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Isolation and Identification of Cellulase Producing and Sugar Fermenting Bacteria for Second-Generation Bioethanol Production

1Department of Zoology, Centre for Water Quality and Algae Research, Faculty of Applied Sciences, University of Sri Jayewardenepura, Gangodawila, Nugegoda, 10250, Sri Lanka

2Faculty of Graduate Sciences, University of Sri Jayewardenepura, Gangodawila, Nugegoda, 10250, Sri Lanka

Received: 5 Jan 2021; Revised: 29 Mar 2021; Accepted: 10 Apr 2021; Available online: 20 Apr 2021; Published: 1 Nov 2021.
Editor(s): Rock Keey Liew
Open Access Copyright (c) 2021 The Authors. Published by Centre of Biomass and Renewable Energy (CBIORE)
Creative Commons License This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.

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Over the last decades, the negative impacts of fossil fuel on the environment and increasing demand for energy due to the unavoidable depletion of fossil fuels, has transformed the world’s interests towards alternative fuels. In particular, bioethanol production from cellulosic biomass for the transportation sector has been incrementing since the last decade. The bacterial pathway for bioethanol production is a relatively novel concept and the present study focused on the isolation of potential “cellulase-producing” bacteria from cow dung, compost soil, and termite gut and isolating sugar fermenting bacteria from palm wine. To select potential candidates for cellulase enzyme production, primary and secondary assays were conducted using the Gram’s iodine stain in Carboxy Methyl Cellulose (CMC) medium and the Dinitrosalicylic acid (DNS) assays, respectively. Durham tube assay and Solid-Phase Micro-Extraction (SPME) coupled with Gas Chromatography-Mass Spectrometry (GC-MS) was used to evaluate the sugar fermenting efficiency of the isolated bacteria. Out of 48 bacterial isolates, 27 showed cellulase activity where Nocardiopsis sp. (S-6) demonstrated the highest extracellular crude enzyme activity of endoglucanase (1.56±0.021 U) and total cellulase activity (0.93±0.012 U). The second-highest extracellular crude enzyme activity of endoglucanase (0.21±0.021 U) and total cellulase activity (0.35±0.021 U) was recorded by Bacillus sp. (T-4). Out of a total of 8 bacterial isolates, Achromobacter sp. (PW-7) was positive for sugar fermentation resulting in 3.07% of ethanol in broth medium at 48 h incubation. The results of the study revealed that Nocardiopsis sp. (S-6) had the highest cellulase enzyme activity. However, the highest ethanol percentage was achieved with by having both Bacillus sp. (T-4) and Achromobacter sp. (PW-7) for the simultaneous saccharification and fermentation (SSF) method, as compared to separate hydrolysis and fermentation (SHF) methodologies. 

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Keywords: Bioethanol; Carboxy methyl cellulose; Cellulase producing bacteria; Solid phase micro-extraction; Sugar fermenting bacteria

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  1. Aditiya, H.B., Mahlia, T.M.I., Chong, W.T., Nur, H. and Sebayang, A.H., (2016). Second generation bioethanol production: A critical review. Renewable and sustainable energy reviews, 66, 631-653;
  2. Ahmadi, P., Dincer, I. and Rosen, M.A., (2015). Performance assessment of a novel solar and ocean thermal energy conversion based multigeneration system for coastal areas. Journal of Solar Energy Engineering, 137(1);
