatis using sugarcane bagasse (SCB) hydrolysate as a low-cost, renewable carbon source. Key fermentation parameters including temperature, pH, agitation, inoculum size and nitrogen supplementation were optimised to enhance biomass growth and PHB accumulation. Under optimised conditions (37 °C, pH 7, 100 rpm, 3% inoculum), M. smegmatis achieved a maximum PHB content of 64% of DCW from SCB hydrolysate, corresponding to a PHB titre of ~ 1.0 g L⁻¹ and a volumetric productivity of 0.014 g L⁻¹ h⁻¹ after 72 h of cultivation, with yeast extract identified as the most effective nitrogen source. Thin-layer chromatography (TLC) analysis demonstrated that M. smegmatis could not only co-utilise glucose and xylose simultaneously but preferentially consumed xylose, which is a major advantage when processing lignocellulosic biomasses, where xylose is abundant and typically underutilised by many microorganisms. Whole-genome sequencing with Oxford Nanopore Technologies (ONT) confirmed the presence of PHB production genes (phbA, phbB, phbC) and a xylose-utilisation pathway, supporting its metabolic capability. These findings establish M. smegmatis as a promising candidate for converting agricultural waste into biodegradable bioplastics, contributing to circular bioeconomy strategies.

Graphical abstract

Keywords

Biopolymers / Hydrolysate / Lignocellulosic bioconversion / Polyhydroxyalkanoates / Sugar co-utilisation / Whole genome sequencing

References

[1] Albuquerque PBS, Malafaia CB. 2018/02/01/). Perspectives on the production, structural characteristics and potential applications of bioplastics derived from polyhydroxyalkanoates. Int J Biol Macromol, 2018, 107: 615-625

[2] Ali S, Lodhi FS, Ahmad MU, Khan QF, Asad ur R, Ahmed A, Liaqat I, Aftab MN, Shah TA, Salamatullah AM, Wondmie GF, Bourhia M. 2024/04/22). Kinetics and synthesis of poly(3-hydroxybutyrate) by a putative-mutant of Bacillus licheniformis. Bioresources Bioprocess, 2024, 11(1): 41

[3] Alsafadi D, Al-Mashaqbeh O, Mansour A, Alsaad M. 2020/08/01). Optimization of nitrogen source supply for enhanced biosynthesis and quality of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by extremely halophilic archaeon Haloferax mediterranei. MicrobiologyOpen, 2020, 9(8): e1055

[4] Apiyo D, Mouton JM, Louw C, Sampson SL, Louw TM. 2022/01/07/). Dynamic mathematical model development and validation of in vitro Mycobacterium smegmatis growth under nutrient- and pH-stress. J Theor Biol, 2022, 532: 110921

[5] Arora NK, Mishra I. 2019/12/01). United Nations Sustainable Development Goals 2030 and environmental sustainability: race against time. Environ Sustain, 2019, 2(4): 339-342

[6] Ashby RD (2020) Xylose Utilization for polyhydroxyalkanoate biosynthesis. in sustainability & green polymer chemistry Volume 2: Biocatalysis and Biobased Polymers.1373:125–143. American Chemical Society. https://doi.org/10.1021/bk-2020-1373.ch007

[7] Baloni P, Padiadpu J, Singh A, Gupta KR, Chandra N. Nov 18). Identifying feasible metabolic routes in Mycobacterium smegmatis and possible alterations under diverse nutrient conditions. BMC Microbiol, 2014, 14: 276

[8] Barrameda HJJrRequiso PJ, Alfafara CG, Nayve FRP, Ventura RLG, Ventura J-RS. Hydrolysate production from sugarcane bagasse using steam explosion and sequential steam explosion-dilute acid pretreatment for polyhydroxyalkanoate fermentation. Bioactive Carbohydr Diet Fibre, 2023,

[9] Benesova P, Kucera D, Marova I, Obruca S. Chicken feather hydrolysate as an inexpensive complex nitrogen source for PHA production by Cupriavidus necator on waste frying oils. Lett Appl Microbiol, 2017, 65(2): 182-188

[10] Briassoulis D, Tserotas P, Athanasoulia I-G. 2021/10/10/). Alternative optimization routes for improving the performance of poly(3-hydroxybutyrate) (PHB) based plastics. J Clean Prod, 2021, 318: 128555

[11] 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. 2020/03/01/). Metabolic engineering for the synthesis of polyesters: A 100-year journey from polyhydroxyalkanoates to non-natural microbial polyesters. Metab Eng, 2020, 58: 47-81

[12] Crowley PD, Vaillant JJ, Shirley JD, Wengenack NL, Kasten J. 2024/12/01/). Rapidly growing knowledge of Mycobacterium smegmatis: A case series and review of antimicrobial susceptibility patterns. J Clin Tuberculosis Other Mycobact Dis, 2024, 37: 100489

[13] de Paula CBC, de Paula-Elias FC, Rodrigues MN, Coelho LF, de Oliveira NML, de Almeida AF, Contiero J. Polyhydroxyalkanoate Synthesis by Burkholderia glumae into a Sustainable Sugarcane Biorefinery Concept. Front Bioeng Biotechnol, 2020, 8: 631284

