Recent evolutionary trends in the production of biofuels

Abstract

In recent years considerable attention has been focused on lowering biofuel costs, greenhouse
emission emissions, and land and water resource needs, and on improving compatibility with fuel
distribution systems and vehicle engines. Fossil fuels as energy sources are non-renewable sources
whose demand is increasing day-by-day and their supply is decreasing. Thus, Biofuels can be the best
alternative. Biofuel converts inorganic matter into organic. The necessity for long-term energy
security has increased the public and scientific attention on the production of biofuels. A biofuel is
safer, less harmful to the environment, less expensive. Generally, they are classified into four
generations based on feedstock. First-generation biofuels utilize oil biomass which sparked
controversy because it competes with global food needs. Second-generation biofuels use non-edible
(lignocellulose) biomass but still, it does not solve the problem of greenhouse gases. Third-generation
biofuels use microorganisms as feedstock, while Fourth-generation biofuel focuses on modifying
these microorganisms genetically to achieve preferable hydrogen to carbon (HC) yield to minimize
the carbon footprint. These last two generations of biofuel are still in the early development stage,
they require genetic modification for the more compatible in yielding a good amount of green diesel.
The aim to provide an overview of four generations with the latest developments. This research
concludes that the present production methods of biofuel in the first and second generations will
sooner or later fail to satisfy the increasing demand for biofuel. Therefore, development efforts are
necessary for third and fourth generations, specifically genetic modification of algae or bacterial
strains and co cultivation of various microorganisms.

Content

1. Introduction
2. Generations of biofuels
2.1. First generation
2.1.1.First generation bioethanol
2.1.2.First generation biodiesel
2.1.3.First generation bio ethers
2.2. Second generation
2.2.1.Second generation biodiesel
2.2.2.Second generation bioethanol
2.2.3.Second generation butanol
2.3. Third generation
2.3.1.Third generation bioethanol
2.3.2.Third generation biodiesel
2.4. Fourth generation
2.4.1.Cyanobacteria
2.4.2.Eukaryotic microalgae
3. Conclusions
4. References
 1. Introduction

The demand of energy is increasing as time moves but, due to the depletion of the fossil fuels which
are used primarily as energy sources because of their high abundance, high energy value, and cheap
prices as source of energy the world wants a better alternative. As of now we can say biofuels can be
the best alternative. Biofuel, any fuel that is derived from biomass that is, plant or algae material or
animal waste. Since such feedstock material can be replenished readily, biofuel is considered to be a
source of renewable energy, unlike fossil fuels such as petroleum, coal, and natural gas. [1] Fossil
fuels also come from biomass but they take many years to produce. So, Biofuel is commonly
advocated as a cost-effective and environmentally benign alternative to petroleum and other fossil
fuels, particularly within the context of rising petroleum prices and increased concern over the
contributions made by fossil fuels to global warming.[2] Biofuels are developed as a substitute for
petroleum because of their nontoxic, sulphur-free, biodegradable nature, originating from the
renewable sources. [3]

There are four types of biofuels: 1 st, 2nd, 3rd, and 4th generation biofuels. They are classified based on
the feedstock, technological progress involved, and the drawbacks as renewable sources of energy.
First generation biofuels also called conventional biofuels, use edible sugar, starch or vegetable oil.
Oils are extracted from the biomass and converted into bioethanol or biodiesel or bioether through
well-understood technologies and processes, like distillation, fermentation, and transesterification.
These processes have been used for several decades in many uses, such as production of alcohol. But
the drawback of first-generation biofuels is that they are available from biomass that's also a food
source. This presents a drag when there's not enough food to feed everyone. Second-generation
biofuels made from lignocellulose are often portrayed as a promising alternative to using food crops
as feedstock for biofuel production [4] Biomass of trees as second-generation fuels is claimed to
contain more carbohydrate and therefore the staple for biofuel than that of food crops. Cellulosic
ethanol is taken from non-food crops or inedible waste products that have less impact on food like
switch gases, rice hulls, paper pulp, wood chips etc., But Second-generation biofuels use non-edible
(lignocellulose) biomass but still, it cannot solve the problem of greenhouse gases.
Fig.1. First and second generation biofuels [6]

The first-generation biofuels from agricultural yields are currently not considered as a sustainable
energy source. Similarly, the second-generation biofuels produced from non-food feedstock are not
economically viable for its high processing cost. In contrast, the third-generation biofuels from
microalgae are considered as the feasible and sustainable solution for future energy demand by
overcoming the shortcomings of the previous generations of biofuels. [5] Nowadays researchers are
mainly concentrating on the development of 3rd generation biomass from microalgae and at the
present, it's thought to be a plausible option for a sustainable energy source for biofuel production by
overcoming the weaknesses of both first and second generation biofuel sources. Third-generation
biofuels present the simplest possibility for alternative fuel because they don’t compete with food.
But, there exist some challenges in making it beneficial.