  3. Ansari, S. and Karimi, M., (2017). Recent progress, challenges and trends in trace determination of drug analysis using molecularly imprinted solid-phase microextraction technology. Talanta, 164, 612-625; 10.1016/j.talanta.2016.11.007
  4. Anu, Kumar, S., Kumar, A., Kumar, V. and Singh, B., (2020). Optimization of cellulase production by Bacillus subtilis subsp. subtilis JJBS300 and biocatalytic potential in saccharification of alkaline-pretreated rice straw. Preparative Biochemistry & Biotechnology, 1-8; 10.1080/10826068.2020.1852419
  5. Azhar, S.H.M., Abdulla, R., Jambo, S.A., Marbawi, H., Gansau, J.A., Faik, A.A.M. and Rodrigues, K.F., (2017). Yeasts in sustainable bioethanol production: A review. Biochemistry and Biophysics Reports, 10, 52-61;
  6. Balkhair, K.S. and Rahman, K.U., (2017). Sustainable and economical small-scale and low-head hydropower generation: A promising alternative potential solution for energy generation at local and regional scale. Applied Energy, 188, 378-391;
  7. Dahnum, D., Tasum, S.O., Triwahyuni, E., Nurdin, M. and Abimanyu, H., (2015). Comparison of SHF and SSF processes using enzyme and dry yeast for optimization of bioethanol production from empty fruit bunch. Energy Procedia, 68, 107-116;
  8. Dung, N.T.P. and Huynh, P.X., (2013). Screening Thermo-and Ethanol Tolerant Bacteria for Ethanol Fermentation. American Journal of Microbiological Research, 1(2), 25-31;
  9. Dung, N.T.P., Thanonkeo, P. and Phong, H.X., (2012). Screening useful isolated yeasts for ethanol fermentation at high temperature. International Journal of Applied, 2(4), 65-71
  10. Ekanayake, E.M.M.S. and Manage, P.M., (2020). Green approach for decolorization and detoxification of textile dye-CI direct blue 201 using native bacterial strains. Environment and Natural Resources Journal, 18(1),.1-8
  11. Ekanayake, M.S. and Manage, P.M., (2016). Isolation of Textile Dye Decolorizing Bacteria from Environmental Samples. Symposium Proceedings, Fifth International Symposium on Water Quality and Human Health: Challenges Ahead, 05 & 06 August, PGIS, Peradeniya, Sri Lanka
  12. El-Sayed, A.F., Abo-Sereih, N.A., Mahmoud, A.E., El-Kawokgy, T.M. and El-Ghamery, A.A., (2019). Genetic identification and optimization of novel β-glucosidase-producing Lysinibacillus sphaericus QS6 strain isolated from the Egyptian environment. Egyptian Pharmaceutical Journal, 18(4), 341;
  13. Gunawardena.S., (2009). Liquid biofuel for transportation in srilanka., Technical report, University of Moratuwa
  14. Hassan, S.S., Williams, G.A. and Jaiswal, A.K., (2019). Moving towards the second generation of lignocellulosic biorefineries in the EU: Drivers, challenges, and opportunities. Renewable and Sustainable Energy Reviews, 101, 590-599;
  15. Idroos, F.S. and Manage, P.M., (2018). Bioremediation of Microcystins by Two Native Bacteria; Bacillus Cereus and Rahnella Aquatilis. Asian Journal of Microbiology Biotechnology and Environmental Sciences, 20(3), 24-32
  16. Islam, F. and Roy, N., (2018). Screening, purification and characterization of cellulase from cellulase producing bacteria in molasses. BMC research notes, 11(1),1-6;
  17. Jayasekara, S.K., Abayasekara, C.L. and Ratnayake, R.R., (2019). Efficient Microorganisms for Bioethanol Production from the Natural Environment of Sri Lanka. In Proceedings of International Research Symposium of Uva Wellassa University.