[14] Dovbnya DV, Bragin EY, Ivashina TV, Donova MV. Dec 15). Draft Genome Sequence of Mycolicibacterium smegmatis VKM Ac-1171 Contains Full Set of Sterol Catabolic Genes. Microbiol Resour Announc, 2022, 11(12): e0077222

[15] Edson NL. The intermediary metabolism of the mycobacteria. Bacteriol Rev, 1951, 15(3): 147-182

[16] Farag S. Separation and analysis of some sugars by using thin layer chromatography. JASSBT, 1979, 20: 251-254

[17] Getachew A, Woldesenbet F. 2016/12/12). Production of biodegradable plastic by polyhydroxybutyrate (PHB) accumulating bacteria using low cost agricultural waste material. BMC Res Notes, 2016, 9(1): 509

[18] Hahn SK, Chang YK, Kim BS, Chang HN. Optimization of microbial poly(3-hydroxybutyrate) recover using dispersions of sodium hypochlorite solution and chloroform. Biotechnol Bioeng, 1994, 44(2): 256-261

[19] Hassan S, Khudur LS, Nahar K, Dekiwadia C, Ball AS. 2025/07/01). Isolation and Identification of Xylose-Utilising, Polyhydroxyalkanoate-Producing Bacteria from Sugarcane Bagasse: First Report of PHA Production by Mycolicibacterium Smegmatis. J Polym Environ, 2025, 33(7): 3144-3157

[20] Hassan S, Ngo T, Ball AS. Valorisation of Sugarcane Bagasse for the Sustainable Production of Polyhydroxyalkanoates. Sustainability, 2024, 16(5): 2200

[21] Karan H, Funk C, Grabert M, Oey M, Hankamer B. Green Bioplastics as Part of a Circular Bioeconomy. Trends Plant Sci, 2019, 24(3): 237-249

[22] Kourilova X, Pernicova I, Sedlar K, Musilova J, Sedlacek P, Kalina M, Koller M, Obruca S. 2020/11/01/). Production of polyhydroxyalkanoates (PHA) by a thermophilic strain of Schlegelella thermodepolymerans from xylose rich substrates. Bioresour Technol, 2020, 315: 123885

[23] Kovalcik A, Kucera D, Matouskova P, Pernicova I, Obruca S, Kalina M, Enev V, Marova I. 2018/04/01/). Influence of removal of microbial inhibitors on PHA production from spent coffee grounds employing Halomonas halophila. J Environ Chem Eng, 2018, 6(2): 3495-3501

[24] Ladeira-Ázar RIS, Morgan T, Maitan-Alfenas GP, Guimarães VM. 2019/05/01). Inhibitors Compounds on Sugarcane Bagasse Saccharification: Effects of Pretreatment Methods and Alternatives to Decrease Inhibition. Appl Biochem Biotechnol, 2019, 188(1): 29-42

[25] Lyu Q, Dar RA, Baganz F, Smoliński A, Rasmey A-HM, Liu R, Zhang L. Effects of lignocellulosic biomass-derived hydrolysate inhibitors on cell growth and lipid production during microbial fermentation of oleaginous microorganisms—A Review. Fermentation, 2025, 11(3): 121

[26] Mahato RP, Kumar S, Singh P. Optimization of Growth Conditions to Produce Sustainable Polyhydroxyalkanoate Bioplastic by Pseudomonas aeruginosa EO1. Front Microbiol, 2021, 12: 711588

[27] Mattlar T, Ekholm T. 2025/04/01/). The impact of bioplastics production on climate change mitigation, fossil fuels and land-use. Renew Sustain Energy Rev, 2025, 212: 115234

[28] Miller GL. 1959/03/01). Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Anal Chem, 1959, 31(3): 426-428

[29] Mittal M, Kumar N, Yadav A, Aggarwal NK. 2024/03/01). Production and Optimization of Polyhydroxybutyrate by Using Cupriavidus necator with Banana Peels as a Substrate. Circular Econ Sustain, 2024, 4(1): 717-732

[30] Mohanrasu K, Rao RGR, Dinesh GH, Zhang K, Prakash GS, Song D-P, Muniyasamy S, Pugazhendhi A, Jeyakanthan J, Arun A. 2020/07/01/). Optimization of media components and culture conditions for polyhydroxyalkanoates production by Bacillus megaterium. Fuel, 2020, 271: 117522

[31] Morgado SM, Vicente ACP. Comprehensive in silico survey of the Mycolicibacterium mobilome reveals an as yet underexplored diversity. Microb Genom, 2021,

[32] Mostafa YS, Alrumman SA, Otaif KA, Alamri SA, Mostafa MS, Sahlabji T. Production and Characterization of Bioplastic by Polyhydroxybutyrate Accumulating Erythrobacter aquimaris Isolated from Mangrove Rhizosphere. Molecules, 2020, 25(1): 179

[33] Muigano MN, Mauti GO, Anami SE, Onguso JM. 2025/05/01/). Advances and challenges in polyhydroxyalkanoates (PHA) production using Halomonas species: A review. Int J Biol Macromol, 2025, 309: 142850