Fourth-generation biofuel uses recombinant microorganisms such as microalgae, yeast, fungi and
cyanobacteria to enhance the production of biofuel. Research has been administered on genetic
modification and other technologies that aim to extend the productivity of algae strains, only a couple
of them affect the legislative limitations imposed on exploiting and processing genetically modified
algae.

Fig. 2. Third and fourth generation biofuels [7].  

2. Generations of biofuels

2.1 First generation

The most well-known first-generation biofuel is ethanol made by fermenting sugar extracted from
sugar cane or sugar beets, or sugar extracted from starch contained in maize kernels or other starch-
laden crops. Similar processing, but with different fermentation organisms, can yield another alcohol,
butane. Commercialization efforts for butane are on-going [8], while ethanol is already a well-
established industry. Global production of first-generation bio-ethanol in 2006 was about 51 billion
litres [9], with Brazil (from sugar cane) and the United States (from maize) each contributing about 18
billion litres, or 35 per cent of the entire . China and India contributed 11 per cent to global
ethanol production in 2006, and production levels were much lower in other countries (Figure 3), with
feed stocks that include cane, corn, and a number of other sugar or starch crops (sugar beets, wheat,
potatoes). Many countries are expanding or contemplating expanding their first-generation ethanol
production, with Brazil and therefore the us having far and away the most important expansion plans.
Ethanol production is expected to more than double between now and 2013 in Brazil [10], and
production capacity in the United States will double from the 2006 level once new plants currently
under construction are completed [9].
From the attitude of petroleum substitution or carbon emissions mitigation efficiencies, the potential
for many first-generation biofuels is restricted. This is illustrated in Figure 4, which shows that the
United States is projected to produce about 34 billion litres of ethanol in 2007 by using 27 per cent its
corn crop [11]. On an energy basis, this ethanol will still account for fewer than 4 per cent of us
gasoline plus ethanol consumption in 2007. In addition, the many amount of fuel wont to produce this
ethanol substantially offsets the carbon emissions reductions from photosynthetic uptake of carbon by
the corn plants.

