Biotechnological Interventions for the Production of Glycerol-Free Biodiesel

Abstract

Advances in plant biotechnology and microbial genetics are speeding up because of the urgent need to provide a steady supply of resources. Growing cost of crude oil is having a negative impact on economies throughout the globe. Just biodiesel and bioethanol have been recognized as viable fossil fuel replacements. Chemical catalysis is primary way to synthesize biodiesel, besides enzymatic and microbial methods also play important role in biodiesel synthesis. These processes may play a significant part in the replacement of petroleum-based diesel in the future. The growth of sustainable, economically feasible biotechnological tools for the synthesis of biodiesel requires strong collaboration among several disciplines. In this age, lipases are the preferred enzymes for producing methyl esters (FAME), which are significant biological objects in biodiesel, from fatty acid esters (FAE) derived from fats and oils. It has also been shown that designed whole-cell microorganisms may directly produce FAE (MicroDiesel). The expensive cost of the biocatalyst continues to be a barrier to current enzymatic procedures, although advancements have recently been achieved, enabling the first synthetic enzymatic biodiesel synthesis. The fabrication of biodiesel which is enzymatic is primarily desirable due to the initial materials (waste frying oils, oils that were having high water content, etc.), where standard interesterification which is chemical is seldom applicable.

1. Introduction

As a result of climate change-causing greenhouse gas emissions, depleting oil reserves, and skyrocketing crude oil prices, the global community is increasingly turning to biological processes and green technologies to create technological compounds and alternative fuels from renewable resources. Advances in plant biotechnology and microbial genetics are speeding up because of the urgent need to provide a steady supply of resources. There is capability in biotechnology to manufacture a large number of these compounds from inexhaustible sources, at present virtually all the main bulk chemicals, with the exception of ethanol, are produced through the petrochemical approach [1]. Fermentation products typically have substantially smaller production quantities (less than one million tonnes per year) but higher pricing compared to petrochemical-based compounds [2]. However, bio-based processes offer the benefits of using renewable feedstock, producing environmentally friendly emissions, and operating at relatively moderate temperatures and pressures. Although biotechnology has shown its worth in the fabrication of fine chemicals like organic acids, vitamins, and medicinal compounds, there has been a rising push to optimise green technologies, which is slowly shifting the situation. For example, oil which is a fossil fuel much likely to run out first. Unrefined oil is a multi-component mixture that consists of around 50–95% hydrocarbons. Nearly all glasshouse gas releases are caused by the burning of fossil fuels. In addition to lowering pollution levels, cutting down on fossil fuel consumption would significantly decrease the quantity of carbon dioxide generated. Inquisitiveness in the maintainable generation of chemical raw materials and fuels has increased in light of the known scarcity of crude oil compared to the vast availability of biomass. The quantity of biofuel produced is massive and is anticipated to produce exponentially in the coming years. The growing cost of crude oil is having a negative impact on economies throughout the globe, especially those of the industrialised countries that rely heavily on the commodity. Over the last decade, consumers have seen a quadrupling in the price of essential fuels including natural gas, gasoline, and diesel. Even though the United States and Europe are a wide range of users of fossil fuels, these innovations will also help Asia’s rapidly expanding economy. As a result, there has been a rise in the study of potential substitutes for fossil fuels. Just biodiesel and bioethanol, however, have been widely recognised as viable fossil fuel replacements. When customers shop around for ways to save their energy bills, innovative technologies and efficiency improvements become hot commodities. Due to the dramatic changes in the cost of crude oil in recent months, various major oil importers have been actively looking for alternatives. More than only the oil shortage and environmental impact, biofuel’s significance cannot be overstated. Feedstock and fuels which are liquids derived from resources that can be renewed will allow us to tap into the vast, as-yet-untapped potential of agricultural and forestry waste products. Other significant arguments in favour of the biofuel alternative include its positive effect on worldwide climate, increased security related to the environment and economics, and practically better and improved goods. Environmental difficulty and political will are necessary, but they are not sufficient to usher in a sustainable age, which is nevertheless hampered by economic constraints. Until recently, biotechnology was seen by the chemical industries as an excessively costly high-tech technique that was not suitable for use on a commercial scale. The current research efforts of large chemical firms to create bulk compounds like 1,3-propanediol using biological approaches show that this perspective is gradually changing. Despite government support, bioethanol has become the biggest fermentation product in use today, demonstrating biotechnology’s potential for large-scale chemical production.

