Sciknow Publications Ltd. RSE 2014, 1(2):29-34
Open Journal of Renewable and Sustainable Energy DOI: 10.12966/rse.09.03.2014
©Attribution 3.0 Unported (CC BY 3.0)
Advances and Biotechnological Applications in Biofuel Production:
Tenzin Tseten and Thirupathihalli Pandurangappa Krishna Murthy*
Department of Biotechnology, Sapthagiri College of Engineering, Bangalore-560057, India
*Corresponding author (email: email@example.com )
Abstract - This paper assesses the global and sectoral implications of the growing demand for bio-based inputs for energy and
fuel production. This paper also pinpoints the importance of relative prices between bio-based and fossil inputs in the petroleum
and electricity sector and current advances in the production of biofuel. First generation biofuels continue to be substantially
subsidized, and this has contributed to the increasing use of such fuel. This report reviews the current status of second and third
generation biofuels. Second generation biofuels are made from cellulose, which is in more abundant supply than the first
generation biofuel feedstock. Whereas third generation biofuels have become an innovative alternative that offers a wide variety
of exceptional benefits. Nowadays the production of biodiesel from microalgae is an option that has attracted strong interest of
the scientific community and should be evaluated to determine the technical, technological, economic and environmental
sustainability of the process. Industrial biotechnology with its competitive, clean and clever use of bio -based technologies can
play a key role in making biofuels more sustainable.
Keywords - Biofuel, Fossil Fuels, Biomass Feedstock, Cellulose, Microalgae
Each and every solution is crucial to shift towards future with
a sustainable energy resources and healthy goods production.
Hence the energy source has to be more efficient and
processes for the production of sustainable energy resource
have to be improved with complete new technologies.
Biotechnological applications with its clean and chicanery use
of bio-based technologies can play a vital role in making
biofuels more sustainable . Fossil fuels are hydrocarbons,
primarily coal, fuel oil or natural gas, formed from the
remains of dead plants and animals. Fossil fuels because of its
high heating potential, availability and ignition properties and
it has been used as a source for transportation and other
energy purposes . Due to speedy fossil fuel depletion, the
global warming, rising future energy requirement and demand,
and climatic changes because of emission of the fossil fuel,
we are forced to search for alternative source of energy.
Among various alternative sources available today, biofuel is
the one of the best alternative source of energy for
diminishing dependency on fossil fuel by replacing it fully or
partially . Through sustainable biofuel production and
continuous speedy improvements in both bio feedstock and
processes for biofuel production, it is possible to reduce the
dependency on non- renewable fossil fuels and to provide
national energy security . Biofuel is any solid, liquid or
gaseous fuels derived from organic biomass which is any
living matter such as field crops, wood products, water plants,
and municipal solid waste that is converted into energy . As
an alternative for fossil fuel in the transportation part, biofuel
can become critical for solving environmental troubles
because of these reasons: it minimizes greenhouse gases
(GHG) emission, and does not need much engine
modifications, it also improves national security and provides
employment and finally supports rural development . To
move successfully towards biofuel future, the use of modern
biotechnology is extremely important and the cost of
conversion from bio feedstock into biofuel has already been
reduced using modern biotechnology [4, 6].
Ever since human discovered fire, charcoal, woodchips and
cattle dung have been used as a source of energy and still
today people used these solid fuels for heating and cooking in
many parts of the world. In mid 1700s and early 1800s, Oil
extracted from whale was broadly used for lighting purposes
. From more than a century, biofuel has been around us
Rudolph Diesel in late 19th century used peanut oil to generate
power and he is the one who started using vegetable oil for the
production of energy source. Diesel also developed first
working engine that runs on peanut oil at the World’s
Exhibition in Paris in 1900 [8, 9]. Henry Ford was also an
initial proponent of biofuel and he developed the Model T car
in 1903 which was totally designed to use hemp derived
30 Open Journal of Renewable and Sustainable Energy (2014) 29-34
biofuel as fuel .
Fig. 1. Evolution of world biofuel production in million tons 
Because of the features such as productivity, obtain ability,
low greenhouse gas content, biodegradability and
renewability makes biofuel more advantageous over other
non-renewable fossil fuel and production of these sustainable
biofuel is rapidly increasing all over the world every year .
As long as fossil oil prices remained close to US $20 per
barrel (bbl), biofuel production stagnated around 10 Mtoe.
When fossil oil prices started to soar as from 2000, biofuel
production followed the same pattern. It is however
noteworthy that the 2009 decline in fossil oil prices had no
effect on the upward trend of biofuel production (Figure 1)
. In 2007, around 90% of global biofuel production was
from US, Brazil and Europe .
