fermentation

Review
Production of Bioethanol—A Review of Factors Affecting
Ethanol Yield
Timothy J. Tse 1, * , Daniel J. Wiens 1 and Martin J. T. Reaney 1,2,3, *

1 Department of Plant Sciences, University of Saskatchewan, 51 Campus Drive,

Saskatoon, SK S7N 5A8, Canada; [email protected]
2 Prairie Tide Diversified Inc., 102 Melville Street, Saskatoon, SK S7J 0R1, Canada
3 Guangdong Saskatchewan Oilseed Joint Laboratory, Department of Food Science and Engineering,
Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China
* Correspondence: [email protected] (T.J.T.); [email protected] (M.J.T.R.)

Abstract: Fossil fuels are a major contributor to climate change, and as the demand for energy
production increases, alternative sources (e.g., renewables) are becoming more attractive. Biofuels
such as bioethanol reduce reliance on fossil fuels and can be compatible with the existing fleet
of internal combustion engines. Incorporation of biofuels can reduce internal combustion engine
(ICE) fleet carbon dioxide emissions. Bioethanol is typically produced via microbial fermentation of
fermentable sugars, such as glucose, to ethanol. Traditional feedstocks (e.g., first-generation feedstock)
include cereal grains, sugar cane, and sugar beets. However, due to concerns regarding food
sustainability, lignocellulosic (second-generation) and algal biomass (third-generation) feedstocks
have been investigated. Ethanol yield from fermentation is dependent on a multitude of factors.
This review compares bioethanol production from a range of feedstocks, and elaborates on available





 technologies, including fermentation practices. The importance of maintaining nutrient homeostasis
of yeast is also examined. The purpose of this review is to provide industrial producers and policy
Citation: Tse, T.J.; Wiens, D.J.;
Reaney, M.J.T. Production of
makers insight into available technologies, yields of bioethanol achieved by current manufacturing
Bioethanol—A Review of Factors practices, and goals for future innovation.
Affecting Ethanol Yield. Fermentation
2021, 7, 268. https://doi.org/ Keywords: bioethanol; fermentation; biofuels; solid-state; submerged; very high gravity; yeast
10.3390/fermentation7040268

Academic Editors: Nhuan Nghiem

and Tae Hyun Kim 1. Introduction
Continued growth of the global economy has increased both energy consumption and
Received: 15 October 2021
concern regarding the accumulation of atmospheric greenhouse gases, and their effects on
Accepted: 15 November 2021
climate change. In response, many countries are developing renewable energy, including
Published: 18 November 2021
biofuel production. Biofuels are any fuels produced from biomass, such as organic waste
materials [1], and such fuels can have a significantly reduced ecological footprint compared
Publisher’s Note: MDPI stays neutral
to traditional fossil fuels [2]. One such biofuel is bioethanol, the production of which is
with regard to jurisdictional claims in
projected to surpass 130 billion liters/year worldwide [3], with the United States and Brazil
published maps and institutional affil-
iations.
supplying most of the world’s ethanol [4]. Bioethanol is ethanol (an alcohol) produced
through microbial fermentation of carbohydrates from plants or algae (e.g., corn, sugarcane,
wheat, lignocellulosic biomass, etc.).
Microbial fermentation is a natural process used to break larger organic molecules
into simpler ones. Prior to alcoholic fermentation, pretreatment processes may be required
Copyright: © 2021 by the authors.
to prepare the biomass for extraction and fermentation. After preparation, enzymatic
Licensee MDPI, Basel, Switzerland.
hydrolysis can then release fermentable monosaccharide and disaccharide sugars. Yeast
This article is an open access article
then converts these sugars (e.g., glucose, galactose, and fructose) to ethanol, carbon dioxide,
distributed under the terms and
conditions of the Creative Commons
and other by-products in metabolic processes that can occur under both aerobic and
Attribution (CC BY) license (https://
anaerobic conditions. For example, glucose molecules produce two molecules of pyruvate
creativecommons.org/licenses/by/ during glycolysis. The two molecules of pyruvic acid are then reduced to two molecules of
4.0/). ethanol and carbon dioxide [5]. Under anaerobic conditions, pyruvate can be metabolized

Fermentation 2021, 7, 268. https://doi.org/10.3390/fermentation7040268 https://www.mdpi.com/journal/fermentation

Fermentation 2021, 7, 268 2 of 18

to acetaldehyde with the release of carbon dioxide. Subsequently, acetaldehyde can then
be reduced to ethanol by alcohol dehydrogenase [6].
Traditional alcoholic fermentation (first-generation bioethanol production) has used
food crops as feedstocks (e.g., wheat, corn, potatoes, beets, sugarcane), as these materi-
als are superior sources of easily accessible starch and sugar required for fermentation.
However, as the global population grows and the amount of arable land remains limited,
there has been increasing concern regarding fuel production from food crops. Therefore,
non-edible sources of biomass, such as lignocellulosic materials and algae, are being ex-
plored as resources for environmentally sustainable bioethanol production. As a result,
bioethanol production can be accomplished using an increasingly wide array of feedstock
materials. With improved ethanol production technology, it has become possible to pro-
duce ethanol from a greater range of biomass resource materials. Fermentation technology
that allows bioethanol production from a previously untapped biomass resource is often
designated as a new generation. Approaches for biomass production are also grouped
based on factors related to the fermentation conditions. The concentration of water and
sugar in the fermentation media and the use of batch or continuous processes are used
in grouping fermentation technology. Additional techniques can also be applied to the
fermentation media to further optimize ethanol yields. The purpose of this review is to
describe current knowledge of fermentable materials and fermentation technologies used
in bioethanol production. In addition, this review considers various other factors that
influence ethanol yield.