fig., 3 Global fuel production of country

2.1.1 First generation bioethanol

Bioethanol fuel is liquid ethyl alcohol (C2H5OH or EtOH) produced from feed stocks such as wheat,
sugar beet, and corn through fermentation. Its primary application is in motor vehicles. It can be used
as a transportation fuel in its pure form or by blending it with gasoline in traditional combustion
engines especially in flex-fuel vehicles. It is most commonly blended with gasoline at a low per cent
(10% bioethanol) which known as E10.It can be also used as a feedstock to produce ethyl tertiary
butyl ether (ETBE) which is blended with gasoline to increase its oxygen content for pollution control
[13]. Historically, bioethanol was utilized industrially in Germany and France as early as 1894 [14].
In 1925 Brazil started to use it as a transportation fuel. Its use as fuel was common in Europe and the
United States until the early 1900s. However, due to its high production cost, it was ignored especially
after World War II until the oil crisis of the 1970s [15]. In the last three decades, the use of bioethanol
has gotten more attention as an alternative transportation fuel. Several countries such as Brazil and the
USA have long promoted domestic production of bioethanol. Shifting to the use of bioethanol as a
green energy source could decrease CO2 concentrations in the atmosphere in two ways. First it
reduces dependence on fossil fuels, and second it consumed CO2 in the atmosphere to grow the
feedstock crops. Global bioethanol production reached about 93 billion litres in 2014 which is
approximately four times more than its production a decade earlier [16]. A recent investigation
showed that most bioethanol production is based on sugarcane and maize followed by wheat, sugar-
beet, and sorghum. These crops could have fed 200 million people [17], thus concerns arose about
competition of fuel with food needs. In term of edible biomass, sugarcane is the highest crop used in
bioethanol production and requires less water than maize and wheat [17]. The USA is at the forefront
of the bioethanol market with about 47% of the global bioethanol production [18]. To produce high
bioethanol equality from crops with reference to greenhouse gas (GHG) benefits, two important
requirements should be considered. These are the wise choice of croplands and fertilization strategies
to avoid increasing the carbon concentration in/on ground and minimize nitrous oxide emissions [19,
20]. In addition, by-product production should be emphasized and utilized efficiently to maximize the
cost effectiveness. Several carbohydrate-containing crops have been utilized as feed stocks for ethanol
production using a fermentation process. These feed stocks are classified in two major categories: (a)
Sugar containing crops, such as palm juice, sugar cane, beet root, wheat, fruits. (b) Starch-containing
crops including grain, such as wheat, barely, sweet sorgum, rice, and corn. The direct conversion of
starch to ethanol cannot be done using conventional fermentation technologies due to the long chain
polymer structure of glucose [21]. Therefore, a practical approach involves breaking down the
macromolecular structure first into simpler and smaller glucose molecules. In order to do this, starch
feed stocks are converted to a mash typically containing 15–20% starch. The process involves starch
grinding, mixing with water, and then cooking at or above its boiling point. The process also requires
using two enzymes. The first enzyme, amylase, breaks down starch molecules to short chains and
releases dextrin and oligosaccharides. These components are hydrolysed in a process known as
saccharification which uses enzymes such as pullulanase and glucoamylase. This process converts all
dextrans to glucose, maltose and isomatose. The next step is cooling the mash to 30 C and yeast is
added for fermentation [22]. Bioethanol production from corn can be classified into wet & dry mill
processes [23]. The wet mill ethanol process has usually a higher production capacity than the dry
process and produces some valuable coproducts such as nutraceuticals, pharmaceuticals, organic acids
and solvent [24]. In addition to ethanol, the dry milling process produces distillers’ dried grains and
soluble (DDGS) which is an excellent livestock feed because it contains protein, fats and
carbohydrates. On the other hand, in addition to ethanol the wet milling process produces corn oil, and
two types of animal feed which are corn gluten meal (CGM) and corn gluten feed (CGF) [25].

2.2.2. First generation biodiesel

Diesel fuel, which has a chemical formula range between C10H20 to C15H28 with an average
molecular weight 168 (amu), is an important liquid petroleum fuel that is widely used in