  18. Jayathilaka, M.G.L.W., Henagamage, A.P., Peries, C.M. and Seneviratne, G., (2018). Enhancement of Cellulolytic Activity through Biofilm Action for Bioethanol Production. In Proceedings of International Research Symposium of Uva Wellassa University
  19. Kularathne, I.W., Rathneweera, A.C., Kalpage, C.S., Rajapakshe, S. and Gunathilaka, C.A., (2020). Optimization and kinetic parameter estimation of bioethanol production from freely available Sri Lankan fruits in batch fermentation. Ceylon Journal of Science, 49(3), 283-291;
  20. Ladeira, S.A., Cruz, E., Delatorre, A.B., Barbosa, J.B. and Leal Martins, M.L., (2015). Cellulase production by thermophilic Bacillus sp: SMIA-2 and its detergent compatibility. Electronic journal of biotechnology, 18(2), 110-115;
  21. Liu, C.G., Xiao, Y., Xia, X.X., Zhao, X.Q., Peng, L., Srinophakun, P. and Bai, F.W., (2019). Cellulosic ethanol production: progress, challenges and strategies for solutions. Biotechnology advances, 37(3), 491-504;
  22. Liyanage, G.Y and Manage, P.M., (2016a). Isolation and characterization of oil degrading bacteria from coastal waters and sediments from three locations in Sri Lanka. Journal of the National Science Foundation of Sri Lanka, 44(4), 201-203;
  23. Liyanage, G.Y and Manage, P.M., (2016b). Optimization of Environmental Factors on Oil Degrading Bacteria Isolated from Coastal Water and Sediments in Sri Lanka. Journal of Tropical Forestry and Environment, 5(2), 13-25;
  24. Lu, M., Li, J., Han, L. and Xiao, W., (2019). An aggregated understanding of cellulase adsorption and hydrolysis for ball-milled cellulose. Bioresource technology, 273, 1-7; 10.1016/j.biortech.2018.10.037
  25. Lugani, Y., Singla, R. and Sooch, B.S., (2015). Optimization of cellulase production from newly isolated Bacillus sp. Y3. Journal of Bioprocessing & Biotechniques, 5(11), 1;
  26. Madusanka D. A. T. and Manage P. M., (2018). Optimizing a solvent system for lipid extraction from cyanobacterium Microcystis spp.: Future perspective for biodiesel production. Journal of National Sciences Foundation, 46(2), 219-227;
  27. Magocha, T.A., Zabed, H., Yang, M., Yun, J., Zhang, H. and Qi, X., (2018). Improvement of industrially important microbial strains by genome shuffling: Current status and future prospects. Bioresource technology, 257, 281-289; 10.1016/j.biortech.2018.02.118
  28. Manzum, A.A. and Al Mamun, M.A., (2018). Isolation of Bacillus spp. bacteria from soil for production of cellulase. Nepal Journal of Biotechnology, 6(1), 57-61; 10.3126/njb.v6i1.22338
  29. Maravi, P. and Kumar, A., (2021). Optimization and statistical modeling of microbial cellulase production using submerged culture. Journal of Applied Biology & Biotechnology, 9(2), 142-152; 10.7324/JABB.2021.9213
  30. Mboowa, D., Chandra, R.P., Hu, J. and Saddler, J.N., (2020). Substrate characteristics that influence the Filter paper assay’s ability to predict the hydrolytic potential of cellulase mixtures. ACS Sustainable Chemistry & Engineering, 8(28), 10521-10528;
  31. Mohanty, S.K. and Swain, M.R., (2019). Bioethanol production from corn and wheat: food, fuel, and future. In Bioethanol production from food crops, 45-59;
  32. Nair, R.B. and Taherzadeh, M.J., (2016). Valorization of sugar-to-ethanol process waste vinasse: a novel biorefinery approach using edible ascomycetes filamentous fungi. Bioresource technology, 221, 469-476;
  33. Niegel, J., (2018). Tidal Waves. Trusts & Trustees, 24(6), 463-473;
  34. Onuki, S., Koziel, J.A., Jenks, W.S., Cai, L., Rice, S. and van Leeuwen, J., (2016). Optimization of extraction parameters for quantification of fermentation volatile by‐products in industrial ethanol with solid‐phase microextraction and gas chromatography. Journal of the Institute of Brewing, 122(1), 102-109;
