Abstract
This study investigated the production of levan biopolymer utilizing cane molasses, an agro-industrial waste, as a substrate. The kinetics of growth, substrate consumption, and levan production by Bacillus megaterium KM3 were examined in bioreactor design employing cane molasses-based media. Experiments were conducted in triplicate to ensure reproducibility, first in a 1L shake flask under optimized conditions, followed by scale-up to a 5L bioreactor, achieving a maximum levan yield as 18.5 g/L. The logistic model for microbial growth and Luedeking–Piret equation for product formation and substrate utilization were found to fit the experimental data, with a maximum specific growth rate constant (µm) as 0.6 h−1. The obtained levan was purified, and monosaccharide analysis by HPLC, confirmed the presence of the fructose monomer. Further structural characterization for the presence of functional group was performed using FTIR. Congo red analysis reveals a triple-helix structure. XRD analysis indicated the levan’s non-crystalline amorphous nature, while thermogravimetric analysis demonstrated its high thermal stability. In addition, the in vitro biological activity of levan was evaluated, where it showed strong antioxidant activities to scavenge DPPH radical, hydroxyl radical, and reducing power in dose-dependent manner. The results showcased the promising structural and functional properties of the obtained levan, positioning it as an attractive biopolymer for a wide range of industrial applications. By turning trash into gold, this study provides a model of clean technology’s potential to boost productivity while simultaneously lessening its negative effects on the environment.
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References
Shukla A, Mehta K, Parmar J et al (2019) Depicting the exemplary knowledge of microbial exopolysaccharides in a nutshell. Eur Polym J 119:298–310. https://doi.org/10.1016/j.eurpolymj.2019.07.044
Mehta K, Shukla A, Saraf M (2021) Articulating the exuberant intricacies of bacterial exopolysaccharides to purge environmental pollutants. Heliyon. https://doi.org/10.1016/j.heliyon.2021.e08446
Shukla A, Parmar P, Goswami D et al (2021) Exemplifying an archetypal thorium-EPS complexation by novel thoriotolerant Providencia thoriotolerans AM3. Sci Rep 11:3189. https://doi.org/10.1038/s41598-021-82863-4
Mulani R, Mehta K, Saraf M, Goswami D (2021) Decoding the mojo of plant-growth-promoting microbiomes. Physiol Mol Plant Pathol 115:101687. https://doi.org/10.1016/j.pmpp.2021.101687
Tabuchi SCT, Martiniano SE, Cunha MAA et al (2021) Kinetic study of lasiodiplodan production by Lasiodiplodia theobromae MMPI in a Low-shear aerated and agitated bioreactor. J Polym Environ 29:89–102. https://doi.org/10.1007/s10924-020-01857-x
Moscovici M (2015) Present and future medical applications of microbial exopolysaccharides. Front Microbiol 6:1012. https://doi.org/10.3389/fmicb.2015.01012
Freitas F, Torres CAV, Reis MAM (2017) Engineering aspects of microbial exopolysaccharide production. Bioresour Technol 245:1674–1683. https://doi.org/10.1016/j.biortech.2017.05.092
Valdez AL, Delgado OD, Fariña JI (2021) Cost-effective optimized scleroglucan production by Sclerotium rolfsii ATCC 201126 at bioreactor scale. A quantity-quality assessment. Carbohydr Polym 260:117505. https://doi.org/10.1016/j.carbpol.2020.117505
Schmid J (2018) Recent insights in microbial exopolysaccharide biosynthesis and engineering strategies. Curr Opin Biotechnol 53:130–136. https://doi.org/10.1016/j.copbio.2018.01.005
Tsioptsias C, Lionta G, Deligiannis A, Samaras P (2016) Enhancement of the performance of a combined microalgae-activated sludge system for the treatment of high strength molasses wastewater. J Environ Manag 183:126–132. https://doi.org/10.1016/j.jenvman.2016.08.067
