The Subtle Art Of Thermophilic Bacteria
The dwindling of the present fuel sources creates a need to produce Biofuels from microorganisms. Biofuels are extracted from organic matter like plants or animal waste and represent a potentially eco-friendly alternative for oils and gases. Biofuels like bioethanol and biodiesel increase fuel efficiency in cars, buses, and other transport instead of traditional fuel. Currently, fuels like bioethanol are marginally less expensive to produce than petrol. One of the remarkable properties of biofuel is that it can be cultivated and harvested in lesser time, making them renewable and widely acceptable. Synthetic biology provides a great pathway to engineer microorganisms that can produce biofuels akin to petroleum-based power.
The current situation calls for the need for biofuel to be used instead of petroleum products. The strengthening of metabolic engineering techniques and advances in synthetic biology will provide new apparatus for a better interpretation of how to engineer cells that generate desired physical composition for producing economically feasible biofuels. Today the most significant amount of biofuel generated worldwide is ethanol. The most crucial characteristic of ethanol production is that the organism should have good ethanol tolerance. For the economic recovery to occur using downstream processes, the microorganism should grow and generate ethanol in the presence of 4 per cent (v/v) ethanol. So, the production of ethanol is now carried out using engineered thermophiles. Thermophilic bacteria (which can thrive in high-temperature conditions) have a fantastic ability to tolerate fluctuations in pH, temperature, and environmental changes.
Furthermore, thermophilic industrial fermentation is less susceptible to microbial contamination and requires lower energy input due to reduced cooling steps needed between the fermentation steps. These thermophilic bacterias are taken into consideration due to several other factors. Some microbes of this group have a remarkable ability to ferment biomass containing cellulose without any added enzymes rapidly. These thermophilic microbes can process even at elevated temperatures, reducing heat exchange. But the problem with these microbes is that they naturally do not carry out homo-ethanol fermentation and do not exhibit high product tolerance. To address these deficiencies, scientists often do strain development involving metabolic engineering techniques.
Recent advancements in gene transfer systems and genome sequencing have resulted in a new generation of engineered thermophilic ethanologens. Scientists have focused on two main strategies to engineer thermophilic organisms for consolidated metabolic bioprocessing. The first approach achieves increased ethanol production by stamping out other fermentation products and boosting ethanol tolerance. The second approach involves genetic manipulation of good ethanol-producing bacteria to incorporate cellulolytic genes into their genome. One such genetically modified strain of Thermoanaerobacter mathranii is modified and widely explored. The first variant generated by knocking the ldh gene from this species was BG1L1, which resulted in a more than two-fold increase in ethanol production compared to the wild variant. Upon further manipulation in this stain, it involved overexpression of NAD(P)H-dependent alcohol dehydrogenase, which resulted in another variant called BG1E1. The overexpression of this enzyme thus resulted in higher ethanol production. Later, scientists finally developed the BG1G1 strain, where the gene encoding for NAD+ -dependent glycerol dehydrogenase was inserted. This increased ethanol production by 40 per cent compared to the wild-type strain. Thus recent advancements in genetic engineering techniques and overall efforts to engineer thermophilic anaerobes to increase ethanol titers have resulted in a modest gain in ethanol output with the elimination of undesired end products. Future targets to increase ethanol production may include the cellulolytic machinery of C.thermocellum into highly ethanologenic thermoanaerobacter and thermoanaerobacterium strains.
Similarly, the advancement in synthetic biology techniques enabled biodiesel production with desirable pathways. Biodiesel is environmentally friendly, renewable, has a low emission rate and high level of safety, and hence is a better established and preferable petrochemical. Chemically it is composed of fatty acid alkyl esters (FAAEs). In-vitro transesterification is the most common process to produce biodiesel, where methanol is combined with triacylglycerides of vegetable oils to form fatty acid methyl esters and byproduct glycerol. The reaction can be catalysed in the presence of enzymes or alkali acids. However, the high cost and limited availability of vegetable oil are a matter of concern for the commercial viability of biodiesel production. Also, the in vitro transesterification reaction has unresolved issues, such as the high usage of toxic compounds and the high cost of isolation and immobilisation of enzyme catalysts. These problems are now being explored by developing an interest in microbial processes where a wide range of new materials other than vegetable oils can be used to generate biodiesel. Nowadays, technologies exist where living cells manufacture products that are more readily biodegradable, require less energy and create less waste during production compared to those obtained by chemical synthesis. The target molecule should have high productivity and yield to compete with existing petroleum-based products. These goals can be challenging to achieve by naturally existing microbes; therefore, metabolic engineering techniques are used, which help in redesigning the pathway for efficient production of target molecules, including biofuels. Synthetic biology tools enable us to create new biological functions that are not present in nature by emphasising the design of microbes either by modification in the existing pathways or by heterologous expression of the natural path to allow efficient biodiesel production.
Synthesis of biodiesel using microbes is currently an up-and-coming alternative to conventional technologies. There are two different possibilities to approach the production of biodiesel. One is the indirect synthesis of microbial oils by the traditional in-vitro transesterification processes, and the other is the direct synthesis of biodiesel by redesigning the cell factories to increase the synthesis of alcohol or free fatty acids, which subsequently lead to biodiesel production. The indirect synthesis includes using oleaginous microbes as the oil derived from them represents a promising raw material to produce biodiesel through transesterification using plant-based processes. For example, a photoautotrophic microorganism, microalgae, contains oil content ranging between 15 to 70 per cent by weight of dry biomass. These algae are used for the generation of biodiesel, but a lot of harvesting is required to obtain a substantial amount of fuel. Therefore tools from synthetic biology are being used efficiently to engineer these microorganisms, allowing cultivators to use organic carbon sources instead of photosynthesis from sunlight by introducing non-natural metabolic pathways into autotrophic microalgae.
Similarly, direct synthesis involves using ethanologenic microorganisms such as S.cerevisiae and Z.mobilis to produce high ethanol concentrations. These ethanologens are being introduced to the pentose catabolic pathway so that they can ferment 6-carbon sugar and 5-carbon sugar like arabinose and xylose, which will lead to more ethanol production.
As noted in this article, there is an increasing interest in the microbial fuel production of biodiesel and ethanol through synthetic biological techniques. The advancement in metabolic engineering and synthetic biology as the latest approach has been essential for developing new technologies to refine the microbial cell factories amenable to industrial application. The research cited in this article clearly states the potentiality of direct production of biofuel by microbes. Therefore investing significant time and effort will help develop a synthetic host for efficient target molecule production and open doors for future biofuel research.
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