Author’s Accepted Manuscript

Yeasts in sustainable bioethanol production: A

review

Siti Hajar Mohd Azhar, Rahmath Abdulla, Siti

Azmah Jambo, Hartinie Marbawi, Jualang Azlan
Gansau, Ainol Azifa Mohd Faik, Kenneth Francis
Rodrigues
www.elsevier.com/locate/bbrep

PII: S2405-5808(16)30242-4
DOI: http://dx.doi.org/10.1016/j.bbrep.2017.03.003
Reference: BBREP405
To appear in: Biochemistry and Biophysics Reports
Received date: 4 November 2016
Revised date: 10 February 2017
Accepted date: 4 March 2017
Cite this article as: Siti Hajar Mohd Azhar, Rahmath Abdulla, Siti Azmah Jambo,
Hartinie Marbawi, Jualang Azlan Gansau, Ainol Azifa Mohd Faik and Kenneth
Francis Rodrigues, Yeasts in sustainable bioethanol production: A review,
Biochemistry and Biophysics Reports,
http://dx.doi.org/10.1016/j.bbrep.2017.03.003
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Yeasts in sustainable bioethanol production: A review

Siti Hajar Mohd Azhara, Rahmath Abdullaa,b*, Siti Azmah Jamboa, Hartinie Marbawia,

Jualang Azlan Gansaua, Ainol Azifa Mohd Faika, Kenneth Francis Rodriguesc

a
Faculty of Science and Natural Resources, Universiti Malaysia Sabah, Jalan UMS, 88400

Kota Kinabalu, Sabah, Malaysia

b
Energy Research Unit, Universiti Malaysia Sabah, Jalan UMS, 88400 Kota Kinabalu, Sabah,

Malaysia
c
Biotechnology Research Institute, Universiti Malaysia Sabah, Jalan UMS, 88400 Kota

Kinabalu, Sabah, Malaysia

*
Corresponding author. Address: Faculty of Science and Natural Resources, Universiti

Malaysia Sabah, Jalan UMS, 88400 Kota Kinabalu, Sabah, Malaysia Tel.: +6-088 320000

(ext: 5592) ; rahmahabdulla@gmail.com (Rahmath Abdulla)

Abstract

Bioethanol has been identified as the mostly used biofuel worldwide since it significantly

contributes to the reduction of crude oil consumption and environmental pollution. It can be

produced from various types of feedstocks such as sucrose, starch, lignocellulosic and algal

biomass through fermentation process by microorganisms. Compared to other types of

microoganisms, yeasts especially Saccharomyces cerevisiae is the common microbes

employed in ethanol production due to its high ethanol productivity, high ethanol tolerance

and ability of fermenting wide range of sugars. However, there are some challenges in yeast

fermentation which inhibit ethanol production such as high temperature, high ethanol
concentration and the ability to ferment pentose sugars. Various types of yeast strains have

been used in fermentation for ethanol production including hybrid, recombinant and wild-type

yeasts. Yeasts can directly ferment simple sugars into ethanol while other type of feedstocks

must be converted to fermentable sugars before it can be fermented to ethanol. The common

processes involves in ethanol production are pretreatment, hydrolysis and fermentation.

Production of bioethanol during fermentation depends on several factors such as temperature,

sugar concentration, pH, fermentation time, agitation rate, and inoculum size. The efficiency

and productivity of ethanol can be enhanced by immobilizing the yeast cells. This review

highlights the different types of yeast strains, fermentation process, factors affecting

bioethanol production and immobilization of yeasts for better bioethanol production.

Keywords: Yeasts, fermentation, bioethanol, immobilization

1. Introduction

The improvement of living standard urges the hunt for sustainable energy in order to

meet energy consumption across the world [1]. On the other hand, the use of fossil fuels as the

main energy resources caused the arising of worldwide problems such as environmental

pollution and global warming [2,3]. These led to the finding of environmentally friendly,

renewable and sustainable energy by government, industrial and energy sector [4,5]. Among

renewable energies, priority was given to liquid biofuels as it represents about 40% of the

total energy consumption in the world [6]. The use of liquid biofuels contributes to the

reduction of greenhouse gas emissions, creation of job opportunities, regional development

and supply security [5,7].

Bioethanol is known as the most widely used biofuel in transportation sector and have

a long history as alternative fuels. In 1984, Germany and France started to use bioethanol as a
fuel in internal combustion engines (ICEs) [8]. Utilization of bioethanol by Brazil was

initiated since 1925. In Europe and United States, bioethanol was widely used until the early

1900s. After World War II, the use of bioethanol was neglected due to its expensive

production cost compared to petroleum fuel until the oil crisis in the 1970s [5]. The interest in

using bioethanol has been increasing since the 1980s and it has been considered as an

alternative fuel in many countries. Global ethanol production increased from 13.12 billions of

gallons in 2007 to 25.68 billions of gallons in 2015 with a slight decreased in 2012 and 2013

[9]. United States is the largest ethanol producer with the production of nearly 15 billion

gallons in 2015. The production of ethanol by United States and Brazil contribute to 85%

world’s ethanol production.

Bioethanol is also known as ethyl alcohol or chemically C2H5OH or EtOH. It can be

used directly as pure ethanol or blended with gasoline to produce “gasohol” [10]. It can be

used as a gasoline improver or octane enhancer and in bioethanol-diesel blends to reduce the

emission of exhaust gasses [11]. Bioethanol offers several advantages over gasoline such as

higher octane number (108), broader flammability limits, higher flame speeds and increased

heats of vaporization [12]. In contrast to petroleum fuel, bioethanol is less toxic, readily

biodegradable and produces lesser air-borne pollutants [13]. A variety of feedstocks from the

first, second and third generation has been used in bioethanol production. The first-generation

bioethanol involves feedstocks rich in sucrose (sugar cane, sugar beet, sweet sorghum and

fruits) and starch (corn, wheat, rice, potato, cassava, sweet potato and barley). Second-

generation bioethanol comes from lignocellulosic biomass such as wood, straw and grasses.