Fig. 2. World biofuel production in 2007 
3. Advancement in Biofuel Production
3.1. Primary biofuel
Fuelwood such as woodchips, pellets, animal waste and crop
residues have been used in an unprocessed form by man
primarily for cooking and heating purposes ever since human
discovered fire . Fuelwoods, generally unprocessed
biomass of wood are commonly used for producing fire
specially for cooking and the most common form of fuelwood
is charcoal that mainly comprises of carbon and produces
more heat and energy . Because of an ever-expanding
world population, requiring more food, more resources, more
production and more infrastructures, there was huge increase
in the growth of oil demand from 1996 to 2012 as shown in
Fig. 3. World oil demand.
(Source available: http://azizonomics.com/2011/08/30/hunger)
3.2. First generation biofuel
Ethanol is the one of the most renowned first generation
biofuel which is prepared from sugar cane or sugar beets or
maize by fermentation process. Different alcohol can be made
by using different fermentation organism such as butanol.
Overall production of bioethanol in 2006 was about 51 billion
litres with Brazil and the United States both of which
contributes around 18 billion litres, or 35 per cent of the total.
China and India contributed 11 per cent to global ethanol
production in 2006. Figure 4 clearly shows increased in world
bioethanol production from about 9 Mtoe (in 1990) to 40
Mtoe (in 2009). There is also steady increase in the world
share for the production of various biofuel as shown in Figure
Fig. 4. World ethanol and biodiesel production, 1975-2010.
(Source available: http://cdn.intechopen.com/pdfs-wm/18291.pdf)
Open Journal of Renewable and Sustainable Energy (2014) 29-34 31
Fig. 5. Shares of world production of cereals, vegetable oils
and sugar plant used for biofuel production.(Source available:
The major reasons behind the rapid increase in the
production of 1st generation biofuel in OECD countries are
national energy security, support rural development and
agricultural industries and reduction of GHG emissions.
Though first generation biofuel shows net benefit in terms of
above reasons, it also have few disadvantages which includes
higher production cost, competing with food crops causing
increase in food price, causing water scarcity in some region
and limited reduction in GHG emission . Bioalcohol and
other first generation biofuels are generally made from
biofeedstock which can also be used for the production for
human food . Fast increase in demand for and production
of biofuels, mainly bioethanol from maize and sugarcane, has
had several effects on grain supply-and-demand systems
because of which the price of rice and wheat and other crops
have increased (Figure. 6).
Fig. 6. Simulated Real Grain Prices, 2000-2007 (US$/metric ton)
Note: Grain price is the production-weighted average of rice, wheat, maize,
and other coarse grains. (Source: IFPRI IMPACT).
In near future, utilization of first generation biofuel is
expected to cause many challenges due to which
biotechnologists are forced to look for alternative or
advancement in biofuel and hence with the help of advance
biotechnology, second generation biofuel came into existence
. Bioethanol, biodiesel, biogas, syngas and solid biofuel
are the examples of first generation biofuel.
3.3. Second generation biofuel
World production of second generation biofuel has been
increased rapidly in last few years because of its additional
sustainable features over first generation biofuel which is
primarily made from food crops but commercially these
biofuels are not yet produced. According to a survey
conducted by IEA, speedy increase in second generation
biofuel demand has been noticed for stabilizing concentration
of CO2 in the atmosphere at 450 parts per million(ppm) .
Second generation biofuels are generally produced from
lignocellulosic biofeedstocks which seems to have more
potential to reduce GHG emissions and supply for these
feedstocks are more compared to first generation biofuel .
Lignocellulosic biofeedstock enables the use of non-food
crops and less expensive biomass which completely replaces
the direct use of food crops for biofuel production which has a
strong adverse effect on agriculture worldwide .
Second-generation biofuels can be further classified in terms
of the process used to convert the biomass to fuel:
biochemical or thermochemical.
3.3.1. Second-generation biochemical biofuel
Second-generation biochemically-produced alcohol fuels are
often referred to as “cellulosic ethanol” and “cellulosic
biobutanol”. Figure. 7 shows the basic steps for producing
bioethanol which includes pre-treatment, saccharification,
fermentation, and distillation. Pretreatment is designed to help
separate cellulose, hemicellulose and lignin so that the
complex carbohydrate molecules constituting the cellulose
and hemicellulose can be broken down by enzyme-catalyzed
hydrolysis (water addition) into their constituent simple
sugars.The sugar molecules are easily fermented to
ethanol using well-known micro-organisms, and some
micro-organisms for fermentation to butanol are also known.
