Determination of hydrolyzing and ethanolic potential of cellulolytic bacteria isolated from fruit waste

Main Article Content

Areeba Shehzadi
Asma Chaudhary
Ayesha Aihetasham
Nageen Hussain
Sumaira Naz
Tariq Aziz
Abdullah F. Alasmari

Keywords

Cellulolytic bacteria, ethanol yield, CMCase activity, Lignocellulosic Biomass, Reducing Sugar Content

Abstract

Fruit wastes containing cellulose can be made valuable by cellulolytic bacteria in biofuel production. This study aimed to assess the potential of isolated cellulolytic bacteria to produce bioethanol and its fermentation efficiency. Seven out of 16 bacterial isolates were selected on the basis of their cellulose-degrading potential by providing cellulose as the only carbon source. Their potential to degrade cellulose was determined by different biochemical tests. All strains produced bubbles as indicators of carbon dioxide production in Durham tubes. The maximum hallow on Congo red staining was shown by CA2, CG2, as 54, 40 mm with cellulolytic index 16.3 and 19. Cellulose degradation was evaluated as light pink to maroon color in triphenyl tetrazolium in all strains except CA4 and CB1. Cellulose (2%) added medium was provided to the isolated strains for the period of 10 days to allow fermentation. CG2 and CA2 yielded maximum ethanol as 0.42±0.005 and 0.43±0.011 (g of ethanol/ g of reducing sugar consumed), respectively. Their percent fermentation efficiency was observed as 79.71±0.059% and 75.58±0.011% correspondingly. All strains showed cellulose activity, and the maximum was seen in CA2 and CG2 as 0.265±0.05, 0.27±0.011 μmol/min/L. Molecular characterization suggested that the CA2 and CG2 strains were Bacillus manliponesis CA2 and Bacillus sp. CG2 (Accession Nos. ON324120 and OM974175). This study elaborates on the capability of bacteria to produce bioethanol by degrading cellulose.

Abstract 230 | PDF Downloads 273 HTML Downloads 0 XML Downloads 64

References

Abate G, Aseffa A, Selassie A, et al. Direct colorimetric assay for rapid detection of rifampin-resistant Mycobacterium tuberculosis. J Clin Microbiol. 2004;42(2):871–873. 10.1128/JCM.42.2.871-873.2004

Abedinifar S, Karimi K, Khanahmadi M, Taherzadeh MJ. production by Mucor indicus and Rhizopus oryzae from rice straw by separate hydrolysis and fermentation. Biomass Bioenergy. 2009;33(5):828–833. 10.1016/j.biombioe.2009.01.003

Ali SS, Nugent B, Mullins E, Doohan FM. Fungal-mediated consolidated bioprocessing: the potential of Fusarium oxysporum for the lignocellulosic ethanol industry. AMB Express. 2016;6:1–13. 10.1186/s13568-016-0185-0

Arooj, S., Iftikhar, T., Mustafa, S. et al. Optimization of critical medium components for lipase production by Neurospora crassa in solid state fermentation. Biomass Conv. Bioref. 2023. 10.1007/s13399-023-05081-0

Aziz, T., Shah, Z., Sarwar, A. et al. Production of bioethanol from pretreated rice straw, an integrated and mediated upstream fermentation process. Biomass Conv. Bioref. 2023. 10.1007/s13399-023-04283-w

Barbosa FC, Silvello MA, Goldbeck R. Cellulase and oxidative enzymes: new approaches, challenges and perspectives on cellulose degradation for bioethanol production. Biotechnol Letters. 2020;42:875–884. 10.1007/s10529-020-02875-4

Behera BC, Sethi BK, Mishra RR, Dutta SK, Thatoi HN. Microbial cellulases–Diversity & biotechnology with reference to mangrove environment: A review. J Genetic Eng Biotechnol. 2017;15(1):197–210. 10.1016/j.jgeb.2016.12.001

Bergey DH. Bergey’s manual of determinative bacteriology. Lippincott Williams & Wilkins; 1994.

