Antonie van Leeuwenhoek

, Volume 101, Issue 2, pp 331–346

Cellulase production from agricultural residues by recombinant fusant strain of a fungal endophyte of the marine sponge Latrunculia corticata for production of ethanol

  • Ahmed M. A. El-Bondkly
  • Mervat M. A. El-Gendy
Original Paper


Several fungal endophytes of the Egyptian marine sponge Latrunculia corticata were isolated, including strains Trichoderma sp. Merv6, Penicillium sp. Merv2 and Aspergillus sp. Merv70. These fungi exhibited high cellulase activity using different lignocellulosic substrates in solid state fermentations (SSF). By applying mutagenesis and intergeneric protoplast fusion, we have obtained a recombinant strain (Tahrir-25) that overproduced cellulases (exo-β-1,4-glucanase, endo-β-1,4-glucanase and β-1,4-glucosidase) that facilitated complete cellulolysis of agricultural residues. The process parameters for cellulase production by strain Tahrir-25 were optimized in SSF. The highest cellulase recovery from fermentation slurries was achieved with 0.2% Tween 80 as leaching agent. Enzyme production was optimized under the following conditions: initial moisture content of 60% (v/w), inoculum size of 106 spores ml−1, average substrate particle size of 1.0 mm, mixture of sugarcane bagasse and corncob (2:1) as the carbon source supplemented with carboxymethyl cellulose (CMC) and corn steep solids, fermentation time of 7 days, medium pH of 5.5 at 30°C. These optimized conditions yielded 450, 191, and 225 units/gram dry substrate (U gds−1) of carboxylmethyl cellulase, filter-paperase (FPase), and β-glucosidase, respectively. Subsequent fermentation by the yeast, Saccharomyces cerevisiae NRC2, using lignocellulose hydrolysates obtained from the optimized cellulase process produced the highest amount of ethanol (58 g l−1). This study has revealed the potential of exploiting marine fungi for cost-effective production of cellulases for second generation bioethanol processes.


