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Saccharomyces cerevisiae Bio-Ethanol Production, A Sustainable Energy

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Article  in  Asian Journal of Microbiology, Biotechnology and Environmental Sciences · December 2017

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Asian Jr. of Microbiol. Biotech. Env. Sc. Vol. 20 (Feb. Suppl.) : 2018 : S202-S206
© Global Science Publications
ISSN-0972-3005

SACCHAROMYCES CEREVISIAE BIO-ETHANOL PRODUCTION, A

SUSTAINABLE ENERGY ALTERNATIVE
NAHID AKHTARA, ARUN KARNWALA, ATUL KUMAR UPADHYAYA,
SANJOY PAULB, AND M. AMIN-UL MANNANC
a
Lovely Professional University, School of Bioengineering and Biosciences
Department of Molecular Biology and Genetic Engineering, Phagwara 144 411, Punjab, India
b
Molecular Physiology & Biophysics, Carver College of Medicine, University of Iowa,
Iowa City, IA – 52242, USA

(Received 10 July, 2017; accepted 1 September, 2017)

Key words : Bioethanol, Ethanol fermentation, Sustainable energy, Yeast

Abstract –Current world energy demand is based on fossil fuels, which will vanish in coming decades.
Renewable energy especially biofuels has attracted great interest as solutions to the current energy problem.
Among available biofuel resources, bioethanol seems to be an efficient alternative. Saccharomyces cerevisiae
a well-established organism for bioethanol production. However, during the fermentation process, yeast
cells experience various stress conditions and inhibitors hampering its efficacy for commercial bioethanol
production. To overcome these yeast cells, adopt different signal transduction pathways. In this review,
common and least explored carbon feedstocks which can be readily converted into bioethanol are
highlighted. The various protectants, genes, and pathways which can be tempered to engineer yeast strains
are discussed. We have suggested strategies to utilize this lucrative alternative for sustainable bioethanol
production.

INTRODUCTION hemicellulose are used for bioethanol production.

Sugarcane juices, molasses, and corn are primary
Human population has dramatically increased in feedstock used worldwide for bioethanol (Wilkie,
the past decades, stretching the finite fossil fuels 2000). Starch, a polysaccharide of glucose is
resources. Fossil fuels, coal, natural gas, oils account obtained from corn, barley, wheat, rye, potato,
for 90% of total energy demand in the world. sorghum, and cassava. For the production of
However, fossil fuels are limited and are major bioethanol starch-containing feedstock must be first
contributors of greenhouse gasses. Renewable converted to sugar or dextrin by an enzymatic
energy is alternative and among available resources, process, often enzyme used is amylase. Other
biofuels seem to be an efficient and sustainable complex sugars are converted to simple sugars by
energy. Current biofuels for bioethanol and saccharification process and are fermented to
biodiesel production are based on sugar crops. ethanol (Naik, 2010).
However, food versus fuel dilemma jeopardizes its The biofuel produced from starch, sugars, animal
long-term usage. Non-food crops (switchgrass, fats and vegetable oils are referred as ‘first
poplar, and willow), algae and genetically modified generation biofuel’. Food vs fuel dilemma
organisms are other sources for biofuel production. jeopardizes its large-scale commercial production.
Till date, yeast, Saccharomyces cerevisiae is considered In coming decades, world population is expected to
to be ideal microorganism for ethanol fermentation be 9 billion and around 2.5 billion more people will
(Lam et al., 2014). It can utilize various feedstocks for be added by 2050 (Godfray et al., 2010), thus,
bioethanol production which are discussed below. hampering sustainability of food crops for biofuel
production. Moreover, insufficient supply of these
Bioethanol Production From Carbohydrates
crops hampers its long-term usage and its
Carbohydrates present in starch, cellulose, and commercialization. As an alternative, lignocellulose

*Corresponding author’s email: mohammad.20597@lpu.co.in, maminulmannan@gmail.com

 Saccharomyces cerevisiae Bio-ethanol Production, A Sustainable Energy Alternative S203