3.3.2. Second-generation thermochemical biofuels.
In this method, conversion of biomass into biofuel involves
high temperature and pressure than biochemical process.
Conversion of biomass begins with gasification or pyrolysis
where the biomass is heated at high temperature converting
biomass into a mixture of gases followed by the removal of
impurities such as carbon dioxide which are present in the gas
mixture. Carbon monoxide (CO), hydrogen(H2) and small
quantity of methane(CH4) are major constituents of clean gas
commonly known as syngas after the removal of CO2. With
the help of catalyst, the CO and H2 reacts with each other to
give liquid biofuel as shown below in Figure 8 .
32 Open Journal of Renewable and Sustainable Energy (2014) 29-34
Fig. 7. Simplified depiction of process steps for production of
second generation fuel ethanol 
Fig. 8. Simplified depiction of process steps for
thermochemical biofuels production .
3.4. Third generation biofuels
In response to the problems of second generation biofuel, new
approach came up with the solution of utilizing algae biomass
with the help of microbial enzymes to achieve better quality
and more efficient sustainable biofuel .The elevated cost
of oil, the trend to continue growing or to remain at soaring
levels and the exhaustion of reserves has affected global
energy security. The third-generation biofuels are emerging as
a promising alternative to using microalgae biomass avoid
making use of raw materials that come from food sources .
Different bioconversion processes such as biochemical,
thermochemical, chemical and direct combustion can be used
to obtain various types of biofuel as shown in Figure 9. The
demand for liquid fuels in transport is increasing at an
alarming rate. Currently, fatty acid methyl esters (FAME) are
considered to be used as liquid biofuels for diesel engines.
They are typically prepared from vegetable oils or animal fats.
Since these materials are mainly meant for consumption,
other renewable sources of natural triacylglycerols (TAG) are
sought. One of the most prominent alternatives is microalgae
. Most of the microalgae species are photoautotrophic,
that is, they convert solar energy into chemical forms through
photosynthesis. Their photosynthetic mechanism is
comparable to land based plants, but due to a simple cellular
structure, and their underwater habitat, where they have
efficient access to water, CO2 and other nutrients, they are
generally more efficient in converting solar energy into
biomass . Microalgae have a very good prospective as
biodiesel precursors since many of them are very rich in oils,
at times with oil contents over 80% of their dry weight, even
though all species are not suitable as biodiesel production oils
. There are many advantages of using microalgae as a
source of biofuel production. They can double their biomass
in less than 24 hours. Additionally they can be grown in
wastewater, or any non-potable water. The synthesis of
microalgae biodiesel could be integrated with the CO2
removal from power generation facilities for waste water
treatment from which microalgae would remove NH4
3-. However, there are certain limitations in using
microalgae for synthesis of biofuel. Microalgae
biomass-based biofuel have several problems which include
the optimization of high density and large surface units of
production. The location of the microalgae production unit
may also pose a difficulty .
Fig. 9. Conversion process for biofuel production from algal
Another alternative which can be considered as third
generation biofuel is cellulose biomass. Discarded cellulosic
biomass obtained from forestry, agriculture, and municipal
sources are prospective raw materials for the synthesis of
biofuel. An efficient way of producing it is consolidated
bioprocessing (CBP).In this technique, cellulose production,
substrate hydrolysis, and fermentation are accomplished in a
single process step by microorganisms that express
cellulolytic (and hemicellulolytic) enzymes . Apart from
microalgae, there are few other sources to produce biofuel
from oily biomass. Multiple prokaryotes and eukaryotes can
accumulate high amounts of lipids. But, as is the case with
microalgae, not all species are suitable for biodiesel
production due to the differences in the kind of storage lipids.
Thus, as stated by  many prokaryotes produce polymeric
compounds such as poly (3-hydroxybutyrate) (PHB) or other
Open Journal of Renewable and Sustainable Energy (2014) 29-34 33
polyhydroxyalkanoates (PHAs), whereas only a few genera
show accumulation of certain triacylglycerols (TAGs) and
wax esters (WEs) in the form of intracellular lipid bodies.