2. Bioethanol Production Processes

Currently, industrial bioethanol production is divided into three generations based on
the type of feedstock used (Figure 1) [7]. The processes involved in all biofuel generations
include: (1) pretreatment, (2) hydrolysis (although not required in the fermentation of
sugar cane), and (3) conversion of sugars to bioethanol via fermentation. Some feedstocks
require pretreatment conditions (i.e., lignocellulosic feedstock and algal biomass) to release
fermentable sugars into the media. Without pretreatment, fermentation progress can be
slowed due to limited availability of fermentable sugars for metabolism. Furthermore,
genetics of feedstocks can contribute to variations in sugar content and influence fermen-
tation ethanol yield [8]. Currently, fourth-generation bioethanol production methods are
being investigated, which utilize genetically engineered organisms to enhance fermentation
efficiency. However, these approaches are not yet implemented at an industrial scale.
First-generation bioethanol is derived through the fermentation of biomass contain-
ing high levels of starch (e.g., wheat, corn) and/or sugar (e.g., sugar cane, sugar beet).
The industrial production of fuel and potable ethanol using first-generation technology is
widely practiced commercially in many countries, although the preferred feedstock varies.
The most common feedstock in the United States is corn [9], while in Canada both corn
and wheat are widely used [10]. In Brazil, sugarcane is the common feedstock [11], and in
Europe, the ethanol industry most commonly uses potatoes, wheat, and sugar beets [12].
These high-quality inputs require little pretreatment to afford relatively high ethanol yields
(Table 1). Production of ethanol by first-generation technology and feedstocks is criticized
for the consumption of crops which might otherwise be used as food for human or feed
for animal consumption [13]. Nevertheless, bioethanol production can offer a means to
process crops into useful products and to recover damaged grain that might otherwise
be wasted [14]. For example, due to the presence of mycotoxins (e.g., deoxynivalenol),
grain infected with Fusarium head blight is potentially toxic to humans or other animals.
However, contaminated grain can be detoxified by yeast-based fermentation followed by
nutrient recovery using insects (e.g., black soldier fly larvae; Hermetia illucens) [15] and
lactic acid bacteria [16]. Approaches that reclaim grain products that would otherwise
be lost can minimize economic loss, especially due to this fungal disease in cereal grains
such as wheat and barley [15,16]. After detoxification, insects and lactic acid bacteria could
potentially be used in producing protein feed supplements for domestic animals. Fermen-
 Fermentation 2021, 7, x FOR PEER REVIEW 3 of 19

Fermentation 2021, 7, 268 3 of 18

grains such as wheat and barley [15,16]. After detoxification, insects and lactic acid bacte-
ria could potentially be used in producing protein feed supplements for domestic animals.
Fermentation
tation byproductsbyproducts fromcrops
from edible edible
arecrops are also
also seen seen as improved
as improved feed products.
feed products. Wet
Wet distillers’
distillers’ grain produced from fermenting cereal grains has a much higher protein
grain produced from fermenting cereal grains has a much higher protein content on a dry content
on a than
basis dry basis than thegrain.
the original original
Wetgrain. Wetgrain
distillers’ distillers’
can begrain can be
blended blended
directly intodirectly
animalinto
feed
animal feed or mixed with distillers’ solubles, another fermentation by-product,
or mixed with distillers’ solubles, another fermentation by-product, and further dried and fur-
to be
ther as
sold dried to be sold asfeed
an inexpensive an inexpensive
for livestock.feed for livestock.

Figure1.1. General
Figure General flowchart
flowchartofofbioethanol
bioethanol production, providing
production, a comparison
providing of the
a comparison ofpre-fermentation processing
the pre-fermentation of feed-of
processing
stocks for the
feedstocks firstfirst
for the three generations
three of bioethanol
generations production.
of bioethanol The blue
production. Thehighlighted area provides
blue highlighted an example
area provides of a value-
an example of a
added process that can enhance the value of bioethanol production.
value-added process that can enhance the value of bioethanol production.

In contrast to the high starch or sugar content found in first-generation feedstocks,

second-generation bioethanol typically utilizes non-edible feedstocks [7], such as lignocel-
lulosic materials and agricultural forest residues (e.g., wood) [13,17]. Although the use of
these feedstocks for ethanol production does not directly compete with food production,
Fermentation 2021, 7, 268 4 of 18

second-generation feedstocks require more advanced technologies and facilities [16] to