2. Bioethanol from glycerol as raw material in biodiesel production

Most delegates to the UN Climate Change Conference in Doha, Qatar, in December 2012 committed to even deeper cuts in carbon dioxide emissions and established the latest objectives and restrictions to be executed under the Kyoto Protocol Extension (2012–2020). In addition, the price of a barrel of Brent oil has risen as high as 87.19 Euros in the last year (www.indexmundi.com). As alternatives to fossil fuels, biofuels like bioethanol and biodiesel also help the environment. As a result, governments in many developed and developing nations are establishing and expanding research and policy initiatives to boost biofuels’ production and consumption. EU leaders have decided to prioritise biofuels in their quest to meet their renewable energy goal of 20% of total energy consumption by 2020. The European Commission has set a target of 10% of total fuel consumption in transportation by 2020. In contrast to fossil fuels, biodiesel may be replenished again and over again. It may be made using either animal or plant-based fats or oils. Since the inception of diesel engines in 1893, the potential of utilising vegetable oils as fuel has been recognised. Vegetable oil has the potential to be an operational substitute fuel oil, but its high viscosity prevents its usage in most conventional diesel engines. Several techniques exist for reducing the thickness of vegetable oils. Methods such as diluting, microemulsifying, pyrolysing, and transesterifying are used to lessen the thickness of a substance. Most biodiesel producers employ transesterification, a process that lowers oil viscosity. The chemical process by which oil is changed into its fatty ester is termed transesterification, although it is also known as alcoholysis. Triglycerides (1) are converted into ethyl esters of fatty acids (3) and glycerol (4) by the transesterification process, in which alcohol (ethanol or methanol) (2) is reacted with a catalytic base (1), (4) (Figure 1).

Figure 1.

Transesterification of triglycerides with alcohol.

Adding a catalyst speeds up and increases the yield of a reaction. Since the reaction might go either way, an excess of alcohol is utilised to tip the scales in favour of the product side of the equilibrium. An oil-splitting catalyst like NaOH or KOH and an alcohol-like methanol or ethanol are needed for the biodiesel process. Glycerol is the primary output. You may recognise this kind of crude glycerine by its dark colour and syrupy consistency. In contrast to petro-diesel, biodiesel is superior in many ways: flash point, sulphur content, biodegradability, and aromatic content [3]. Transesterification of vegetable oils typically employs homogeneous catalysts. Under moderate reaction conditions, base homogeneous catalysts like NaOH and KOH are the most effective. However, the reaction time for using acid homogenous catalysts is much higher. On the other hand, the price of the production of biodiesel is inflated by the need of treating the waste produced by homogeneous catalysts, which is itself difficult to recover. Biodiesel may be made from vegetable oils, and heterogeneous catalysts are a viable option for this process. Researchers are looking at the transesterification activity of several heterogeneous catalysts [4]. Heterogeneous catalysts have many advantages over their homogeneous counterparts, including the fact that they can be reused multiple times without degrading their performance and the ability to use an uninterrupted procedure without the need for additional decontamination steps, not to mention the possibility of being relatively inexpensive. Financial incentives for both producers and consumers are still needed to increase biofuel usage. In this way, many governments subsidise the biofuel industry via tax credits and other measures. In the beginning, the EU subsidised biofuels like bioethanol and biodiesel. The United States government offers tax breaks to domestic producers in the amount of $0.51 per gallon for bioethanol and $1.00 per gallon for biodiesel. Presently, Germany incentivizes the use of biodiesel by taxing it at a lower rate than regular fuel. The yearly fabrication and use of biodiesel in Germany exceeds 2.5 billion litres [5]. The usage of biodiesel has spread to other EU nations, often as an additive to petroleum fuel. However, using objectives to stimulate the future production and use of biofuels are significantly more essential than tariffs and subsidies. Spain’s Renewable Energy Plan (REP) 2011–2020 sets goals in conformity with the European Parliament’s Directive 2009/28/EC on the encouragement of the utilisation of energy from sources which can be renewed. The goal of the REP is to meet the target set by the EU Directive, which calls for the use of renewable sources to account for at least 20% of gross final energy exhaustion by the year 2020. The International Energy Agency (IEA) reports that in 2010, biofuels substituted for 2% of global oil production. The amazing expansion of renewable energy sources over the last several years may be attributed in large part to the government assistance provided under the Renewable Energy Plan 2005–2010 (Figure 2).

