## Kobe Univ. researchers develop yeast capable of direct fermentation from cellulosic materials to produce ethanol

##### 19 May 2011

In a proof-of-concept study, a team from Kobe University (Japan) has developed a diploid yeast strain optimized for expression of cellulolytic enzymes, which is capable of directly fermenting from cellulosic materials to produce ethanol without the use of additional enzymes to break down the cellulose. Their open access paper is published in the journal Biotechnology for Biofuels.

The engineered strain, which contains multiple copies of three cellulase genes integrated into its genome, displayed approximately six-fold higher phosphoric acid swollen cellulose (PASC) degradation activity than the parent haploid strain. When used to ferment PASC, the diploid strain produced 7.6 g/L ethanol in 72 hours, with an ethanol yield that achieved 75% of the theoretical value, and also produced 7.5 g/L ethanol from pretreated rice straw in 72 hours.

Although this is a proof-of-concept study, it is to our knowledge, the first report of ethanol production from agricultural waste biomass using cellulolytic enzyme-expressing yeast without the addition of exogenous enzymes. Our results suggest that combining multigene expression optimization and diploidization in yeast is a promising approach for enhancing ethanol production from various types of lignocellulosic biomass.

Although lignocellulosic biomass (such as rice straw, which is one of the most abundant lignocellulosic waste materials), is seen a promising starting material for bioethanol production for a number of factors, it is still much more expensive to process than grains because of the need for extensive pretreatment and relatively large amounts of cellulases for efficient hydrolysis of the biomass, the authors note.

Hydrolysis of cellulose requires the synergistic action of the cellulolytic enzymes endoglucanase, cellobiohydrolase and ß-glucosidase. The expression ratios and synergetic effects of these enzymes significantly influence the extent and specific rate of cellulose degradation, the authors note. In this study, they used a previously developed method (cocktail δ-integration) to optimize cellulase-expression levels in yeast.

In cocktail δ-integration, several kinds of cellulase-expression cassettes are integrated into yeast chromosomes simultaneously in one step, and strains with high cellulolytic activity (that is, expressing the optimum ratio of cellulases) can be easily obtained, they note.

In they study, the cocktail δ-integration method was used to optimize cellulase expression in two yeast strains of opposite mating types. These strains were mated to produce a diploid strain with enhanced cellulase expression, which was then evaluated for its efficiency in converting cellulose to ethanol from PASC and pretreated rice straw.

As expected from the PASC hydrolysis reaction results, high ethanol production and yield from PASC was achieved using the diploid strain prepared in molasses medium. When we compared our results with those from different cellulase-expression systems of S. cerevisiae published previously, our diploid strain clearly achieved the highest ethanol production and yields. In addition to these promising findings, the cellulolytic enzyme-expressing diploid yeast strain was also able to produce ethanol from pretreated rice straw.

Although the ethanol production rate from rice straw was nearly identical to that from PASC, the ethanol yield from rice straw was relatively low. This result suggests that highly crystalline regions of cellulose in rice straw were not effectively degraded, thus reducing these regions by improving the efficiency of pretreatment or further optimizing cellulolytic enzyme-expression ratios in recombinant diploid yeast may lead to improved bioethanol yields from agricultural waste biomass. Using the cocktail δ-integration method, cellulase expression in yeast could be optimized for degradation of rice straw. Furthermore, hemicelluloses and a lignin matrix surrounding cellulose would also prevent effective degradation of lignocellulose; however, these could be removed by pretreatment or with use of additional exogenously expressed enzymes.

Resources

• Yamada et al. (2011) Direct ethanol production from cellulosic materials using a diploid strain of Saccharomyces cerevisiae with optimized cellulase expression. Biotechnology for Biofuels 4:8 doi: 10.1186/1754-6834-4-8

rice straw, which is one of the most abundant lignocellulosic waste materials

I would imagine Japan has quite a bit of that. I know central California does and could use this method, rather than burning it and causing pollution.

This technology appears to have the potential for being applicable for on-farm use, thus obviating the need for transport of bulk materials to a distant processing center.

It'll need a large increase in cellulose-cleaving capacity, and a high ethanol tolerance both for the yeast itself and the cellulase it uses. Producing 1 %/vol of EtOH doesn't give you fuel, it gives you a wastewater disposal problem.

Speaking of which, wastewater will be an issue regardless. I seem to recall reading about a fungus which captured low concentrations of EtOH, dead yeast and such from the distillation bottoms and made them useful. I'm not sure, but this might be it. We'll need something like that.

I would just gasify the rice straw and make synthetic gasoline.

If you could do it cheaply, so would I. But cheapness seems to be lost in the process of gasification, gas cleanup, F-T synthesis, and cracking of F-T wax to product.

There seems to be several companies doing it. They can make methanol and then make gasoline. Syntec makes several alcohols using gasification of biomass.

http://syntecbiofuel.com/process.php

"MTG converts crude methanol directly to low sulfur, low benzene gasoline that can be sold directly or blended with conventional refinery gasoline. Although the original application of the MTG technology processed methanol from natural gas, the same technology can be used for methanol from other sources such as coal, petcoke or biomass."

"The Sundrop process is expected to be able to create gasoline, without subsidies, for less than $2 per gallon. The company is constructing a pilot plant and aims to have a full, commercial-scale plant with a capacity of 100 million gallons by 2015." http://www.sundropfuels.com/ by Cathy Proctor Jan 29 2010 Green Oil Exxon Mobil recently spent$600 million on biofuels. But that's just a tiny fraction of what oil firms spend on looking for crude. Are they really looking to find an alternative to black gold? Read More
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As a result, Sundrop can produce 100 to 125 gallons of fuel per ton of dry biomass, about twice what conventional gasification plants are getting. It also needs just a half gallon of water—and its hydrogen molecules—to produce a gallon of fuel, compared to six gallons or more needed by traditional gasification technology

This was all I wanted to paste.

"As a result, Sundrop can produce 100 to 125 gallons of fuel per ton of dry biomass, about twice what conventional gasification plants are getting. It also needs just a half gallon of water—and its hydrogen molecules—to produce a gallon of fuel, compared to six gallons or more needed by traditional gasification technology"

This shows that you can make gasoline using biomass and make money.

I still say that there's no point in the MTG step (either its cost or energy loss), because we could very quickly have more methanol-capable vehicles than we'd have methanol to run them, but if Sundrop can do BTL for that price that's terrific.

The proof of the pudding is in the eating.

The Open Fuel Standard would provide millions more cars capable of running methanol every year, but there is no way of knowing if the bill will pass and there are all of those other cars out there now.

The MTG process is fairly efficient. They go from methanol to DME to gasoline. If it is an 87 octane gasoline that can blend with the refined product and has less sulfur and benzene than the refined product, then I would say give it a go.

There's a fair amount of energy loss in 2 MeOH -> DME + H2O. The reaction is exothermic.

3 billion GPY of MeOH is about 2% of US gasoline consumption. That could be blended without problems. If the OFS bill passes, there would be millions of methanol-capable vehicles by the time MeOH capacity gets high enough to need a separate fuel stream to consume it. We really don't need MTG... except as a PR move and possible subsidy-capturing measure.

Well, we will see what happens. I don't say we do or don't need something, that is absolute and closes the door and closes minds.

You can also go from synthesis gas directly to DME and then to gasoline. The heat generated can be captured and used in other parts of the process.

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