Microbial Biofuel Mechanisms and Diversity
By Owen McCloskey
Introduction
“The fuel of the future is going to come from fruit like that sumac out by the road, or from apples, weeds, sawdust – almost anything. There is fuel in every bit of vegetable matter that can be fermented.” – Henry Ford, 1925
Henry Ford was an innovator who had his eye set on the future, and as the search for fossil fuel alternatives continues, scientists are being to find that there is potential fuel in vegetable matter. As the green initiative expands, an emerging and developing topic in biology has come to the front. Microbial biofuels are a field of research that is expanding and reaching depths never before reached. Biofuels can be produced in a number of ways originating from living organisms or metabolic byproducts and contain over 80 percent renewable materials. The metabolic byproducts important in biofuel product are organic and food waste products. Biofuels provide many positives and are essential in shifting away from the usage of fossil fuels. They provide a high amount of energy security in that they are constantly available and sustainable which allows for a solid supply of affordable energy for consumers and industry. Countries that depend largely on the importation of fossil fuels could be weaned off with the idea of producing biofuels locally. Additionally, in developing countries, the concept of biofuel production is a very attractive option because a high percentage of the population is involved in agriculture, thus the development of biofuels can provide new sources of income for current farmers as well as creating new agricultural jobs. For this reason, biofuels could be an attractive option for governments to pursue to further economic development. Lastly, the continued expansion of biofuels should help reduce greenhouse gas admissions.
As research becomes more and more highly diversified, new studies have come to the forefront that show many more microbes than originally thought are cable of producing biofuel. Bioethanol, for example, is the most common biofuel, and is produced from plants by microbes such as Saccharomyces cerevisiae (commonly yeast).
Ethical Issues
Before delving deeper into the mechanisms involved with the production of biofuels, it is important to highlight the ethical issues that include the violation of human rights, solidarity, sustainability, stewardship, and justice. In an April 2011 report titled Biofuels: ethical issues, published by the Nuffield Council on Bioethics, two important ethical principles were established to allow policy makers to evaluate biofuel technologies and guide ethical development. These principles state that “Biofuels should be environmentally sustainable” and “Biofuels should contribute to a net reduction of total greenhouse gas emissions and not exacerbate global climate change”[7]. These two principles are keys to continue the progress of a greener and more sustainable 21st century while avoiding many ethical issues that could arise. By making sure the biofuels are environmentally sustainable and contribute to a net reduction of total greenhouse gas emissions, the Council has established guidelines for the near future that could lead to great reductions in the amount of pollution on Earth[7]. For example, new types of biofuels, such as lignocellulosic and algal biofuels, could vastly reduce greenhouse emissions, although commercial-scale production of those biofuels is still years away.
Looking to the future, the Council is urging further research on the economic and social impacts of rights and property pertaining to the development of microbial biofuels in order to ensure that violations of human rights and solidarity are minimized as much as possible. To further encourage sustainability and economic growth, the Council urged that initiatives should be instituted that should encourage local and small-scale biofuel production. These initiatives would be directed at fuel-deficient developing countries to possibly help alleviate some of the fuel poverty. Lastly, looking deeper into the future, the Council is encouraging policy makers to somehow make it possible that biofuel production consumes less land and resources by the development of policies that will incentivize the research and development of new biofuel technologies.
Ethanol Production
via Sugar Monomers
The most widespread and tradition method for the production of ethanol is that of utilizing pyruvate broken down from glucose by glycolysis and ethanol, or alcoholic, fermentation. Ethanol fermentation is an anaerobic process that allows cells to continue producing energy in the absence of oxygen.
Saccharomyces cerevisiae
S. cervisiae is a eukaryotic, globular-shaped fungus commonly known as yeast. The natural strains of this fungus have a vast array of livable environments ranging from the surfaces and gastrointestinal tracts of insects and other warm-blooded animals, in soils, and on plants all around the world.
Ethanol fermentation in S. cerevisiae is mainly done via the metabolic, EMP (Embden-Meyerhof-Parnas) pathway, commonly known as glycolysis, and involves the metabolization of one molecule of glucose to produce two molecules of pyruvate, which then produces two molecules of ethanol. With the release of CO2, pyruvate can be reduced to ethanol under anaerobic conditions[5].
Outside of ethanol and CO2, there are many byproducts of ethanol fermentation, with glycerol being the main one. In much smaller quantities, organic acids and higher alcohols are produced. The production of these byproducts combined with the growth of yeast cells decrease the percent yield of ethanol from glucose. This is because some intermediates will inevitably be directed to other metabolic pathways preventing them from continuing glycolysis until they are reduced to pyruvate. The removal of the intermediates means that on average in the industry ethanol yield based on sugar feeding into the system can only be as high as 90-93% efficiency[5].