[34] New England Biolabs (2025) from https://www.neb.com/en-au/-/media/nebus/files/manuals/manualt3010.pdf?rev=f566aa6d43b240bebfc49cf0b7dae63a&hash=54AAB2B687D516BD4657B6239A0DA529

[35] Nieves L, Panyon L, Wang X. 02/18). Engineering Sugar Utilization and Microbial Tolerance toward Lignocellulose Conversion. Front Bioeng Biotechnol, 2015, 3: 17

[36] Oxford Nanopore Technologies (2025) Ligation sequencing gDNA - Native Barcoding Kit 24 V14 (SQK-NBD114.24). Retrieved 2025 from https://nanoporetech.com/document/ligation-sequencing-gdna-native-barcoding-v14-sqk-nbd114-24

[37] Raposo RS, de Almeida MCMD, de Oliveira MD, da Fonseca MM, Cesário MT. A Burkholderia sacchari cell factory: production of poly-3-hydroxybutyrate, xylitol and xylonic acid from xylose-rich sugar mixtures. New Biotechnol, 2017, 34: 12-22

[38] Saratale RG, Cho SK, Saratale GD, Ghodake GS, Bharagava RN, Kim DS, Nair S, Shin HS. 2021/03/01/). Efficient bioconversion of sugarcane bagasse into polyhydroxybutyrate (PHB) by Lysinibacillus sp. and its characterization. Bioresour Technol, 2021, 324: 124673

[39] Siripurapu A, Kvn V, Shivshetty N, Poosarla VG. 2022/12/01/). Production and characterization of biodegradable polymer-polyhydroxybutyrate from agricultural waste-sugarcane bagasse by the novel marine bacterium Klebsiella pneumoniae G1. Bioresource Technol Rep, 2022, 20: 101268

[40] Sirohi R, Prakash Pandey J, Kumar Gaur V, Gnansounou E, Sindhu R. 2020/09/01/). Critical overview of biomass feedstocks as sustainable substrates for the production of polyhydroxybutyrate (PHB). Bioresour Technol, 2020, 311: 123536

[41] Sparks IL, Derbyshire KM, Jacobs WRJrMorita YS. Jan 26). Mycobacterium smegmatis: The Vanguard of Mycobacterial Research. J Bacteriol, 2023, 205(1): e0033722

[42] Sriyapai T, Chuarung T, Kimbara K, Samosorn S, Sriyapai P. 2022/03/01/). Production and optimization of polyhydroxyalkanoates (PHAs) from Paraburkholderia sp. PFN 29 under submerged fermentation. Electron J Biotechnol, 2022, 56: 1-11

[43] Titgemeyer F, Amon J, Parche S, Mahfoud M, Bail J, Schlicht M, Rehm N, Hillmann D, Stephan J, Walter B, Burkovski A, Niederweis M. Aug). A genomic view of sugar transport in Mycobacterium smegmatis and Mycobacterium tuberculosis. J Bacteriol, 2007, 189(16): 5903-5915

[44] Tyagi B, Gupta B, Khatak D, Meena R, Shekhar Thakur I. 2022/06/01/). Genomic analysis, simultaneous production, and process optimization of extracellular polymeric substances and polyhydroxyalkanoates by Methylobacterium sp. ISTM1 by utilizing molasses. Bioresour Technol, 2022, 354: 127204

[45] Vale FF, Lehours P, Yamaoka Y (2022). Editorial: The role of mobile genetic elements in bacterial evolution and their adaptability [Editorial]. Front Microbiol.13 https://doi.org/10.3389/fmicb.2022.849667

[46] Vasylyshyn R, Kurylenko O, Ruchala J, Shevchuk N, Kuliesiene N, Khroustalyova G, Rapoport A, Daugelavicius R, Dmytruk K, Sibirny A. 2020/04/25). Engineering of sugar transporters for improvement of xylose utilization during high-temperature alcoholic fermentation in Ogataea polymorpha yeast. Microb Cell Fact, 2020, 19(1): 96

[47] de Vieira S, Barros MV, Sydney ACN, Piekarski CM, Francisco AC, Vandenberghe LP, Sydney EB. Sustainability of sugarcane lignocellulosic biomass pretreatment for the production of bioethanol. Bioresour Technol, 2020, 299: 122635

[48] Wang J, Liu S, Huang J, Qu Z. 2021/12/01/). A review on polyhydroxyalkanoate production from agricultural waste Biomass: Development, Advances, circular Approach, and challenges. Bioresour Technol, 2021, 342: 126008

[49] Zhao Z, Xian M, Liu M, Zhao G. Biochemical routes for uptake and conversion of xylose by microorganisms. Biotechnol Biofuels, 2020, 13(1): 21

[50] Zytner P, Kumar D, Elsayed A, Mohanty A, Ramarao BV, Misra M. A review on polyhydroxyalkanoate (PHA) production through the use of lignocellulosic biomass. RSC Sustain, 2023, 1(9): 2120-2134

Funding

Royal Melbourne Institute of Technology

RIGHTS & PERMISSIONS

The Author(s)