Mikrobiol. Z. 2021; 83(4):3-14.
Ethanol Production by Co-Cultivation of Yeast and Lactic Acid Bacteria on Starch
M.O. Fomina, O.D. Ianieva, M.V. Havrylenko, T.M. Golovach, V.S. Pidgorskyi
Zabolotny Institute of Microbiology and Virology, NAS of Ukraine
154 Akad. Zabolotny Str., Kyiv, 03143, Ukraine
The co-cultivation of GRAS amylolytic bacteria together with ethanol-producing yeast Saccharomyces cerevisiae in starch-containing media might be one of the ways solving the problem of starch-containing waste disposal with simultaneous formation of ethanol as a potential biofuel for increasing octane number of gasoline. The aim of the study was to test the combination of microorganisms (amylolytic lactic acid bacteria and yeast) suitable for co-cultivation on starch and to optimize the conditions for starch cofermentation. Methods. Conventional microbiological, biochemical and statistical methods, including serial dilution technique with counting colony forming units (CFU) for growth assessment of mixed cultures, Gas Chromatograph/Mass Spectrometer (GC/MS) for measuring ethanol concentration and Box-Behnken experimental design (Statistica 10) for bioethanol production optimization, were used in this work. Results. The combination of microorganisms for mixed cultures co-cultivation in single-stage starch fermentation was established: the strain of ethanol-producing yeast S. cerevisiae UCM Y-527 and the amylolytic strain of lactic acid bacteria Streptococcus bovis IMV B-7151. Mathematical simulation using a Box-Behnken (3k-p) design determined the optimal parameters for the fermentation of starch in the process of co-cultivation of yeast and bacteria: 10 g/L of starch in the medium at simultaneous inoculations of both cultures and co-cultivation for 72 hours. The theoretically obtained parameters data were experimentally verified: the maximum ethanol yield 1.95 g/L in the experiment corresponded to the theoretically calculated values. Conclusions. It was suggested and optimized a method of starch cofermentation using strains of amylolytic lactic acid bacteria S. bovis IMV B-7151 and yeast S. cerevisiae UCM Y-527, which can be used for one-stage process of hydrolysis and fermentation of starch and starchcontaining wastes with the production of bioethanol and microbial biomass.
Keywords: ethanol, starch, co-cultivation, Saccharomyces cerevisiae, Streptococcus bovis.
Full text (PDF, in English)
- Girotto F, Alibardi L, Cossu R. Food waste generation and industrial uses: A review. Waste Manag. 2015; 45:32–41. https://doi.org/10.1016/j.wasman.2015.06.008
- Toksoy Oner E, Oliver SG, Kirdar B. Appl Environ Microbiol. 2005; 71(10):6443–5. https://doi.org/10.1128/AEM.71.10.6443-6445.2005
- Van Zyl WH, Bloom M, Viktor MJ. Engineering yeasts for raw starch conversion. Appl Microbiol Biotechnol. 2012; 95(6):1377–88. https://doi.org/10.1007/s00253-012-4248-0
- Bai FW, Anderson WA, Moo-Young M. Ethanol fermentation technologies from sugar and starch feedstocks. Biotechnol Adv. 2008; 26(1):89–105. https://doi.org/10.1016/j.biotechadv.2007.09.002
- Kosseva MR. Sources, Characterization, and Composition of Food Industry. In: Kosseva MR, Webb C, editors. Food Industry Wastes. USA: Elsevier; 2013. p. 37–60. https://doi.org/10.1016/B978-0-12-391921-2.00003-2
- Lounglawan P, Khungaew M, Suksombat W. Silage production from cassava peel and cassava pulp as energy source in cattle diets. J Anim Vet Adv. 2011; 10(8):1007–1011. https://doi.org/10.3923/javaa.2011.1007.1011