  35. Pandey, A.K., Kumar, M., Kumari, S., Kumari, P., Yusuf, F., Jakeer, S., Naz, S., Chandna, P., Bhatnagar, I. and Gaur, N.A., (2019). Evaluation of divergent yeast genera for fermentation-associated stresses and identification of a robust sugarcane distillery waste isolate Saccharomyces cerevisiae NGY10 for lignocellulosic ethanol production in SHF and SSF. Biotechnology for biofuels, 12(1), 1-23; 10.1186/s13068-019-1379-x
  36. Phuog, N.T., Lena, N.T. and Thang, N.D., (2015). Isolation of cellulose degrading bacteria from the gut of the termite Coptotermes gestroi. Global Science Publications, 17(4), 931-936
  37. Pramanik, S.K., Mahmud, S., Paul, G.K., Jabin, T., Naher, K., Uddin, M.S., Zaman, S. and Saleh, M.A., (2021). Fermentation optimization of cellulase production from sugarcane bagasse by Bacillus pseudomycoides and molecular modeling study of cellulase. Current Research in Microbial Sciences, 2,100013;
  38. Rajwade, J.M., Paknikar, K.M. and Kumbhar, J.V., (2015). Applications of bacterial cellulose and its composites in biomedicine. Applied microbiology and biotechnology, 99(6), 2491-2511;
  39. Rastogi, M. and Shrivastava, S., (2017). Recent advances in second generation bioethanol production: An insight to pretreatment, saccharification and fermentation processes. Renewable and Sustainable Energy Reviews, 80, 330-340; 10.1016/j.rser.2017.05.225
  40. Rawway, M., Ali, S.G. and Badawy, A.S., (2018). Isolation and identification of cellulose degrading bacteria from different sources at Assiut governorate (Upper Egypt). J. Ecol. Heal. Environ. Int. J, 6, 15-24; 10.18576/jehe/060103
  41. Rebière, L., Clark, A. C., Schmidtke, L. M., Prenzler, P. D., & Scollary, G. R. (2010). A Robust Method for Quantification of Volatile Compounds within and between Vintages Using Headspace-Solid-Phase Micro-Extraction Coupled with GC–MS – Application on Semillon Wines. Analytica Chimica Acta, 660(1–2), 149-157;
  42. Saeed, M.A., Ma, H., Yue, S., Wang, Q. and Tu, M., (2018). Concise review on ethanol production from food waste: development and sustainability. Environmental Science and Pollution Research, 25(29), 28851-28863;
  43. Saleem, A., Hussain, A., Chaudhary, A., Iqtedar, M., Javid, A. and Akram, A.M., (2020). Acid hydrolysis optimization of pomegranate peels waste using response surface methodology for ethanol production. Biomass Conversion and Biorefinery, 1-12; 10.1007/s13399-020-01117-x
  44. Sarkar, D., Prajapati, S., Poddar, K. and Sarkar, A., (2020). Ethanol production by Klebsiella sp. SWET4 using banana peel as feasible substrate. Biomass Conversion and Biorefinery, 1-13;
  45. Sarsaiya, S., Awasthi, S.K., Awasthi, M.K., Awasthi, A.K., Mishra, S. and Chen, J., (2018). The dynamic of cellulase activity of fungi inhabiting organic municipal solid waste. Bioresource technology, 251, 411-415;
  46. Senarathna, D.B.G.M., Rupasinghe, C.P. and Bandara, W.B.M.A.C., (2019). Bioethanol Production from Lignocellulosic Materials. In Proceedings of EdHat International Research Conference on Technology and Innovation (IRCTECiN)
  47. Sharma, A., Choudhary, J., Singh, S., Singh, B., Kuhad, R.C., Kumar, A. and Nain, L., (2019). Cellulose as potential feedstock for cellulase enzyme production: versatility and properties of various cellulosic biomasses. In New and Future Developments in Microbial Biotechnology and Bioengineering, 11-27;
  48. Singh, S., Jaiswal, D.K., Sivakumar, N. and Verma, J.P., (2019). Developing efficient thermophilic cellulose degrading consortium for glucose production from different agro-residues. Frontiers in Energy Research, 7, 61;
  49. Sirohi, R., Singh, A., Tarafdar, A. and Shahi, N.C., (2018). Application of genetic algorithm in modelling and optimization of cellulase production. Bioresource technology, 270, 751-754;