Third-generation bioethanol has been derived from algal biomass including microalgae and

macroalgae [14].

Microorganisms such as yeasts play an essential role in bioethanol production by

fermenting a wide range of sugars to ethanol. They are used in industrial plants due to
valuable properties in ethanol yield (>90.0% theoretical yield), ethanol tolerance (>40.0 g/L),

ethanol productivity (>1.0 g/L/h), growth in simple, inexpensive media and undiluted

fermentation broth with resistance to inhibitors and retard contaminants from growth

condition [15]. As the main component in fermentation, yeasts affect the amount of ethanol

yield. In this review, the role of yeasts in bioethanol fermentation and its immobilization

techniques will be discussed in order to enhance the production of ethanol for the benefits of

mankind.

2. Yeasts

Yeasts are defined as ascomycetous or basidiomycetous fungi that are capable of

reproducing by budding or fission and form spores which are not enclosed in a fruiting body

[16]. They are first classified based on its sexuality (Ascomycotina or Basidiomycotina) or the

lack of sexual phase in the life cycle (Deuteromycotina). The lower taxonomic subdivisions

(families, subfamilies, genera, species and strain) are determined by its morphological,

physiological and genetic characteristics including sexual reproduction [17].

2.1 Yeasts diversity

The number of discovered yeasts has been increasing from year to year. More than

2500 yeast species were published by 2005. It is assumed that only 1% of yeast species is

currently known which represents approximately 1500 species. The total numbers of yeast

species on earth are expected to reach 150,000 [18]. The diversity of yeast species in

particular niches is determined by its capability of utilizing different carbon source and its

nutritional selectivity as it exhibits great specialization for habitat [19]. Yeasts can be isolated

from the terrestrial, aquatic and aerial environment. Plant is the preferred habitat of yeasts

community. A few species are found to have commensalism or parasitic relationships with
animals. Extreme environments like low water potential (high sugar or salt concentration) and

low temperature may be inhabited by yeasts [20,21]. The natural habitats of yeasts are

summarized in Table 1.

There are a broad diversity of yeast cells including its size, shape and colour. Cell

sizes of yeasts are influenced by its species and growth condition. The length of some yeast

cells are only 2-3 µm while the other species may reach the length of 20-50 µm [19]. Most

yeasts have a width in the range of 1-10 µm. Generally, the sizes of brewing strains of S.

cerevisiae are larger than laboratory strains [22]. Many yeast species including

Saccharomyces spp. are ellipsoidal or ovoid in shape and have creamy colour colonies

[20,21].

2.2 Molecular genetics of yeasts

The production of bioethanol is founded on the ability of yeasts to catabolize six-

carbon molecules such as glucose into two carbon components, such as ethanol, without

proceeding to the final oxidation product which is CO2. Crabtree positive yeasts such as S.

cerevisiae accumulate ethanol in the presence of oxygen, however Candia albicans which is a

crabtree-negative yeast catabolizes sugars into CO2 in the presence of oxygen [23]. The

presence of six carbon carbohydrates represses the oxidative respiration pathway in Crabtree

positive yeasts and energy for growth is generated via glycolysis. Upon depletion of six

carbon molecules, the catabolism shifts to oxidation of two carbon molecules into CO2 [24].

This phenomenon is termed at the ‘diauxic shift’. The process of bioethanol production via

fermentative metabolism and the diauxic shift is dependent upon the enzyme Alcohol

Dehydrogenase (EC 1.1.1.1) which is encoded on the ADH1locus. ADH1 catalyzes the

reduction of acetaldehyde to ethanol during the fermentation of glucose, it can also catalyze
the reverse reaction which is the conversion of ethanol into acetaldehyde, albeit with a lower

catalystic efficiency [25].

The yeast S. cerevisiae contain two genes that encode ADH, ADH1 is expressed

constitutively, while the expression of ADH2 is induced by the reduction in the intracellular

concentration of glucose. The substrate for the enzyme ADH2 is ethanol [26]. The expression

of ADH2 gene is governed by transcription factors and genome sequencing and transcriptome

analysis has revealed the structure and DNA binding elements of these regulatory proteins

[27]. Recent advances in synthetic biology have focused on re-engineering the ADH gene for

greater substrate specificity and improvement of catalytic activity as well as engineering the

yeast genome with protein coding genes [28] which improve tolerance to ethanol and catalysis

of a wide range of carbon sources [29]. Molecular biologists are actively seeking novel genes

encoding ADHs using metagenomic approaches, and this had yielded a number of unique

variants [30].

2.3 Yeasts in bioethanol production

Since thousands of years ago, yeasts such as S. cerevisiae have been used in alcohol

production especially in the brewery and wine industries. It keeps the distillation cost low as it

gives a high ethanol yield, a high productivity and can withstand high ethanol concentration

[31]. Nowadays, yeasts are used to generate fuel ethanol from renewable energy sources [32].

Certain yeast strains such as Pichia stipitis (NRRL-Y-7124), S. cerevisiae (RL-11) and

Kluyveromyces fagilis (Kf1) were reported as good ethanol producers from different types of

sugars [33].

S. cerevisiae is the most commonly employed yeast in industrial ethanol production as

it tolerates a wide range of pH [34] thus making the process less susceptible to infection.