process them prior to fermentation [18]. Lignocellulosic biomass sources are predom-
inantly composed of cellulose, hemicellulose, and lignin. These molecules often form
highly recalcitrant structures due to their strong covalent bonds and extensive van der
Waal and hydrogen bonding [19]. This makes lignocellulosic biomass more resistant to
chemical and biological breakdown, and therefore, pretreatment processes must be imple-
mented to disrupt lignocellulose structures prior to beginning biorefinery and fermentation
processes [19]. Typical pretreatments can include physical (e.g., milling, temperature,
ultrasonication), chemical (e.g., acid and alkaline treatments, organic solvent treatments),
physicochemical (e.g., steam or CO2 explosion treatments), or biological (e.g., enzymatic hy-
drolysis) processes. Cellulose, hemicellulose, and lignin content vary among feedstocks [19].
This variability might necessitate different approaches for pretreatments [20]. After suc-
cessful pretreatment, cellulose can be hydrolyzed to sugars and converted to bioethanol
via fermentation [21]. Ethanol yield for second-generation bioethanol feedstocks is also
highly variable, and feedstock dependent (Table 1).
Third-generation bioethanol utilizes algal biomass for ethanol production [22]. Em-
ploying algae as a bioethanol feedstock can be advantageous, as algae can rapidly absorb
carbon dioxide, accumulate high concentrations of lipid and carbohydrates, be easily culti-
vated, and require less land than terrestrial plants [23]. Like second-generation bioethanol,
third-generation bioethanol production also requires pretreatment to disrupt algal cells.
Such treatments can involve chemical (e.g., acid treatments) or physical (e.g., mechanical
forces) pretreatment processes that destroy or disrupt algal cell walls. After pretreatment,
complex carbohydrates are more readily converted to fermentable sugars via enzymatic
hydrolysis, through a process known as saccharification [24]. However, inadequate pre-
treatment and saccharification conditions can result in the formation of side products
(e.g., formic acid, acetic acid, and furanic compounds) [24–27]. This approach is further
complicated by the highly variable composition of neutral sugars, amino sugars, and
uronic acids in different algal species [28]. Pretreatment processes are, therefore, highly
dependent on the algal species used and their composition. In general, large species
of algae (macroalgae), which naturally grow anchored to the seafloor and can be many
meters in length, contain fibrous material which requires extensive physical/chemical
treatments before starches/sugars are released. In contrast, microalgae are much smaller,
often unicellular, and can contain much higher amounts of sugar. However, they cannot
easily be recovered from water like macroalgae, which can lead to significant inputs to
dewater the algal biomass prior to processing [29]. Ethanol yields per gram algal dry
matter are variable, and some have observed lower bioethanol quantities compared to first-
and second-generation bioethanol feedstocks. Although production of third-generation
bioethanol is less significant than the first two, the technology involved is also more recent,
and algal species are being surveyed and engineered to identify and breed more productive
species or mixtures of species. Third-generation bioethanol production may continue to
grow through future improvements to existing technology, or in combination with other
applications such as producing bioethanol from algae also used to treat wastewater.
Technoeconomic evaluations, (e.g., economic benefits, process design, etc.) [30], among
the three generations of biofuel processes demonstrated that first-generation biofuels are
currently the preferred option for commercial biofuel production [31]. Second-generation
biofuel processes are becoming competitive. Further process developments will lower produc-
tion costs (e.g., lower cost of enzymes) and increase coproduct utilization (e.g., electricity) [32].
Furthermore, integrating both first- and second-generation biofuels can maximize ethanol
yields and revenue, and would also require less capital investment to integrate lignocellu-
loses processes into an industrial design [33]. Finally, third-generation biofuels using mi-
croalgae are being considered as the best alternative for biofuel production due to the lower
land requirement and high potential to capture CO2 . However, utilization of these feed-
stocks requires improvements to reduce costs and increase economic sustainability [34].
Fermentation 2021, 7, 268 5 of 18

Currently, fourth-generation bioethanol production methods are in development

and utilize genetically engineered organisms (e.g., yeasts and algae) in combination with
other methods of improving fermentations such as high-yielding biomass (with low lignin
and cellulose contents) [35]. Although fourth-generation methods vary wildly, some
fourth-generation bioethanol production methods capture CO2 emissions throughout the
production process using oxy-fuel combustion (a process in which fossil fuels are burned
with an oxygen-enriched gas mixture instead of air) [36–38]. This produces flue gas mix-
tures comprised primarily of CO2 and H2 O. This method provides an opportunity to
capture CO2 directly by physical compression and cooling (e.g., distillation processes) [38].
Unfortunately, current commercial processes require high energy inputs and are econom-
ically infeasible [35]. Another example of fourth-generation bioethanol production is
electro-fermentation, in which electrical energy is used to help regulate respiration in
genetically engineered algae through the transfer of electrons [36]. These methods are not
currently used by industry and represent a substantial shift away from the more traditional
bioethanol production processes.
Table 1. Approximate ethanol yields from different feedstocks [39–44].

Bioethanol Generation Biomass Source Ethanol Yield (L/t)

First Sugar beet 110 (L/t) [40]
First Sugar cane 70–75 (L/t) [40]
First Cassava 137–180 (L/t) [40]
First Maize 400 (L/t) [40]
First Rice 430 (L/t) [40]
First Wheat 340 (L/t) [40]
Second Corn stover 362–456 (L/t) [39,41]
Second Wheat straw 406 (L/t) [39,41]
Second Sugarcane bagasse 318–500 (L/t) [39,41]
Second Switchgrass 392–457 (L/t) [39]
Second Sorghum 268–380 (L/t) [39,41]
Second Poplar 419–456 (L/t) [39]
Second Agave 347 (L/t) [39]
Second Agave Americana 347 (L/t) [39]
Second Agave tequilana 401 (L/t) [39]
Second Agave tequilana leaves 401 (L/t) [39]
Second Juice from Agave americana leaves 34 (L/t) [39]
Second Juice from Agave tequilana leaves 30 (L/t) [39]
Second Corn grain 470 (L/t) [39]
Second Rice straw 416 (L/t) [39]
Second Cotton gin trash 215 (L/t) [39]
Second Forest thinnings 308 (L/t) [39]
Second Hardwood sawdust 381 (L/t) [39]
Second Mixed paper 439 (L/t) [39]
Third Microalgae 167–501 (L/t) [42] *
Third Brown seaweeds (macroalgae) 12–1128 (L/t) [43] **
Third Seagrass (macroalgae) 747 (L/t) [43] **
Third Green seaweeds (macroalgae) 72–608 (L/t) [43] **
Third Red seaweeds (macroalgae) 12–595 (L/t) [43] **
* Denotes conversion of L/ha to L/tonne assuming 8–10% energy conversion efficiency (taken from
Benedetti et al., 2018) [44]. ** Denotes conversion from g/g to L/t.