Bilgili L. A systematic review on the acceptance of alternative marine fuels. Renew Sust Energ Rev. 2023;182:113367. 10.1016/j.rser.2023.113367

Bischof RH, Ramoni J, Seiboth B. Cellulases and beyond: the first 70 years of the enzyme producer Trichoderma reesei. Microb Cell Factories. 2016;15(1):1–13. 10.1186/s12934-016-0507-6

Byadgi SA, Kalburgi PB. Production of bioethanol from waste newspaper. Procedia Environment Sci. 2016;35:555–562. 10.1016/j.proenv.2016.07.040

Caviedes L, Delgado J, Gilman RH. Tetrazolium microplate assay as a rapid and inexpensive colorimetric method for determination of antibiotic susceptibility of Mycobacterium tuberculosis. J Clin Microbiol. 2002;40(5):1873–1874. 10.1128/JCM.40.5.1873-1874.2002

Chandel AK., Chandrasekhar G, Lakshmi Narasu M, Venkateswar Rao L. Simultaneous saccharification and fermentation (SSF) of aqueous ammonia pretreated Saccharum spontaneum (wild sugarcane) for second generation ethanol production. Sugar Tech, 2010;12:125–132. 10.1007/s12355-010-0025-5

Chaudhary A, Hussain A, Shehzadi A, Manzoor M, Shahbaz M, Deepanraj B. Production of ethanol from xylan by indigenous xylanolytic and ethanologenic bacteria isolated from fruit wastes. Sustain Energy Technol Assessments. 2023;57:103216. 10.1016/j.seta.2023.103216

Chien F, Kamran HW, Albashar G, Iqbal W. Dynamic planning, conversion, and management strategy of different renewable energy sources: a sustainable solution for severe energy crises in emerging economies. Int J Hydrogen Energy. 2021;46(11):7745–7758. 10.1016/j.ijhydene.2020.12.004

Chu LK. The role of energy security and economic complexity in renewable energy development: evidence from G7 countries. Environ Sci Pollut Res. 2023;30(19):56073–56093. 10.1007/s11356-023-26208-w

Dang Z, Wang Y, Wang M, et al. The FRAGILE CULM19 (FC19) mutation largely improves plant lodging resistance, biomass saccharification, and cadmium resistance by remodeling cell walls in rice. J Hazardous Mater. 2023;132020. 10.1016/j.jhazmat.2023.132020

Darwesh OM, El-Maraghy SH, Abdel-Rahman HM, Zaghloul RA. Improvement of paper wastes conversion to bioethanol using novel cellulose degrading fungal isolate. Fuel. 2020;262:116518. 10.1016/j.fuel.2019.116518

Dubey R, Gupta DK, Radhakrishnan SK. Biomass: sustainable energy solution from agriculture. In Handbook of energy management in agriculture. Singapore: Springer Nature; pp. 1–29. 10.1007/978-981-19-7736-7_11-1

Florencio C, Couri S, Farinas CS. Correlation between agar plate screening and solid-state fermentation for the prediction of cellulase production by Trichoderma strains. Enzyme Res. 2012;2012. 10.1155/2012/793708

Fujimoto N, Kosaka T, Nakao T, Yamada M. Bearing a high cellulose-degrading activity, which was isolated as a heat-resistant and micro-aerophilic microorganism from bovine rumen. Open Biotechnol J. 2011;5(1). 10.2174/1874070701105010007

Ghezelbash A, Khaligh V, Fahimifard SH, Liu JJ. A comparative perspective of the effects of CO2 and non-CO2 greenhouse gas emissions on global solar, wind, and geothermal energy investment. Energies. 2023;16(7):3025. 10.3390/en16073025

Gnanasekaran L, Priya AK, Thanigaivel S, Hoang TK, Soto-Moscoso M. The conversion of biomass to fuels via cutting-edge technologies: Explorations from natural utilization systems. Fuel. 2023;331:125668. 10.1016/j.fuel.2022.125668

Gupta P, Samant K, Sahu A. Isolation of cellulose-degrading bacteria and determination of their cellulolytic potential. Int J Microbiol. 2012;2012. 10.1155/2012/578925

Hidayat MR. Isolation and identification of cellulolytic bacteria symbiont from various termites on different nest type in Bukit Baka Bukit Raya National Park, West Kalimantan, Indonesia. Walailak J. Sci Technol (WJST). 2021;18(14):12708–12. 10.48048/wjst.2021.12708

Jalandoni-Buan AC, Decena-Soliven ALA, Cao EP, Barraquio VL, Barraquio WL. Characterization and identification of Congo red decolorizing bacteria from monocultures and consortia. Philipp. J. Sci. 2010;139(1):71–78.

Juturu V, Wu JC. Microbial cellulases: engineering, production and applications. Renew Sustain Energ Rev. 2014;33:188–203. 10.1016/j.rser.2014.01.077

Keshavarzi M, Mohammadi P, Rastegari H. Investigation of ketal-acetin mixture synthesized from glycerol as a renewable additive for gasoline-ethanol fuel blend: Physicochemical characterization and engine combustion, performance, and emission assessment. Fuel. 2023;348:128519. 10.1016/j.fuel.2023.128519

Khan MZA, Khan HA, Ravi SS, Turner JW, Aziz M. Potential of clean liquid fuels in decarbonizing transportation–An overlooked net-zero pathway? Renew Sustain Energ Rev. 2023;183:113483. 10.1016/j.rser.2023.113483

Lakra A, Bano S. Biofuels as an opportunity to sustainable energy. World. 2023;1:5.