Baker’s yeast was traditionally used as a starter culture in ethanol production due to its low
cost and easy availability. However, baker’s yeast and other S. cerevisiae strains were unable

to compete with wild-type yeast which caused contamination during industrial processes.

Stressful conditions like an increase in ethanol concentration, temperature, osmotic stress and

bacterial contamination are the reasons why the yeast cannot survive during the fermentation

[35]. Flocculent yeasts were also used during biological fermentation for ethanol production

as it facilitates downstream processing, allows operation at high cell density and gives higher

overall productivity [36,37]. It reduces the cost of cells recovery as it separate easily from the

fermentation medium without centrifugation [38].

There are common challenges to yeasts during sugar fermentation which are rise in

temperature (35-45oC) and ethanol concentration (over 20%) [39]. Yeasts growth rate and

metabolism increase as the temperature increases until it reaches the optimum value. Increase

in ethanol concentration during fermentation can cause inhibition to microorganism growth

and viability [40,41]. Inability of S. cerevisiae to grow in media containing high level of

alcohol leads to the inhibition of ethanol production [42]. The other problems in bioethanol

fermentation by yeast are the ability to ferment pentose sugars. S. cerevisiae is the most

commonly used in bioethanol production. However, it can only ferment hexoses but not

pentoses [43]. Only some yeasts from genera Pichia, Candida, Schizosaccharomyces and

Pachysolen are capable of fermenting pentoses to ethanol [33].

The efficiency of ethanol production on an industrial scale will be increased by using

yeasts that are tolerant to inhibitors [39]. The common challenges of yeasts can be overcome

by using ethanol-tolerant and thermotolerant yeast. Ethanol-tolerant and thermotolerant

strains which can resist stresses can be isolated from natural resources such as soil, water,

plants and animals. This is because cells adapt to their environment over time by natural

selection. Ethanol fermentation at high temperature is a beneficial process as it selects

thermo-tolerant microorganisms and does not require cooling costs and cellulase [44]. For
example, K. Marxianus is thermotolerant yeast which is capable of co-fermenting both hexose

and pentose sugars and can survive the temperature of 42-45oC [45].

The problems of pentose fermentation can be solved by using hybrid, genetically

engineered or co-culture of two yeast strains. Hybrid yeast strains are used simultaneously to

ferment pentose and hexose sugars to ethanol. The hybrid strain has been developed by fusing

protoplast of S. cerevisiae and xylose-fermenting yeasts like P. tannophilus, C. shehatae and

P. stipitis [46]. Genetically engineered S. cerevisiae and co-culture of two strains have been

developed to produce bioethanol from xylose with high yield. Genetic engineering use

recombinant DNA technology to up-regulate the stress tolerance genes in order to overcome

the inhibitory situations [47]. Xylose reductase and xylitol dehydrogenase genes from S.

stipitis were introduced into S. cerevisiae to develop strain with the ability of fermenting

xylose. The engineered yeast strains can convert cellulose to ethanol more rapidly compared

to unmodified yeast strains. Co-culture process simultaneously culture and grow two different

yeasts in the same reactor [48]. Co-culture shows better ethanol production as compared to its

pure culture [49]. In co-culture, pentose utilizing yeasts like Pichia fermentans and Pichia

stipitis are combined together with S. cerevisiae so that hexose and pentose sugars can be

efficiently utilized [50,51].

Yeast strains that have been used in bioethanol production are summarized in Table 2.

S. cerevisiae was the most widely studied yeasts. Different types of feedstock were used for

the production of bioethanol. Kim et al. [52] reported the highest ethanol concentration of

96.9 g/L with a productivity of 3.46 g/L/h. It was contributed by the wild-type yeast strain

used, S. cerevisiae KL17 which is capable of utilizing both glucose and galactose

simultaneously. It shows that wild-type yeasts has high potential in fermenting sugars to

ethanol. Moreover, Silva-filho et al., [53] reported that wild-type strains could be more

efficient to the industrial process than commercial strains. Fermentation of giant reed using S.
stipitis CBS 6054 obtained the lowest ethanol concentration of 8.2 g/L with a productivity of

0.17 g/L/h [54]. At the optimum condition for sugars release, the levels of toxic degradation

products exceed the critical level and made the condition unsuitable for yeast fermentation.

3. Process in bioethanol production

The process of ethanol production depends on the types of feedstocks used. Generally,

there are three major steps in ethanol production: (1) obtaining solution that contains

fermentable sugars, (2) converting sugars to ethanol by fermentation and (3) ethanol

separation and purification [108]. Feedstocks are usually pretreated in order to reduce its size

and facilitate subsequent processes. Then, the hemicellulose and cellulose will be hydrolyzed

to fermentable sugars. Yeasts are given the responsibility to ferment these sugars into ethanol.

Separation technologies are used to recover ethanol before it can be used as fuel [55].

3.1 Pretreatment

Pretreatment has a significant effect on the overall process which makes the hydrolysis

easier and produces higher amount of fermentable sugars. It influences the amount of ethanol

yield and production cost [56]. Methods that are currently used for pretreatments are physical,

chemical, biological and physicochemical. Physical pretreatment uses mechanical milling to

ground the substrate. The common chemical pretreatment includes ozonolysis, acid

hydrolysis, alkaline hydrolysis [57] and organosoly based process [58]. Different fungal

species are involved in biological pretreatment while physicochemical pretreatment includes

ammonia fibre explosion [59] and steam [60]. Dehydration of hexose and pentoses during

pretreatment release furan compounds like 5-hydroxymethyl-2-furaldehyde (HMF) and 2-

furaldehyde. These furan derivatives induce the inhibition of cell growth and reduce ethanol

productivity [61]. Yeasts fermentation is inhibited by the weak acid stress induced from
lignocellulosic materials. However, the low concentration of weak acids can increase ethanol

production by cellular division. It was reported that the presence of weak acids can improve

glucose utilization, ethanol production and tolerance to HMF and furfural in S. cerevisiae

[62].

