BIOFUELS SUPPLEMENT
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A bi-weekly summary of world developments on biofuels, produced by the Global Knowledge Center on Crop Biotechnology, International Service for the Acquisition of Agri-biotech Applications SEAsiaCenter (ISAAA)
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October 14, 2011

In This Issue:

News and Trends
- Scientists Develop "Lignin-Lite" Switchgrass for Use as Dedicated Bioenergy Crop 
- New Pine Tree Breeding Technique May Help Forests Adapt to Climate Change and Bioenergy Use 
- By-products from Fungal Pretreatment of Corn Stalks Enhance Saccharification to Ethanol-fermentable Sugars 

Energy Crops and Feedstocks for Biofuels Production
- Mutated Corn Gene Expressed in Switchgrass Improves Digestibility and Starch Content 

Biofuels Processing
- Interaction of Xylanase in Enzyme Cocktails for Saccharification of Pretreated Biomass 
- Improvement of L-Arabinose Utilization in Pentose-fermenting Saccharomyces cerevisiae 

Biofuels Policy and Economics
- Scheme for Mitigating Indirect Land Use Change (ILUC) Risks in the Use of Biofuels Proposed 





* NEWS AND TRENDS *

Scientists Develop "Lignin-Lite" Switchgrass for Use as Dedicated Bioenergy Crop
(complete access to journal article may require payment or subscription)
http://www.pnas.org/content/early/2011/02/04/1100310108

Switchgrass (Panicum virgatum L.) is a perennial prairie grass, considered as a "leading" (lignocellulosic, "second generation") biofuel feedstock for "cellulose-ethanol"production in the United States. Among the positive features of this feedstock are: (1) high biomass yields, (2) "broad cultivation range", and (3) low agricultural inputs. The biomass is usually processed by extracting the complex carbohydrates from the biomass (i.e.the cellulose and hemicellulose fractions), breaking them down (i.e. "saccharification"or "hydrolysis") into simple sugars, and eventually fermenting these sugars to ethanol. As with most lignocellulosic feedstocks, however, the main challenge against reducing the production cost, is "biomass recalcitrance". This is the property of the biomass (attributed to lignin) which makes the "extraction" of the carbohydrates in the biomass difficult. Lignins are "tough" molecules (resisting chemical attack) that tightly wraps around the cellulose/hemicellulose fractions. This "tight wrapping" prevents the carbohydrate portions of the biomass from being saccharified into simple sugars for ethanol fermentation.

Pretreatment methods to "delignify" the biomass (and to break biomass recalcitrance) often involve extreme conditions of thermal and/or chemical treatment, and this contributes a large portion of the production cost. Instead of focusing on development of more cost-effective pretreatment methods, one approach is to focus on the plant by addressing the lignin associated with biomass recalcitrance. Molecular biology methods can be used to develop low-lignin plants which can be used as "dedicated bioenergy crops".

Scientists from the Samuel Roberts Noble Foundation, Georgia Tech and Oak Ridge National Laboratory (United States) report the development of a "lignin-lite" transgenic switchgrass, with a biomass recalcitrance(indicated by lignin content) reduced by about one-eighth. They used a concept called "downregulation" , where the production of a key cellular component is reduced by genetic engineering techniques. Here, they targeted on a key enzyme involved in lignin biosynthesis called, "caffeicacid 3-O-methyltransferase" (COMT). By downregulating the COMT gene, the researchers were able to decrease the plant's lignin content by one-eighth, and increased ethanol production by about 33 percent. The full report of their method is published in the journal, Proceedings of the National Academy of Sciences (PNAS) (URL above).

The Bioenergy site reports of a new pine tree breeding technique which was developed by a collaborative team of scientists led by researchers from the University of Florida (United States). The pine breeding technique is said to accelerate the period of development of pine tree varieties with desired traits, which could include those that might actually help forests adapt to climate change and bioenergy use.