Ethanol yield can also be reduced by the various stresses that the cells suffer from during ethanol fermentation. Environmental stresses that could arise include nutrient deficiency, high temperatures, and contamination. Internal stresses could arise within the yeast cell metabolism, for example, ethanol accumulation has an inhibitory affect on yeast cell growth and ethanol production, so as more yeast accumulates the lower yield per theoretical weight of glucose.
Escherichia coli
E. coli is a proteobacteria that prefers to live in high temperature environments. Similarly to S. cerevisiae, E. coli has the ability to live and thrive in many environments including animal feces, the lower intestines of mammals, and at the edges of hot springs.
The production of ethanol in E. coli is done very similarly to Saccharomyces cerevisiae in that glucose is metabolized to pyruvate via the glycolysis pathway, and then undergoes the process of ethanol fermentation. It is important to note that a net of 2 ATP are produced in glycolysis for both aforementioned microbes because 2 ATP are consumed in the early steps of glycolysis as an energy input is required for glycolysis to occur.
E. coli and S. cerevisiae require two steps to produce ethanol from pyruvate, with the first reaction being to release the CoA to form acetaldehyde. The same enzyme, alcohol dehydrogenase, then catalyzes the reduction of acetaldehyde into ethanol. In each, theoretically 2 molecules of ethanol are produced per glucose original molecule.
Zymomonas mobilis
Similarly to E. coli, Z. mobilis is a proteobacteria, but the habitat of this bacterium is very unique and interesting because it is found in sugar rich plant saps. Also, it can be isolated from sugar cane and some alcoholic beverages.
Differing from both S. cerevisiae and E. coli, Zymomonas mobilis utilizes the Entner-Doudoroff (ED) pathway to produce ethanol. Two enzymes, pyruvate decarboxylase and alcohol dehydrogenase, are key to produce ethanol from pyruvate in Z. mobiilis. Pyruvate decarboxylase reduces pyruvate into acetaldehyde and a CO2 molecule. Alcohol dehydrogenase then reduces the acetaldehyde into ethanol[3].
The ED pathway produces only 1 ATP molecule per glucose; whereas, glycolysis produces 2 molecules of ATP. As a result of using the ED pathway, Z. mobilis produces less biomass to produce pyruvate which allows for more carbon to be funneled into ethanol fermentation. As a result of this, the ethanol yield from glucose of Z. mobilis had been reported to be near theoretical levels, possibly as high as 97%[3].
Despite the advantages and higher yields of ethanol, Z. mobilis is not suitable for industrial ethanol production for a few reasons. First of note, is that unlike laboratory environment, the ethanol fermentation industry cannot use pure glucose as its raw material. This is important because Z. mobilis cannot form ethanol from anything but monosaccharides, which means the starches the ethanol fermentation industry utilizes will not be effectively broken down by this species. Z. mobilis not just able to utilize glucose, but also the other monosaccharides fructose and sucrose, but growth by sucrose feed is accompanied by subsequent formation of fructose oligomers and sorbitol, which significantly decrease the ethanol yield[3]. Also of note is the deposal of the biomass, as Z. mobilis biomass is not commonly acceptable to use as animal feed, unlike S. cerevisiae which is commonly accepted for animal feed, so disposal poses a problem.
Moving forward, of the 3 aforementioned microbes utilizing glucose for ethanol fermentation, Saccharomyces cerevisiae seems to be the logical industry leader for the near future. With ethanol being the most highly produced biofuel, alternatives to S. cerevisiae that increase the percent yield could very well be on the horizon .Interestingly, as research into alternate means of ethanol production continues, other microbes have been shown to produce biofuels.
via Cellulose
Cellulose ethanols are produced from lignocelluloses. Lignocellulose is plant biomass that accumulates to 200 billion metric tons worldwide and is a mixture of lignin, hemicellulose, and cellulose, and together these three materials form the cell walls of plants. The carbohydrate polymers (cellulose and hemicellulose) are tightly bound to lignin and together they give a plant its rigidity. The production of ethanol from lignocellulose is highly advantageous to that from other sources in that the raw material is not only diverse, but abdundant around the globe. The only drawback is that the cellulose requires greater processing before becoming available to the microbes as usable sugar monomers such as glucose. Due to their high density around the world, the biomass from Switchgrass and Miscanthus are the major lignocelluloses used in studies today.
Looking into the uses of lignocelluloses in ethanol production, scientists have focused their study on microbes containing cellulase enzymes that can break down hemicellulose and cellulose into its monosaccharide glucose which can then be used in anaerobic conditions to product ethanol. It is important to note that commercial production of ethanol via the breakdown of cellulose is expected within the next few years.