3.2 Hydrolysis

Hydrolysis process takes place after pretreatment to break down the feedstocks into

fermentable sugar for bioethanol production. The two most commonly used hydrolysis

methods are acidic and enzymatic. Acid hydrolysis is considered as the oldest and most

commonly used method [63]. Acidic hydrolysis can be divided into two types namely dilute

and concentrated. Dilute acid hydrolysis is performed at higher temperature using low acid

concentration while concentrated acid hydrolysis is carried out at lower temperature using

high acid concentration. Dilute acid hydrolysis is the most commonly used process. However,

it generates large amount of inhibitors compared to concentrated acid hydrolysis. Acid

hydrolysis of lignocellulosic biomass is conducted in two-stage process as the pentose sugars

degrade more rapidly compared to hexose sugars. Hemicellulose is hydrolyzed in the first

stage using dilute acid while cellulose is hydrolyzed in the second stage using concentrated

acid. Concentrated acid process generates high sugar recovery (90%) in shorter period of time

[64]. The disadvantages of acid hydrolysis are the difficulty of performing acid recovery and

recycling process which increases the production cost.

Enzymatic hydrolysis requires enzymes to hydrolyse the feedstocks into fermentable

sugars. Three types of enzymes that are commonly used for cellulose breakdown such as

endo-β—1,4-glucanases, cellobiohydrolases and β-glucosidases. The activity of cellulase

enzyme is influenced by the concentration and source of the enzyme. Cellulose will be

degraded into reducing sugars under mild reaction conditions (pH: 4.8-5.0, temperature: 45-
50oC). Moreover, it does not cause corrosion problem in the reactors which can result in high

sugar yields. The efficiency of enzymatic hydrolysis is influenced by optimized conditions

such temperature, time, pH, enzyme loading and substrate concentration [65]. The amount of

fermentable sugar obtained increases as the enzyme load increases while cellulose load

decreases. Enzymatic saccharification of cellulose can be enhanced by using surfactants

which function to block lignin. The efficiency of cellulose hydrolysis can be improved by

adding Polyethylene glycol (PEG) or Tween 20 to increase enzymatic saccharification and

reduce the adsorption of cellulase on lignin [64]. The limitation of using enzymes in

hydrolysis is because they are too expensive for the economical production of ethanol from

biomass.

3.3 Fermentation process

There are three processes that are commonly used in bioethanol production which are

separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation

(SSF) and simultaneous saccharification and co-fermentation (SSCF). In SHF, hydrolysis of

lignocellulosic materials is separated from ethanol fermentation. The separation of enzymatic

hydrolysis and fermentation allows enzyme to be operated at high temperature for better

performance while fermentation organisms can be operated at moderate temperature for

optimizing sugar utilization. SSF and SSCF have a short overall process as the enzymatic

hydrolysis and fermentation process occur simultaneously to keep the concentration of

glucose low. For SSF, the fermentation of glucose is separated from pentoses while SSCF

ferment glucose and pentoses in the same reactor [65]. Both SSF and SSCF are preferred over

SHF because the operation can be performed in the same tank. The benefits of both processes

are lower cost, higher ethanol yield and shorter processing time [66].
 Fermentation of bioethanol can be carried out in batch, fed-batch, repeated batch or

continuous mode. In batch process, substrate is provided at the beginning of the process

without addition or removal of the medium [67]. It is known as the simplest system of

bioreactor with multi-vessel, flexible and easy control process. The fermentation process is

carried out in a closed-loop system with high sugars and inhibitors concentration at the

beginning and ends with high product concentration [68]. There are several benefits of batch

system including complete sterilization, does not require labour skills, easy to manage the

feedstocks, can be can be control easily and flexible to various product specifications [69,70].

However, the productivity is low and need intensive and high labour costs. The presence of

high sugar concentration in the fermentation medium may lead to substrate inhibition and

results in the inhibition of cell growth and ethanol production [71].

Cell recycle batch fermentation (CRBF) is a strategic method for effective ethanol

production as it reduce time and cost for inoculum preparation. The other advantages of

repeated-batch process are easy cell collection, stable operation and long-term producitivity

[72,73]. Sugar materials and immobilized yeast cells are used to facilitate cell separation for

cell recycling [74,75]. Combination of SSF and repeated-batch fermentation has been

successfully applied on the fermentation of cassava starch using flocculating yeast [76].

However, its application in SSF process of lignocellulosic materials is extremely difficult

because lignocelluosic residue remain in the fermentation medium together with yeast cells

[77]. The use of free cells in this system reduces yeast cell concentration and results in lower

ethanol production in the subsequent batches. Repeated-batch fermentation can be performed

by replacing free cells with the immobilized cells [78].

Fed-batch fermentation is a combination of batch and continuous mode which

involves the addition of substrate into the fermentor without removing the medium. It has

been used to overcome the problem of substrate inhibition in batch operation. Volume of
culture in fed-batch processes can vary widely but it must be fed properly at certain rate with

the right component composition. Productivity of fed-batch fermentation can be increased by

maintaining substrate at low concentration which allows the conversion of sufficient amount

of fermentable sugars to ethanol [70]. This process has higher productivity, higher dissolved

oxygen in medium, shorter fermentation time and lower toxic effect of the medium

components compared to other types of fermentation [71]. However, ethanol productivity in

fed-batch is limited by feed rate and cell mass concentration [79]. Fed-batch operation has

been applied successfully in non-uniform SSF system by continuously adding a pretreated

substrate in order to achieve relatively high sugar and ethanol concentration [80].

