Background
We have decided to research a very imposing ecological problem that is becoming more and more important due to steady growth of energy consumption and fossil depletion. Fossil fuels, such as oil and gasoline, have gained popularity in the beginning of the 20th century. It is estimated that we have spent between 100 and 135 billion tons of oil since 1850 and the demands are still increasing. Given that our society is largely dependent on oil, with the stocks being limited and, according to some projections, sufficient only for the next 40 years, it is becoming necessary to find an alternative method of obtaining fuels. In many countries, is becoming an important compensation for fossil fuels. Biogas, especially hydrogen, can be obtained during either aerobic or anaerobic fermentation of biomass with the help of mixed bacterial cultures. In the process of anaerobic fermentation, biomass is converted via carbon acids intermediates into hydrogen with so-called decarboxylation. In the process of anaerobic fermentation, the reforming process takes place. The final product is biogas.
The Laboratory for Environmental Sciences and Engineering of the National Institute of Chemistry in Ljubljana is already engaged in the development of the processes transforming waste and renewable substances into energy, especially biogas, and we have been well informed about the anaerobic fermentation since our first meetings included the extension of knowledge on biotechnological and biosynthetic fields. In anaerobic fermentation the main products are hydrogen, which is used as a source of energy, and carbon dioxide. The side products of anaerobic fermentation are ethanoic acid, butyric acid and other organic acids. They are produced after the processes of hydrolysis and acid genesis take place, in the third step of anaerobic fermentation known as acetogenesis.
Thinking about global problem, that our team is going to deal, we said to ourselves: “Hey, why do not we use the remaining products, for example butyric acid as a source of carbon and initial substance in the new devised pathway? Why discarding something, when it represents a potential benefit?” The efficiency of the fermentation increases and there is no need to buy or otherwise obtain butyric acid as the reactant of the biosynthetic process, because all necessary butyric acid is produced from inoculum, obtained from the Central waste water treatment plant in Ljubljana. The maximum amount of butyric acid is produced at the end of acidogenesis, when it starts to convert into other substances at the beginning of acetogenesis. To exploit the maximum amount of butyric acid the fermentation must be followed by days.
We decided to convert butyric acid into so-called biofuel of future, biobutanol. The biological pathway that synthesizes biobutanol primarily occurs when the input substance is glucose, however biobutanol can be produced via plenty of intermediates in this pathway. One of these intermediates is butyrate, the conjugate base of butyric acid. The biological pathway from butyrate to butanol is synthesized by 2 enzymes, called CoA-transferase and dehydrogenase.
One of the living organisms possessing these two enzymes and having the ability to carry out butanol production via butyrate conversion is a bacterium Clostridium acetobutylicum. C. acetobutylicum is a Gram-positive bacterium, which can digest sugar, whey, starch, cellulose and other organic substances. It is most likely to be found in dwelling soil, although it has been found in a number of very diverse environment. However, C. acetobutylicum is not laboratory standardized organism and is quite complexive for biosynthetic processes, for that reason we decided to use the bacteria Escherichia coli for their well-explored characteristics on the genetic basis. For our project we have chosen DH5alpha strain, a pathogen strain which is not found in the human intestine but it is used only in laboratories (safety class 1). E.coli DH5 alpha is a Gram-negative bacterium commonly found in the lower intestine of endotherms. It is a facultatively anaerobe, which means that it uses aerobic conditions for reproduction and anaerobic conditions for growth. E.coli requires macroelements ( C,H,N,O,P and S) and microelements (Fe,Se,Ca, Na…) in its growing environment.
We decided to isolate and transfer three genes, coding for two previously mentioned enzymes that are primarily present in the bacterium Clostridium acetobutylicum, into E. coli. Thus E.coli bacteria must be modified by synthetic biology and genetic engineering methods. We used E. coli DH5 alpha strain with PSB1C3 plasmid. 1-butanol can be produced via two enzymatic steps. In the first step butyrate is transformed into butyryl CoA by CtfAB gene. CtfAB gene is very large, thus we have to separate it into two polypeptide chains: CtfA and CtfB. Butyryl CoA is then finally transformed into butanol by BdhB gene.The reprogrammed E.coli would be able to convert butyric acid into butanol.
In 2008, a strain of Escherichia coli was genetically engineered to synthesize butanol for the first time, which was a considerable step toward the production of gasoline. In our opinion, this mechanism could provide a solution to a worldwide threatening problem associated with the lack and reduction of oil supplies. Looking from the perspective of biotechnologists, the process of butanol production with genetically modified bacteria must be optimized and our job is additionally to compose the optimum medium and the sufficient environment in the bioreactor, in which modified E.coli DH5 alpha converts as much butanol from butanoic acid as possible.
And why biobutanol?
A lot of research has been done in the field of fossil fuels in recent years. In recent years an the fact that there will be a shortage of fossil fuels in the future was exposed, thus forcing the world to search for alternatives to fossil fuel. One such replacement is biobutanol.
Butanol is a four-carbon alcohol, which is very energetically efficient due to its longer hydrocarbon chain in comparison to that of ethanol or propanol. Butanol has high energy density (29,2 MJ/L), which is closer to the energy density of gasoline than to that of ethanol. It can be produced by biomass fermentation in the A.B.E process, where such produced butanol is called biobutanol. One amongst the possibilities of butanol production is also the production from algae. Although biobutanol can be made entirely with solar energy and other algae nutrients, the current yield seemes to be very low. Butanol, produced via A.B.E fermentation, would offer a possibility of expanding the production and use of fuel to the countries that primarily do not posses any oil sites. In addition to being supplied with fuel, such countries would also become economically more independable and gain self-sufficiency. The management with biobutanol would positively impact on the country’s input and would aid in the improvements in the area of construction and traffic.