INDEXED IN 

ISSN 2456-0235

International Journal of Modern Science and Technology

​​​​​​​May 2019, Vol. 4, No. 5, pp 128-137. 

​​​A Novel Strain Developed through Protoplast Fusion for Consolidated Bioprocessing of Lignocellulosic Waste Mixture

R. Navnitkumar, T. R. Sambavi, S. Renganathan*
Biofuels Laboratory, Centre for Biotechnology, Anna University, Chennai – 600025. India.

​​​*Corresponding author’s e-mail: rengsah@rediffmail.com

​Abstract

​A consolidated bioprocessing (CBP) strain is capable of producing cellulolytic enzymes to saccharify the lignocellulosic biomass and fermenting the produced monosaccharides to ethanol. In this work, protoplasts of Trichoderma harzanium ATCC® 20846™ and Saccharomyces cereviseae were obtained and they were fused using a PEG fusion method. The fused protoplasts were regenerated and screened for their cellulolytic activity and fermentative activity. An optimized Consolidated Bioprocessing was carried out for 160 hours using the fused protoplasts showing a regeneration frequency of 5 x 10-4. Around 0.04 g/L ethanol was produced by the CBP strain with a percentage cellulose conversion of 1.96%. SDS PAGE analysis was done to observe the proteins secreted by the CBP strain. The enzyme activities of the produced cellulase observed at the end of 160 hours were FPases: 0.9 IU/mL, CMCases: 6.30 IU/mL, Xylanases: 352.9 IU/mL and Beta-glucosidases 1491.2 IU/mL.

Keywords: Protoplast fusion; Consolidated bioprocessing; Lignocellulosic waste; Regeneration frequency; Cellulase.