feedstocks ‘second generation biofuel’, can be used

(Kumar, 2009). Lignocellulose includes agricultural
waste (straw of rice, wheat and corn, and sugarcane
bagasse), nonfood plants like poplar, napiergrass,
switches grass, paper waste, agro-industrial waste,
water hyacinth (Yasuda et al., 2014). Being non-food
crops it seems to be sustainable energy resource.
Bioethanol from Agricultural Waste Materials
Crops like corn, wheat, and sugarcane are primarily
used for food. So, the sufficient production of these
crops for fuel remains the major obstacle in the
production of bioethanol (Cheng, 2011). The major
agricultural wastes are straws of corn, wheat, and
rice and sugarcane bagasse. These waste material
don’t have any nutritional value, easily available and
are cheap. Moreover, it does not require separate
agricultural land, water supply, fertilizers, and Fig. 1. Ethanol fermentation: One molecule of glucose
energy sources. Most of the agricultural waste (C 6 H 12O 6 ) is converted into two molecules of
materials are either left to rot in the field for pyruvic acid (C 3 H 4 O 3 ) during the process of
composting or burnt in the fields. Rather than just glycolysis. Pyruvic acid is further decarboxylated
disposing or burning these wastes it can be to generate two molecules of acetaldehyde
judiciously used as biomass for bioethanol (CH 3 CHO), which is reduced to ethanol
production. Besides this feedstocks like vegetable or (C2 H5OH). During the process there is a net gain
of 2 molecules of ATP and one molecule of glucose
fruit processing wastes can also be used for
is converted to two molecules of ethanol and two
bioethanol or biodiesel production. molecules of carbon dioxide (CO2).
Fermentation Stress Tolerance Mechanism
The yeast, S. cerevisiae is widely used in ethanol that overexpressed genes like ARG4 and CAR1
fermentation industry owing to its efficient responsible for the synthesis of arginine, showed to
conversion of sugars to ethanol (Fig. 1). However, maintain the stability of cell wall and cell membrane
during fermentation, it experiences numerous stress (Cheng et al., 2016). Our group has also shown that
conditions. Stress conditions and an adaptive overexpression of RPI1 increases ethanol tolerance
mechanism to overcome can be collectively called as by over 50 fold compared to WT. RPI1 over
‘Fermentation Stress Tolerance’ (FST). expression strain is highly resistant to cell wall lytic
enzyme Zymolyase (Puria et al., 2009) suggesting,
Tolerance to Ethanol perhaps RPI1 improves the cell viability by
S. cerevisiae ferments sugar, starch, lignocellulose to strengthening the yeast cell wall.
ethanol but when the ethanol accumulates above a Apart from affecting plasma membrane, ethanol
threshold level, it inhibits growth, causes also denatures functional proteins and protein
mitochondrial loss and eventually kills the yeast present in the cell membrane. In order to survive
cells (Bai et al., 2004; Ibeas and Jimenez, 1997). different environmental fluctuations, and to
Increased ethanol level affects membrane stability, maintain the internal steady state homeostasis, cells
damages protein and destroys cell membrane. There have developed adaptive stress tolerance
are several studies which have shown the major mechanisms. These cellular responses lead to
pathways and genes involved in ethanol stress change in gene expression and require signal trans-
tolerance. The knockout strains developed by You et duction pathways to communicate from the sensors
al; showed tolerance to ethanol when supplemented on the cell surface or cytoplasm to transcriptional
with monounsaturated fatty acid (You KM, 2003). machinery located in the nucleus to elicit a stress
Inoue et al; demonstrated that strains lacking response, (Fig. 2) (Gasch and Werner-Washburne,
ergosterol were sensitive to the moderate level of 2002).
intracellular ethanol (Inoue et al., 2000). Yeast strains
S204 AKHTAR ET AL

Exposure of ethanol to yeast cells affects the activity

of Pma1 membrane protein, an H-ATPase, necessary
to preserve intracellular pH and membrane
potential.
Genetic Engineering to Improve Yeast Strain
C6 and C5 Carbon Substrate
Yeast can efficiently metabolize glucose and have
less affinity for other carbon sources such as
galactose. Moreover, in presence of glucose other
metabolic genes are repressed by a process called as
‘glucose repression’(Le Borgne, 2011). In this regard
various genetically engineered strains of S. cerevisiae
have been developed. Improved galactose uptake
and yield of ethanol were obtained when the genes
encoding phosphoglucomutase and positive
regulator of Gal4p was overexpressed (Ostergaard et
al., 2000). Overexpression of truncated TUP1 gene
Fig. 2. Fermentation Stress Tolerance Mechanism: Yeast encoding repressor of transcription showed
cells experience a plethora of stress conditions
improved fermentation rates. Besides this lactose
during fermentation namely high initial substrate
concentration, nutrient deprivation, gradual present in whey can be used to produce ethanol. But
accumulation of ethanol, the temperature rise of the commonly used S. cerevisiae for industrial
the fermentation medium, a decrease in pH, ethanol production is not able to metabolize lactose.
generation of reactive oxygen species (ROS). The For metabolizing lactose Kluyveromyces fragilis
cell sensing these signals by receptors over cell (Guimaraes et al., 2008) or genetically engineered S.
surface or intracellular receptors and transduce cerevisiae can be used. Starch rich materials are cheap
the signals for expression of certain genes under and abundantly available, they can be used as
general or stress-responsive conditions. This feedstock for bioethanol productions. For
expression of protective genes or detoxifier brings
fermentation of five carbon sources like xylose,
the adaptive stress responsive conditions called as
fermentation stress tolerance
yeasts with higher xylose fermentation rates like
Pichia stipitis and Pachysolen tannophilus can be used
(Jeffries, 1985; Jeffries et al., 2007). Xylose can be
Ethanol Toxicity in Yeast converted to xylulose by using enzyme xylose
isomerase, xylose reductase and xylitol
The cell wall of S. cerevisiae is made of about 85% dehydrogenase (Klimacek et al., 2014). After
polysaccharides and 15% proteins. The main conversion of xylose to xylulose, xylulose is
functions of the cell wall are to stabilize osmotic phosphorylated to xylulose-5-phosphate and
homeostasis, protect cells against physical damage, metabolized to ethanol. Klimacek et al., (2014) have
maintain cell shape and act as a scaffold for developed an evolutionarily engineered strain of S.
glycoproteins (Klis et al., 2006). The main targets of cerevisiae (IBB10B05) which can efficiently convert
ethanol stress are yeast plasma membrane and xylose to ethanol (Klimacek et al., 2014). Konishi et
hydrophilic and hydrophobic proteins (Stanley et al., al; developed a genetically modified strain of S.
2010). On exposure of yeast to ethanol the fluidity of cerevisiae by using endogenous xylose digesting
cell membrane increases and the stability of genes coding for sorbitol dehydrogenase, aldose
membrane decrease (Mishra, 1989). Thus, ethanol reductase, and xylulose kinase to ferment xylose to
affects the structure and function of the cell ethanol (Konishi et al., 2015). Besides xylose,
membrane. Ethanol also denatures various proteins arabinose is also not utilized by yeast, for
present in the plasma membrane. Ethanol economical ethanol production from lignocellulose
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