3. Very High Gravity, Solid-State, and Submerged/Liquid Fermentation

There are three main strategies for fermentation used in commercial bioethanol pro-
duction: submerged/liquid state fermentation, solid-state fermentation, and very high
gravity fermentation. A single type of feedstock might be fermented using any of those
approaches, however, some strategies are more suited to a particular feedstock than others
based on its properties (Table 2).
Fermentation 2021, 7, 268 6 of 18

Table 2. Comparison of three fermentation strategies.

Submerged/Liquid-State Fermentation Solid-State Fermentation Very High Gravity Fermentation

• Uses liquid medium to
grow microorganisms • Uses solid substrate to
• Requires larger grow microorganisms • Uses increased concentrations of
operational footprint • Smaller vessels sugar substrate to increase final
• Increased usage of water and energy • Less water and energy requirements ethanol concentration
• Better monitoring and ease • Not easy to monitor or in the medium
of handling change parameters • Less water and energy requirements
• Shorter fermentation time • Longer fermentation time
• High waste generation • Reduced waste generation
• High ethanol yield

3.1. Submerged/Liquid State Fermentation

Submerged fermentation refers to processes where the fermentable substrate is sub-
stantially liquefied, and microbes are grown in that liquid substrate. This type of fer-
mentation is commonly used in first-generation bioethanol production [45], as ground
starch/sugar rich materials can be mixed with water and the starchy materials further lique-
fied via cooking and enzymatic hydrolysis. This results in a liquid medium in which sugars
and various nutrients are either dissolved or suspended as particulate solids. Submerged
fermentation is utilized in other bio-industrial processes including enzyme production as
this process can quickly afford a high yield of bioactive metabolites (Table 1). Unfortunately,
this process can have disadvantages including requirements for inputs of energy and wa-
ter, requirements for large volume bioreactors and distillation columns, and generation
of large volumes of waste or low-value coproducts (e.g., thin stillage and wet distillers’
grains). Fortunately, the waste by-product wet distillers’ grains can be centrifuged to
remove the excess thin stillage, the thin stillage can be dried with modest efficiency to
distillers’ solubles, and the solids dried to distillers’ dried grain. These drying processes
lead to three products that are used as feed ingredients: distillers’ solubles, distillers’ dried
grains, and distillers’ dried grain with solubles (the latter being a combination of the former
two products). Thin stillage can also be provided as a water substitute for cattle in nearby
feed lots or be processed via further microbial fermentation to produce a high-quality
protein feed. A benefit of this latter technology is the conversion of low-value glycerol to
the higher-value compound 1,3-propanediol [46,47].

3.2. Solid-State Fermentation

Solid-state fermentation (SSF) is a process in which organisms grow on non-soluble
material or solid substrates in the absence of near absence of free water [48]. Solid-state
fermentation is currently used for a wide range of applications in addition to bioethanol,
including the production of enzymes, antibiotics, bioactive compounds, organic acids, and
biodiesel [49]. The SSF process is affected by many factors including type of microorganism,
substrate used, water activity (to prevent the growth of nuisance organisms), temperature,
aeration, and bioreactor used [50]. The most common organisms used for SSF are filamen-
tous fungi (e.g., Trichoderma and Aspergillus), as solid matrices better simulate the natural
habitat of some fungi [51]. Nevertheless, SSF is also used with single-celled organisms
such as yeast and bacteria [52]. Second-generation bioethanol production often involves
solid-state fermentation of waste material and other feedstocks. The second-generation
bioethanol feedstocks listed in Table 1 are all fermented using SSF technologies, except
for agave.
SSF is frequently used to process large quantities of waste produced by agricultural-
based industries [50], which may have poor nutritive value (e.g., low digestibility, crude
protein, and mineral content) [53]. These residues are often disposed of via burning or
dumping [50], which can lead to greenhouse gas release and other environmental im-
pacts. Many of these substrates contain lignin, cellulose, and hemi-cellulose molecules,
Fermentation 2021, 7, 268 7 of 18

which can be used to produce ethanol when fermented (Table 3). However, due to
the complex lignocellulosic structures, saccharification of these materials to make them
suitable as substrates for fermentation requires significantly more processing than for
starchy materials. Cellulose is derived from linkages of D-glucose subunits which are
linked by β-1,4 glycosidic bonds [54], whereas hemi-cellulose is a polysaccharide com-
posed of D-xylose, D-mannose, D-galactose, D-glucose, L-arabinose, 4-O-methyl-glucuronic,
D -galacturonic, and D -glucuronic acids linked by β-1,4 and sometimes β-1,3 glycosidic
bonds [54]. To make these sugar linkages accessible, the recalcitrant structure of ligno-
cellulosic must be disrupted via mechanical or physiochemical pretreatment processes
(e.g., steam explosion and acid/alkaline treatments). Acid prehydrolysis followed by enzy-
matic hydrolysis is then required to saccharify the substrate. Implementation of these pre-
treatment processes is feedstock dependent as the composition of cellulose, hemi-cellulose,
and lignan depend on the agro-industrial waste used [50].
Table 3. Examples of fermentable agro-industrial residues.

