As an essential component of life, energy has run the
global economy. The adverse effects of fossil fuels urge to find a clean and
sustainable source of energy. Biodiesel has gained considerable attention as a
similar conventional diesel for its chemical structure and energy content. The
non-toxic biodiesel can be produced commercially as a result of the transesterification
of fats (Yusuf et al., 2011). As the current biofuel production faces challenges
of stability, inconsistency, and economic challenges, genetically modified
microbes have the potential to overcome the scale-up procedure, seasonal
issues, climate change, and the labour force (Lin et al., 2013).
Genetically modified bacteria for bioethanol
The concept of recombinant technology refers to the
molecular cloning of other DNA molecules, mainly foreign into bacterial DNA
elements also called plasmids and propagated into bacterial host usually Escherichia
coli (E. coli). Various bacterial species have plasmid in it, and it
can be transferred from one to another by conjugation, and transformation. This
technology is harboured for the more massive production of essential protein
and other biomolecules (Hacker and Wurm, 2011).
This technology has ushered into a new era for
harnessing biological processes, including genetically modified crops, biofuels,
and chemicals (Burrill, 2014). With E. coli as the best host for genetic
engineering for higher yield, the first report of the de novo biosynthetic
pathway of diesel was carried out on it. The overexpression of bioethanol was
performed with the production genes from Zymomonas mobilis and wax ester
synthase/acyl-CoA-diacylglycerol acyltransferase (WS/DGAT) gene from the Acinetobacter
baylyi strain ADP1, resulting into 95% ethanol without any redox
imbalance (Kalscheuer et al., 2006).
Bioethanol Production process in bacteria
In the presence of glucose and oleic acid and under
aerobic conditions, bioethanol production was carried out together with
esterification. Bioethanol production takes place as a result of an endogenous
process and anaerobic condition where one mole of glucose metabolizes into two
moles of formate, two moles of acetate, and one mole of ethanol. The endogenous
process of ethanol production involves the reduction of acetyl-CoA into ethanol
by catalyst aldehyde-alcohol dehydrogenase (Adh).
The reaction consumes two “nicotinamide adenine dinucleotide (NAD) +
hydrogen (H)” (NADH) molecules
which causes an imbalance as initial glycolysis only produces one NADH. Hence,
to overcome the imbalance, E. coli balances the production of ethanol by
oxidation of acetyl-CoA into acetate that does not require NADH.; this process
of fermentation produces a sub-optimal level of ethanol (Koppolu and Vasigala,
Yeast gene manipulation for enhanced biofuels
Similarly, Saccharomyces cerevisiae is one of
the potential yeast for genetic engineering. It is considered a good ethanol
producer with its ability to accumulate fatty acids with a long chain of 16-18
carbon atoms (Tehlivets et al.,
2007). In general, the microbial pathways for the production of biofuels are
based on the production of non-fermentative alcohol, isoprenoid-derived
hydrocarbons, fatty acid-derived hydrocarbons, and fermentative alcohol.
As the primary component for bioenergy comes to fatty
acid, oleaginous microorganisms (grease) are used to produce biofuel as a fatty
acid source for the transesterification process, as shown in equation 1. The
grease strains are found in bacteria, yeasts, algae, and molds (Kosa and Ragauskas,
2011). The critical characteristics with more than 50% lipids accumulated in
the cells, industrialization culture with simple apparatus, faster growth rate,
and easy extraction make the use of grease microorganisms for biofuels more
popular (Lin et al., 2013).
Triglyceride+Methanol ↔ Glycerol+Methyl Esters (Biodiesel)….(1)
Genetic modification of
microalgae biomass for biofuels
Photosynthetic microorganisms such as microalgae are also
looked at as a potential source for modification of lipids, alcohols,
polysaccharides, and other sources through genetic modification. The higher
photosynthetic conversion, diverse metabolic pathway, and faster growth rate
contribute to the secretion of energy-rich hydrocarbons (Radakovits et al.,
2010). The role of enzymes in the production of biofuel is enormous, and β-glucosidase
(BGL) enzymes hydrolyze the biomass by producing monomeric sugars. The cost-effective
model to produce BGL has been done recently on recombinant E. coli with a
yield of 13g/L (Srivastava et al.,
Optimization of microbial biofuel production
To perform the genetic engineering of microorganisms
for biofuel production, the metabolic pathways and the essential and
non-essential genes of bacterial life is essential. It helps in the modulation
of the efficiency of production and balances the optimization of bioproduction
The advanced sequencing technique of microorganisms
has resulted in a genome database which could be a guide for understanding the
metabolic and physiologic pathway namely KEGG pathway,Embden–Meyerhof–Parnas
(EMP) pathway, oxidative tricarboxylic acid (TCA) cycle, etc. This necessary
information provides an essential methodology to endogenous regulation of
biofuel-producing pathway for higher yield (Lee et al., 2013).
The most critical way to design the synthetic
microorganism for biofuel production is to increase the efficiency of
biosynthesis by reducing the unused energy transducing components. Implementing
the non-indigenous pathway by which renewable energy converts to biofuels with a
The ability of any microorganism in the industrial
production of fuel depends on the conversion rate of raw material with higher
productivity at a lower price. The prevalence of genetic and molecular tools
has shown an environment-friendly way of synthesizing biofuels and E. coli
has been the best choice so far.
The strains of E.coli used in state-of-the-art
bioethanol production have shown titers range of 40-55 g/L with a yield of
approximately 100%. However, the raw materials used as a source (cellulosic and
hemicellulosic hydrolysates) contain toxic acids and other phenolic compounds
that inhibit the growth of E. coli. Thus, improved pretreatment is
necessary, and approaches to genetic engineering would be useful (Koppolu and Vasigala,
2016). Moreover, the addition of the osmolyte supplements reduces the osmotic
stress of high concentration sugars on E. coli.
The genetic modification of microorganisms has
significantly enhanced the biofuel yield and production process. The
manipulated genes when ligated to the specific promoters get desirable
outcomes, and the future generation of biofuel will rely on the metabolic and
genetic engineering of microorganisms to reduce the dependency on conventional
While most biodiesel production follows the non-microbial
process, the lower toxicity of microbial production of diesel stands out as the
best replacement. However, more studies and research are needed to improve the energy
content and productivity of bioenergy.
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C. 2020. Genetic engineering of microbial biomass for biofuel production.
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