Continuous operation is carried out by constantly adding substrates, culture medium

and nutrients into a bioreactor containing active microorganisms. Culture volume in

continuous operation must be constant and the fermentation products are taken continuously

from the media. Various type of products can be obtained from the top of the bioreactor such

as ethanol, cells and residual sugar [69]. The advantages of continuous system over batch and

fed-batch system are higher productivity, smaller bioreactor volumes and less investment and

operational costs [70]. At high dilution rate, ethanol productivity is increased while ethanol

yield is decreased due to incompletely substrate consumption by yeasts [81]. However, the

possibility for contamination to occur is higher than other types of fermentation [66].

Moreover, the ability of yeasts to produce ethanol in continuous process are reduced due to

long cultivation time.

Processes involved in bioethanol production are summarized in Table 3. Enzymatic

hydrolysis is the preferred saccharification method because of its higher yields, higher

selectivity, lower energy cost and milder operating condition than chemical processes [82].

The most commonly used pretreatment method is steam explosion. This is contributed by the

attractive features of steam explosion which has less environmental impact, low capital
investment, high energy efficiency, less hazardous process chemicals and conditions and

complete sugar recovery [83]. Fermentation of Miscanthus by S. cerevisiae CHY1011

reported the highest ethanol concentration of 69.2 g/L with a productivity of 1.24 g/L/h [84].

The pretreatment method applies a high shearing force to increase the biomass surface and

results in increased enzymatic digestibility. Moreover, the continuous SSF feeding system

increased the biomass concentration thus providing sufficient time for liquefaction of the

substrate by the enzyme. Scordia et al., [85] reported the lowest ethanol concentration of 12.1

g/L with a productivity of 0.13 g/L/h by fermenting Miscanthus x giganteus using S. stipitis

CBS 6054. This is because sugar recovery from the water soluble fraction (WSF) which has

been used as the feedstock for fermentation was low.

3.4 Factors affecting bioethanol production

There are several factors which influence the production of bioethanol including

temperature, sugar concentration, pH, fermentation time, agitation rate, and inoculum size

[86]. The growth rate of the microorganisms is directly affected by the temperature [87]. High

temperature which is unfavorable for cells growth becomes a stress factor for microorganisms

[88]. The ideal temperature range for fermentation is between 20 and 35 oC. Free cells of S.

cerevisiae have an optimum temperature near 30oC whereas immobilized cells have slightly

higher optimum temperature due to its ability to transfer heat from particle surface to inside

the cells [89]. Moreover, enzymes which regulate microbial activity and fermentation process

are sensitive to high temperature which can denature its tertiary structure and inactivates the

enzymes [90]. Thus, temperature is carefully regulated throughout the fermentation process.

The increase in sugar concentration up to a certain level caused fermentation rate to

increase.. However, the use of excessive sugar concentration will cause steady fermentation

rate. This is because the concentration of sugar use is beyond the uptake capacity of the
microbial cells. Generally, the maximum rate of ethanol production is achieved when using

sugars at the concentration of 150 g/L. The initial sugar concentration also has been

considered as an important factor in ethanol production. High ethanol productivity and yield

in batch fermentation can be obtained by using higher initial sugar concentration. However, it

needs longer fermentation time and higher recovery cost [86].

Ethanol production is influenced by pH of the broth as it affects bacterial

contamination, yeast growth, fermentation rate and by-product formation. The permeability of

some essential nutrients into the cells is influenced by the concentration of H+ in the

fermentation broth [86]. Moreover, the survival and growth of yeasts is influenced by the pH

in the range of 2.75 to 4.25 [91]. In fermentation for ethanol production, the optimum pH

range of S. cerevisiae is 4.0-5.0 [34]. When the pH was below than 4.0, a longer incubation

period is required but the ethanol concentration was not reduced significantly. However, when

then pH was above 5.0, the concentration of ethanol reduced substantially [10].

Fermentation time affect the growth of microorganisms. Shorter fermentation time

causes inefficient fermentation due to inadequate growth of microorganisms. On the other

hand, longer fermentation time gives toxic effect on microbial growth especially in batch

mode due to the high concentration of ethanol in the fermented broth. Complete fermentation

can be achieved at lower temperature by using longer fermentation time which results in

lowest ethanol yield [86].

Agitation rate controls the permeability of nutrients from the fermentation broth to

inside the cells and removal of ethanol from the cell to the fermentation broth. The greater

the agitation rate, the higher the amount of ethanol produced. Besides, it increases the amount

of sugar consumption and reduces the inhibition of ethanol on cells. The common agitation

rate for fermentation by yeast cells is 150-200 rpm. Excess agitation rate is not suitable for

smooth ethanol production as it causes limitation to the metabolic activities of the cells [86].
 Inoculum concentration does not give significant effects on the final ethanol

concentration but it affects the consumption rate of sugar and ethanol productivity [92]. The

production of ethanol was seen to be increased with the increase in cell numbers from 1 x 104

to 1 x 107 cells per ml but there was no significant ethanol production found between 107 and

108 cells per ml. This is because the increase in cell concentration within certain range

reduces fermentation time as the cells grow rapidly and directly consumes sugars into ethanol

[86].

Factors affecting the production of bioethanol are shown in Table 4. Most of

fermentation process using S. cerevisiae was carried out the at 30oC whereas fermentation

using K. marxianus was performed at 42oC. The ideal temperature for bioethanol production

depends on the ideal temperature of the yeasts. Most of the fermenting medium used for

bioethanol production has pH in the range of 4.5 to 5.5 with various sugar concentration.