Lee S, Kong DH, Yun, SH, et al. Evaluation of a modified antimycobacterial susceptibility test using Middlebrook 7H10 agar containing 2, 3-diphenyl-5-thienyl-(2)-tetrazolium chloride. J Microbiol Methods.2006;66(3):548–551. 10.1016/j.mimet.2006.02.004

Lee S, Kong DH, Yun SH. Evaluation of a modified antimicrobial susceptibility test using Middlebrook 7H10 agar containing 2,3-diphenyl-5-thienyl-(2)-tetrazolium chloride, J Microbiol Biotechnol. 2006;43:412–415. 10.1016/j.mimet.2006.02.004

Lu WJ, Wang HT, Nie YF, et al. Effect of inoculating flower stalks and vegetable waste with ligno-cellulolytic microorganisms on the composting process. J Environ Sci Health, Part B. 2004;39(5–6):871–887. 10.1081/PFC-200030896

LI, Z., Tian-tian, L., Aziz, T. et al. Purification of Galacto-oligosaccharide (GOS) by fermentation with Kluyveromyces lactis and Interaction between GOS and casein under simulated acidic fermentation conditions. World J Microbiol Biotechnol 39, 342 (2023). 10.1007/s11274-023-03791-1

Luo AG, Wang YY, Xue SS, et al. Screening, identification, and optimization of enzyme-producing conditions for cellulose-degrading bacteria in distillery lees. Applied Sci. 2023;13(13):7693. 10.3390/app13137693

Ma L, Lu Y, Yan H, et al. Screening of cellulolytic bacteria from rotten wood of Qinling (China) for biomass degradation and cloning of cellulases from Bacillus methylotrophicus. BMC Biotechnol. 2020;20(1):1–13. 10.1186/s12896-019-0593-8

Malik A, Abdieva G, Ualieva P. Microbial diversity of soils contaminated with persistent organic pollutants and POP-degrading strains. Water Air Soil Pollut. 2023;234(5):290. 10.1007/s11270-023-06193-z

Meyer SA, Yarrow D. Validation of the names of three Candida species. Mycotaxon 1998;66:170–9.

Miller GL, Blum R, Glennon WE, Burton AL. Measurement of carboxymethylcellulase activity. Analyt Biochem. 1960;1(2):127–132. 10.1016/0003-2697(60)90004-X

Mshana RN, Tadesse G, Abate G, Miörner H. Use of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide for rapid detection of rifampin-resistant Mycobacterium tuberculosis. J Clin Microbiol. 1998;36(5):1214–1219. 10.1128/JCM.36.5.1214-1219.1998

Najeeb U, Rehman MU, Sarwar A, Nadeem M, Nelofer R, Shakir HA, Irfan M, Idrees M, Naz S, Nabi G, et al. Purification, Characterization, and Application of Alkaline Protease Enzyme from a Locally Isolated Bacillus Cereus Strain. Fermentation. 2022; 8(11):628. 10.3390/fermentation8110628

Narjis K, Najeeb U, Abid S, Tariq A, Metab A, Abdulrahman A. Isolation and Identification of Protease Producing Bacillus strain from Cold Climate Soil and Optimization of its Production by applying Different Fermentation Conditions. Appl Ecol Environ Res. 2023, 21(4):3391-3401. 10.15666/aeer/2104_33913401

Payne CM, Knott BC, Mayes HB, et al. Fungal cellulases. Chem Rev. 2015;115(3):1308–1448. 10.1021/cr500351c

Peristiwati, Natamihardja YS, Herlini H. Isolation and identification of cellulolytic bacteria from termites gut (Cryptotermes sp.). J Phys Conf Ser. 2018;1013:12173. 10.1088/1742-6596/1013/1/012173

Rajnish KN, Samuel MS, Datta S, et al. Immobilization of cellulase enzymes on nano and micro-materials for breakdown of cellulose for biofuel production-a narrative review. Int J Biol Macromol. 2021;182:1793–1802. 10.1016/j.ijbiomac.2021.05.176

Riskawati R, Natsir H, Dali S, Baharuddin M. Isolation and identification of cellulolytic bacteria from gut of horn beetle larvae (Oryctes rhinoceros L.). Molekul. 2023;18(2):210–217. 10.20884/1.jm.2023.18.2.6848

Rudolf A, Alkasrawi M, Zacchi G, Lidén G. A comparison between batch and fed-batch simultaneous saccharification and fermentation of steam pretreated spruce. Enzyme Microb Technol. 2005;37(2):195–204. 10.1016/j.enzmictec.2005.02.013

Scheffers WA. Alcoholic fermentation. Stud Mycol. 1987;30:321–332.