Conversely, storage TAGs are frequently found in eukaryotes,
while PHAs are absent, and WE accumulation has only been
reported in jojoba. All these lipids are carbon and energy
storage compounds that guarantee the metabolism for
viability during starvation stage. Analogous to the formation
of PHAs, TAGs and WE, synthesis is promoted by cellular
stress and during imbalanced growth; for instance, by
nitrogen insufficiency along with the abundance of a carbon
4. Application of Biotechnology in
Biotechnology uses eye-catching way of producing biofuel
which increases the yield without much increase in the energy
needed for production. In the past few decades, significant
improvement has been made with the help of molecular
biology so as to improve the microbial activity and enzymes
. The use of Genetically Modified Organisms (GMOs) is
found to be most efficient and quick method to improve
biofuel conversion, particularly in case of lignocellulosic
biomass . With the help of biotechnology, structure of cell
wall and composition of lignocellulosic in plant cell can be
modified to enhance ethanol yield per acre .
Biotechnology can influence yield density by varying plant
physiology, their architecture, along with their photosynthetic
efficiency and it has also shown its ability to lessen agronomic
inputs for instance herbicide and pesticides. Advancement is
rapidly being made on characters which enable crops to take
up and consume nutrients more resourcefully, thus equipping
them to be grown with less amount of fertilizer. Producing
biomass crops on supposed “marginal” acres, such as land
that is highly dry, or with deprived soil characteristics, can
raise the scale of biofuel production without any influence on
food production acres. Biotechnology is focusing on the
development of drought, cold, salt and heat plants as well as
plants that can survive on a wide range of soil conditions. For
a biomass feedstock plant, a higher level of cellulose and
hemi-cellulose content would give better fermentation yield
and hence gallons of ethanol per ton of biomass. This results
in added net energy per acre and more revenue. Research has
already been carried out successfully for the cloning of genes
that code for cellulases and polygalacturonase enzymes to
develop low-cost effective biorefinery strategy to achieve
maximum biomass conversion and improved gas
chromatography-mass spectrophotometry method that has
been developed by researchers at TAMUK in understanding
the catalytic action of the expressed enzymes in the
bioconversion process . Difficulties faced by the biofuel
manufacturers in processes such as microbial digestion and
fermentation can effectively reduce by enhanced
biotechnological processes .
According to Current state-of-the-art knowledge, Green
House Gas can be abridged within the next 30-40 years and
the demand for low carbon dioxide emission fuel in all forms
of automobile is enormous. Because of fast growing price of
crude oil with several adverse effects on environment, the
demand for the biofuel production has increased. Liquid
biofuel such as bioethanol, biodiesel and gaseous biofuel such
as biomethane, biohydrogen have developed as an effective
alternative to the fossil fuel resource. To reduce the
dependency on fossil fuel, biofuel was found to be most
efficient alternative among various existing source of energy.
Biofuel can replace the effective use of fossil fuel which is
known reason for the greenhouse effect. Researches have
shown that first generation biofuel that is crop biofuel has a
durable influence on agriculture world-wide. Second
generation biofuels are generally made from non-crop
biomass by two different fundamental approaches
(biochemical and thermochemical second generation biofuel).
Cellulose because of its easy availability, price and ability to
be proficiently degraded by cellulolytic bacteria, it has
become an eye-catching biofeedstock for the production of
second generation biofuel. A wide range of agricultural and
industrial residues, from lignocellulosic forestry and domestic
waste can be used as precursors of biofuels with the help of
microbial enzymes. Biofuels production plant design,
heterogeneous catalysts and enzyme immobilization
techniques, protein engineering of lipases, alcohol
dehydrogenases or hydrolases to increase their activity and
reusability, genetic engineering of microbes to facilitate both
the pretreatment of precursors, and the synthesis and
purification of the biofuels. Biotechnology plays a crucial role
in biofuel production. It helps in decreasing agronomic inputs,
in optimizing process characteristics and in engineering the
plants to produce high yield and hence high energy. With
progresses in the field of biotechnology, improvements in
synthesis of biofuels can be expected.
 Biofuel production technologies: status, prospects and implications for
trade and development. United Nations Conference on Trade and
 Hassan, M. H., and Kalam, M. A. (2013). An overview of biofuel as a
renewable energy source: development and challenges. Procedia
Engineering, 56, 39-53.
34 Open Journal of Renewable and Sustainable Energy (2014) 29-34
 Masjuki, H. H., Kalam, M. A., Mofijur, M., and Shahabuddin, M.
(2013). Biofuel: Policy, Standardization and Recommendation for
Sustainable Future Energy Supply. Energy Procedia, 42, 577-586.
 Wolt, J. D. (2009). Advancing environmental risk assessment for
transgenic biofeedstock crops. Biotechnol Biofuels, 2, 27.
 Dragone, G., Fernandes, B. D., Vicente, A. A., and Teixeira, J. A.