Fermentation process is commonly performed at 24 and 72 hours with rotation at 120 and 150

rpm. The common inoculum size employed in bioethanol production are 5 and 10%. Zhang et

al. [93] reported the highest ethanol concentration (128.5 g/L) and ethanol productivity (4.76

g/L/h) probably due to favourable conditions for the yeast to produce bioethanol. The lowest

ethanol concentration (9.5 g/L) and ethanol productivity (0.31 g/L/h) was produced from

water hyacinth due to its low sugar concentration which limits substrate for bioethanol

production [94].

A large amount of ethanol must be produced in order to fulfill the increasing

worldwide demand. However, the production of ethanol using free yeast cells is still

inefficient due to its higher cost of cell cycling, greater contamination risk, limitation of the

dilution rate and susceptibility to environmental variations [95]. Moreover, free cells cause

substrate or product inhibition from direct contact between the cells and medium. Most of the

problems occurred in free-cell systems are reduced by the immobilization method.

4. Immobilization

Immobilized cell technology is commonly applied in fermentation process. The

benefits of immobilized cells over free cells include higher cell density per volume of reactor,

easier separation from the reaction medium, higher substrate conversion, less inhibition by

products, shorter reaction time and control of cell replication [96]. The immobilization of

yeast cells and its productivity are influenced by several factors such as the surface

characteristics of the carrier, pore size, water content, hydrophilicity and magnetism [97].

Immobilization should be performed under mild condition to maintain the activity of the cells

[98].

4.1 Immobilization of yeast cells

Cells can be immobilized by different types of methods like adsorption, crosslinking,

encapsulation and entrapment. Entrapment is carried out by the polymerization of an aqueous

solution of acrylamide monomers in which microorganisms are suspended. It is commonly

used to overcome the problems of degradation and limitation of mass transfer. It avoids the

release of cells while allowing diffusion of substrates and products [99]. This method allows

high biomass loading which results in high ethanol productivity. Entrapment method is

widely used due to its simplicity, non-toxic, less expensive, reversible and good mechanical

properties. Entrapment can be operated at extremely high dilution rates without causing

washout of cells. Most of the researches involving the immobilization of microbial cells were

focused on gel entrapment. The most commonly used gels are in the form of spherical beads

with diameters in the range of 0.3 to 5 mm. However, gel has limited mechanical stability

which can be easily damaged by the growth of the microbial cells and carbon dioxide

production. Moreover, the presence of phosphates causes the weakening of calcium alginate

gels [79].
 Adsorption is a very popular way of cell immobilization due to its simple, cheap and

fast method. Cells are attached to the surface of the material by electrostatic force such as Van

der Waals forces, ionic bonds, hydrogen bridges or covalent interactions. Ionic attraction is

used to immobilize yeast cells. The supporting material used must have a high affinity in

order for the yeast strain to withstand the environmental conditions present within the

bioreactor. In most cases of continuous ethanol production, adsorption is carried out by

circulating a concentrated suspension of yeast cells through the bioreactor for several hours.

Adsorption technique does not require the use of toxic chemical and the yeast cells can be

maintained in a viable state. The absorbed-cell system is limited by lower biomass loading

and lower feed flow rates compared to entrapped-cell system. This is because the number of

yeast cells that can be absorbed on the carrier is limited by the surface area of the carrier [79].

The other commonly used method for cells immobilization is encapsulation which

encloses cells within a thin semi-permeable membrane. The cells are free to move in the inner

liquid core inside the capsule. However, the space is limited by the outer membrane [100]. In

fermentation, the molecular dimensions of the microcapsules limit the growth of cells and the

size of both nutrients and products. The rate of substrate transfer into the capsules will

determine the rate of reaction. Encapsulation method gives several advantages such as

mechanical and chemical stability of the membrane system, possibility of high loading and

regulation of the fermentation reaction by selective diffusion of substrate and products [101].

There are many types of supporting materials that have been used in yeast cells

immobilization such as calcium alginate, sugarcane bagasse, delignified cellulosic materials,

orange peel, spent grains, corn cobs, k-carrageenan, wood blocks, porous cellulose, zeolite,

loofa sponges and sorghum bagasse [102]. The support used in immobilization must be

conducive to cell viability and have proper permeability for the diffusion of oxygen, essential

nutrients, metabolic waste and secretory products across the polymer network. There are two
types of polymers that are used as carrier in yeasts immobilization which are natural and

synthetic polymers. The benefits of using natural polymers are low price and no impurities

produced from chemical reaction. Synthetic polymers exhibit high chemical and biological

stability, mechanical resistance to abrasion, permeable to reagents, and have large surface,

capacity and porosity [103].

4.2 Immobilized yeasts in bioethanol production

Immobilization of yeast strains for bioethanol production is presented in Table 5. The

commonly employed method for yeast immobilization is adsorption because the cells are not

affected and yeast can be added or washed out from the fermentation medium [104]. Calcium

alginate is the most preferred carrier due to its good biocompatibility, low cost, ease of

availability [96]. Ariyajaroenwong et al., [105] reported the highest ethanol concentration of

98.48 g/L by fermenting sweet sorghum juice using S. cerevisiae NP 01. Sorghum stalk which

was used as the carrier showed an important function as the source of inoculum for ethanol

production while the sweet sorghum juice which was used as the feedstocks contained

essential nutrients for yeast growth. Singh et al., [106] used S. cerevisiae MTCC 174 to

ferment sugarcane bagasse and produced only 15.4 g/L of ethanol concentration. The low

amount of sugar obtained from sugarcane bagasse could be the reason for low amount of

ethanol concentration. Zheng et al. [107] used S. cerevisiae to ferment sugar molasses and

obtained the highest ethanol productivity of 6.55 g/L/h. The adsorption and covalent binding

of MCM-41 zeolite with the embedding of alginate caused the cell in the MCM-41

mesoporous zeolite composite carrier to grow better than in pure carrier. Behera et al., [108]

used S. cerevisiae CTCRI to ferment mahula flower and achieved ethanol productivity of only

0.27 g/L/h. The lowest ethanol productivity obtained probably due to mahula flower which

contains low amount of fermentable sugars compared to other types of feedstocks.