transportation. Several technologies have been well established to produce biodiesel (fatty acid esters)
from different feedstocks. Biodiesel fuel depends mainly on oil crops and 75% of its production cost
is due to the feedstock production cost [26]. More than 350 oil-bearing crops, both edible and non-
edible, have been suggested as promising feedstock for biodiesel manufacturing [29]. The most
common food crop sources are rapeseed, soybean, palm, sunflower, peanut, safflower, corn, rice bran,
coconut, olive, castor, milkweed seed, and linseed. Jatropha curcas, Pongamia glabra, Madhuca
indica, Salvadora oleoides, cotton seed oil, Tobacco, Calophyllum Eruca Sativa Gars, inophyllum,
terebinth, rubber seed, desert date, Jojoba, neem oil, leather pre-fleshings, apricot seed, Pistacia
chinensis Bunge Seed, sal (Shorea robusta) and fish oil, Moringa oleifera and croton megalocarpus
are common non-edible oil sources. Several technologies have been established to produce high
quality biodiesel such as direct use and blending, pyrolysis of vegetable oil micro-emulsions, and
transesterification. The direct use of vegetable oils blended with diesel fuel is used to overcome the
drawbacks of using the high viscous pure vegetable oil. These drawbacks include coking and trumpet
formation on the engine injectors after long-term use, as well as carbon deposits, thickening and
gelling of the lubricant, and oil ring sticking [26]. Another common solution is decreasing the
vegetable oil’s viscosity by preheating it and that also improve the atomisation and mixing process to
achieve better combustion [28]. Micro-emulsification can be used to solve the high viscosity issue of
vegetable oils. Micro-emulsion is defined as clear thermodynamically stable isotropic liquid mixtures
of oil with dimensions range between 1–150 nm created spontaneously from two normally immiscible
fluids and one or more ionic or non-ionic amphiphiles [29]. Microemulsions consist of three phases,
which are surfactant, oil, and aqueous phase. Methanol and ethanol are the common solvents used in
this process. The standard viscosity limitation for diesel engines can be achieved by all micro-
emulsions with butanol, hexanol and octanol [30, 31]. The pyrolysis process is also used to enhance
the quality of biodiesel by thermal and catalytic means. In pyrolysis a conversion of one substance
into another can be achieved by using heat or with the assistance of a catalyst in the absence of
oxygen. Compared with other cracking processes, pyrolysis is very simple, waste-free, pollution free,
and very efficient [32]. Transesterification of oils (triglycerides) with alcohol is the most developed
and promising method of biodiesel production which produces glycerine as a by-product. Figure 1
which is adapted from Ghazali et al. shows the transesterification reaction of triglycerides [27].
Transesterification, or alcoholysis, is replacing alcohol from an ester with another alcohol in a similar
way to hydrolysis, but using alcohol instead of water [33]. Figure 2 shows the process diagram of
biodiesel produced by the transesterification reaction using alkali catalyst [34]. Catalysts usually
speed the completion of the transesterification reaction. Several operating variables such as reaction
temperature, time, and pressure, as well as the molar ratios of alcohol to oil, catalyst concentration
and type, mixing intensity and kind of feedstock can affect the transesterification process [35].
Albayati and Doyle recently reported using the incipient wetness impregnation method to
manufacture nonporous catalyst, SBA-15, from encapsulated of alkali metals and their hydroxides to
produce biodiesel from sunflower oil [36]. Specifically, they used Na, NaOH, Li, and LiOH to
prepare the catalyst which showed promising results with yields of fatty acid methyl ester (FAME) in
the range between 96 to 99% at moderate process conditions (1 atm and 65 C). The catalyst also
showed high production stability and easy recovery through seven production cycles under the same
conditions with better fuel properties than those of fossil fuels. Also, Doyle et al. reported that using
zeolite Y, with a Si/Al ratio 3.1 for the biodiesel production from oleic acid esterification with ethanol
increased the oleic acid conversion to 85% comparing with 76% using commercial catalyst of HY
zeolite [39]. Later, the same research group reported that using FAU-type zeolites showed similar
oleic acid conversion to that reported for commercial HY zeolite [38], while addition of Co-Ni-Pt to
the FAU-type zeolites enhanced its efficiency in biodiesel production in the same process to achieve
93% and 89% for batch and continuous reactors, respectively [39].