Sulfolobus sulfataricus
S. sulfataricus is a crenarchaeota that requires a low pH and a constant presence of environmental sulfur, as a result of this S. sulfataricus grows in volcanic hot springs.
The usage of Sulfolobus enzymes in cellulose degradation has been shown to have many advantages including economical degradation of cellulose that can be readily brought to the industrial scale, an increased function at higher temperatures as well as an increased function in a highly acidic environment. The enzymes of Sulfolobus family microbes are essential in the conversion of lignocellulosic biomass into fermentable sugars, which is a key step in the production of second-generation biofuels. A gene sequence encoding a putative extracellular endoglucanase (sso1354) was identified as the gene necessary to produce cellulase in S. sulfataricus[2]. The enzyme was shown to be endowed with endo-beta-1-4-glucanase activity and specifically to hydrolyze cellulose[2]. Researchers have attempted to genetically modify and include the sso1354 gene in main other microbes, with the focus being mainly on incorporating this gene into E. coli.
If the gene could be successfully incorporated into E. coli, the fitness of E. coli could increase immensely to the point where it may be the industrial leader in the production of ethanol.
Trichoderma reesei
Trichoderma species are the most culturable fungus that is frequently isolated from forest and agricultural soils in most environments.
T. reesei contains the main enzymes necessary for cellulose degradation, with those being cellulases and hemicellulases. This species first degrades the crystalline cellulose structure with the use of endoglucanases and cellobiohydrolases. Cellobiohydrolases and endoglucanases work in tandem to start the breakdown of cellulose. Endoglucanases hydrolyze and cleave internal cellulose bonds randomly increasing accessibility for cellobiohydrolases to attach to the free cellulose ends and liberate the glucoses from the long chains[6]. Swollenin is another protein that aids in the degradation of cellulose by disrupting the crystalline structure much like endoglycanases, allowing cellobiohydrolases to bind. T. reesei has two CBH enzymes, with CEL6A cleaving the non-reducing end of the cellulose chain and CEL7A cleaving the reducing end[6].
Once cleaved into cellodextrins and cellobioses, b-glucosidases degrade them further into D-glucose. At this point, the d-glucose is suitable to undergo glycolysis and ethanol fermentation to produce bioethanol.
Gene Manipulation of E. coli
Synthetic biology and gene manipulation are of importance in making E. coli useful in producing biofuel at a greater rate and efficiency. E. coli is the model organism many researchers use to try and place certain beneficial genes from other microbes into a user-friendly host. A specific example involves incorporating the genes necessary to produce isopropanol and n-butanol from the acetone pathway. Isopropanol and n-butanol are of importance because petroleum is used to make isopropanol and n-butanol has an energy content similar to that of gasoline[]. These advanced biofuels offer significant advantages over ethanol; for example,n-butanol's vapor pressure is roughly 11 times left than that of ethanol, explosion[8]. which greatly decreases risks of instability and By eliminating the use of petroleum we can conserve fuel, and through the utilization of n-butanol we could completely replace the use of gasoline with a more sustainable fuel. Not to mention, the reduction of gasoline use would lower greenhouse emissions, while conserving the environment at the same time.
To achieve high-efficiency production, the incorporation of the genes to produce isopropanol and n-butanol are isolated from Clostridium. The result of utilizing E. coli as a host cell resulted in a production rate of acetone and isopropanol at levels much higher than native Clostridium strains. The same was not the case with n-butanol, very low levels were detected [8]. Initially, this may be seen as a failure, but the results were at least slightly encouraging in that they demonstrated it would be possible to produce n-butanol in vitro.
Algal Biofuels
Looking to the future, the great diversity of aquatic microorganisms that could be used for biofuels is encouraging. It is common knowledge that oil can be extracted from algae and used as fuel, but the extraction of the oil does not immediately mean it can be utilized as such. After extraction, the oil must undergo transesterification, which includes the addition of alcohol and a chemical catalyst. The reaction that occurs as a result of these additives leads to the creation of a mixture of biodiesel and glycerol. To fully harness the effectiveness of the biodiesel, the glycerol must then removed from the mixture.
Many algal species are currently being tested to assess how suitable they would be to produce the biodiesel on an industrial scale. The unique combinations of fatty acids within each algal species influence the quality of the resulting biodiesel, so it is important to see which unique fatty acids result in the highest quality biodiesel. Botryococcus braunii, Chlorella, Dunaliella tertiolecta, Gracilaria, Pleurochrysis carterae, and Sargassum are a sampling of the species currently undergoing study. Of these species, initial results point to Botryococcus braunii being the choice for industrial scale production having the highest oil yield at 75%, but a risk arises because the fluctuation of oil yield can produce results as low as 29%[1]. The outcome is so scattered because the process has yet to be perfected or fully understood, but this is an actively expanding area of research with mainly large US universities actively taking part.