(2010). Third generation biofuels from microalgae.
 Lynd, L. R., Laser, M. S., Bransby, D., Dale, B. E., Davison, B.,
Hamilton, R., ... and Wyman, C. E. (2008). How biotech can transform
biofuels. Nature biotechnology, 26(2).
 A brief history of biofuels: from ancient history to todayby Annie
Webb, Wednesday, July 31st.
 Biodiesel: A Review Monisha J, Harish A, Sushma R, Krishna Murthy
T P, Blessy B Mathew, Ananda S.
 Bryn mayesapril (2009). The biofuel debate: fuel, food, and the future
of the planet.
 New York Times, Sept. 20, (1925) Ford Predicts Fuel from Vegetation,
 Hervé, G., Agneta, F., and Yves, D. (2011). Biofuels and world
agricultural markets: outlook for 2020 and 2050. Economic Effects of
Biofuel Production, 129-162.
 Coyle, W. (2008). The future of biofuels: A global perspective.
 Agarwal, Bina. (1986). Cold hearths and barren slopes: the woodfuel
crisis in the third world. Allied Publishers Private Ltd.,
 Sims, R. E., Mabee, W., Saddler, J. N., and Taylor, M. (2010). An
overview of second generation biofuel technologies. Bioresource
technology, 101(6), 1570-1580.
 Eggert, H., Greaker, M., and Potter, E. (2011). Policies for second
generation biofuels: current status and future challanges.
 Balat, M., and Balat, H. (2009). Recent trends in global production and
utilization of bio-ethanol fuel. Applied Energy, 86(11), 2273-2282.
 Eisentraut, A. (2010). Sustainable production of second-generation
biofuels: potential and perspectives in major economies and
developing countries (No. 2010/1). OECD Publishing.
 Banse, M., van Meijl, H., and Woltjer, G. (2008, June). The impact of
first and second generation biofuels on global agricultural production,
trade and land use. In GTAP Conference Paper, June.
 Goldemberg, J., Johansson, T. B., Reddy, A. K., and Williams, R. H.
(2004). A global clean cookin g fuel initiative. Energy for Sustainable
Development, 8(3), 5-12.
 Carere, C. R., Sparling, R., Cicek, N., and Levin , D. B. (2008). Thi rd
generation biofuels via direct cellulose fermentation. International
journal of molecular sciences, 9(7), 1342-1360.
 Peralta, Y., Sanchez, E., and Kafarov, V. (2010). Exergy analysis for
third generation biofuel production from microalgae biomass. Chem.
and Cvengroš,J.(2012).Biofuelsfromalgae. Procedia
Engineering, 42, 231-238.
 Micro-and Macro-algae: Utility for Industrial Applications: Outputs
from the EPOBIO Project, September (2007). CPL Press, 2007.
 Chisti, Y. (2007). Biodiesel from microalgae. Biotechnology
advances, 25(3), 294-306.
 Aslan, S., and Kapdan, I. K. (2006). Batch kinetics of nitrogen and
phosphorus removal from synthetic wastewater by algae. Ecological
Engineering, 28(1), 64-70.
 Picazo-Espinosa, R., González-López, J., and Manzanera, M. (2011).
Bioresources for third-generation biofuels. Biofuel's Engineering
 Demain, A. L., Newcomb, M., and Wu, J. D. (2005). Cellulase,
clostridia, and ethanol. Microbiology and molecular biology
reviews, 69(1), 124-154.
 Wältermann, M., and Steinbüchel, A. (2005). Neutral lipid bodies in
prokaryotes: recent insights into structure, formation, and relationship
to eukaryotic lipid depots. Journal of bacteriology, 187(11),
 Kalscheuer, R., Stölting, T., and Steinbüchel, A. (2006). Microdiesel:
Escherichia coli engineered for fuel production. Microbiology, 152(9),
 Lee, D., Chen, A., and Nair, R. (2008). Genetically engineered crops
for biofuel production: regulatory perspectives. Biotechnology and
Genetic Engineering Reviews, 25(1), 331-362.
 Gressel, J. (2008). Transgenics are imperative for biofuel crops. Plant
science, 174(3), 246-263.
 Carpita, N. C. (1996). Structure and biogenesis of the cell walls of
grasses.Annual review of plant biology, 47(1), 445-476.
 Ibrahim, E., Jones, K. D., and Hossenya, E. N. (2013). Molecular
Cloning and Expression of Cellulase and Polygalacturonase Genes in
E. coli as a Promising Application for Biofuel Production. Journal of
Petroleum & Environmental Biotechnology.