Contribution of proteases and cellulases produced by solid-state fermentation to the improvement of corn ethanol production

Abstract

By cultivating a strain of Aspergillus tubingensis on agro-industrial by-products using solid-state fermentation technology, a biocatalyst containing more than 130 different enzymes was obtained. The enzymatic complex was composed mainly of hydrolases, among which a protease, an aspergillopepsin, accounted for more than half of the total proteins. Cell-wall-degrading enzymes such as pectinases, cellulases and hemicellulases were also highly represented. Adding the biocatalyst to corn mash at 1 kg/T corn allowed to significantly improve ethanol production performances. The final ethanol concentration was increased by 6.8% and the kinetics was accelerated by 14 h. The aim of this study was to identify the enzymes implicated in the effect on corn ethanol production. By fractionating the biocatalyst, the particular effect of the major enzymes was investigated. Experiments revealed that, together, the protease and two cellulolytic enzymes (an endoglucanase and a β-glucosidase) were responsible for 80% of the overall effect of the biocatalyst. Nevertheless, the crude extract of the biocatalyst showed greater impact than the combination of up to seven purified enzymes, demonstrating the complementary enzymatic complex obtained by solid-state fermentation. This technology could, therefore, be a relevant natural alternative to the use of GMO-derived enzymes in the ethanol industry.

Keywords

Solid-state fermentation / Enzymes / Protease / Cellulases / Bioethanol / Corn / Purification / Characterization

References

[1] Albers E, Larsson C, Lidén G, Niklasson C, Gustafsson L. Influence of the nitrogen source on Saccharomyces cerevisiae anaerobic growth and product formation. J Appl Environ Microbiol, 1996, 62(9): 3187-3195.

[2] Ashokkumar B, Kayalvizhi N, Gunasekaran P. Optimization of media for β-fructofuranosidase production by Aspergillus niger in submerged and solid state fermentation. Process Biochem, 2001, 37: 331-338.

[3] Bansal N, Tewari R, Soni SK, Soni T. Production of cellulases from Aspergillus niger NS-2 in solid state fermentation on agricultural and kitchen waste residues. Waste Manag, 2012, 32: 1341-1346.

[4] Bhargav S, Panda BP, Ali M, Javed S. Solid-state fermentation: an overview. Chem Biochem Eng Q, 2008, 22(1): 49-70.

[5] Brahmachari G. Biotechnology of microbial enzymes: production, biocatalysis and Industrial applications, 2017, Amsterdam: Academic Press

[6] Carboué Q, Tranier M-S, Perraud-Gaime I, Roussos S. Production of microbial enzymes by solid-state fermentation for food applications. Microbial enzyme technology in food applications, 2016, New York: CRC Press, 437-451.

[7] Chutmanop J, Chuichumcherm S, Chisti Y, Srinophakun P. Protease production by Aspergillus oryzae in solid-state fermentation using agroindustrial substrates. J Chem Technol Biotechnol, 2008, 83: 1012-1018.

[8] de Castro RJS, Sato HH. Production and biochemical characterization of protease from Aspergillus oryzae: an evaluation of the physical-chemical parameters using agroindustrial wastes as supports. Biocat Agric Biotechnol, 2014, 3: 20-25.

[9] de Castro AM, De Andréa TV, Carvalho DF, Teixeira MMP, Dos Reis Castilho L, Freire DMG. Valorization of residual agroindustrial cakes by fungal production of multienzyme complexes and their use in cold hydrolysis of raw starch. Waste Biomass Valoriz, 2011, 2: 291-302.

[10] de Castro RJS, Nishide TG, Sato HH. Production and biochemical properties of proteases secreted by Aspergillus niger under solid state fermentation in response to different agroindustrial substrates. Biocat Agric Biotechnol, 2014, 3(4): 236-245.