Agricultural Residues Industrial Residues

Field Residues Process Residues
Straw Husks Potato peels
Stalks Seeds Orange peels
Leaves Bagasse Cassava peels

Another difference between submerged fermentation and SSF is related to enzyme

use. Submerged fermentations typically rely on large initial doses of enzymes for sacchari-
fication, whereas SSF processes releases reducing sugars continuously through enzymatic
cellulose hydrolysis. Reducing sugars are fermented to ethanol in a process referred to
as simultaneous saccharification and fermentation, where enzymatic hydrolysis and fer-
mentation occur in a single step, thereby increasing ethanol yields by minimizing product
inhibition and reducing the need for separate saccharification and fermentation reactors.
However, the optimum temperature for enzymatic hydrolysis is typically greater than the
fermentation temperature; thus, to fully incorporate this hybrid method it is important to
identify a temperature range that is compatible with both hydrolysis and fermentation [55].
To achieve simultaneous saccharification and fermentation, a combination of filamentous
and thermotolerant fungi (e.g., Trichoderma and Aspergillus) or bacteria (e.g., Streptomyces) [56]
and yeast (e.g., Saccharomyces cerevisiae) is often utilized [57]. Thermotolerant yeasts and
bacteria are compatible with higher temperatures needed to improve enzymatic hydrol-
ysis [58], which is often the rate-limiting step during the SSF process [59]. Microbial sac-
charification and simultaneous fermentation can reduce the need for expensive enzymes,
although longer incubation times may be required and monitoring the internal tempera-
ture and maintaining the appropriate process conditions can be challenging. Solid-state
fermentation strategies show great promise in utilizing agricultural wastes for bioethanol
production [60], with simultaneous saccharification and fermentation helping to decrease
costs and improve SSF ethanol yields for many feedstocks. Solid-state fermentation has
been accomplished without supplementary nutrients [61,62].
Another hybrid approach is simultaneous saccharification and cofermentation.
This technology primarily involves simultaneous consumption of two different substrates
by some microorganisms [55]. However, this approach is challenging, as many organisms
utilize substrates sequentially [63]. For example, a microorganism grown in the presence
of both xylose and glucose might initially metabolize glucose more readily than xylose and
will only begin consuming xylose when glucose concentrations are depleted. The sequential
depletion of substrates can slow fermentation. Methods to alleviate this phenomenon
include initial acclimatization of the microorganism to low glucose substrate and forcing
the microorganism to utilize both substrates simultaneously [64]. Genetic engineering has
also been investigated to explore this avenue in biofuels production [65].
Fermentation 2021, 7, 268 8 of 18

Nonetheless, sequentially conducting solid-state fermentation for enzyme generation

followed by hydrolysis on a second medium for submerged/liquid state fermentation is
also being explored [66]. Combining these two technologies (SmF and SSF) can result in
synergistic advantages as elaborated in López-Gómez and Venus (2021) [67].

3.3. Very High Gravity Fermentation

Very high gravity (VHG) fermentation is an emerging strategy using high-sugar musts
or feedstock to increase ethanol production while minimizing production costs [68]. In
general, sugar concentrations for ethanol production can be divided into normal gravity
(<180 g/L total sugars), high gravity (180–240 g/L of total sugars), and very high gravity
(≥250 g/L of total sugars) [69,70]. In VHG fermentations, more than 30% of solids are
consumed to achieve high ethanol concentrations [71]. Very high gravity fermentation can
achieve more than 15% (v/v) of ethanol, compared to the average of 10–12% (v/v) that is
observed in most distilleries [72]. The benefits of VHG fermentation include decreased
waste production, energy consumption, and water consumption, resulting in reduced
production costs as well as improved environmental sustainability. However, under VHG
fermentation conditions, yeasts undergo multiple stresses due to increased metals and
sodium ions, nutrient stresses (e.g., nutrient limitations, such as free amino nitrogen and
dissolved oxygen), increased temperatures, acidic conditions, osmotic stress, and increased
ethanol concentrations [72,73]. The increased osmotic pressure exerted on the yeast cells can
result in intracellular ethanol accumulation, leading to decreased production efficiency and
yeast-cell viability as fermentation progresses [72]. Although some yeasts (e.g., S. rouoxii)
are more tolerant to these osmotic stresses, their ability to produce ethanol is lower than
S. cerevisiae [72]. Very high gravity fermentation usually involves feedstocks used in first-
and third-generation bioethanol production and has the greatest potential for high ethanol
yields compared to SmF and SSF (Table 1).