Figure 2.

The total energy used in 2010 (REP 2011/2022). The US and Brazilian markets account for the bulk of bioethanol sales, but biodiesel use has surged in the EU. At the end of 2010, Spain has more than 47 biodiesel facilities and four bioethanol plants with a combined installed production capacity of over 4 million tep (Per 2011/2020). The biofuel industry is experiencing a period of the technical revolution that is largely influencing the range of materials amenable to use in production processes. As such, the European Industrial Initiative on Bioenergy’s implementation strategy prioritises thermochemical and biochemical processes for material conversion. Rapid expansion is predicted for the biodiesel industry. Its popularity will rise steadily over the next decade, helped by new regulations requiring the labelling of some mixtures. From 2011 to 2020, bioethanol use is predicted to roughly treble. The anticipated increase is due in part to the elimination of gas price subsidies and the standardisation of the labelling of gasoline blends. However, fresh environmental and economic questions regarding the feasibility of alternative fuels have been raised due to the increasing demand for biofuel, in particular biodiesel.

3. Glycerol, from major commodity to waste effluent

Pure 1,2,3-propanediol (glycerol) is a colourless, odourless, hygroscopic, viscous liquid having the chemical formula OCH2CHOHCH2OH. It is a member of the alcohol family of organic compounds. The glycerol molecule’s space-filling model is seen in Figure 3.

Figure 3.

Space-filling model for glycerol molecule.

Glycerol interacts with inorganic and organic acids to produce ethers, aldehydes, esters, and numerous derived molecules because it has one secondary and two primary alcohol groups per molecule. When there are several alcohol groups present, it is easier to make polymers and coatings (polyesters, polyether, and alkyd resins). Glycerol is a versatile chemical that may be used in a wide variety of industries due to its solubility in water, its biocompatibility, its lack of toxicity when applied topically, and its legal status in the food and drug industries. Also, glycerol’s non-toxic qualities enable it for a wide variety of applications. It is safe to say that the cosmetics, explosives, food, pharmaceutical, polymer, and printing sectors are not the only ones that put this chemical component to good use (Figure 4). In the construction, automotive, and textile sectors, it is used to make gums and resins. In addition to its uses as a stabiliser in ice cream and a softening agent in baked products, mono and diglyceride emulsifiers also include this substance. As an added bonus, glycerol has several applications in the medical and pharmaceutical fields. It has also been put to use as a safe medium for freezing biological cells, which is a relatively new yet crucial.

Figure 4.

Principal glycerine utilisation in industry [6].

Chemical synthesis from petrochemical feedstock or microbial fermentation [7] is both viable methods for glycerol production. In the last 150 years, scientists have learned that glycerol may be made by microorganisms. During World War I, glycerol was manufactured in industries by microbial fermentation. Due to poor glycerol yields and the difficulties of extracting and purifying glycerol from broth, microbial synthesis eventually fell in favour of chemical synthesis from petrochemical feedstocks. At present, roughly 600,000 tonnes of glycerol are generated each year, and most of this comes from the saponification of lipids as a byproduct of soapmaking (Figure 5) [7]. The widespread use of detergents in industrialised countries has reduced the relevance of this step [8].

Figure 5.

Saponification of fats.