[11] de Vries RP, Riley R, Wiebenga A, Aguilar-Osorio G, Amillis S, Uchima CA, Anderluh G, Asadollahi M, Askin M, Barry K, Battaglia E, Bayram O, Benocci T, Braus-Stromeyer SA, Caldana C, Canovas D, Cerqueira GC, Chen FS, Chen WP, Choi C, Clum A, dos Santos RAC, Damasio ARD, Diallinas G, Emri T, Fekete E, Flipphi M, Freyberg S, Gallo A, Gournas C, Habgood R, Hainaut M, Harispe ML, Henrissat B, Hilden KS, Hossain A, Hope R, Karabika E, Karaffa L, Karanyi Z, Krasevec N, Kuo A, Kusch H, LaButti K, Lagendijk EL, Lapidus A, Levasseur A, Lindquist E, Lipzen A, Logrieco AF, MacCabe A, Makela MR, Malavazi I, Melin P, Meyer V, Mielnichuk N, Miskei M, Molnar AP, Mule G, Ngan CY, Orejas M, Orosz E, Ouedraogo JP, Overkamp KM, Park HS, Perrone G, Piumi F, Punt PJ, Ram AFJ, Ramon A, Rauscher S, Record E, Riano-Pachon DM, Robert V, Rohrig J, Ruller R, Salamov A, Salih NS, Samson RA, Sandor E, Sanguinetti M, Schutze T, Sepcic K, Shelest E, Sherlock G, Sophianopoulou V, Squina F, Sun H, Susca A, Todd RB, Tsang A, Unkles SE, van de Wiele N, van Rossen-Uffink D, Oliveira JVD, Vesth TC, Visser J, Yu JH, Zhou MM, Andersen MR, Archer DB, Baker SE, Benoit I, Brakhage AA, Braus GH, Fischer R, Frisvad JC, Goldman GH, Houbraken J, Oakley B, Pocsi I, Scazzocchio C, Seiboth B, vanKuyk PA, Wortman J, Dyer PS, Grigoriev IV. Comparative genomics reveals high biological diversity and specific adaptations in the industrially and medically important fungal genus Aspergillus. Genome Biol, 2017, 18: 28-45.

[12] Diaz-Godinez G, Soriano-Santos J, Augur C, Viniegra-Gonzalez G. Exopectinases produced by Aspergillus niger in solid-state and submerged fermentation: a comparative study. J Ind Microbiol Biotechnol, 2001, 26(5): 271-275.

[13] Gao L, Wang F, Gao F, Wang L, Zhao J, Qu Y. Purification and characterization of novel cellobiohydrolase (PdCel6A) from Penicillium decumbens JU-A10 for bioethanol production. Bioresour Technol, 2011, 102: 8339-8342.

[14] Gross KC. A rapid and sensitive method for assaying polygalacturonase using 2-cyanoacetamide. Hort Sci, 1982, 17: 933-934.

[15] Guillaume A, Carré YA, Drevelle ASD, Thorigné A (2016) Biocatalyst for manufacturing ethanol from corn. WO2017144590

[16] Harris PV, Xu F, Kreel NE, Kang C, Fukuyama S. New enzyme insights drive advances in commercial ethanol production. Curr Opin Chem Biol, 2014, 19: 162-170.

[17] Hölker U, Höfer M, Lenz J. Biotechnological advantages of laboratory-scale solid-state fermentation with fungi. Appl Microbiol Biotechnol, 2004, 64(2): 175-186.

[18] Hoskins B, Lyons M. Improving bioethanol yield: the use of solid-state fermentation products grown on DDGS. J Inst Brew, 2009, 115(1): 64-70.

[19] Idris ASO, Pandey A, Rao SS, Sukumaran RK. Cellulase production through solid-state tray fermentation, and its use for bioethanol from sorghum stover. Bioresour Technol, 2017, 242: 265-271.

[20] Kang SW, Park YS, Lee JS, Hong SI, Kim SW. Production of cellulases and hemicellulases by Aspergillus niger KK2 from lignocellulosic biomass. Bioresour Technol, 2004, 91: 153-156.

[21] Kłosowski G, Mikulski D, Kotarska K, Czupryński B. Characterisation of fermentation of high-gravity maize mashes with the application of pullulanase, proteolytic enzymes and enzymes degrading non-starch polysaccharides. J Biosci Bioeng, 2010, 109(5): 466-471.