Xu M, Pan L, Zhou Z, Han Y (2022) Structural characterization of levan synthesized by a recombinant levansucrase and its application as yogurt stabilizers. Carbohydr Polym 291:119519. https://doi.org/10.1016/j.carbpol.2022.119519
Srikanth R, Reddy CHSSS, Siddartha G et al (2015) Review on production, characterization and applications of microbial levan. Carbohydr Polym 120:102–114. https://doi.org/10.1016/j.carbpol.2014.12.003
Esawy MA, Amer H, Gamal-Eldeen AM et al (2013) Scaling up, characterization of levan and its inhibitory role in carcinogenesis initiation stage’. Carbohydr Polym 95:578–587. https://doi.org/10.1016/j.carbpol.2013.02.079
Du YH, Wang MY, Yang LH et al (2022) Optimization and scale-up of fermentation processes driven by models. Bioengineering 9:473. https://doi.org/10.3390/bioengineering9090473
González-Figueredo C, Alejandro Flores-Estrella R, Rojas-Rejón AO (2019) Fermentation: metabolism, kinetic models, and bioprocessing. Curr Top Biochem Eng. https://doi.org/10.5772/intechopen.82195
Sirajunnisa AR, Vijayagopal V, Sivaprakash B et al (2016) Optimization, kinetics and antioxidant activity of exopolysaccharide produced from rhizosphere isolate, Pseudomonas fluorescens CrN6. Carbohydr Polym 135:35–43. https://doi.org/10.1016/j.carbpol.2015.08.080
Luedeking R, Piret EL (1959) Transient and steady states in continuous fermentaion. Theory and experiment. J Biochem Microbiol Technol Eng 1:431–459. https://doi.org/10.1002/jbmte.390010408
Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428. https://doi.org/10.1021/ac60147a030
Tilwani YM, Lakra AK, Domdi L et al (2021) Optimization and physicochemical characterization of low molecular levan from Enterococcus faecium MC-5 having potential biological activities. Process Biochem 110:282–291. https://doi.org/10.1016/j.procbio.2021.08.021
Kavitake D, Devi PB, Singh SP, Shetty PH (2016) Characterization of a novel galactan produced by Weissella confusa KR780676 from an acidic fermented food. Int J Biol Macromol 86:681–689. https://doi.org/10.1016/j.ijbiomac.2016.01.099
Wang Y, Li C, Liu P et al (2010) Physical characterization of exopolysaccharide produced by Lactobacillus plantarum KF5 isolated from Tibet Kefir. Carbohydr Polym 82:895–903. https://doi.org/10.1016/j.carbpol.2010.06.013
Taylan O, Yilmaz MT, Dertli E (2019) Partial characterization of a levan type exopolysaccharide (EPS) produced by Leuconostoc mesenteroides showing immunostimulatory and antioxidant activities. Int J Biol Macromol 136:436–444. https://doi.org/10.1016/j.ijbiomac.2019.06.078
Kumar R, Bansal P, Singh J, Dhanda S (2020) Purification, partial structural characterization and health benefits of exopolysaccharides from potential probiotic Pediococcus acidilactici NCDC 252. Process Biochem 99:79–86. https://doi.org/10.1016/j.procbio.2020.08.028
Adesulu-Dahunsi AT, Sanni AI, Jeyaram K (2018) Production, characterization and In vitro antioxidant activities of exopolysaccharide from Weissella cibaria GA44. Lwt 87:432–442. https://doi.org/10.1016/j.lwt.2017.09.013
Li W, Ji J, Rui X et al (2014) Production of exopolysaccharides by Lactobacillus helveticus MB2-1 and its functional characteristics in vitro. Lwt 59:732–739. https://doi.org/10.1016/j.lwt.2014.06.063
Valdez AL, Babot JD, Schmid J et al (2019) Scleroglucan production by Sclerotium rolfsii ATCC 201126 from amylaceous and sugarcane molasses-based media: promising insights for sustainable and ecofriendly scaling-up. J Polym Environ 27:2804–2818. https://doi.org/10.1007/s10924-019-01546-4
Qiu Y, Sha Y, Zhang Y et al (2017) Development of Jerusalem artichoke resource for efficient one-step fermentation of poly-(Γ-glutamic acid) using a novel strain Bacillus amyloliquefaciens NX-2S. Bioresour Technol 239:197–203. https://doi.org/10.1016/j.biortech.2017.05.005