​References

  1. Verônica Ferreira, Mariana de Oliveira Faber, Sabrina da Silva Mesquita, Nei Pereira. Simultaneous saccharification and fermentation process of different cellulosic substrates using a recombinant Saccharomyces cerevisiae harbouring the β-glucosidase gene. Electron J Biotechnol 2010;13:1-7.
  2. Simone Brethauer, Michael Hanspeter Studer. Consolidated bioprocessing of lignocellulose by a microbial consortium. Energ Environ Sci 2014;7:1446-53.
  3. Trevor R Zuroff, Salvador Barri Xiques, Wayne R Curtis. Consortia-mediated bioprocessing of cellulose to ethanol with a symbiotic Clostridium phytofermentans/ yeast co-culture. Biotechnol Biofuels 2013;6:5.
  4. Hasunuma, Tomohisa, Kondo Akihiko. Consolidated bioprocessing and simultaneous saccharification and fermentation of lignocellulose to ethanol with thermotolerant yeast strains. Process Biochem 2012;47:1287-94.
  5. Hassan Abdel-Latif A, Mohamed, Wafaa Mohamed Haggag. Mutagenesis and inter-specific protoplast fusion between Trichoderma koningii and Trichoderma reesei for biocontrol improvement. American J Sci Indus Res 2010;1:504-15.
  6. John Wright D, Charles Wyman E, Karel Grohman. Simultaneous saccharification and fermentation of lignocellulose: process evaluation. Appl Biochem Biotechnol 1988;18:75-90.
  7. Manika Sharma, Pratibha Sharma, Ranbir Singh, M. Raja, Pankaj Sharma. Fast Isolation and Regeneration Method for Protoplast  Production in Trichoderma harzianum. Inter J Current Microbiol Appl Sci 2016;5:891-7.
  8. Svoboda. A. Fusion of Yeast Protoplasts Induced by Polyethylene Glycol. J Genrl Microbiol 1979;109:169-75.
  9. Sikandar Hayat, Christos Christias. Isolation and Fusion of Protoplasts from the Phytopathogenic Fungus Sclerotium Rolfsii. Brazilian J Microbiol 2010;41: 253-63.
  10. Kao K N, Michayluk M R. Fusion of Plant Protoplasts – Techniques. Biotechnology in Agriculture and Forestry 1989;8:277-88.
  11. Muralidhar R V, Panda T. Fungal Protoplast Fusion – A Revisit. Bioprocess Eng 2000;22:429-31.
  12. Sundberg E, Landgren M, Glimelius K. Fertility and chromosome stability in Brassica napus resynthesised by protoplast fusion. Theor Appl Genet 1987;75:96-104.
  13. Surbhi Vaid, Parushi Nargotra, Bijender Kumar Bajaj. Consolidated bioprocessing for biofuel-ethanol production from pine needle biomass. Environ Prog Sustain Energ 2017;37:546-52.
  14. Tanmayee Mishra, Arvind Kumar Goyal, Malay Bhattacharya, Pallab Kar, Arnab Sen. Polyethylene glycol mediated protoplast fusion of medicinally important Canna. Res Plant Biol 2015;5:20-24.
  15. Nighat Aslam, Munir A Shiekh, Muhammad Ashraf, Amer Jamil. Expression of Trichoderma Cellulases under Different Carbon Sources. Pak J Bot 2010;42:2895-902
  16. Eyini M, Rajkumar K, Balaji P. Isolation, Regeneration and PEG-Induced Fusion of Protoplasts of Pleurotus pulmonaris and Pleurotus florida. Mycobiol. 2006;34:73-8.
  17. Kasana RC, Salwan R, Dhar H, Dutt S, Gulati A. A rapid and easy method for the detection of microbial cellulases on agar plates using gram's iodine. Current Microbiol 2008;57:503-7.
  18. Fexina Tomé Mariana, Lima Antônio. Direct Cost of Reprocessing Cotton-woven Surgical Drapes: a Case Study. Revista da Escola de Enfermagem da USP 2015;49: 488-94.
  19. Rajaa A S M, Arputharaj A, Sujata Saxena, P G Patil. Single bath enzymatic scouring and bleaching process for preparation of absorbent cotton. Indian J Fibre Text Res 2017;42:202-8.
  20. Ma Y, Hummel M, Maattanen M, Sarkilahti A, Harlin Sixta. Upcycling of waste paper and cardboard to textiles. RSC Adv J 2015;18:858-66.
  21. Ghose TK. Measurement of cellulase activities. Pure Appl Chem 1987;59:257-68.
  22. Aftab Ahamed, Patrick Vermette. Culture-based strategies to enhance cellulose enzyme production from Trichoderma reesei RUT-C30 in bioreactor culture conditions. Biochem Eng 2008;40:399-407.
  23. Hardik N Lakhani, Dinesh N Vakharia, Abeer H Makhlouf, Ragaa A Eissa, Mohamed M Hassan. Influence of Protoplast fusion in Trichoderma Spp. on Some Soil Borne Dieases. J Plant Pathol Microbiol 2016;7:1-7.
  24. Couteaudier Y, Viaud M, Riba G. Genetic Nature, Stability, and Improved Virulence of Hybrids from Protoplast Fusion in Beauveria. Microbiol Ecol 1996;32:1-10.  
  25. Anjani Kumari J, Panda T. Intergeneric hybridization of Trichoderma reesei QM9414 and Saccharomyces cerevisiae NCIM 3288 by protoplast fusion. Enzyme Microb Technol 1994;16:870-82.
  26. Xiao Bin Yu, Joo-Heon Nam, Hyun Shik Yun, Yun Mo Koo. Optimization of Cellulase Production in Batch Fermentation by Trichoderma resei. Biotechnol Bioprocess Eng 1998; 3:44-7.
  27. Bakri Y, Akeed Y, Thonart P. Comparison between Continuous and Batch Processing to Produce Xylanase By Penicillium canescens 10-10c. Brazilian J Chem Eng 2012;29:441-8.
  28. Yi-Heng Percival Zhang, Lee R. Lynd. Regulation of Cellulase Synthesis in Batch and Continuous Culture of Clostridium thermocellum. J Bacteriol 2005;187:99-106.
  29. Yan Jinping Wei Zhilei, Wang, Qiaoping, He Manman, Li Shumei, Chagan Irbis. Bioethanol production from sodium hydroxide/hydrogen peroxide-pretreated water hyacinth via simultaneous saccharification and fermentation with a newly isolated thermotolerant Kluyveromyces marxianu strain. Bioresour Technol 2015;193:103-9.