[22] Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970, 227: 680-685.

[23] Luangthongkam P, Fang L, Noomhorm A, Lamsal B. Addition of cellulolytic enzymes and phytase for improving ethanol fermentation performance and oil recovery in corn dry grind process. Ind Crops Prod, 2015, 77: 803-808.

[24] McAloon AJ, Johnston DB. Protease increases fermentation rate and ethanol yield in dry-grind ethanol production. Bioresour Technol, 2014, 154: 18-25.

[25] Mikulski D, Kłosowski G, Rolbiecka A. Influence of phytase and supportive enzymes applied during high gravity mash preparation on the improvement of technological indicators of the alcoholic fermentation process. Biomass Bioenergy, 2015, 80: 191-202.

[26] Mukhtar H, Ikram-Ul-Haq. Production of acid protease by Aspergillus niger using solid state fermentation. Pak J Zool, 2009, 41(4): 253-260.

[27] Oda K, Kakizono D, Yamada O, Iefuji H, Akita O, Iwashita K. Proteomic analysis of extracellular proteins from Aspergillus oryzae under submerged and solid-state culture conditions. Appl Environ Microbiol, 2006, 72(5): 3448-3457.

[28] Ortiz GE, Noseda DG, Ponce Mora MC, Recúpero MN, Blasco M, Albertó E. A comparative study of new Aspergillus strains for proteolytic enzymes production by solid state fermentation. Enzyme Res, 2016, 2016: 1-11.

[29] Panda S, Mishra S, Kayitesi E, Ray R. Microbial-processing of fruit and vegetable wastes for production of vital enzymes and organic acids: biotechnology and scopes. Environ Res, 2016, 146: 161-172.

[30] Pandey A, Selvakumar P, Soccol CR, Nigam P. Solid state fermentation for the production of industrial enzymes. Curr Sci, 1999, 77: 149-162.

[31] Perez-Carrillo E, Serna-Saldivar SO, Chuck-Hernandez C, Cortes-Callejas ML. Addition of protease during starch liquefaction affects free amino nitrogen, fusel alcohols and ethanol production of fermented maize and whole and decorticated sorghum mashes. Biochem Eng J, 2012, 67: 1-9.

[32] Rose AH, Harrison JS. The yeasts, vol 3. Metabolism and physiology of yeasts, 1989, 2, London: Academic Press.

[33] Singh SK, Sczakas G, Soccol CR, Pandey A. Production of enzymes by solid-state fermentation. Current developments in solid-state fermentation, 2008, New York: Springer, 183-204.

[34] Singhania RR, Saini R, Adsul M, Saini JK, Mathur A, Tuli D. An integrative process for bio-ethanol production employing SSF produced cellulase without extraction. Biochem Eng, 2015, J102: 45-48.

[35] Subramaniyam R, Vimala R. Solid state and submerged fermentation for the production of bioactive substances: a comparative study. Int J Sci Nat, 2012, 3(3): 480-486.

[36] Vidal BC, Rausch KD, Tumbleson ME, Singh V. Protease treatment to improve ethanol fermentation in modified dry grind corn processes. Cereal Chem, 2009, 86(3): 323-328.

[37] Yamane Y, Fujita J, Izuwa S, Fukuch IK, Shimizu R, Hiyoshi A, Fukuda H, Mikami S, Kizaki Y, Wakabayashi S. Properties of cellulose-degrading enzymes from Aspergillus oryzae and their contribution to material utilization and alcohol yield in sake mash fermentation. J Biosci Bioeng, 2002, 93(5): 479-484.

[38] Zimbardi AL, Sehn C, Meleiro LP, Souza FH, Masui DC, Nozawa MS, Guimarães LH, Jorge JA, Furriel RP. Optimization of β-glucosidase, β-xylosidase and xylanase production by Colletotrichum graminicola under solid-state fermentation and application in raw sugarcane trash saccharification. Int J Mol Sci, 2013, 14: 2875-2902.

Funding

Bpifrance(OSIRIS Program)