Niknezhad SV, Kianpour S, Jafarzadeh S et al (2022) Biosynthesis of exopolysaccharide from waste molasses using Pantoea sp. BCCS 001 GH: a kinetic and optimization study. Sci Rep 12:10128. https://doi.org/10.1038/s41598-022-14417-1
Ragab TIM, Malek RA, Elsehemy IA et al (2019) Scaling up of levan yield in Bacillus subtilis M and cytotoxicity study on levan and its derivatives. J Biosci Bioeng 127:655–662. https://doi.org/10.1016/j.jbiosc.2018.09.008
Wu FC, Chou SZ, Shih IL (2013) Factors affecting the production and molecular weight of levan of Bacillus subtilis natto in batch and fed-batch culture in fermenter. J Taiwan Inst Chem Eng 44:846–853. https://doi.org/10.1016/j.jtice.2013.03.009
Hamid KRA, Elsayed EA, Enshasy HAE et al (2018) Bioprocess optimization for levan production by Bacillus subtilis B58. J Sci Ind Res 77:386–393
Erkorkmaz BA, Kırtel O, Ateş Duru Ö, Toksoy Öner E (2018) Development of a cost-effective production process for Halomonas levan. Bioprocess Biosyst Eng 41:1247–1259. https://doi.org/10.1007/s00449-018-1952-x
Mehta K, Shukla A, Saraf M (2023) From waste to wonder : harnessing the potential of agro-industrial waste (Cane Molasses) in systemic optimization for the levan type of exopolysaccharide by Bacillus megaterium KM3 and physiochemical characterization. Waste Biomass Valoriz. https://doi.org/10.1007/s12649-023-02236-y
Öner ET, Hernández L, Combie J (2016) Review of Levan polysaccharide: from a century of past experiences to future prospects. Biotechnol Adv 34:827–844. https://doi.org/10.1016/j.biotechadv.2016.05.002
Domżał-Kędzia M, Ostrowska M, Lewińska A, Łukaszewicz M (2023) Recent developments and applications of microbial levan, a versatile polysaccharide-based biopolymer. Molecules 28:5407. https://doi.org/10.3390/molecules28145407
Kovanda L, Zhang W, Wei X et al (2019) In vitro antimicrobial activities of organic acids and their derivatives on several species of Gram-negative and Gram-positive bacteria. Molecules 24:3770. https://doi.org/10.3390/molecules24203770
Kim KH, Chung CB, Kim YH et al (2005) Cosmeceutical properties of levan produced by Zymomonas mobilis. J Cosmet Sci 56:395–406. https://doi.org/10.1111/j.1467-2494.2006.00314_2.x
Pantelić I, Lukić M, Gojgić-Cvijović G et al (2020) Bacillus licheniformis levan as a functional biopolymer in topical drug dosage forms: from basic colloidal considerations to actual pharmaceutical application. Eur J Pharm Sci 142:105109. https://doi.org/10.1016/j.ejps.2019.105109
El Halmouch Y, Ibrahim HAH, Dofdaa NM et al (2023) Complementary spectroscopy studies and potential activities of levan-type fructan produced by Bacillus paralicheniformis ND2. Carbohydr Polym 311:120743. https://doi.org/10.1016/j.carbpol.2023.120743
Lakra AK, Ramatchandirane M, Kumar S et al (2021) Physico-chemical characterization and aging effects of fructan exopolysaccharide produced by Weissella cibaria MD2 on Caenorhabditis elegans. Lwt 143:1111000. https://doi.org/10.1016/j.lwt.2021.111100
Abid Y, Casillo A, Gharsallah H et al (2018) Production and structural characterization of exopolysaccharides from newly isolated probiotic lactic acid bacteria. Int J Biol Macromol 108:719–728. https://doi.org/10.1016/j.ijbiomac.2017.10.155
Mathivanan K, Chandirika JU, Vinothkanna A et al (2021) Characterization and biotechnological functional activities of exopolysaccharides produced by Lysinibacillus fusiformis KMNTT-10. J Polym Environ 29:1742–1751. https://doi.org/10.1007/s10924-020-01986-3
Xu X, Gao C, Liu Z et al (2016) Characterization of the levan produced by Paenibacillus bovis sp. nov BD3526 and its immunological activity. Carbohydr Polym 144:178–186. https://doi.org/10.1016/j.carbpol.2016.02.049
