Research into alternative energy sources has been a more relevant issue as petroleum will be limited in the future. Finding a suitable replacement that is close enough to petroleum is extremely difficult. Ethanol from corn grain and biodiesel from soybeans have been particularly successful. However, as one study states: Ethanol yields 25% more energy than the energy invested in its production, whereas biodiesel yields 93% more. Compared with ethanol, biodiesel releases just 1. 0%, 8. %, and 13% of the agricultural nitrogen, phosphorus, and pesticide pollutants, respectively, per net energy gain. Relative to the fossil fuels they displace, greenhouse gas emissions are reduced 12% by the production and combustion of ethanol and 41% by biodiesel. Biodiesel also releases less air pollutants per net energy gain than ethanol. (Hill) It is clear from this research that biodiesel is the much more efficient and clean energy source. It must still be considered that there may be times in the future where sources of energy become limited, and so all resources must be accounted for.
In a study by Dr. Knothe, frying oils used, before and after cooking in restaurants, were tested for their fatty acid profile using gas chromatography and proton nuclear magnetic resonance spectroscopy (Knothe). This study was invalidated by the study by Canakci stating, “Using food-grade vegetable oils is not economically feasible since they are more expensive than diesel fuel” (Canakci). According to Dr. Canakci, “the main obstacle for commercialization of biodiesel is its high cost. Nonetheless, both Knothe and Canakci focused on aims of conserving energy and waste by considering potential feedstocks such as cooking oils, restaurant greases, soapstocks and animal fats for the production of biodiesel. Rising energy costs and apprehension over global and environmental damage have prompted research for producing sustainable renewable fuels and chemicals. It has been found that using microbial conversion of sustainable, mass produced feedstocks, particularly biomass carbohydrates, has the prospect of being a cost efficient and high energy fuel source.
It is necessary to begin taking proactive steps to find alternative sources of energy that are both more environmentally friendly as well as efficient. The growing shortage of fossil fuel deposits has placed urgency on this research. Although the production of fuels from the processing of fatty acids from plant and animal oils has been in use since the beginning of the 20th century, the need for new sources of energy has been a relatively contemporary issue. A wide range of more modern chemical products are in demand.
With this rise in demand have come a more competitive and higher priced food market as well as issues with environmental consequences and controversial land usage. The most efficient solution to this problem is the development of a renewable energy source by a fermentation process that will be both economical and lasting. Fatty acids consist of long alkyl chains and are the basis of most fuels. Fatty acids are a primary metabolite that provide chemical and energy storage for cells. E. coli is a microorganism that can easily be mass produced and can be used, through fermentation, to produce fatty acid metabolites at a rate of 0. g l-1 h-1. E. coli consists of about 9. 7% lipid and generates a product-dependent mass of 30-35%. E. coli, therefore, can be used to synthetically redirect fatty acid metabolism to produce fuel and other necessary chemicals.
Fatty acyl-ACP is produced from microbial fatty acid biosynthesis and is used by cells as structural or storage lipids. However, fatty acid biosynthesis is impeded by the buildup of fatty acyl-ACP feedback. Cytoplasmic thioesterase has the function of hydrolyzing the acyl-ACPs, deregulating fatty acid biosynthesis, and overproducing and secreting high levels of free fatty acids. E. oli thioesterase, or tesA, can be manipulated in cytosolic expression to yield a free fatty acid production of about 0. 32 gl-1. tesA targets in particular C14 fatty acyl ACPs, but also a wider range of free fatty acids. By manipulating other thioesterases from plants, the lengths of the fatty acid chains can be managed. Eradicating enzymes related to ?-oxidation, such as FadD and FadE, raised the titre to about 1. 2 g l-1, which is three to four times the production level of free fatty acid. From 2% glucose in shake flasks, the TesA-?fadE produced a 6% yield of fatty acid which comes to be about 14% of the theoretical limit.
Free fatty acids are important chemicals, but the more urgent necessity is the production of biodiesel. Biodiesel is a compatible and sustainable alternative to diesel. Although gasoline supplements have been at the forefront of microbial production of biofuels, diesel has a growth rate three times faster than that of gasoline. More than 2 billion gallons of biodiesel, produced through the chemical transesterification of plant and animal oils, are used each year. Biodiesel consists of fatty acid methyl esters, FAMEs, and fatty acid ethyl esters, FAEEs.
As fatty acids cannot reasonably be used commercially, glucose and ethanol are used to produce FAEEs through the biosynthesis of E. coli. AtfA, which encodes for a wax-ester synthase combined with the expression of fadD and tesA with 2% ethanol yielded about 400 mg l-1 of FAEEs in 48 hours. The first two steps of fatty acid degradation, the overexpression of fadD and deletion of fadE, has a noticeable impact on FAEE production. Other chemicals such as fatty alcohols, aldehydes, and wax esters are used in making soaps, detergents, cosmetic additives, pheromones, flavoring compounds, and in limited circumstances, biofuels.
There is a huge market for these products valued at $1,500 per ton in a $3 billion market. These products are developed through the hydrogenation of FAMEs developed from FAMEs as well as the combination of petrochemical precursors. Acr1 from Acinetobactercalcoaceticus BD413 encodes the expression of fatty alcohol-forming fatty acyl-CoA reductases. The expression of acr1 yielded medium chain fatty alcohols at or below 60 mg l-1. FadD contains less alcohol than FAEE pointing Acr1 as a limiting factor in the slightly improved production that FadD provides.
The cetane number and melting points of fuel and chemicals have a direct relationship to the chain length and saturation of the fatty acid. By modifying fatty ester composition, the effectiveness of certain plant derived biodiesels can be enhanced. To enhance the FAEE and fatty alcohol chain length composition, a replacement of tesA in the FAEE and fatty alcohol strains with plant genes programmed for thioesterases that choose more different chain length fatty acyl-ACPs. Thus, the FAEE and fatty alcohol chain lengths can be controlled though the genetic treatment of fatty acids.