FIGURE 4 Transestrification reaction of triglycerides with alcohol.

FIGURE 5 A schematic diagram of biodiesel production according to the transesterification process.

2.2.3 First generation bio ethers

Bio ethers (also known as fuel ethers) are used to enhance the octane number of fuels. They can
replace petro-ethers and improve engine performance [40]. Furthermore, bio ethers can greatly reduce
engine wear and toxic exhaust emissions [40]. They are produced by the reaction of bioethanol with
iso-olefins, such as isobutylene. The usual source of bio ethers is wheat and sugar beet [41]. However,
their main drawback is low energy density. There are six ether additives that are commonly used to
enhance transportation fuel quality: dimethyl ether (DME), methyl tertiary butyl ether (MTBE),
diethyl ether (DEE), ter-amyl methyl ether, ethyl ter-butyl ether (ETBE), and tar-amyl ethyl ether
(TAEE) [42]. Ethers have been used in Europe since the 1970s to replace highly toxic compounds
such as lead especially MTBE and ETBE. However, bio ethers are no longer used in the USA as fuel
additives. [43]

1.1. Second generation

In this generation, a more sustainable protocol is employed to supply biofuels. internet carbon
(emitted–consumed) from combusting second-generation biofuels is neutral or maybe negative. The
feedstock is lignocellulosic material which include the cheap and abundant nonedible biomass
available from plants [84]. The costeffectiveness of this generation of biofuels still needs development
because there are severaltechnical barriers thatneed to be overcome [85]. the utilization of waste plant
biomass has attracted researchers for a wide variety ofuses suchas feedstock to generateheat and
electricity by direct burning [91,86] or as a staple for wastewater treatments [87]. However, utilizing
it as a cheap source of biofuel is very attractive [91]. a good sort of abandoned materials are often
used as biofuel feedstock like agriculture waste, poplar trees, willow and eucalyptus, miscanthus,
switchgrass, reed birdseed grass , and wood and that they mostly contains plant cell walls whose
primary components is polysaccharides (75%) [89,90]. These polysaccharides have a high sugar
content whichis preferred for biofuel production. However, agricultural by-products can provide only
a limited proportion of the increased demand for biofuels [91].
Second generation biodiesel
Second generation bioethanol Bioethanol are often produced from lignocellulosic biomass through
hydrolysis and subsequent fermentation. It also can be produced by thermochemical processes which
include gasification followed by either fermentation or a catalyzed reaction [92]. However, these
processes are complicated thanks to (1) the problem of biomass breakdown, (2) the discharge of
various sorts of sugars after hemi cellulose and cellulose polymers breakdown and therefore the got to
ferment these sugars with suitable organisms which may require gene-splicing , and (3) the value of
collection and storage of rarity lignocellosic feedstocks [93]. There are four main operational steps
within the lignocellosic con version process to ethanol: (1) pretreatment, (2) hydrolysis, (3)
fermentation, and (4) product separation/ distillation [94]. The hydrolysis step increases the
complexity of the fermentation of sugar which is released from the cellulosic a part of the biomass,
and fermentation converts these sugars to bioethanol. to market the hydrolysis step, a pretreatment
step is required that softens the biomass and breaks down its cell structures. An efficient pretreat ment
must meet the subsequent standards: (1) enhance the forma tion of sugars by hydrolysis, (2) avoid the
degradation or loss of carbohydrate; (3) avoid the formation of undesired by-products that reduce the
hydrolysis and fermentation process efficiencies, and (4) be economically feasible [95].
Second generation bioethanol
Second generation biodiesel Several sorts of second-generation feedstocks are often utilized to supply
biodiesel like energy crops, agricultural remains, and wood residual wastage. the foremost common
energy crops for this purpose are Jatropha, candlenut , animal oil , Rubber tree Madhuca longifolia,
tobacco seed, sea mango, and jojoba oil. additionally , waste from cooking oils, non-edible oil crops,
restaurant grease, tallow , animal fats, and pork lard also can be utilized as biodiesel feedstocks [96].
Animal fats are preferable over first generation feedstocks thanks to properties like higher-octane
numbers, non-corrosiveness, lack of waste and sustainability. However, the most drawback of this
generation of feedstocks is that the lack of active technologies for the commercial exploitation of
waste generated by biodiesel production. Furthermore, most ani mal fats possess a high concentration
of saturated fatty acids, which increases the transesterification complexity [98]. the most limitation of
biodiesel is its comparatively low performance in cold temperatures which hinders their ability to
completely replace petroleum transport fuels [97]. Furthermore, bio-safety issues can present in cases
of contaminated animal feedstocks [98].
Second generation butanol
Butanol alcohol (C4H10O) consists mainly of hydrogen and car bon, so it are often easily blended
with gasoline and other hydrocar bon products [99]. Butanol has more heat than ethanol, which
increases the harvestable energy gains (around 25%) [100,101]. The gross heat value of butanol is
110,000 BTU per gallon which is closer thereto of diesel oil (115,000–138,700 BTU per gallon) .
Butanol is safer to handle than gasoline and ethanol thanks to its low Reid Value of 0.33 psi. The Reid
Value may be a measure ment indicator of a fluid’s rate of evaporation, and it's values of 8–15 and a
couple of .3 psi, for gasoline and ethanol, respectively [102]. one more reason why butanol is taken
into account safer than ethanol and gasoline is its low production of volatile compound (VOC), which
is additionally helps to its low evaporation rate. additionally , the corrosiveness of butanol is a smaller
amount than that of ethanol which helps with shipping and distribution it through existing pipelines
and filling stations.
Third Generation Biofuels

Due to Limitations of – 1st and 2nd generation such as highmaintaining cost but low return, these
long process technique has led to the basement for 3rd generation . The feedstocks for third
generation biofuels are photosynthetic microorganisms like algae, micro algae and cynobacteria. The
main benefit of algae is that it is having a rapid growth, Low area requirements and tolerance of algae
to harsh conditions makes them a best fit for biofuel production and algae have the capability to
mitigate CO2(Liu et al., 2017; Schenk et al., 2008)[44].Algae can grow in almost all types of water
like saline water, Fresh water, even in industrial waste waters. In the case of growth and oil content,
the growth rate of algae is approximately 20- 30 times faster than food yielding crops and the oil
content of algae is around 30 times more than the conventional first and second generation feed
stocks. The algae residue after oil extraction can be used as fertilizers or as fish feed in fish. Algal
based biofuel source is completely biodegradable and virtually Sulphur free, the oil quality is better.
(Ullah et al., 2015)[45],[46].