There are a number of ways to convert propylene into glycerol. Chlorinating propylene yields allyl chloride, which is oxidised with hypochlorite to form dichlorohydrin, which interacts with a robust base to yield epichlorohydrin, the most essential step in the epichlorohydrin process. Glycerol is produced once epichlorohydrin is hydrolysed. The oxidation or chlorination of propylene accounted for around 25% of 2001 global glycerol production in the chemical sector [7]. However, environmental concerns and the rising cost of propylene have caused this route’s popularity to decline. Glycerol is a byproduct of biodiesel production, therefore the rising popularity of this alternative fuel has led to a slump in the glycerol market and rendered the epichlorohydrin technique for glycerol synthesis unprofitable on a commercial scale. By 2020, it is expected that there would be a glycerol surplus of six times the yearly need. Glycerol, on the other hand, might be used as a substrate in emerging commercial fermentation processes. Bioconversion of glycerol has resulted in a wide variety of useful byproducts. chotIn particular, the microbial synthesis of 1,3-propanediol from glycerol has received a lot of attention because of the diol’s various potential uses in the creation of novel polymers. Only around 62–85% (w/w) of the crude stream is glycerol [9, 10]. Fats (soaps), water, methanol (often used in Europe) or ethanol (typically used in the United States) and catalyst leftovers make up the rest (salts). In addition to carbon, hydrogen, and oxygen, raw glycerol also includes trace amounts of elements including calcium, magnesium, phosphorus, and sulphur [10]. Producers of biodiesel employ a variety of feedstocks, each of which contributes a unique range of purity values. Raw glycerol levels were observed to be greatest (76.6%) in waste vegetable oil, with lower levels (62%) being produced by mustard, rapeseed, canola, crambe, and soybean. Raw glycerol is mostly accumulated by biodiesel factories, which create 10 g of glycerol for 100 g of biodiesel produced [11]. The concentration of raw glycerol is also attributable to the bioethanol sector. Common industrial methods provide 4 g of glycerol for every 48 g of ethanol [12]. Pure glycerol quantities are quite large and growing. Glycerol costs around $1200 per tonne in 2003. In 2006, the price per tonne was approximately $600 and declining [13]. Glycerine spot prices in Europe ranged from €260 to €350 per tonne in the month of November 2009. Even crude glycerol is no longer worth what it used to. US$0–$70 per tonne was the stated price range in 2006 [13]. Raw glycerol is worthless to those who manufacture it on a small basis. In these situations, the crude stream is often transported to a purification plant at the expense of the producers, disposed of in a landfill, or held in containers until a solution is sought. Crude glycerine is currently considered a contaminant that must be disposed of at a cost since it is unfit for most glycerol markets. The dumping of polluted waste glycerol from biodiesel production has caused environmental issues and driven down global glycerol market prices.

4. Method and approaches for glycerol-free biodiesel production

4.1 Emerging trends of microorganism in the biodiesel production

A variety of feedstocks (such as lipids, starch, and sugars) that may be utilised for the generation of biofuels are harvested and processed via the photosynthetic activities of plants (including cyanobacteria, algae, trees, grasses, and crops). Biofuels such as methane, ethanol, and diesel are already widely produced using already accepted “first generation” biofuel systems based on crop plants like sugar beet (Beta vulgaris), rapeseed (Brassica napus), sugarcane (Saccharum spp.), wheat (Triticum spp.), oil palm (Elaeis oleifera), soya beans (Glycine max), and corn (Zea mays). Pressure on food supply has resulted in increased worry and has sparked a heated “food versus fuel” debate, as a consequence of an expanding global population and significant droughts in key grain exporting countries (such as Australia). So, scientists are working on a new generation of land-free biofuel technologies. Most significantly, lignocellulosic methods are being developed to transform plant-based cellulose materials into liquid fuels. The most promising “non-food” plant possibilities for these methods include sorghum, miscanthus, camelina, switchgrass (Panicum virgatum), and poplar trees (Populus spp.). However, these systems can only be effective if scientists discover and implement energy-saving production techniques, such as enzymatic lignin digesting procedures (although chemical digestion techniques are also being investigated). Even if the resulting need for enzymes seems like a manageable obstacle, this technique may eventually add to food versus fuel dilemmas because of the existing reliance on appropriate land, most of which is already forested. Unless only waste products from existing agricultural or forestry systems are utilised, or feedstocks grown on non-arable land can be created, this might lead to a forest versus fuel dilemma.

