Bioethanol Production from Lignocellulose, Whey and Starch Using Yeasts Isolated from Ethiopian Traditional Beverage and Other Sources
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Date
2018-05-04
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Addis Ababa University
Abstract
Ethanol is produced by yeasts and used for different applications. The ever-increasing demand
for energy necessitates the production of ethanol as biofuel to supplement fossil fuels as a source
of energy and protect the environment from environmental pollution. This requires for selection of effective ethanol producing local yeasts to produce ethanol from easily available agricultural
wastes. To this end, indigenous yeasts were isolated from yeast habitats such as traditional
fermented beverages, soil, flower, and compost samples collected from different part of Ethiopia.
The isolates were screened for their glucose fermentation and ethanol production.. The selected
yeast isolates with better ethanol production from glucose were identified using 26S rRNA
sequence analysis using NL1 and NL4 primers. They were further evaluated for their ability to
ferment different carbon sources, sedimentation rate, tolerance to sugar and ethanol
concentration. Production of ethanol by selected yeasts was optimized based on variables such as
inoculum size, temperature, pH, and incubation time using response surface methodology based
on central composite design. Enzymatic, acid, and alkali hydrolysis, and coculture were
employed to produce ethanol from grass pea and wild oat straws and starch. In order to produce
ethanol from whey, experimental runs such as molasses and external nutrient supplementation,
effect of whey pH, sterilized and non-sterilized whey were evaluated. Five isolates were
identified as Saccharomyces cerevisae and the remaining three were grouped into Kluveromyces
marxianus, Pichia fermentans and Candida humilis. The pattern of sugar utilization showed that
only K. marxianus ETP87 and P. fermentans ETP22 were able to grow on xylose; and K.
marxianus ETP87 was the only yeast that fermented lactose and, therefore, was selected to
produce ethanol from whey. All the 8 yeasts other than C. humilis were able to flocculate a
feature that makes the biomass separation easy for industrial applications. Regarding tolerance to
ethanol, S. cerevisae ETP53, K. marxianus ETP87, P. fermentans ETP22 and C. humilis ETP122
were tolerant to 10% extraneous ethanol but the percentage of ethanol tolerance considerably
decreased at 15% and 20% ethanol shock treatment. S. cerevisae ETP53 produced ethanol
optimally at pH 5.0, 60 hours, and 34oC; whereas the optimal growth and fermentation by K
marxianus ETP87 was at pH 4.8, temperature 36oC, and incubation time of 65 hours. Highest
reducing sugar was released from 1% (v/v) H2SO4 and 1% (w/v) NaOH treated straws; where
sulfuric acid yielded higher amount of sugars than NaOH, and more ethanol was obtained from
alkali hydrolysates. Significant amount of furfural was liberated from acid hydrolyzed straw that
was reduced by treatment with activated carbon and overliming. The data also showed that
significant amount of sugars was released from fungi (Pleurotus ostreatus M2191, Pleurotus
sajor-caju M2145, Trichoderma reesei JCM22676, and Aspergillus niger JCM22344) grown on
straws solid state media than enzymatic and chemically treated straws. However, the ethanol
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production from fungal-treated straws was small. It was also established that higher ethanol
production was obtained from acid-hydrolyzed sorghum flour than the one produced from crude
amylase enzyme treated substrate and ethanol derived from coculturing, indicating that
simultaneous saccharification and fermentation is a promising method to produce ethanol from
starch. The data also showed that K. marxianus ETP87 was capable of producing ethanol from
non-sterilized and non-deproteinized substrates. The effect of nutrient supplementation to whey
was variable depending on the kind of nutrient added. Generally, it could be concluded that S.
cerevisiae ETP53, K. marxianus ETP87, and P. fermentans ETP22 are promising yeasts to
produce ethanol from different substrates at acidic pH, near 35oC and late fermentation time.
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Keywords
Ethanol Fermentation, Saccharomyces Cerevisiae, Kluyveromyces Marxianus, Candida Humilis, Lignocellulose, Sorghum Flour, Whey