- Gao M-T, Yano S, Inoue H, Sakanishi K. Production of ethanol from potato pulp: Investigation of the role of the enzyme from Acremonium cellulolyticus in conversion of potato pulp into ethanol. Process Biochem. 2012; 47(12):2110–2115. https://doi.org/10.1016/j.procbio.2012.07.031
- Kroukamp H, Mert M, Viljoen-Bloom M, van Zyl WH, Gorgens JF, Haan RD. Engineering Saccharomyces cerevisiae for next generation ethanol production. J Chem Technol Biotechnol. 2013; 88(6):983–991. https://doi.org/10.1002/jctb.4068
- Shigechi H, Koh J, Fujita Y, Matsumoto T, Bito Y, Ueda M, Satoh E, Fukuda H, Kondo A. Direct production of ethanol from raw corn starch via fermentation by use of a novel surface-engineered yeast strain codisplaying glucoamylase and 25 alpha-amylase. Appl Environ Microbiol. 2004; 70(8):5037–40. https://doi.org/10.1128/AEM.70.8.5037-5040.2004
- Yamada R, Tanaka T, Ogino C, Fukuda H, Kondo A. Novel strategy for yeast construction using delta-integration and cell fusion to efficiently produce ethanol from raw starch. Appl Microbiol Biotechnol. 2010; 85(5):1491–1498. https://doi.org/10.1007/s00253-009-2198-y
- Buttner R, Bode R, Birnbaum D. Alcoholic fermentation of starch by Arxula adeninivorans. Zbl Mikrobiol. 1992; 147(3–4):225–230. https://doi.org/10.1016/S0232-4393(11)80333-1
- Kurtzman C, Fell JW. The Yeasts – A Taxonomic Study. 4th Edition. Amsterdam: Elsevier.
- Golovach TN, Kovalenko NK. [Microflora of silage: amylolytic lactic acid bacteria]. Mikrobiol Z. 1994; 56(2):3–7. Russian.
- Rukhlyadeva AP, Polygina GV. [Methods for determining the activity of hydrolytic enzymes]. Moscow: Pischevaya promyshlennost; 1981. Russian.
- Kvasnikov E, Nesterenko O. [Lactic acid bacteria and ways to use them]. Moscow: Nauka; 1975. Russian.
- Sneath PHA, Mair NS, Sharpe ME, Holt JG, editors. Bergey’s Manual of Systematic Bacteriology. 1st ed., vol. 2. Baltimore: Williams & Wilkins; 1986.
- Nagornaya SS, Zharova VP, Kotlyar AN. [Yeastantagonists in the normal microflora of the intestine tract in long-living people of Abkhazia]. Mikrobiol Z. 1989; 51(5):34–39. Russian.
- Chandel AK, Chandrasekhar G, Radhika K, Ravinder R, Ravindra P. Bioconversion of pentose sugars into ethanol: A review and future directions. Biotechnol Mol Biol Rev. 2011; 6(1):008–020.
- Darku ID, Richard TL. Biofuels: ethanol producers in eLS. Chichester: John Wiley & Sons.
- Verma G, Nigam P, Singh D, Chaudhary K. Bioconversion of starch to ethanol in a single-step process by coculture of amylolytic yeasts and Saccharomyces cerevisiae 21. Biores Technol. 2000; 72(3):261–266. https://doi.org/10.1016/S0960-8524(99)00117-0
- Horn CH, du Preez JC, Kilian SG. Fermentation of grain sorghum starch by co-cultivation of Schwanniomyces occidentalis and Saccharomyces cerevisiae. Bioresour Technol. 1992; 42(1):27–31. https://doi.org/10.1016/0960-8524(92)90084-B
- Jeon BY, Kim DH, Na BK, Ahn DH, Park DH. Production of ethanol directly from potato starch by mixed culture of Saccharomyces cerevisiae and Aspergillus niger using electrochemical bioreactor. J Microbiol Biotechnol. 2008; 18(3):545–51.
- Rath S, Singh AK, Masih H, Kumar Y, Peter JK, Singh P, Mishra SK. Bioethanol production from waste potatoes as an environmental waste management and sustainable energy by using cocultures Aspergillus niger and Saccharomyces cerevisiae. Int J Adv Res. 2014; 2(4):553–563.