The effective synthesis of starch, sugars, and oils by many microalgae makes them ideal feedstocks for the manufacture of biofuels such as biodiesel, ethanol, butanol, methane, and hydrogen. These microalgae may be cultivated in salt water. Microalgae may help with carbon capture because they take up carbon dioxide (CO₂) during growth from the atmosphere and, in certain circumstances, from industrial sources. To store carbon, the leftover waste biomass from fuel production may be pyrolysed to create a charcoal-like product (Biochar) that is stable over time. Biochar may replace coal as a fuel source, or it can be sold to the public as a soil amendment. While in principle microalgal biofuel systems may solve both the food versus fuel and the prospective forest versus fuel issues, no such system has yet reached commercial viability. In spite of widespread and overwhelming excitement, which Emily Waltz calls “algae ardour,” there are firms actively pushing these technologies towards commercial operation, as Waltz revealed recently. Investment in microalgal biofuels has actually increased after the first failure of a start-up rather than decreasing. This investment makes sense in light of recent economic case studies on standalone microalgal biofuel production models and on a model that co-produces high-value products (HVPs). Key economic determinants, such as building costs, biomass productivity, and cost of the dominating output, were found via sensitivity analysis in these models (and its production in the case of high-value co-products) (Figure 6).

Figure 6.

Conversion of raw material into ethanol by photosynthesis.

4.2 Biotechnological production of biodiesel fuel using biocatalysed transesterification

Under supercritical circumstances, Saka and Kusdiana found that methanol facilitated the transesterification of triglyceride (TG) into fatty acid methyl ester (FAME). It is well-known that three sequential reversible processes may be used to convert methanol into biodiesel. First, triglycerides are broken down into smaller molecules called diglycerides (DGs), and then the DGs are broken down further into monoglycerides (MGs). The last process is the transformation of MGs into glycerol. In every stage of the reaction, FAME is the end product. Transesterification results in the formation of three FAMEs, as seen in. The biodiesel generation in supercritical MTBE process is quite similar, consisting of three successive reversible reactions. Triglyceride combines with MTBE to form mono tert-butyl ether (DGE), and DGE then undergoes further reactions to form monoglyceride di tert-butyl ether (MGE). The end result of MGE reacting with MTBE is the production of FAME GTBE. Without the need for a catalyst, MTBE can transform TG into FAME. Although the supercritical MTBE approach operates at a higher temperature, its FAME yield is lower than that of the supercritical methanol route under identical reaction conditions. When utilising supercritical MTBE, however, the yield of FAME reaches 95.4 wt% after a 12-minute residence period, which is almost identical to that achieved using the supercritical methanol approach. Surprisingly, the supercritical MTBE approach has a larger FAME yield than the supercritical methyl acetate route. Since MTBE is less polar than methyl acetate, it is more reactive. Thus, at ambient temperature and pressure, MTBE is more miscible with oil than methyl acetate. In this case, MTBE’s miscibility helps it get around the mass transfer issue.

4.3 Glycerol-free biodiesel using methyl acetate as acyl acceptor with bio-enzymes

Using enzymes as the biocatalyst might potentially reduce downstream separation costs since they eliminate the requirement for a separate solvent. Novozyme 435 has been used in almost all published reports of enzymes for synthesis utilising dimethyl carbonate as an acyl acceptor, either as a free enzyme in solution or inactive on a poly-acrylic macro-porous substrate. Novozymes 435 includes lipase B from Candida antarctica, which is an approximate lipase. The summary of recently published research on the use of dimethyl carbonate to catalyse the enzymatic transesterification of vegetable oil is given here (Figure 7, Table 1).

Figure 7.

The use of dimethyl carbonate to catalyse the enzymatic transesterification of vegetable oil.