 While it usually takes about 13 years to develop a new pine tree variety, the new method is said to cut the period down to 6 or 8 years. The bioenergy site describes the discovery of the technique (using "genome selection") as follows: "The finding came when the researchers bypassed uncovering every bit of genetic code behind pine tree traits. Instead they used the parts of the genetic code they already knew to develop a  trait prediction model. The model allows the researchers to predict with great accuracy traits that will appear in a tree without having to first grow it in a field test, which can take about eight years". "Accuracies of prediction models are reported to range from 0.65 to 0.75 for diameter, and 0.63 to 0.74 for height". Accuracies were also reported to remain high across environments "as long as they were used within the same breeding zone". The research results are published in the journal, New Phytologist (URL above).

The removal of lignin by pretreatment of lignocellulosic biomass is the first phase in the production of cellulose-ethanol. This usually requires extreme use of heat and chemicals, and many of these extreme processes generate toxic/inhibitory compounds. Recently, the use of biological pretreatment using lignin-degrading fungi (usually white-rot fungi) is regaining interest as a milder, less toxic and less-costly pretreatment alternative. There are also indications that by-products produced from fungal pretreatment could enhance the second step of cellulose-ethanol production (the saccharification step, or the conversion of carbohydrates in the pretreated biomass to ethanol-fermentable sugars).

Researchers from Huazhong University of Science and Technology (Wuhan, China) report the pretreatment of corn stalk with a fungus, called Irpex lacteus, and found that its by-products stimulate saccharification. The pretreated biomass samples were exposed to water extracts containing by-products of fungal pretreatment, in combination with commercial saccharification enzymes (cellulases). The study showed that Irpex lacteus can be a promising white-rot fungus for lignocellulosic biomass pretreatment. About 82 percent of glucan hydrolysis yield was obtained after pretreatment. The results also showed that the saccharification efficiency of commercial cellulose preparations was higher when extracts of fungal pretreatment were added to the biomass. The reducing sugar yield was higher by 31 percent. The full results are published in the open-access journal, Biotechnology for Biofuels.

(complete access to journal article may require payment or subscription)
http://www.pnas.org/content/early/2011/10/04/1113971108.abstract?sid=d94a0389-4be0-4059-a434-508a5616707e
http://biofuelsdigest.com/bdigest/2011/10/12/mutaged-corn-gene-triples-starch-content-in-switchgrass-researchers

Researchers from the Plant Gene Expression Center of the United States Department of Agriculture (USDA) and University of California (UC) Berkeley improved the digestibility, and increased the starch content of switchgrass (Panicum virgatum L). The resulting "tailored switchgrass" is reportedly one that is a better bioenergy crop for biofuel-ethanol production. Their strategy is based on the observation that plants in the "juvenile phase" are "less lignified" and displays differences in biomass accumulation/character that point to less biomass recalcitrance (tendency of biomass to resist pretreatment and processing to ethanol, due to the high lignin content). 

They studied and harnessed the genes that regulated the transition of plants from juvenile to adult phase, to make tailored, dedicated bioenergy switchgrass. What they did was to express a key gene from corn (Corngene1 or Cg1 gene) into switchgrass. The mutant Cg1 gene, according to the study, "fixes plant development in the juvenile phase". In corn, these mutant genes produce biomass with reduced adult characteristics, and the leaves have less lignin and higher sugar levels compared to the wild type. When the mutant Cg1 gene was expressed in switchgrass, the plants were "found to have up to 250% more starch, resulting in higher glucose release from saccharification assays with or without biomass pretreatment". Furthermore, the modified switchgrass did not show evidence of flowering during the two-year trial period, indicating that the "transfer of genetic modification was low". These results show the potential for lowering the production cost of cellulose-ethanol by reducing pretreatment inputs. The full study is published in the Proceedings of the National Academy of Sciences (URL above).