Yang Z, Zeng Y, Hu Y et al (2023) Comparison of chemical property and in vitro digestion behavior of polysaccharides from Auricularia polytricha mycelium and fruit body. Food Chem X 17:100570. https://doi.org/10.1016/j.fochx.2023.100570
Li S, Xia H, Xie A et al (2020) Structure of a fucose-rich polysaccharide derived from EPS produced by Kosakonia sp. CCTCC M2018092 and its application in antibacterial film. Int J Biol Macromol 159:295–303. https://doi.org/10.1016/j.ijbiomac.2020.05.029
Zhao D, Jiang J, Liu L et al (2021) Characterization of exopolysaccharides produced by Weissella confusa XG-3 and their potential biotechnological applications. Int J Biol Macromol 178:306–315. https://doi.org/10.1016/j.ijbiomac.2021.02.182
Shimazu A, Miyazaki T, Ikeda K (2000) Interpretation of d-spacing determined by wide angle X-ray scattering in 6FDA-based polyimide by molecular modeling. J Membr Sci 166:113–118. https://doi.org/10.1016/S0376-7388(99)00254-9
Krishnamurthy M, Jayaraman Uthaya C, Thangavel M et al (2020) Optimization, compositional analysis, and characterization of exopolysaccharides produced by multi-metal resistant Bacillus cereus KMS3-1. Carbohydr Polym 227:115369. https://doi.org/10.1016/j.carbpol.2019.115369
Lakra AK, Domdi L, Tilwani YM, Arul V (2020) Physicochemical and functional characterization of mannan exopolysaccharide from Weissella confusa MD1 with bioactivities. Int J Biol Macromol 143:797–805. https://doi.org/10.1016/j.ijbiomac.2019.09.139
Domżał-Kędzia M, Lewińska A, Jaromin A et al (2019) Fermentation parameters and conditions affecting levan production and its potential applications in cosmetics. Bioorg Chem 93:102787. https://doi.org/10.1016/j.bioorg.2019.02.012
Mendonça CMN, Oliveira RC, Freire RKB et al (2021) Characterization of levan produced by a Paenibacillus sp. isolated from Brazilian crude oil. Int J Biol Macromol 186:788–799. https://doi.org/10.1016/j.ijbiomac.2021.07.036
Nambiar RB, Sellamuthu PS, Perumal AB et al (2018) Characterization of an exopolysaccharide produced by Lactobacillus plantarum HM47 isolated from human breast milk. Process Biochem 73:15–22. https://doi.org/10.1016/j.procbio.2018.07.018
Pei F, Ma Y, Chen X, Liu H (2020) Purification and structural characterization and antioxidant activity of levan from Bacillus megaterium PFY-147. Int J Biol Macromol 161:1181–1188. https://doi.org/10.1016/j.ijbiomac.2020.06.140
Bouallegue A, Casillo A, Chaari F et al (2020) Levan from a new isolated Bacillus subtilis AF17: purification, structural analysis and antioxidant activities. Int J Biol Macromol 144:316–324. https://doi.org/10.1016/j.ijbiomac.2019.12.108
Xiao L, Han S, Zhou J et al (2020) Preparation, characterization and antioxidant activities of derivatives of exopolysaccharide from Lactobacillus helveticus MB2-1. Int J Biol Macromol 145:1008–1017. https://doi.org/10.1016/j.ijbiomac.2019.09.192
Acknowledgements
Authors are thankful to Department of Microbiology and Biotechnology, School of Sciences, Gujarat University, DST-FIST Sponsored Department, for providing necessary facilities to perform experiments. We acknowledge Education Department, Government of Gujarat, India for the providing research fellowship to Krina Mehta under the ScHeme Of Developing High-quality research (SHODH).
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KM and AS contributed in the conception and experiment design. KM performed the experiment and wrote the manuscript. KM prepared tables and figures. MS and AS reviewed the manuscript.
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Mehta, K., Shukla, A. & Saraf, M. Production Kinetics and Structural Characterization of Levan Derived from Bacillus megaterium KM3 Using Pretreated Cane Molasses. J Polym Environ 32, 1602–1618 (2024). https://doi.org/10.1007/s10924-023-03054-y
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DOI: https://doi.org/10.1007/s10924-023-03054-y