4. Batch, Fed-Batch, and Continuous Fermentation Modes

Another factor affecting fermentation outcomes is the batch type, which includes
batch, fed-batch, and continuous fermentations (Table 4). The optimal batch type for a
fermentation depends on the kinetics of the microorganisms used and the feedstock.
In batch fermentation, the microorganisms are typically inoculated to a fixed volume
of medium in the fermenter. As the nutrients are consumed and the microorganisms
reproduce, by-products accumulate. Once the nutrients are depleted, the fermentation is
complete. As a result of the fixed initial nutrient input and continuous consumption of
nutrients by microorganisms, the culture environment is continuously changing [74]. This
type of fermentation typically produces a standard growth curve consisting of a lag phase,
exponential phase, stationary phase, and death phase. The lag phase is the first major
phase of microbial growth in batch fermentation when the organism begins to adapt to the
new environment. During the exponential phase the organisms reproduce at a constant
rate, resulting in an exponential increase in microbial growth (logarithmic growth phase).
The cell growth rate is often substrate limited and can be due to products missing from the
media (e.g., nutrient imbalance) or high substrate concentrations (e.g., excess sugar) [75],
resulting in prolonged fermentation times and reduced ethanol yields [76]. Following the
exponential phase, the microorganisms will enter the stationary phase where the number of
cells reproducing and those dying reach an equilibrium, due to depletion of nutrients in the
media (e.g., sugar) or accumulation of toxic by-products (e.g., ethanol toxicity) [77]. Once
fermentation completes, a death phase may occur as the density of viable cells decreases.
However, some industries avoid lag phases by pre-growing yeasts in smaller tanks with
favorable conditions, then adding a large innoculum to the main fermentation [78]. This
truncates the exponential phase and can improve fermentation efficiency. Nonetheless,
batch fermentation has the advantages of typically being inexpensive, having low risk
of contamination, and easier sterilization and management of feedstocks than other fer-
mentation types. However, compared to fed-batch and continuous fermentations, batch
Fermentation 2021, 7, 268 9 of 18

fermentations exhibit lower cell density [74], as nutrients are not supplemented during the
exponential growth phase. There is also increased downtime due to frequent cleaning and
sterilization of the vessels between subsequent fermentation batches. Batch fermentation is
most used in long-term, small-scale, or solid-state fermentation processes [75].

Table 4. Comparison between batch, fed-batch, and continuous fermentation under a submerged/liquid state [74,79].

Batch Fed-Batch Continuous

Microorganisms are provided with a Media is inoculated with microorganisms Fresh media is continuously added to the
fixed volume of medium (nutrients and which then grow under a batch regime fermenter, replacing the
other ingredients). for a certain amount of time, then consumed nutrients.
Culture environment is consistently nutrients are added incrementally Ethanol, used media, and toxic
changing as nutrients are consumed. throughout the fermentation. metabolites are continuously removed.
Advantages: Advantages: Advantages:

• Maintenance of maximum viable • Less downtime for vessel cleaning

• Low cost cell concentration • Increased productivity
• Low risk of contamination • Extended lifespan of cells • Lower cost
• Less control required • Higher ethanol accumulation • Higher degree of control
• Easier sterilization • By-product accumulation is limited • Ability to automate, more
• Control of factors (e.g., pH, cost-efficient and less sensitive to
temperature, dissolved oxygen) human error.
Disadvantages: Disadvantages: Disadvantages:
• Less control for
non-growth-related products
• Lower cell densities, • Cell aggregation can prevent
• Increased costs for process control optimum steady-state growth
ethanol production • Longer downtime between batches
• Longer downtime between batches • Long growth periods can increase
due to cleaning, vessel setup, risk of contamination
due to cleaning, vessel setup, and and sterilization
sterilization • Can be difficult to maintain
filamentous organisms due to
viscosity and heterogeneity of
the medium

Fed-batch fermentation is like batch fermentations, except nutrients are incrementally

added to the fermenter throughout the fermentation [74]. The consistent addition of nu-
trients results in increased cell density during the exponential phase and thus enhances
product yields. For example, continuous supply of sugars to yeast cells in the stationary
phase can maximize ethanol yield. However, the maximum working volume of the fermen-
tation vessel can limit the amount of fresh media/nutrient input. Yeast alcohol production
is maintained by nutrient additions, which also reduce the risk of overflow metabolism or
the risk of excreting metabolic by-products that could otherwise be used for catabolism or
anabolism [77]. This type of fermentation is exceptionally useful when the desired product
is correlated with microbial growth, such as bioethanol [74].
Finally, during continuous fermentation, fresh media/nutrients are continuously
added to the fermenter at the same rate as ethanol, by-products, and toxic metabolites are
removed from the culture [74,80]. During these processes, yeast is often recovered and
returned to the fermentation vessel. If the medium is continuously fed at a suitable rate,
a steady state is eventually achieved in the fermentation broth [80]. As a constant volume
is achieved in continuous fermentations, the maximum working volume of the bioreactor
does not limit the amount of fresh medium which can be added to the culture over the
duration of the fermentation [74], unlike in fed-batch fermentations. Cultures in a steady
state can last for extended periods of time (up to months) [74], thus reducing equipment
downtime between batches, and improving economic yields by keeping the culture at an
ideal state for ethanol production. However, due to the long fermentation time, continuous
fermentation methods are prone to contamination and downstream processing can be
difficult. Traditional methods of distilling ethanol from fermented media through heating
Fermentation 2021, 7, 268 10 of 18

would result in the destruction of the microbial culture, so methods such as settling and/or
filtering of yeast from the product stream before distillation are employed [75].