Algae based biofuels

3.1 Bioethanol potentials of algae
The world population is increasing every year, thus the demand of energy is increasing day by
day[59].A demand for bioethanol as a transportation fuel is on the rise around world and many
countries have taken initiative towards production of bioethanol as a commercial fuel (Lee and Lee,
2016).There is a much effort has been taking place on research of plant and microbial biomass for
biofuel production. microalgal are best and efficient candidates because they meet the prerequisites
and because their synthesized and accumulated bioproducts e.g. lipids and carbohydrates[60].
Bioethnol produced by microalgae is called 3rd Generation biofuel[56]. Bioethanol is ethanol or ethyl
alcohol can be produced by any of three algal processes (mixotrophic, heterotrophic, and autotrophic).
It can be used as a substitute or an additive to petrol (Nahak et al., 2013)[7]. It contains low amounts
of Sulphur compared to petrol thus reducing its harmful emission of greenhouse gases on combustion.
It contains about 66% of the energy contained by petrol of the same quantity. Considering its
renewable nature, scope for use of bioethanol is high. Bioethanol is manufactured by breaking down
of starch or other sugars from first and second Journal Pre-proof generation feedstocks such as corn,
lignocellulosic biomass (sugarcane waste), wheat, etc. (John et al., 2011)[8][6].
Advantages
 High volume of biomass can be grown in smaller areas.
 Algae can be cultivated in and productive areas.
Disadvantages
 variables of production process i.e.cultivation, harvesting, cell destruction and conversion are
not all optimised.
Cell disruption
Cell disruption methods can be physical, chemical, or biological. Physical methods are shaking,
ultrasound, autoclaving, microwave, and others. These methods cause mechanical breakdown through
contact, pressure, or denaturation (Lakatos et al., 2019)[56][57]. one among the advantages of those
methods is that they're efficient and generate no waste, but their disadvantage that they require
energy. On the opposite hand, chemical methods are characterized by the utilization of acid or
alkaline chemical reagents for cell disruption. The advantage of this process is that it simplifies two
processes since cell disruption and polysaccharide hydrolysis are often achieved by using chemical
reagents (mainly sulfuric acid)(Chng, Lee, & Chan, 2017)[58]. The disadvantages are that waste is
generated which the process represents operational risks because sulfuric acid is very corrosive.
Saccharification
Saccharification processes are indispensable due to the range of polysaccharides found in microalgal
biomass. The sugars accumulated in microalgae biomass include xylose, mannose, arabinose,
mannose, galactose, and glucose(Kim et al., 2014; Kim, Oh, & Bae, 2017).The most common
saccharification processes for the discharge and hydrolysis of polysaccharides contained in
microalgae are chemical and enzymatic hydrolysis. Chemical hydrolysis is usually performed with
sulfuric acid, but some studies have reported using acid and even acetic acid (Chng et al., 2017;Phwan
et al., 2019)[62].
Fermentation
Bioethanol is produced by fermenting poly-saccharides found in microalgal biomass [63]. Concerning
the fermentationprocess, the choice may be to perform separatehydrolysis and fermentation (SHF) or
to per-form simultaneous saccharification and fermentation (SSF). The SSF process could also be
preceded by physical treatment, or it's going to involve the use of enzymes to perform cell disruption.
Most studies have found that the bioethanol yieldwithin the SSF process is higher than in SHF,
because there is a synergistic effect between carbohydrate digestion (with the enzyme or acid) and
bioethanol production [58][62][64]. The SHF process involves two operations, i.e., hydrolysis
followed by fermentation.
In both processes, fermentation occurs using two main microorganisms, Zymomonas mobilis[65] and
Saccharomyces cerevisiae[61][62]. To confirm the efficiency of carbohydrate conversion in ethanol,
we use the following equation described by Mussatto[66]:
Theoretical ethanol yield ;%
Ethanol(g)
=
Total sugar (g)*0.511*100
where 0.511 is the maximum theoretical conversion of sugar into ethanol.
Flowchart of bioethanol production process variables[57].