In order to maximize sugar yield in the enzymatic saccharification (i.e. conversion of complex carbohydrates to simple sugars) of pretreated biomass for cellulose-ethanol production, the use of "enzyme cocktails" has been recommended. The reason is that the complex carbohydrates in the biomass contain glucose and xylose as the main sugar units, plus a few other simple sugars such as arabinose and mannose; thus, enzymes to unlock these sugars from the complex carbohydrates must be appropriate for a particular sugar. For example, cellulases are a group of enzymes which attack cellulose, and xylanases are enzymes which attack part of the hemicellulose. Little is known, however, about how these enzymes interact in a cocktail mixture, when used for enzymatic saccharification. Research studies have been done to understand these interactions, in order to find an "optimum" mix of enzyme cocktail.

Researchers from the University of British Columbia "assessed the interaction between cellulase and xylanase enzymes and their potential to improve the hydrolysis (saccharification) efficiency of various pretreated lignocellulosic substrates when added at low protein loadings". The results showed that xylanase can have a "blocking effect', which can limit the accessibility of cellulases on cellulose. However, "the synergistic interaction of the xylanase and cellulase enzymes was also shown to significantly improve cellulose accessibility through increasing fiber swelling and fiber porosity and also plays a major role in enhancing enzyme accessibility". The full results are published in the open access journal, Biotechnology for Biofuels (URL above)

In the production of ethanol from lignocellulosic biomass, the major simple sugars after biomass pretreatment (lignin removal) and saccharification (breakdown of complex carbohydrates in pretreated biomass) are usually (1) glucose (a 6-carbon sugar or hexose), (2) xylose,and (3) arabinose (the latter two are 5-carbon sugars, or pentoses). These sugars are usually contained in a liquid stream called, "hydrolyzates", which are fermented to ethanol by the yeast, Saccharomyces cerevisiae. In this system, only the glucose is effectively utilized, since Saccharomyces cerevisiae cannot utilize pentoses. Hence, not all carbohydrates in the biomass are converted to ethanol. To address this problem, pentose-utilizing strains of this yeast have been developed, but many are limited by the slow transport of these pentoses into the cells during fermentation. One strategy is to find ways to increase pentose transport in pentose-fermenting Saccharomyces cerevisiae.

Researchers from Institute of Molecular Biosciences, Goethe-University Frankfurt am Main (Germany) report the "cloning and characterization of two sugar transporters, AraT from the yeast Scheffersomyces stipitis and Stp2 from the plant Arabidopsis thaliana, which mediate the uptake of L-arabinose but not of D-glucose into S. cerevisiae cells". From their new screening system, they were able to identify two heterologous sugar transporters which can support uptake and utilization of L-arabinose in L-arabinose fermenting S. cerevisiae cells, especially at low L-arabinose concentrations. The full study is published in the open-access journal, Biotechnology for Biofuels (URL above).

The risk associated with "Indirect Land Use Change (ILUC) Risk" in the production and use of biofuels has been a contentious issue in the assessment of biofuels sustainability. Within the context of biofuels production, the International Union for Conservation of Nature describes ILUC risk as: "the risk that expanding biofuel production could displace some agricultural production activities onto land with high natural carbon stocks, such as grasslands and forests, leading to significant greenhouse gas emissions, biodiversity loss and potentially threatening food security". Indirect land use change is also said to be difficult to observe or measure directly.

The European Energy Review website mentions a report published by Ernstand Young, (and commissioned by a consortium of industry/NGO partners) which aimed to: (1) establish the facts surrounding the issue of ILUC by examining existing literature, and (2) investigate "issues concerning implementation of practical ILUC mitigation measures and their effectiveness in biofuels production". The report indicated that "indirect land use change (ILUC) risks can be mitigated by incentives that encourage existing and additional sustainable practices in biofuels production, as well as other sectors that use agricultural commodities". The proposed scheme is said to be different from the options being considered by the European Commission (EU) to address ILUC issues, but it provides opportunities to combine incentives with penalizing provisions for those who do not take action. The proposal involves the application of an "ILUC mitigation credit scheme", which could work alongside with, and remain subject to the existing polices of the EU Renewable Energy Directive. This may include the extension of the application of carbon incentives established under the Renewable Energy Directive. The complete report can be accessed from the URL posted above.

Related information on Land Use Change: http://www.isaaa.org/kc/cropbiotechupdate/article/default.asp?ID=8230.