5. Yeast Stress
Most yeasts can convert a range of hexose sugars to ethanol via glycolysis. However,
Saccharomyces cerevisiae is by far the most used yeast organism for alcoholic fermentation
due to its robustness and tolerances. S. cerevisiae has several advantages over other yeasts as
it is a facultative anaerobe capable of growing under both aerobic and anaerobic conditions
in the presence of glucose [81] and is tolerant of elevated ethanol concentrations [82]. Under
anaerobic conditions, S. cerevisiae will produce acetaldehyde, which is further reduced
to ethanol [82].
During inoculation and fermentation, yeast cells are subjected to several stressors that
can affect bioethanol yields, including biological (e.g., cellular ageing, microbial competi-
tion), chemical (e.g., toxicity from ethanol and its metabolites, pH), and physical stressors
(e.g., temperature shock, osmotic pressure) [83]. Stress can result in increased mutations,
microbial contamination, altered yeast flocculation, increased glycerol production, de-
creased ethanol production, and production of undesired compounds (e.g., flavor and
aromatic compounds in fermented beverages) [84,85]. Poor activity and declines in yeast
viability from stress can also cause stuck or sluggish fermentations. Fortunately, several
methods have been developed to reduce these stresses including increasing fermentation
temperature and pitching rate [86–88], nutritional supplementation [89–92], using mutant
yeast strains [93], immobilizing yeast [94–98], and enhancing aeration efficiency [90,99].
Fermentation success is also influenced by various additional factors, including nutrition
imbalances (e.g., nitrogen, vitamins, mineral deficiencies), medium composition (e.g., sugar
concentration), and inoculum size.
Biotic stress factors (e.g., microbial contamination) can also affect fermentation effi-
ciency. These factors primarily involve the presence of contaminating microorganisms,
such as lactic acid bacteria (LAB) [100]. Lactic acid bacteria can not only compete to utilize
available sugar, but also produce lactic acid, and other metabolites that can suppress fermen-
tation [83]. These LAB are also capable of producing naturally antimicrobial compounds
called bacteriocins. Bacteriocins can suppress the growth of other bacteria by disrupting
transmembrane potential and forming pores in the membranes of sensitive cells [101]. This
can provide LAB with competitive advantages against other bacterial organisms. There
are also risks associated with competing yeasts producing toxins (e.g., ionophore-acting
compounds) [83], which can contaminate the fermentation broth. Microbial contamination
in an industrial fermentation can be highly problematic requiring extended shutdown of
facility operations for cleaning and sterilization before the next fermentation.
Yeasts have developed various mechanisms that help them adapt to chemical and
physical stresses. For example, in response to temperature stress yeast cells will produce
the disaccharide trehalose to help stabilize their plasma membrane [84,102]. In high-sugar
or -salt environments, yeasts will produce glycerol as an osmoprotectant, to reduce osmotic
stress and protect the cells against lysis [103–105]. Glycerol is also produced to maintain
the balance between the NAD+ /NADH ratio during cell growth [106]. Production of
these metabolites can reduce ethanol synthesis efficiency, as more time is required for
acclimatization to the fermentation media. Therefore, minimizing the acclimation period,
by providing optimal growth media, can maximize ethanol yield [107].
The composition of the media and nutrients (e.g., concentration and type of sugars) can
also influence fermentation efficiency. Saccharomyces cerevisiae is more effective at using glu-
cose than fructose [108–110]. The presence of sugars that are slowly metabolized can affect
the fermentation ethanol yield [111]. Accumulation of fructose can result in stuck or slug-
gish fermentations. Problems with fructose concentrations are more common with sugar
cane and fruit-based feedstocks, such as in wine fermentation [112]. To address stuck fer-
mentations, reinoculation of non-Saccharomyces yeast [113,114] (e.g., Zygosaccharomyces bailii)
Fermentation 2021, 7, 268 11 of 18

capable of utilizing fructose [115,116] and tolerating elevated ethanol concentrations is