5. Conclusion

Yeasts which are the most common microorganisms in bioethanol production play

important function in fermenting sugars to ethanol. The influence of yeast strains, processes

of fermentation and immobilization of yeasts on ethanol production has been shown in this

work. Many types of yeast strains have been identified all over the world with the ability of

producing ethanol from different types of feedstocks. This review paper discussed about the

efficiency of different yeast strains in producing ethanol with wild-type strain as the highest

ethanol producer. Fermentation process exhibited significant effect on ethanol production.

Continuous SSF method has shown its ability in producing high ethanol concentration with

high productivity. The application of cell immobilization in ethanol production was evaluated.

Adsorption method is the preferred method of immobilizing yeast cells whereas calcium

alginate is the top choice for yeasts carrier. Immobilized cells give several advantages in

ethanol production such as high cell density, easy separation from the medium, high substrate

conversion, less inhibition, short reaction time and cell recycling. Thus, immobilized yeasts

pave better way for commercialization of bioethanol production from an economical

perspective.

Acknowledgements

This research was financially supported by the Ministry of Higher Education Malaysia

(KPM) through the Fundamental Research Grant Scheme (FRGS0366-SG-1/2014).

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Table 1 Natural yeasts habitats [20,21].

 Habitat Description Yeasts genera
Plants  The common niche of yeasts is the interface Ashbya spp.
between soluble nutrients of plants (sugars) and the Nematospora spp.
septic world
 insects help in spreading yeasts on the phyllosphere

Animals  Several yeasts are pathogenic toward humans and Candida spp.
animals while others are non-pathogenic (can be Cyniclomyces spp.
found in intestinal tract and skin of warm-blooded Pityrosporum spp.
animals)
 Numerous yeasts are commensal to insect which
act as vectors for natural distribution of yeasts.

Soil  Considered as reservoir for yeasts long-term Lipomyces spp.

survival rather than habitat for free growth Schwanniomyces
 Yeasts can be found only in the aerobic soil layers spp.
(10-15 cm)

Water  Yeasts can be found in both fresh water and Rhodotorula spp.
seawater Debaryomyces spp.
 Estuarine regions usually have higher numbers of
yeasts compared to seawater

Atmosphere  Yeasts are dispersed by air currents from the Cryptococcus spp.
vegetative layer above soil surfaces Rhodotorula spp.
 Only a few yeasts may be expected per volume of Sporobolomyces spp.
air Debaryomyces spp.

Extreme  Some halotolerant yeasts can grow in nearly Debaryomyces spp.

environmen saturated brine solution Zygosaccharomyces
t  Osmophilic yeasts were discovered in glacier spp.
horizons
Table 2 Yeast strains used in bioethanol production.

Yeast strain Type of Feedst Sugar Fermenta Ethanol Ethanol Referen

strain ock concentra tion concentra producti ces
tion (g/L) condition tion (g/L) vity
(g/L/h)
o
S. cerevisiae Laborato Spent 195.0 30 C, 11.7 0.49 [33]
RL-11 ry coffee 200 rpm,
ground 48 hr
s
S. cerevisiae Laborato Sorghu 200.0 30oC, 68.0 0.94 [109]
MTCC 173 ry m 120 rpm,
stover 96 hr
S. stipitis CBS Laborato Giant 33.4 30oC, 8.2 0.17 [54]
6054 ry reed 150 rpm,
96 hr
S. cerevisiae Wild- Galact 500.0 30oC, 96.9 3.46 [52]
KL17 type ose 200 rpm,
and 28 hr
glucos
e
S. pombe Wild- Cassav 95.0 32oC, 72.1 1.16 [110]
CHFY0201 type a 120 rpm,
starch 66 hr
S. cerevisiae Wild- Cassav 195.0 32oC, 89.1 1.35 [111]
CHY1011 type a 120 rpm,
starch 66 hr
S. cerevisiae Recombi Corn 99.0 30oC, 41.2 0.57 [112]
ZU-10 nant stover 180 rpm,
72 hr
S. cerevisiae Mutated Ipome 72.1 30oC, 29.0 1.03 [46]
RPRT90 hybrid a 150 rpm,
carnea 28 hr
S. cerevisiae Hybrid Cassav 195.0 32oC, 89.8 1.38 [38]
CHFY0321(pro a 120 rpm,
toplast fusant) starch 65 hr

Table 3 Processes involved in bioethanol production

Yeast Feedstock Pretreatme Fermentati Ethanol Ethanol Referenc
strain nt on concentrati Productivi es
condition on (g/L) ty (g/L/h)
S. Miscanthus CHEMET Continuous 69.2 1.24 [84]