3.2Biodiesel potentials of algae

Biodiesel, like bioethanol, is additionally a highly sought-after alternative to fossil fuels. Biodiesel is
produced by the transesterification of lipids obtained from algae, to make methyl esters of long chain
fatty acids. The length of the chain depends on the source of the lipid. The sources of biodiesel are the
oils from palms, soybeans, canola, sunflower, rapeseed, etc. which are more expensive than fossil
fuels (Demirbas and Demirbas, 2011)[51][46]. However, these sources too cause the fuel versus food
conflict, high usage of arable land and poor economy, thus making algae together of the foremost
feasible sources. Also, from the environmental point of view, biodiesel from algae is more preferable
as it has lesser emission of carbon dioxide, NOx and other greenhouse gases (Scott et al., 2010)[52]
[46].
Biodiesel, like bioethanol, is additionally a highly sought-after alternative to fossil fuels. Biodiesel is
produced by the transesterification of lipids obtained from algae, to make methyl esters of long chain
fatty acids. The length of the chain depends on the source of the lipid. The sources of biodiesel are the
oils from palms, soybeans, canola, sunflower, rapeseed, etc. which are more expensive than fossil
fuels (Demirbas and Demirbas, 2011)[51][46]. However, these sources too cause the fuel versus food
conflict, high usage of arable land and poor economy, thus making algae together of the foremost
feasible sources. Also, from the environmental point of view, biodiesel from algae is more preferable
as it has lesser emission of carbon dioxide, NOx and other greenhouse gases (Scott et al., 2010)[52]
[46]. Microalgae includes fatty acids with 14–22 carbon atoms, which is analogous to the vegetable
oils utilized in biodiesel production. Moreover, their physicochemical characteristics are similar to
those of traditional biomasses and diesel, however, their environmental impact is significantly lower
than that of petrodiesel (Adesanya, Cadena, Scott, & Smith, 2014)[54][46].
For production of biodiesel, species with higher weight percentage of lipid content are opted. Some
algae have lipid contents as high as 60% of its weight. These lipids are commonly triglycerides or
TAGs. They are commonly stored as membrane components, storage products or metabolites. Fatty
acids or lipids obtained from algae are generally polyunsaturated, which leads to lower melting points
and also instability (Demirbas and Demirbas, 2011)[8]. The yield of biodiesel from every batch of
algae can be increased by optimizing different parameters. The growth characteristics of the algae are
often manipulated such it results in more accumulation of fatty acids. This can be done by nitrogen
starvation, controlled supply of nutrients and other parameters that effect the lipid formation and
accumulation in the algae.

M. Branco-Vieira, D. Costa, T.M. Mata et al. / Energy Reports 6 (2020) 397–402[53]

S.H. Shah, I.A. Raja, M. Rizwan, N. Rashid, Q. Mahmood, F.A. Shah, A. Pervez, Renew. Sustainable
Energy Rev. 81 (2018) 76–92[54].

Fourth generation
Fourth-generation biofuels include genetically modified microorganisms such as microalgae, yeast,
fungi and cyanobacteria are utilized as sources. The ability of microorganisms to convert CO2 to fuel
through photosynthesis is utilized [67]. The multiple advantages of microalgae such as their high
growth rate and oil content and low structural complexity enhance their numerous commercial
applications [68]. In addition to genetic modification, some fourth-generation technologies involve
pyrolysis (in a temperature range between 400 to 600 C) [68], gasification, upgrading, and solar-to-
fuel, pathways [69]. The general purpose of these modifications is to improve the HC yield and create
an artificial carbon sink to eliminate or minimize carbon emission [70]. These technologies are still in
early developmental stages [70].
Production of biofuel from gene-splicing of algae is discussed under the fourth generation biofuel
term. Microalgae and macroalgae are eukaryotes – i.e. containing a nucleus surrounded by a
membrane – belonging to kingdom Protista. Cyanobacteria are prokaryotes – i.e. lacking membrane-
bound organelles – belonging to the dominion bacteria that's mentioned as algae during this study
[80]. Table 2 lists the foremost researched microalgae strains within the field of biofuel production
supported their lipid, protein, and carbohydrate contents, also as their attributed classifications.
Bacillariophyceae, also referred to as diatoms, are mostly unicellular with silicate cell walls which
have carbohydrates and triglycerides (TAGs) storage compounds. A golden brown unicellular algae
Chrysophycea, inhabit both in freshwater as well as marine environments. Chrysophyceae’s main
storage compounds are oils and carbohydrates. Eustigmatophytes(are a small group of eukaryotic
algae are yellow-green unicellular eukaryotic algae) that have promising potential to be utilized in
biofuel production. Eustigmatophytes have mostly small cell sizes and high amounts of essential fatty
acids. Chlorophyceae may be a major group of chlorophyte with rigid cell walls. Most of the
Chlorophyceae algae contain proteins and starch as storage compounds [81]. The cultivation of GM
microalgae are often administered in contained and uncontained systems. However, the challenges
created by each of those methods are significantly different from one another . The contained
cultivation system features a more tightly controlled condition, while contamination and
environmental exposure are minimized. Thus , it is still expensive cultivation system.