- Yeunyaw P.-N, Yuwa-amornpitak T. Bioconversion of cassava starch to bio-ethanol in a single step by co-cultures of Amylomyces rouxii and Saccharomyces cerevisiase. Songklanakarin J Scie Technol. 2018; 40(4):776–783.
- Tantipaibulvut S, Pinisakul A, Rattanachaisit P, Klatin K, Onsriprai B, Boonyaratsiri K. Ethanol production from desizing wastewater using co-culture of Bacillus subtilis and Saccharomyces cerevisiae. Energy Procedia. 2015; 79:1001–1007. https://doi.org/10.1016/j.egypro.2015.11.600
- Lacerda IC, Miranda RL, Borelli BM, Nunes AC, Nardi RM, Lachance MA, Rosa CA. Lactic acid bacteria and yeasts associated with spontaneous fermentations during the production of sour cassava starch in Brazil. Int J Food Microbiol. 2005; 105(2):213–219. https://doi.org/10.1016/j.ijfoodmicro.2005.04.010
- Arroyo-López FN, Bautista-Gallego J, Domínguez-Manzano J, Romero-Gil V, Rodriguez-Gómez F, García-García P, Garrido-Fernández A, Jiménez-Díaz R. Formation of lactic acid bacteria-yeasts communities on the olive surface during Spanish-style Manzanilla fermentations. Food Microbiol. 2012; 32(2):295–301. https://doi.org/10.1016/j.fm.2012.07.003
- Ponomarova O, Gabrielli N, Sévin DC, Mülleder M, Zirngibl K, Bulyha K, Andrejev S, Kafkia E, Typas A, Sauer U, Ralser M, Patil KR. Yeast creates a niche for symbiotic lactic acid bacteria through nitrogen overflow. Cell Syst. 2017; 5(4):345–357. https://doi.org/10.1016/j.cels.2017.09.002
- Hirai S, Kawasumi T. Enhanced lactic acid bacteria viability with yeast coincubation under acidic conditions. Biosci Biotechnol Biochem. 2020; 84(8):1706–1713. https://doi.org/10.1080/09168451.2020.1756213
- Scheirlinck T, Mahillon J, Joos H, Dhaese P, Michiels F. Integration and expression of alpha-amylase and endoglucanase genes in the Lactobacillus plantarum chromosome. Appl Environ Microbiol. 1989; 55(9):2130–2137. https://doi.org/10.1128/aem.55.9.2130-2137.1989
- Scheirlink T, de Meutter J, Arnaut G, Joos H, Claeyssens M, Michiels F. Cloning and expression of cellulase and xylanase genes in Lactobacillus plantarum. Appl Microbiol Biotechnol. 1990; 5(33):534–541. https://doi.org/10.1007/BF00172547
- Narvhus JA, Gadaga TH. The role of interaction between yeasts and lactic acid bacteria in African fermented milks: a review. Int J Food Microbiol. 2003; 86(1–2):51–60. https://doi.org/10.1016/S0168-1605(03)00247-2
- Cai Y, Benno Y, Ogawa M, Ohmomo S, Kumai S, Nakase T. Influence of Lactobacillus spp. from an inoculant and of Weissella and Leuconostoc spp. from forage crops on silage fermentation. Appl Environ Microbiol. 1998; 64(8):2982–2987. https://doi.org/10.1128/AEM.64.8.2982-2987.1998
- Syadiah EA, Haditjaroko L, Syamsu K. Bioprocess engineering of bioethanol production based on sweet sorghum bagasse by co-culture technique using Trichoderma reesei and Saccharomyces cerevisiae. IOP Conf Ser: Earth Environ Sci. 2018; 209(1). https://doi.org/10.1088/1755-1315/209/1/012018
- Pidgorskyi VS, Ianieva OD, Fomina MO, Goaclovh TM, Ogirchuk KS. A method of starch fermentation by the co-cultivation of yeast and bacteria. Patent for utility model N126775, Zabolotny Institute of Microbiology and Virology of National Academy of Sciences of Ukraine, Kyiv, Ukraine, 2018.