  50. Song, B., Li, B., Wang, X., Shen, W., Park, S., Collings, C., Feng, A., Smith, S.J., Walton, J.D. and Ding, S.Y., (2018). Real-time imaging reveals that lytic polysaccharide monooxygenase promotes cellulase activity by increasing cellulose accessibility. Biotechnology for biofuels, 11(1), 1-11;
  51. Szilvia, D., Antalné, T., Tamás, E. and Horská, E., (2015). The Change of Residents’ Attitudes Towards Renewable Energy Sources In 2006–2014 As A Reflection Of Primary Research. Visegrad Journal on Bioeconomy and Sustainable Development, 4(1), 17-21;
  52. Tacias-Pascacio, V.G., García-Parra, E., Vela-Gutiérrez, G., Virgen-Ortiz, J.J., Berenguer-Murcia, Á., Alcántara, A.R. and Fernandez-Lafuente, R., (2019). Genipin as an emergent tool in the design of biocatalysts: mechanism of reaction and applications. Catalysts, 9(12), 1035;
  53. Tabssum, F., Irfan, M., Shakir, H.A. and Qazi, J.I., (2018). RSM based optimization of nutritional conditions for cellulase mediated Saccharification by Bacillus cereus. Journal of biological engineering, 12,1-10; 10.1186/s13036-018-0097-4
  54. Thakshika, G., Peries, C.M. and Henegamage, A.P., (2019). Development of Bioethanol from Water Hyacinth (Eichhornia crassipes) Using Cellulose Degrading Microbial Biofilm. In Proceedings of International Research Symposium of Uva Wellassa University
  55. Tsegaye, B., Balomajumder, C. and Roy, P., (2019). Isolation and characterization of novel lignolytic, cellulolytic, and hemicellulolytic bacteria from wood-feeding termite Cryptotermes brevis. International Microbiology, 22(1), 29-39; 10.1007/s10123-018-0024-z
  56. Torres-Ortega, C.E. and Rong, B.G., (2016). Intensified separation processes for the recovery and dehydration of bioethanol from an actual lignocellulosic fermentation broth. In Computer Aided Chemical Engineering, 38, 727-732;
  57. Weerasinghe, W.M.L.I., Madusanka, D.A.T. and Pathmalal, M.M., (2019), November. Isolation of Cellulase Producing Bacteria: Future Perspective for Bio-Ethanol Production. In Proceedings of International Forestry and Environment Symposium. 24;
  58. Weerasinghe W.M.L.I., Madusanka D.A.T. and Pathmalal M.M., (2020), December. Isolation and Optimization of cellulase producing bacteria for Second-generation bio-ethanol production. In Proceedings of International Conference on Multi-Disciplinary Approaches (iCMA). 116
  59. Westman, J.O. and Franzén, C.J., (2015). Current progress in high cell density yeast bioprocesses for bioethanol production. Biotechnology journal, 10(8), 1185-1195;
  60. World Bank. (2017). Sri Lanka Development Update, June 2017. Washington, DC. © World Bank [online]. Available at: CC BY 3.0 IGO [October 2019]
  61. World bioenergy association. (2018). WBA Global Bioenergy Statistics 2018 Summary Report [online]. Available at: [December 2019]
  62. Yadav, S.K., (2017). Technological advances and applications of hydrolytic enzymes for valorization of lignocellulosic biomass. Bioresource technology, 245, 1727-1739; 10.1016/j.biortech.2017.05.066
  63. Zeng, Z., Ziegler, A.D., Searchinger, T., Yang, L., Chen, A., Ju, K., Piao, S., Li, L.Z., Ciais, P., Chen, D. and Liu, J., (2019). A reversal in global terrestrial stilling and its implications for wind energy production. Nature Climate Change, 9(12), 979-985;
  64. Zhao, X., Xiong, L., Zhang, M. and Bai, F., (2016). Towards efficient bioethanol production from agricultural and forestry residues: exploration of unique natural microorganisms in combination with advanced strain engineering. Bioresource technology, 215, 84-91;
  65. Zou, G., Jiang, Y., Liu, R., Zhu, Z. and Zhou, Z., (2018). The putative β-glucosidase BGL3I regulates cellulase induction in Trichoderma reesei. Biotechnology for biofuels, 11(1), 1-14;

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