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Researchers Engineer Thermophilic Bacterium to Produce Ethanol at High Yield
9 September 2008
A team of researchers from Dartmouth’s Thayer School of Engineering and Mascoma Corporation in Lebanon, N.H., have genetically engineered Thermoanaerobacterium saccharolyticum, a thermophilic anaerobic bacterium that ferments xylan and biomass-derived sugars, to produce ethanol at high yield as its only fermentation product. A paper on their work was published online during the week of 8 September in the journal Proceedings of the National Academy of Science.
The knockout of genes involved in organic acid formation (acetate kinase, phosphate acetyltransferase, and L-lactate dehydrogenase) resulted in an engineered strain (ALK2) able to produce ethanol as the only detectable organic product and substantial changes in electron flow relative to the wild type.
Ethanol formation in ALK2 utilizes pyruvate:ferredoxin oxidoreductase with electrons transferred from ferredoxin to NAD(P), a pathway different from that in previously described microbes with a homoethanol fermentation. The homoethanologenic phenotype was stable for >150 generations in continuous culture.
The growth rate of strain ALK2 was similar to the wild-type strain, with a reduction in cell yield proportional to the decreased ATP availability resulting from acetate kinase inactivation. Glucose and xylose are co-utilized and utilization of mannose and arabinose commences before glucose and xylose are exhausted.
Using strain ALK2 in simultaneous hydrolysis and fermentation experiments at 50°C allows a 2.5-fold reduction in cellulase loading compared with using Saccharomyces cerevisiae at 37°C. The maximum ethanol titer produced by strain ALK2, 37 g/liter, is the highest reported thus far for a thermophilic anaerobe, although further improvements are desired and likely possible, according to the researchers.
Our discovery is one potential avenue for research to facilitate turning inedible cellulosic biomass, including wood, grass, and various waste materials, into ethanol. In the near term, the thermophilic bacterium we have developed is advantageous, because costly cellulase enzymes typically used for ethanol production can be augmented with the less expensive, genetically engineered new organism.
—Professor Lee Lynd at the Thayer School of Engineering
Lynd says that this discovery is only the first step, a proof of concept, for future development of ethanol-producing microbes that can make ethanol from cellulosic biomass without adding enzymes. Lynd is the corresponding author on the study and the chief scientific officer and co-founder of Mascoma Corporation, a company working to develop processes to make cellulosic ethanol.
Other authors on this study include: A. Joe Shaw, the lead author on the paper, Kara Podkaminer, Sunil Desai, and Stephen Rogers with the Thayer School; and John Bardsley, Philip Thorne, and David Hogsett with Mascoma Corporation.
Resources
A. Joe Shaw, Kara K. Podkaminer, Sunil G. Desai, John S. Bardsley, Stephen R. Rogers, Philip G. Thorne, David A. Hogsett, and Lee R. Lynd (2008) Metabolic engineering of a thermophilic bacterium to produce ethanol at high yield, PNAS doi: 10.1073/pnas.0801266105
September 9, 2008 in Biotech, Cellulosic ethanol | Permalink | Comments (7) | TrackBack (0)
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Comments
This looks promising for lowering the cost of cellulose ethanol.
Posted by: Kit P | Sep 9, 2008 6:50:41 AM
4.7% ethanol is output solution makes for a light beer. That pretty impressive for a proof of concept experiment.
Posted by: Ben | Sep 9, 2008 7:10:01 AM
The reason they are still wrestling with getting the concentration they max out at above about 9 proof is that the next step is to remove the ethanol from the brew, which is energy-intensive and a practicality barrier. You can't use membranes on the first step I don't think because they'll clog too easily, or degrade. Presumably though the limit comes from the microbes suffering from their own alcohol's toxicity. Pushing the limit harder would also slow the reaction and maybe reduce conversion efficiency.
However, this innovation suggests another solution. If these bugs can live happily at 50C, that's not far below the boiling point of ethanol already. The vapor pressure is almost a third of an atmosphere. If you can arrange to be pumping off the vapor continuously during the fermenting, the ethanol never has to get very high in concentration and you still have high efficiency. The rest of the water can be removed efficiently using membranes and finally zeolites or something.
Putting the still in the high mountains and/or getting the bacteria to tolerate an even higher temperature would minimize the pumping energy and financial costs.
Also, reactions go fast at 50C and so there is lots of promise with this type of microbe as a catalytic tool, for earlier parts of the conversion process.
In any case plants built for this (or any other new ethanol plant) should get their process heat from solar. Especially now that the oil boys have won a massive propaganda victory, just by pounding on it so hard, convincing everyone that biofuels (especially the most threatening one right now, ethanol) are the terror. An environmental, etc. loser. High-efficiency conversion is needed to create a comeback with an exclamation point.
Posted by: P Schager | Sep 9, 2008 8:52:23 AM
Clarification: 37 g/liter ethanol is about 4.7% by volume.
Continuous vacuum distillation would have difficulties due to CO2, requiring much power to vacuum pumps. But if the still bottoms can be recirculated back to fermentation, the waste stream is eliminated and requires no treatment.
If this process can be run on crop wastes, green waste or municipal garbage, it will be a runaway win.
Posted by: Reality Czech | Sep 9, 2008 11:08:22 AM
I hope this company has it's science right, a bacteria that digests non edible cellulose fiber, I think I'd rather have the enzyme. What is the result of this bacteria in the wild?
Posted by: Mark M | Sep 9, 2008 12:29:39 PM
Mark M,
This bacteria is from a wild cellulose consuming species, many such bacteria have been known to exist for centuries, so why haven't we started using these guys if they have existed all this time? Because they are usually ABE fermenters that produce acetone and butanol as well as ethanol (and hydrogen). what been done by these researchers is engineer a wild cellulose consuming ABE fermenter into a cellulose consuming ethanol only fermenter, thus producing only one product and greatly simplifying and improving production of that product.
This modified bacterium is also weakened by being engineered only to produce ethanol, it less efficient then the wild types and would thus be uncompetitive with them if escaped into nature.
Posted by: Ben | Sep 9, 2008 6:52:18 PM
A 50C bug is definitely useful in lowering the cellulase enzyme dosage requirement during simultaneous hydrolysis and fermentation as 50C is the typical optimum for a cellulase complex derived from T. reesei. However, a real cellulosic substrate that is pretreated chemically will produce many degradation products that at 50C will cause significantly higher inhibitions to this bug than regular yeast at 32C.
Posted by: Tom H | Sep 12, 2008 11:54:15 PM