In this way, the appropriate fuels and chemicals can be produced to match the structure and efficiency of production. It is extremely efficient to be able to perform microbial catalysis on the complicated set of biosynthetic pathways and place this in a single cell. This development is effective and reduces waste and abridges the overall process. It was necessary to prove that FAEEs are produced by feeding ethanol. A FAEE-producing strain was created to construct ethanol through the expression of Zymomonasmobilis genes pdc and adhB. The pdc calls or pyruvate decarboxylase while the adhB encodes alcohol dehydrogenase. About 5 g l-1 ethanol was generated after 24 hours of the expression of pdc and adhB from E. coli. When provided with a nominal biosynthetic pathway of FAEE, the strain that produces ethanol is limited to a production of FAEEs of 37 mg l-1.
This was due to the restricted production of native acyl-CoA ligase. FAA2, which is an acyl-CoA ligase gave a 250% boost in the production of FAEE generating 96 mg l-1. An additional 250% boost yielding 233 mg l-1 occurred with the overexpression of an E. oli-derived fadD. AtfA was expressed with an additional copy giving 427 mg l-1 FAEE. A technique implemented was covering dodecane in an organic phase to attempt to inhibit FAEE evaporation. The E. coli produced 674 mg l-1 FAEE equaling 9. 4% of the theoretical yield. It was found that up to 100 g l-1 did not restrict FAEE growth. Thus, joining the processes for ethanol and FAEE biosynthesis, and using glucose as the only source of carbon, was a very efficient method of producing FAEE. Another experiment was run where an E. coli strain expressed tesA, fadD, mFarl, and atfA.
These generated a collection of wax esters from glucose. It will take more research and time for plans for biochemical processing of cellulosic biomass to come to fruition. It is still an expensive method as the enzymes needed to free sugars are costly. FAEEs and fatty alcohols are much more efficient sources of renewable energy over the previous fuels like corn ethanol and plant oil biodiesel. It is helpful that the bioprocessing in a biofuel producing organism can be merged to generate glycosyl hydrolases. This benefit is more cost efficient and no further enzymes are needed in the biochemical process.
More techniques have developed to further increase the efficiency and tediousness of these processes. Hemicellulases were merged on the amino termini to OsmY, an E. coli protein. This process allowed the hemicelluloses to hydrolyze into zylose to be transported and metabolized by the E. coli. The E. coli was able to produce hemicelluloses because of the OsmY-xylanase fusions. This allowed the E. coli to grow without the addition of enzymes. The particular strains DH1 and C41 (DE3) of E. coli were wild strains used in the study, and artificial fadD and fadE in DH1 were enacted. The plasmids sed were assembled using the standard molecular biology methods. The growth and production of the strains was maintained in flasks at 37 °C in M9 medium that was supported by 50 mg l-1 ampicillin, 20 mg l-1 chloroamphenicol, 5 mg l-1 tetracycline, 100 mg l-1carbenicillin, 100 mg l-1 spectinomycin, and 50 mg l-1 kanamycin. Using 1mM isopropylthiogalactoside with a wavelength of 600 nm allowed the pathways to be initiated. During the tests for thioesterase, 2% ethanol at 25°C allowed for the production of FAEEs. The metabolites produced were found and measured using gas chromatography and mass spectrometry.
An additional experiment to supplement those performed in this study could be to test the environmental safety of biofuels. According to Charles Stillman, writer for AlternativeEnergyNews, the biofuel industry has been banned in Texas for the harmful effects it has on the environment. The biodiesel used in Texas has been found to exceed the nitrogen oxide limits deemed hazardous by the Texas Commission on Environmental Quality. Texas at that point was the country’s largest producer of biodiesel, yet the nitrogen oxide emissions have contributed to the formation of ground-level ozone in Eastern Texas.
For the additional experiment, it would be helpful to test the nitrogen oxide levels contained in the final product of biodiesel generated through microbial production. A thorough approach should be taken to guarantee that the levels of toxins would not exceed the limits placed by the government. If the problem of environmental safety is not addressed, the efforts to make a sustainable, renewable, and cost-efficient fuel would be in vain. The future of biofuels is very open to development and further research. It is a fascinating and prospectively very profitable field of research.
Already, there is a large market for biofuels and the particular technique of using microbe production to generate fuels and chemicals from plant biomass has a very promising future. In the next few years, the use of biofuels will probably be a priority for the United States. The efficiency and further safety of these products will be improved upon. Published works on this topic might include effects that biofuels have on the environment as well as other methods of producing biofuels that will be more efficient and profitable.
One of the principal researchers of this article, Jay D. Keasling, is a Professor of Chemical Engineering and Bioengineering at the University of California, Berkeley. Raised in Harvard, Nebraska, he completed his undergrad at the University of Nebraska-Lincoln and received his Ph. D from the University of Michigan in 1991. He is the Acting Deputy Laboratory Director of the Lawrence Berkeley National Laboratory, the Founding Director of the Synthetic Biology Department at UC Berkeley, and CEO of the Joint BioEnergy Institute.
He is at the forefront in research for synthetic biology, particularly in metabolic engineering as well as systems biology and environmental biotechnology. He is currently researching the process of metabolism in E. coli to make the anti-malarial drug artemisinin. Keasling received the first annual Biotech Humanitarian Award by the Biotechnology Industry Organization in 2009. He received a large shared grant of $42. 5 million grant to the Keasling Lab, Amyris Biotechnologies, and the Institute for OneWorld Health from the Bill and Melinda Gates Foundation. It was awarded to fund his efforts develop low-cost malaria treatment.
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