Lignocellulosic residues Endophytic fungi Protoplast fusion Cellulases Ethanol 


  1. Attyia SH, Ashour SM (2002) Biodegradation of agro-industrial orange waste under solid state fermentation and natural environmental conditions. Egyptian J Biol 4:23–30Google Scholar
  2. Bailey MJ, Nevalainen KMH (1981) Induction, isolation and testing of stable Trichoderma reesei mutant with improved production of solubilizing cellulase. Enzyme Microb Technol 3:153–157CrossRefGoogle Scholar
  3. Bansal N, Tewari R, Gupta JK, Soni R, soni SK (2011) A novel strain of Aspergillus niger producing a cocktail of hydrolytic depolymerising enzymes for the production of second generation ethanol. Bioresources 6(1):552–569Google Scholar
  4. Bigelow P, Wyman E (2004) Production of cellulytic on bagasse pretreated with chemicals. Appl Biochem Biotechnol 102:78–82Google Scholar
  5. Bissett J (1991a) A revision of the genus Trichoderma. II. Infrageneric classification. Canad J Botany 69:2357–2372CrossRefGoogle Scholar
  6. Bissett J (1991b) A revision of the genus Trichoderma. III. Sect. Pachybasium. Canad J Botany 69:2373–2417CrossRefGoogle Scholar
  7. Blomquist G, Andersson B, Andersson K, Brondz I (1992) Analysis of fatty acids. A new method for characterization of moulds. J Microbiol Methods 16(1):59–68CrossRefGoogle Scholar
  8. Camassola M, Dillon AJP (2007) Production of cellulases and hemicellulases by Penicillium echinulatum grown on pretreated sugar cane bagasse and wheat bran in solid-state fermentation. J Appl Microbiol 103:2196–2204PubMedCrossRefGoogle Scholar
  9. Caputi AJR, Ueda M, Brown T (1968) Spectrophotometric determination of ethanol in wine. Am J Enol Vitic 19:160–165Google Scholar
  10. Chahal DS (1985) Solid-state fermentation with Trichoderma reesei for cellulase production. Appl Environ Microbiol 49(1):205–210PubMedGoogle Scholar
  11. Cowan D (1996) Industrial enzyme technology. Trends Biotechnol 14(6):177–178CrossRefGoogle Scholar
  12. El-Bondkly AM (2006) Gene transfer between different Trichoderma species and Aspergillus niger through intergeneric protoplast fusion to convert ground rice straw to citric acid and cellulases. Appl Biochem Biotechnol 135(2):117–132PubMedCrossRefGoogle Scholar
  13. El-Bondkly AM, El-Gendy MMA (2010) Keratinolytic activity from new recombinant fusant AYA2000, an endophytic Micromonospora spp. Can J Microbiol 56:748–760PubMedCrossRefGoogle Scholar
  14. El-Bondkly AM, Aboshosha AAM, Radwan NH, Dora SA (2010) Successive construction of β-glucosidase hyperproducers of Trichoderma harzianum using microbial biotechnology techniques. J Microbial Biochem Technol 2(3):070–073CrossRefGoogle Scholar
  15. El-Gendy MMA (2010) Optimization of process parameters for keratinase produced by endophytic Penicillium spp. Morsy1 under solid state fermentation. Appl Biochem Biotechnol 162:780–794PubMedCrossRefGoogle Scholar
  16. El-Gendy MMA, El-Bondkly AM (2011) Genome shuffling of marine derived bacterium Nocardia sp ALAA 2000 for improved ayamycin production. Antonie van Leeuwenhoek 99(4):773–780PubMedCrossRefGoogle Scholar
  17. Ghose TK (1987) Measurement of cellulase activities. Pure Appl Chem 59:257–268CrossRefGoogle Scholar
  18. Girio FM, Fonseca C, Carvalheiro F, Duarte LC, Marques S, Bogel-Łukasik R (2010) Hemicelluloses for fuel ethanol: a review. Bioresource Technol 101:4775–4800CrossRefGoogle Scholar
  19. Gutierrez-Correa M, Tengerdy RP (1997) Production of cellulase on sugarcane bagasse by fungal mixed culture solid substrate fermentation. Biotechnol Lett 19:665–667CrossRefGoogle Scholar
  20. Haq I, Shahzadi K, Hameed U, Javed MM, Qadeer MA (2006) Solid state fermentation of cellulases by locally isolated Trichoderma harzianum for the exploitation of agriculture byproducts. Pakistan J Biol Sci 9(9):1779–1782CrossRefGoogle Scholar
  21. Holler U, Wright AD, Matthee GF, Konig GM, Draeger S, Aust HJ (2000) Fungi from marine sponges: diversity, biological activity and secondary metabolites. Mycol Res 104:1354–1765CrossRefGoogle Scholar
  22. Kohlmeyer J, Kohlmeyer E (1979) Marine mycology: the higher fungi. Academic Press, New YorkGoogle Scholar
  23. Lee CK, Darah I, Ibrahim CO (2011) Production and optimization of cellulase enzyme using Aspergillus niger USM AI 1 and comparison with Trichoderma reesei via solid state fermentation system. Biotechnol Res Intern. Article ID 658493. doi:10.4061/2011/658493
  24. Liu J, Yang J (2007) Cellulase production by Trichoderma koningii AS3.4262 in solid-state fermentation using lignocellulosic waste from the vinegar industry. Food Technol Biotechnol 45(4):420–425Google Scholar
  25. Mathew GM, Sukumaran RK, Singhania RR, Pandey A (2008) Progress in research on fungal cellulases for lignocellulase degradation. J Sci Indu Res 67:898–907Google Scholar
  26. Palmarola-Adrados B, Choteborska P, Galbe M, Zacchi G (2005) Ethanol production from non-starch carbohydrates of wheat bran. Bioresour Technol 96:843–850PubMedCrossRefGoogle Scholar
  27. Pandey A (1991) Effect of particle size of substrate on enzyme production on solid state fermentation. Bioresour Technol 37:169–172CrossRefGoogle Scholar
  28. Patel SJ, Onkarappa DR, Shobha KS (2007) Fungal pretreatment studies on rice husk and bagasse for ethanol production. EJEAFChe 6(4):1921–1926Google Scholar
  29. Rifai MA (1969) A revision of the genus Trichoderma. Mycol Papers 116:1–116Google Scholar
  30. Saha BC, Bothast RJ (1996) Production, purification, and characterization of a highly glucose tolerant novel β-Glucosidase from Candida peltata. Appl Environ Microbiol 62(9):3165–3170PubMedGoogle Scholar
  31. Samson RA (1979) A compilation of the Aspergilli described since 1965. Stud Mycol 18:1–38Google Scholar
  32. Samson RA, Gams W (1984) The taxonomic situation in the hyphomycete genera Penicillium, Aspergillus and Fusarium. Antonie Van Leeuwenhoek 50:815–824PubMedCrossRefGoogle Scholar
  33. Seiboth B, Lukas H, Salovuori N, Karin L, Robson DG, Vehmaanperä J, Penttilä ME, Kubicek CP (2005) Role of the bga1-encoded extracellular β-galactosidase of Hypocrea jecorina in cellulase induction by lactose. Appl Environ Microbiol 71:851–857PubMedCrossRefGoogle Scholar
  34. Stahl PD, Klug MJ (1996) Characterization and differentiation of filamentous fungi based on fatty acid composition. Appl Environ Microbiol 62(11):4136–4146PubMedGoogle Scholar
  35. Sun H, Ge X, Hao Z, Peng M (2010) Cellulase production by Trichoderma sp. on apple pomace under solid state fermentation. African J Biotechnol 9(2):163–166Google Scholar
  36. Zhang Y-HP, Himmel ME, Mielenz JR (2006) Outlook for cellulase improvement: screening and selection strategies. Biotechnol Adv 24:452–481CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Ahmed M. A. El-Bondkly
    • 1
  • Mervat M. A. El-Gendy
    • 2
  1. 1.Applied Microbial Genetics Group, Genetics and Cytology DepartmentNational Research CentreDokki, GizaEgypt
  2. 2.Department of Chemistry of Natural and Microbial ProductsNational Research CentreDokki, GizaEgypt

Personalised recommendations