Future Biofuel Prospects
To tie everything together, we have ranged from widespread currently used microbes to possible future alternatives to options within each realm. As if currently stands, ethanol is the most commonly produced and used biofuel, and will be for the near future, but the idea of biofuels produced from lignocellulose if harnessed could quickly replace ethanol as the most common biofuel. This stems from the fact that there is a great density of lignocellulose all around the world that could be used to produce "green energy". The carbon footprint of ethanol production from lignocellulose, as well as glucose, is far smaller than that from fossil fuels. If the use of lignocellulose is harnessed, the current energy crisis could be stabilized, if not solved all together. The use of oils from algae is another rapidly expanding process that has yet to reach maximum efficiency, but if and when it does, there is the potential that it could replace ethanol and gasoline all together. This field is so vast and widespread that any one of these options could reach a level of production that could supersede anything else currently on the market. The ability to manipulate beneficial genes and make a microbe capable of producing multiple biofuels is a new and intensely researched topic. For example, if E. coli were capable of producing not only ethanol, but isopropanol and n-butanol as well, then this bacteria will pave the way for solving the energy crisis all together, leading to a "super cell" that could power cities. For now though, all of this is a little farfetched and a little ways off, so for the near future we can look for the emergence of lignocellulositic and algal biofuels.
References
[1]"Algal Oil Yields". Oilgae. Retrieved 13 March 2012. Available from: http://www.oilgae.com/algae/oil/yield/yield.html
[2]Girfoglio, Michele, Mose Rossi, and Raffaele Cannio. "Cellulose Degradation by Sulfolobus solfataricus Requires a Cell-Anchored Endo-Beta-1-4-Glucanase." Journal of Bacteriology 194 (18): 5091-5100. Available from: http://jb.asm.org/content/194/18/5091.full.pdf+html
[3]Gunasekaran, P., and K. Chandra Raj. "Ethanol fermentation technology - Zymomonas mobilis." Current Science 70 (): 56-68. Available from: http://www.currentscience.ac.in/Downloads/article_id_077_01_0056_0068_0.pdf
[4]Ohta, Kazuyoshi, D. S. Beall, J. P. Mejia, K. T. Shanmugam, and L. 0. Ingram. "Genetic Improvement of Escherichia coli for Ethanol Production: Chromosomal Integration of Zymomonas mobilis Genes Encoding Pyruvate Decarboxylase and Alcohol Dehydrogenase Il." Applied and Environmental Microbiology 57 (4): 893-900. Available from: http://au8dt3yy7l.scholar.serialssolutions.com/?sid=google&auinit=K&aulast=Ohta&atitle=Genetic+improvement+of+Escherichia+coli+for+ethanol+production:+chromosomal+integration+of+Zymomonas+mobilis+genes+encoding+pyruvate+decarboxylase+and+alcohol+dehydrogenase+II.&id=pmid:2059047
[5]Sawayama, Shigeki. "Ethanol production from xylose in engineered Saccharomyces cerevisiae strains: current state and perspectives." Applied Microbiology and Biotechnology (84): 37-53. Available from: http://0-journals.ohiolink.edu.dewey2.library.denison.edu/ejc/pdf.cgi/Matsushika_Akinori.pdf?issn=01757598&issue=v84i0001&article=37_epfxiecscsap
[6]Bernhard Seiboth, Christa Ivanova and Verena Seidl-Seiboth (2011). Trichoderma reesei: A Fungal Enzyme Producer for Cellulosic Biofuels, Biofuel Production-Recent Developments and Prospects, Dr. Marco Aurelio Dos Santos Bernardes (Ed.), ISBN: 978-953-307-478-8, InTech, DOI: 10.5772/16848. Available from: http://www.intechopen.com/books/biofuel-production-recent-developments-and-prospects/trichoderma-reesei-a-fungal-enzyme-producer-for-cellulosic-biofuels
[7]Albert Weale and Hugh Perry. Biofuels: ethical issues. London: Nuffield Council on Bioethics, 2011. Available from: http://www.nuffieldbioethics.org/sites/default/files/Biofuels_ethical_issues_FULL%20REPORT_0.pdf
[8]Atsumi, Shota and Liao, James (2008). "Metabolic engineering for advanced biofuels production from Escherichia coli." Current Opinion in Biotechnology (19) 5:414-419. Available from: http://ac.els-cdn.com/S0958166908001018/1-s2.0-S0958166908001018-main.pdf?_tid=b42f6152-d797-11e3-885f-00000aacb35d&acdnat=1399653411_7af1437e9f495e6e82e5ba5094a92231