typically employed [117].
As yeast consume medium nutrients, the concentration of ethanol increases. This
increase in ethanol can result in physiological impairment. In the presence of excess ethanol
(>10–20% v/v) [83,118–120], yeast can exhibit reduced cell viability and growth, such as a
decrease in cell volume [121–123]. There can also be effects on yeast metabolism (e.g., stress-
response proteins, lowered protein levels and denaturation) [124–128], cell structure, and
membrane function (e.g., inhibition of endocytosis, loss of electrochemical gradients) [128–134].
Ethanol toxicity to yeast is primarily due to cell membrane damage [83]. However, main-
taining an ion balance (e.g., magnesium and potassium) can provide the membrane with
protective effects from ethanol toxicity and temperature changes [135–139].
Stress in yeast can be mitigated via physiological and genetic strategies. These can in-
clude maintaining nutrient availability and balance during fermentation through adaptive
evolution or genetic modification [85,140]. For example, temperature and ethanol stress
resistance in yeast can be enhanced by prolonged serial culture at elevated temperatures
or with higher concentrations of ethanol [141]. Enhancing ethanol tolerance in yeast via
genetic modification can be more difficult, as stress tolerant phenotypes are influenced
by more than one gene [142], and the genetic background of S. cerevisiae can be complex.
Yeasts can be aneuploids or polyploids, making genetic strategies for improvement chal-
lenging [143]. However, gene editing (e.g., clustered regularly interspaced palindrome
repeats, CRISPR, and CRISPR-associated protein-9 nuclease, cas9) [144] and recombinant
DNA techniques [85,142,145] can be utilized to circumvent these constraints and lead to
greatly improve yeast strains. In fact, CRISPR-cas9 has successfully been utilized to in-
crease yeast tolerance to ethanol [146], acetic acid [147,148], and temperature changes [149].
A commercially available strain of genetically modified S. cerevisiae has also been devel-
oped to secrete a heterologous glucoamylase that enables starch saccharification in SSF
processes. This reduces the dependency on exogenous enzymes, while simultaneously
enhancing the rate of fermentable sugar release for yeast metabolism [150]. Another genetic
approach has been to attempt to construct new S. cerevisiae hybrids with improved stress
tolerances (e.g., acetic acid tolerance) through hybridization, protoplast fusion, rare mating,
and mutagenesis [151].
Nutrient and growth factor imbalances are also factors that affect fermentation effi-
ciency and can result in stuck or sluggish fermentations. For example, S. cerevisiae requires
oxygen for the biosynthesis of key membrane constituents such as sterols (e.g., ergosterol)
and unsaturated fatty acids (e.g., oleic acid) [142], and help with stress tolerance. There-
fore, lack of oxygen can reduce the ability for yeast to synthesize membrane components,
reducing inoculum efficiency and leading to unsuccessful or incomplete fermentations.
Other typical imbalances that can occur include deficiencies in free amino nitrogen,
vitamins, and minerals (e.g., zinc or magnesium). For example, it has been reported that
insufficient free amino nitrogen (<150 mg/L) [152] and trace metals (e.g., zinc <0.1 ppm)
can result in stuck fermentations [153]. This is because the terminal enzyme of fermentation,
alcohol dehydrogenase, is a zinc-dependent enzyme [142]. This enzyme is responsible for
reducing acetaldehyde to ethanol during glucose fermentation [154]. The bioavailability of
these trace metals can be affected by the feedstocks’ physicochemical properties, sometimes
leading to precipitation, chelation, or absorption within the medium [142].
In addition, magnesium is another key element required for efficient fermentation. De-
privation of this essential metal can result in adverse cellular physiological effects (e.g., loss
of protein conformation) [155]. This divalent metal is responsible for the activation of
several enzymes involved in metabolic bioenergetic and biomolecular pathways (e.g., DNA
duplication) [156]. Magnesium is also required in the maintenance of cellular structural
integrity, yeast functionality, heavy-metal detoxification, and stress protection [122,157].
During fermentation, magnesium ions can improve cell viability by promoting tolerances
to dehydration [138], elevated ethanol conditions [136], and heat shock [136,158], during
the exponential and stationary growth phases [156]. These tolerances result from the re-
Fermentation 2021, 7, 268 12 of 18

pression of stress-protein synthesis [158]. Increased biomass and ethanol yields have also
been observed in the fermentation of lignocellulosic material in the presence of Mg2+ [159].
Although Mg2+ is an essential factor for yeast performance, it has also been observed to be
an antagonist for Ca2+ and can influence and destabilize calcium complexes [160–162].
Other trace elements that are important for yeast physiology include Ba2+ , Fe3+ , Co2+ ,
Mo , Ni2+ , and Cu2+ [156]. These trace elements are often required in small amounts
2+

(<10 mM) and are essential for the activation and modulation of several metabolic pro-
cesses involved in yeast performance and survival [156]. Altogether, free amino nitrogen,
minerals and trace metal elements, vitamins, and accessory growth factors are required for
optimizing S. cerevisiae fermentation for ethanol production [142,156].

6. Conclusions
As the global demand for energy increases, bioethanol produced from renewable
feedstock is a valuable and eco-friendly alternative to non-renewable fuels. However, with
growing concerns over the global food supply, lignocellulosic non-edible biomass (second-
generation bioethanol) and algal sources (third-generation bioethanol) are increasingly
attractive feedstocks for bioethanol production. Pretreatment conditions are required for
second-generation and third-generation feedstock to disrupt the recalcitrant lignocellulosic
structure and algal cell wall, to make the fermentable sugars accessible. Fermentation
efficiency and bioethanol yields are dependent on the feedstock, cultivar, and organism
used. Biotic (e.g., microbial contamination) and abiotic factors (e.g., nutrient, trace metal,
and vitamin deficiencies) must also be addressed to ensure optimum fermentation rate
and extent. Different modes of fermentation can be utilized to address some of these
concerns (e.g., fed-batch and continuous fermentation modes) and help alleviate yeast
stress. Furthermore, supplemental additions (e.g., Mg2+ and other micronutrients) and
adaptive responses can increase stress tolerance (e.g., heat shock and ethanol shock) on
yeast organisms and improve fermentation performance. Altogether, it is important for
industrial bioethanol producers to investigate requisite pretreatment conditions when
determining a feedstock candidate, incorporate the different fermentation technological
designs, and determine potential adversities that may occur during fermentation. When
taken together, these steps optimize fermentation performance and maximize ethanol
yield. Technoeconomic aspects should also be evaluated to investigate the feasibility, and
economic impacts of implementing these technologies in the future production of biofuels,
especially in analyzing and promoting the use of third-generation biofuels.

Author Contributions: Conceptualization, T.J.T.; writing—original draft, T.J.T.; writing—review and

editing, T.J.T., D.J.W. and M.J.T.R.; supervision, M.J.T.R.; funding acquisition, M.J.T.R. All authors
have read and agreed to the published version of the manuscript.
Funding: This work was supported by the Saskatchewan Agricultural Development Fund (20190155,
20190154, 20180281, 20180248, 20180255, 20170133); National Sciences and Engineering Research
Council of Canada Discovery Grant (RGPIN-2018-06631); and Mitacs (IT19122, IT16156).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: Martin J. T. Reaney is the founder of, and has an equity interest in, Prairie Tide
Diversified Inc. (PTD, Saskatoon, SK, Canada: previous company name is Prairie Tide Chemicals Inc.).

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