Sillu D, Agnihotri S. Cellulase immobilization onto magnetic halloysite nanotubes: enhanced enzyme activity and stability with high cellulose saccharification. ACS Sustain Chem Eng. 2019;8(2):900–913. 10.1021/acssuschemeng.9b05400

Soni SK, Batra N, Bansal N, Soni R. Bioconversion of sugarcane bagasse into second generation bioethanol after enzymatic hydrolysis with in-house produced cellulases from Aspergillus sp. S 4 B 2 F. BioResources. 2010;5(2):741–758. 10.15376/biores.5.2.741-757

Shah, T.A., Majeed, T., Rahman, S.u, et al. Synergistic treatment of crude enzymes from Bacillus sp. strains to boost anaerobic fermentation of rice straw. Biomass Conv. Bioref. 2023. 10.1007/s13399-023-05090-z

Stolz A. Basic and applied aspects in the microbial degradation of azo dyes. Appl Microbiol Biotechnol. 2001;56:69–80. 10.1007/s002530100686

Suharti S, Novrariani N, Wiryawan KG. Morphological, biochemical, and molecular identification of cellulolytic bacteria isolated from feces of endemic tropical herbivores. Biodivers J Biol Divers. 2023;24(7). 10.13057/biodiv/d240742

Tai HC, Chang CH, Cai W, et al. Wood cellulose microfibrils have a 24-chain core–shell nanostructure in seed plants. Nat Plants. 2023;9(7):1154–1168. 10.1038/s41477-023-01430-z

Thom SM, Horobin RW, Seidler E, Barer MR. Factors affecting the selection and use of tetrazolium salts as cytochemical indicators of microbial viability and activity. J Applied Microbiol. 1993;74(4):433–443. 10.1111/j.1365-2672.1993.tb05151.x

Tyagi U, Anand N. Prospective of waste lignocellulosic biomass as precursors for the production of biochar: application, performance, and mechanism—A review. BioEnergy Res. 2023;16:1335–1360. 10.1007/s12155-022-10560-9

Ullah, N., Mujaddad-ur-Rehman, M., Sarwar, A, et al. Effect of bioprocess parameters on alkaline protease production by locally isolated Bacillus cereus AUST-7 using tannery waste in submerged fermentation. Biomass Conv. Bioref. (2023). 10.1007/s13399-023-04498-x

Wardani AK, Brahmanti AA, Martati E. Leclercia adecarboxylata C12, the newly isolated cellulose-degrading bacteria from Indonesian coffee pulp. HAYATI J Biosci. 2023;30(3):588–595. 10.4308/hjb.30.3.588-595

Wu L, Che S, Qin X, et al. Identification, characteristics and rice growth promotion of a highly efficient cellulolytic bacterial strain, Cellulomonas iranensis ZJW-6, isolated from paddy soil in central China. Front Microbiol. 2023;14:1152966. 10.3389/fmicb.2023.1152966

Yakovlev VA, Belyaev GA. Global climate change, its consequences and ways to solve the problem. E3S Web Conf. 2023;390:4007. 10.1051/e3sconf/202339004007

Yamakawa CK, Rojas ST, Herrera WE, Rossell CE, Maciel MRW, Maciel Filho R. Recovery and characterization of cellulosic ethanol from fermentation of sugarcane bagasse. Chem Eng Res Design. 2023;196:568–576. 10.1016/j.cherd.2023.06.053

Yao J, Yang C, Shi K, Liu Y, Xu G, Pan S. Effect of pulp cell wall polysaccharides on citrus fruit with different mastication traits. Food Chem. 2023;429:136740. 10.1016/j.foodchem.2023.136740

Wang X., Feng H, Chen T, Zhao S, Zhang, Zhang X. (2021). Gas sensor technologies and mathematical modelling for quality sensing in fruit and vegetable cold chains: A review. Trends in Food Science & Technology, 110, 483-492. doi: 10.1016/j.tifs.2021.01.073

Zhang L, Zhao H, Gan M, et al. Application of simultaneous saccharification and fermentation (SSF) from viscosity reducing of raw sweet potato for bioethanol production at laboratory, pilot and industrial scales. Bioresour Technol. 2011;102(6):4573–4579. 10.1016/j.biortech.2010.12.115

Zhang YHP, Cui J, Lynd LR, Kuang LR. A transition from cellulose swelling to cellulose dissolution by o-phosphoric acid: evidence from enzymatic hydrolysis and supramolecular structure. Biomacromolecules. 2006;7(2):644–648. 10.1021/bm050799c