Feedstock	Catalyst	Catalyst loading (g/g oil)	Temperature (°C)	Molar ratio (Oil:DMC)	Time (min)	Yield (wt.% diesel/oil)
Soybean oil	Various	5%	90°C	1:3.7	300	N 99.5
Refined soybean oil	KOCH3	5%	90°C	1:9	300	7.6
			200°C (10 bar)		60	95.8
Refined canola oil	Triazabicyclo[4.4.0]dec-5-ene	2.5%	60°C	1:3	360	100%
Cotton seed oil	Lipase (Novozymes 435)	15% in petroleum ether	40°C	1:4.5	1440	∽93
Waste cooking oil	Immobilised novozymes 435	10%	60°C	1:6	120	∽87
Algae oil extracted from Chlorella sp.	Novozymes 435	50% in DMC	60°C	n/a	360	75.5
Waste cooking oil	Immobilised novozymes 435	20%	40°C	1:6	200	86.61
Purified soybean oil	Immobilised novozymes 435	20%	60°C	1:10	2880	96.4
Purified soybean oil	Immobilised novozymes 435	100 g/L in t-butanol	60°C	1:6	2880	84.9
Corn oil	Novozymes 435	10%	60°C	1:10	900	94
Palm oil	Immobilised novozymes 435	20%	55°C	1:10	1440	90.5
Chlorella sp. KR-1	Novozymes 435	20%	70°C	1:10	1440	36.7

Table 1.

Recent research on dimethyl carbonate as an acyl acceptor in non-supercritical biodiesel manufacturing.

4.4 Glycerol-free biodiesel using dimethyl carbonate as acyl acceptor with bio-enzymes

The environmental friendliness, safe for industrial use (low pressure and temperature is needed), low cost of feedstock (glycerol or glucose), and high theoretical molar yield of the biological conversion of glycerol to 1,3-propanediol are making it a more viable option than the chemical production approach. The sustainability of 1,3-propanediol’s biotechnological production is enhanced by the fact that it may be made from a renewable resource. Although the fermentation by bacteria that converts glycerol to 1,3-propanediol has been investigated for about 120 years, its biotechnological potential has just been recognised and further study has only begun after 1990 [2]. Multiple microbial families have been documented as producing 1,3-propanediol since 1996. Klebsiella, Enterobacter [14], Clostridium butyricum, and Klebsiella pneumoniae stand out as the most promising producers due to their high substrate tolerance, abundant output, and high rates of production. Dual routes ferment glycerol through a dismutation mechanism. One of these routes involves the enzyme glycerol dehydrogenase converting glycerol to dihydroxyacetone, which then goes through conventional glycolysis to generate pyruvate and may be further broken down into a variety of compounds such as acids and alcohols. The second process requires the reduction of 3-hydroxypropionaldehyde (3-HPA) to 1,3-propanediol through the expenditure of decreasing power NADH2 and the catalytic action of 1,3-propanediol:NAD oxidoreductase. This reduction requires coenzyme B12. Glycerol dehydratase is a critical limiting enzyme in the second metabolic pathway, which converts glycerol to 1,3-propanediol and is essential for cellular redox homeostasis. Theoretically, glycerol’s anaerobic fermentation yields its greatest potential when acetate is the sole by-product [15, 16, 17]. The creation of waste substances not only limits the quantity of carbon accessible, but may also stifle the development of microbes.

Since acetic acid is necessary for the NADH2 process, the generation of 1,3-propanediol is reduced in tandem with the formation of all these other byproducts, especially ethanol and butanol. The bioconversion of glycerol to 1,3-propanediol has gained popularity since glycerol might sometimes be a surplus product. In the past 10 years, a lot of work has gone into making this procedure more efficient and raising the yield response. Research into genetically engineered strains for 1,3-propanediol synthesis has been conducted in an effort to boost the efficiency of naturally occurring producers during glycerol fermentations. Another issue with genetically modified microorganisms is that it is difficult to conduct fermentations on an industrial level with these strains since they are so sensitive and fragile. If petroleum supplies are depleted, biotechnology might be the key to continuing 1,3-propanediol manufacturing [18].