Properties of the most widely used microalgae in algal biofuel production[81][82].

Cyanobacteria
Cyanobacteria have attracted a lot of attention in bioenergy and biofuel industries. Recently, the
genomic revolution has greatly developed metabolic engineering for several photosynthetic
organisms. cynobacteria has a special enzyme Aldehyde deformylating oxygenase (ADOs). .
Cyanobacterial ADOs are easy to be manipulated and overexpressed in various expression systems
compared to ADOs from otherv organisms. Synechocystis was the first photosynthetic organism for
which the genome was completely sequenced [71]. Synechocystis, which has the ability for both
photoautotrophic and heterotrophic growth, is a freshwater, non-filamentous, non-nitrogen fixing
organism. The most valuable characteristic of this strain of cyanobacteria as a genetic and
physiological case study of photosynthesis are the available genomic, biochemistry, and physiological
information. It is also well known as a model system for the investigation of oxygenic photosynthesis
in higher plants due to its small genome size compared to higher plant systems [72]. However, further
improvement of its catalytic activity essentially needs to be done. Plus, robust ADOs need to be
engineered for making them applicable and suit the requirements for the renewable fuel industry soon.
Therefore, future studies can be directed on developing a high throughput screening technology for
ADO enzyme to obtain more of their improved versions. Biochemical and biophysical
characterization of this enzyme is also crucially needed to be done to get a better insight into their
structural and functional properties.
Eukaryotic microalgae
Eukaryotic microalgae-based technology has attracted a lot of attention recently due to the
availability of eukaryotic genomic information. Interest in this technology began in the 1980s [73].
Generally, eukaryotic cells are formed in the random integration of exogenous genes into the nuclear
genome. Several kinds of microalgae have been successfully generated by gene transformation into
the cellular nucleus, chloroplasts and mitochondria [74]. The most investigated eukaryotic microalgae
is Chlamydomonas reinhardtii. It is a very common model organism used to investigate the essential
mechanisms of biological processes, such as oxygenic photosynthesis, circadian rhythms, and flagella
biogenesis [75]. The first investigation of the chloroplast DNA map of Chlamydomonas reinhardtii
was in 1978 and led to successful genetic transformation in the Chlamydomonas reinhardtii
chromosome, chloroplast, and mitochondrial genome by 1993 [76–78]. In recent years, marine
diatoms have attracted a lot of attention due to their wide prevalence, ability to adapt to varying
environments, and substantial biomass production in water [79].

Future requirements
Conclusion
Although cyanobacteriaand microalgae have been widely studied to find the optimum conditions to
fabricate multifarious biotechnological products, indigenous strains show little productivity, which in
turn poses serious limitations on production of biofuels,harvesting, and product extraction in an
optimum way from technical and economical viewpoints.Genetic engineering may minimize the costs
of a given process and enhanceits yield, with the aim of exploiting all potential of the selected strains
of cyanobacteria and microalgae which is considered as a multicomponent producer.

3. Conclusions

Experts suggest that current oil and gas reserves would suffice to last only a few more decades. To
exceed the rising energy demand and reducing petroleum reserves, fuels like biodiesel and bioethanol
are at the forefront of alternative technologies. Biofuels have been investigated as alternative
resources to resolve the demanding consumption of conventional fossil fuels, minimize the economic
and environmental impact, and secure sustainability for decades. It is well-known that vehicle
transportation is almost totally dependent on fossil, particularly petroleum-based fuels like gasoline,
diesel oil, liquefied petroleum gas, and gas. Alternative fuel to gasoline and Petro-diesel must be
technically feasible, economically competitive, environmentally acceptable, and easily available.
Accordingly, the viable alternative fuel for compression-ignition engines is biodiesel. Bioethanol and
biodiesel use may improve emission levels of some pollutants and deteriorates others. Usage of
bioethanol and biodiesel will allow a balance to be sought between agriculture, social, economic,
political, and environmental developments in the future.
From the study we conclude that the first generation biofuel has the highest biofuel production and
energy efficiency, but is less effective in meeting the goal of reducing the greenhouse gases emission.
The third and fourth generation biofuel shows the lowest net greenhouse gases emissions, allowing
the reduction of greenhouse gases in the atmosphere.

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