36
cerevisiae sacchariflor with SSF, 33 oC,
CHY1011 us sodium 56 hr, 25 %
hydroxide WIS
S. Wood chips Steam Batch 32.9 0.34 [113]
cerevisiae explosion SSCF, 3 oC,
TMB3400 96 hr, 8 %
WIS
S. Reed Phosphoric Batch SSF, 55.5 0.57 [114]
cerevisiae acid- 38 oC, 150
ATCC248 acetone rpm, 96 hr,
58 36.1 %
WIS
S. Arundo Steam Enzymatic 20.6 0.21 [115]
cerevisiae donax explosion hydrolysis,
VIT C- 45 oC, 72
10880 hr; Batch
SHF, 32 oC,
500 rpm,
96 hr, 10 %
WIS
S. Reed Liquid hot Enzymatic 39.4 0.66 [116]
cerevisiae water hydrolysis,
50 oC, 18
hr; Semi
fed-batch
SSF, 36
o
C, 60 hr,
10 % WIS
S. Wheat meal Steam Enzymatic 53.3 0.44 [117]
cerevisiae and wheat explosion hydrolysis,
TMB 3400 straw 40 oC, 850
rpm, 120
hr; Fed-
batch
SHCF, 32
o
C, 300
rpm, 120
hr, 7.5 %
WIS
S. Liriodendro Acid-free Batch SSF, 29.9 0.42 [118]
cerevisiae n tulipifera organosolv 30 oC, 150
rpm, 96 hr,
1 % WIS

37
Baker’s Corn stover Steam Fed-batch 25.7 0.36 [119]
o
yeast explosion SSF, 30 C,
700 rpm,
72 hr, 10 %
WIS
S. stipitis Miscanthus Dilute SSF, 30 oC, 12.1 0.13 [85]
CBS 6054 x giganteus oxalic acid 96 hr, 10 %
WSF
S. Industrial Steam SSF, 37 oC, 21.3 0.30 [120]
cerevisiae hemp explosion 348 rpm,
72 hr, 7.5%
WIS
Table 4 Factors affecting bioethanol production.

Yeast Feeds Temper p Ti Sugar Agita Inocu Ethanol Ethano Refere

strain tock ature, H me concent tion lum concent l nces
(oC) (h) ration rate size ration Produc
(g/L) (rpm) (%) (g/L) tivity
(g/L/h)
Saccharo Cassa 32 4. 66 585.0 120 5 89.1 2.10 [111]
myces va 5
cerevisia starch
e
CHY101
1
Saccharo Corn 30 5. 72 99.0 120 5 41.2 0.57 [112]
myces stover 5
cerevisia
e ZU-10
Saccharo Instan 30 - 24 84.0 250 5 41.3 1.72 [121]
myces t
cerevisia noodl
e K35 e
waste
Saccharo Wood 30 5. 16 37.47 150 10 18.52 1.16 [122]
myces 5
cerevisia
e
Saccharo Reed 38 5. 96 123.0 150 10 55.0 0.57 [114]
myces 0
cerevisia
e ATCC
#24858
Saccharo Sweet 30 5. 24 240.0 150 7 128.5 4.76 [93]
myces potat 3
cerevisia o
e
Kluyvero Water 42 4. 24 23.3 - 5 7.34 0.31 [94]
myces hyaci 8
38
marxianu nth
s K213
Kluyvero Whea 42 5. 72 - 150 - 36.2 0.50 [123]
myces t 5
marxianu straw
s CECT
10875
Saccharo Paper 33 - 16 27.8 60 6 9.5 0.59 [124]
myces sludg
cerevisia e
e GIM-2
Saccharo Cassa 33 - 42 183.5 100 5 86.1 2.41 [125]
myces va
cerevisia mash
e
CHFY03
21

Table 5 Immobilization of yeasts in bioethanol production

Yeast Feedst Metho Carrier Sugar Ferment Ethanol Ethanol Refere

strain ock d concentr ation concentr product nces
ation conditio ation ivity
(g/L) n (g/L) (g/L/h)
S. Sugar Cross- Alginat 170.0 30 oC, 78.6 6.55 [107]
cerevisi molass linking e-based 115
ae es and MCM- rpm, 12
covale 41 hr
nt mesopo
binding rous
zeolite
compos
ite
S. Cane Cross- Bacteri 220.0 33 oC, 92.0 1.92 [126]
cerevisi molass linking al 150
ae M30 es cellulos rpm, 48
e- hr
alginate
(BCA)
sponge
Mutant Gluco Adsorp Sorghu 200.0 30 oC, 92.7 5.72 [127]
baker’s se and tion m 16 hr
yeast sucros bagasse
3013 e
S. Sugarc Adsorp Sugarc 50.0 30 oC, 15.4 0.43 [106]
cerevisi ane tion ane 72 hr
ae bagass bagasse
MTCC e
39
 174

S. Blacks Adsorp Thin- 240.0 33 oC, 80.6 1.85 [128]

cerevisi trap tion shell 150
ae M30 molass silk rpm, 48
es cocoon hr
S. Sugar Adsorp Sugar 120.0 30 oC, 52.3 1.09 [129]
cerevisi beet tion beet 48 hr
ae thick pulp
DTN juice
S. Sorgh Adsorp Sweet 230.0 30 oC, 98.5 1.37 [105]
cerevisi um tion sorghu 72 hr
ae NP juice m
01 stalks
S. Mahul Entrap Calciu 350.0 30 oC, 25.8 0.27 [108]
cerevisi a ment m 96 hr
ae flower alginate
CTCRI
S. Corn Entrap Calciu 150.0 30 oC, 88.9 2.34 [130]
cerevisi meal ment m 74 hr,
ae var. alginate 150 rpm
ellipsoi
deus
S. Wheat Entrap Calciu 51.4 30 oC, 37.1 0.38 [131]
cerevisi straw ment m 96 hr,
ae alginate 150 rpm
T0936

Highlights

 Yeast strains applied in bioethanol production are highlighted.

 Fermentation in bioethanol production by yeasts are discussed.

 Factors that affect bioethanol production are considered.

 Immobilization of yeasts in bioethanol production are highlighted.

40