4.5 Bioconversion of glycerol by C. butyricum

The age of Clostridium, a kind of bacterium, is estimated to be about 2700 million years. This organism predated Earth’s oxygen atmosphere, which is why Clostridium species are oxygen-sensitive. For almost 60 years, scientists have known that anaerobic bacteria, including Clostridium, may ferment glycerol into 1,3-propanediol. All member microorganisms in this genus have rod-like shape that is Gram-positive, moderately big, heterotrophic, endospore producing, and motile. Some are psychotropic or thermopholic, but mesophilia is the norm for the vast majority. Clostridium thrives in anaerobic settings rich in organic resources. Clostridia bacteria are present everywhere in the environment and may be discovered in a diversity of habitats, including soils, feed, aquatic sediments, and the digestive systems of humans and animals. They can remain alive for extended periods of time in hostile environments because of their spore-forming abilities. As long as they are kept in a medium that provides them with food and the right temperature and humidity, a single bacterial strain may proliferate. Among the roughly 100 species that make up the genus Clostridium are both common, free-living bacteria, and serious diseases. Clostridium produces a wide variety of extracellular enzymes, which contribute to their robust metabolic activity. Sugars may be fermented by these bacteria, leading to the production of hydrogen and organic molecules such as organic acids (particularly acetic acids and butyric), acetone, and butanol. Clostridium produces offensive-smelling breakdown products during the metabolism of amino and fatty acids [19]. Additionally, Clostridia can break down a variety of hazardous compounds and create chiral products, both of which need much effort to get by chemical synthesis. There are several distinct enzymes for breaking down starch and hemicellulose, and they have been found in many non-identical types of bacteria. Clostridium thermocellum is a prototypical cellulolytic Clostridium, producing a multienzyme cellulase complex that can break down cellulose, hemicellulose, and starch [20]. Using clostridial toxins and spores to treat human illness is a great advancement. Dystonias, involuntary muscular problems, pain, and other neurological conditions are treated with botulinum neurotoxin. Therapeutics are being developed to be delivered to tumours using Clostridia spore systems [21]. Clostridia have very basic nutritional needs. Typically, a complex nitrogen supply is necessary for optimal growth and solvent synthesis. There is a good deal of potential commercial interest in the non-pathogenic Clostridia. C. butyricum produces 1,3-PD and other byproducts during the fermentation of glycerol. C. butyricum’s anaerobic fermentation metabolic pathways produced by-products. These byproducts have a negative impact on C. butyricum development because they deplete the available carbon source (Figure 8).

Figure 8.

Pathway of conversion of biomass into biodiesel.

5. Conclusion

Advances in plant biotechnology and microbial genetics are speeding up because of the urgent need to provide a steady supply of resources. The growing cost of crude oil is having a negative impact on economies throughout the globe. Cutting down on fossil fuel consumption would significantly decrease the quantity of carbon dioxide generated. Just biodiesel and bioethanol have been recognised as viable fossil fuel replacements. Biofuel’s significance cannot be overstated.

Feedstock and fuels which are liquids derived from resources that can be renewed will allow us to tap into the vast, as-yet-untapped potential of agricultural and forestry waste products. Bioethanol has become the biggest fermentation product in use today, demonstrating biotechnology’s potential for large-scale chemical production. Environmental difficulty and political will are necessary but not sufficient to usher in a sustainable age, hampered by economic constraints. The price of a barrel of Brent oil has risen as high as 87.19 Euros in the last year. Biofuels like bioethanol and biodiesel also help the environment. EU leaders have decided to prioritise biofuels in their quest to meet their renewable energy goal of 20% of total energy consumption by 2020. Biodiesel is superior to petro-diesel in many ways: flash point, sulphur content, biodegradability, and aromatic content. Transesterification of vegetable oils typically employs homogeneous catalysts, but heterogeneous ones are a viable option for this process.

Financial incentives for both producers and consumers are still needed to increase biofuel usage. The yearly fabrication and use of biodiesel in Germany exceed 2.5 billion litres. The biofuel industry is experiencing a period of technical revolution that is largely influencing the range of materials amenable to use in production processes. The US and Brazilian markets account for the bulk of bioethanol sales, but biodiesel use has surged in the EU. Biodiesel use is predicted to rise steadily over the next decade, helped by new regulations requiring the labelling of some mixtures.

Glycerol is a colourless, odourless, hygroscopic, and viscous liquid with the chemical formula OCH2CHOHCH2OH. It is a versatile chemical that may be used in a wide variety of industries due to its solubility in water and its lack of toxicity when applied topically. In the construction, automotive, and textile sectors, it is used to make gums and resins.