With the increasing concern and rising demand for clean and sustainable energy, biofuels are gaining considerable attention. Genetically modified microbes have the potential to overcome the scale-up procedure, seasonal issues, and biomass availability. Therefore, microorganisms with gene modification could be the primary source of biofuel production in the future.
Genetically modified bacteria for bioethanol production
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 hosts, usually Escherichia coli (E. coli). Various bacterial species have plasmid in them, and they 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 in 95% ethanol without any redox imbalance (Kalscheuer et al., 2006).
Bioethanol Production process
In glucose and oleic acid and under aerobic conditions, bioethanol production was carried out together with esterification. Bioethanol production occurs due to 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 ethanol production process involves reducing 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 ethanol production by oxidation of acetyl-CoA into acetate that does not require NADH.; this fermentation process produces a sub-optimal level of ethanol (Koppolu and Vasigala, 2016).
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 to accumulate fatty acids with a long chain of 16-18 carbon atoms (Tehlivets et al., 2007). The microbial pathways for 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 moulds (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). Biodiesel is produced commercially due to the transesterification of fats (Yusuf et al., 2011).
Triglyceride+Methanol↔Glycerol+Methyl Esters Biodiesel….(1)
Photosynthetic microorganisms gene modification
Photosynthetic microorganisms such as microalgae are also considered a potential source for modifying lipids, alcohols, polysaccharides, and other sources through genetic modification. The higher photosynthetic conversion, diverse metabolic pathways, and faster growth rate contribute to the secretion of energy-rich hydrocarbons (Radakovits et al., 2010). The role of enzymes in biofuel production 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., 2019).
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 and biosynthesis.
The advanced sequencing technique of microorganisms has resulted in a genome database that could guide 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 pathways 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 higher yield.
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. Therefore, the future generation of biofuel will rely on the metabolic and genetic engineering of microorganisms to reduce the dependency on conventional energy.
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.