Ethyl alcohol, or ethanol, is the familiar alcohol responsible for so much pleasure and so much trouble in our lives. Traditionally all alcohol was produced by fermentation of sugars by yeasts, and of course that is still the method used for beer, wine and other beverage production. But ethanol has a very large use in industry, especially as a solvent, and potential use as a liquid fuel. Although fermentation is used for production of industrial alcohol, and for a fuel especially in Brazil, most ethanol is made as a by-product of the petroleum industry. Breaking molecules of crude oil (cracking) to sizes suitable for petrol releases a lot of ethylene (CH2=CH2). This is used for making polyethylene, one of the commonest plastics, but also by adding a molecule of water, it can be converted to ethanol, CH3CH2OH . Ethanol produced this way costs about 30c per litre, whereas by fermentation the costs are about 60c per litre. That sounds as if it should be competitive with petrol --today I had to pay 75.9 c per litre-- but in fact petrol is produced by the oil companies at 25-30 c per litre; most of the rest of what we pay is government taxes.
Fermentation production of ethanol can be economically competitive with the petroleum product in special circumstances. First, in times of war, crude oil can be difficult to get hold of. In both world wars of the 20th century, Germany developed large-scale plants to produce fuel ethanol from sugar and starch. And because of balance-of-payments problems, Brazil which has few oil reserves, runs much of its transport on ethanol produced by fermentation of sugar by yeasts. Sugar cane for fuel is grown on vast plantations in Northern Brazil.
A most important feature of generating ethanol (or any other fuel) from plant material is that it is neutral in greenhouse gas effect. The CO2 released when ethanol is burnt is re-adsorbed by the plants which are to be used for ethanol production. Unfortunately the world's economists are unable to put a money value on this, so we continue to use up oil reserves because it is "cheaper". The main cost of production of ethanol by fermentation is the raw material: sugar or starch (which could be used to feed malnourished populations) costs up to 40 c of the 60 c per litre quoted above. Ethanol is really a very cheap product, and even transporting sugar to a fermentation plant can add a lot to the overall costs. In order to get the costs down, there has been a lot of attention given to using "waste biomass", which costs nothing at source. Such waste includes straw, bagasse (the sugar cane waste), and forestry wastes such as small branches, sawdust, etc. All of these are mainly composed of carbohydrate, which theoretically can be converted to near half its weight of ethanol. The trouble is that the carbohydrate is not in a form suitable for fermentation. First, it must be broken down to its constituent sugars. Then, not all of these sugars are fermented by yeasts. In particular, the sugar xylose, which may be as much as 30% of the total, is untouched. Genetic engineering has been able to modify microorganisms so that they are able to ferment all of the sugars in waste biomass to ethanol.
The main yeast used in ethanol production is Saccharomyces cerevisiae, which is the same species used for bread making and some wines and beers. It can ferment glucose, fructose and sucrose (the main sugars in food), but not xylose. Other less common yeasts do ferment xylose, but only very slowly. Some effort has gone into taking the genes from these other yeasts that are responsible for xylose utilisation, and putting them into S. cerevisiae . This has had only moderate success.
Yeasts are complex organisms of the eukaryote type; bacteria are simpler, called prokaryotes. A few bacteria also produce ethanol from sugars, but only one does so as efficiently. This one is called Zymomonas mobilis . But like S. cerevisiae, it only ferments glucose, fructose and sucrose. Many other bacteria will ferment xylose, but not make ethanol as end products. These include the common gut bacterium Escherichia coli , which we know such a lot about. It is relatively easy to transfer genes between bacteria by genetic engineering, and so there are two possible ways by which we could construct a bacterium that will ferment on all sugars to make ethanol. The first is to take the xylose-utilising genes from, say E. coli , and transfer them to Z. mobilis . This has been done, with reasonable success. However, much simpler is to take the genes that are responsible for Z. mobilis making ethanol, and transfer them to E. coli or a similar xylose-using organism. There are only two genes needed, which catalyse the two last steps of Z. mobilis fermentation. The enzymes that they encode are called pyruvate decarboxylase and alcohol dehydrogenase.
We isolated these two enzymes from Z. mobilis in 1985, and raised antibodies to each of them. A genomic library of Z. mobilis DNA was constructed, and probed with these antibodies. Positive clones were isolated which expressed the two enzymes, and from these a plasmid was constructed which expressed both enzymes in E. coli . This transgenic E. coli was capable of converting both glucose and xylose to ethanol in surprisingly high yield. This work was completed in 1988.
In science it frequently happens that several groups around the world get the same idea at the same time. While we were working on this project, two other groups were doing the same thing, and they eventually published the key stages at about the same time as us. One group, in Florida, was able to continue this work on a large scale with US government funding, and they are now starting up real commercial ethanol production using their transgenic organisms. Their work was considered so important that, when they received a patent for the work, the US patent office decided to award them "Patent number 5,000,000" which was due to be issued at that time.
But it has not been so easy to get funding for this sort of research in Australia. In 1995, our Federal Government made available some funds for renewable fuels from biomass, and on this programme, we have just completed a study of the use of thermophilic bacteria, which grow and ferment at 70°, for production of ethanol. The objective was to introduce the two Z. mobilis enzymes into the thermophile, which is capable of fermenting glucose and xylose, and at this high temperature it should be possible to boil off the ethanol as it is produced. There is a considerable amount of further work to do on this before it can be successful. Unfortunately the present Government has cut all further funding in the area of alcohol fuels, and has even abolished the Energy Research and Development Council. It looks as if all further developments in fuel alcohol will take place overseas.
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