Biomass represents an abundant carbon-neutral renewable resource for the production of bioenergy and biomaterials, and its enhanced use would address several societal needs. Advances in genetics, biotechnology, process chemistry, and engineering are leading to a new manufacturing concept for converting renewable biomass to valuable fuels and products, generally referred to as the biorefinery. The integration of agroenergy crops and biorefinery manufacturing technologies offers the potential for the development of sustainable biopower and biomaterials that will lead to a new manufacturing paradigm.

Switchgrass is a leading dedicated bioenergy feedstock in the United States because it is a native, high-yielding, perennial prairie grass with a broad cultivation range and low agronomic input requirements. Biomass conversion research has developed processes for production of ethanol and other biofuels, but they remain costly primarily because of the intrinsic recalcitrance of biomass. We show here that genetic modification of switchgrass can produce phenotypically normal plants that have reduced thermal-chemical (≤180°C), enzymatic, and microbial recalcitrance. Down-regulation of the switchgrass caffeic acid O-methyltransferase gene decreases lignin content modestly, reduces the syringyl:guaiacyl lignin monomer ratio, improves forage quality, and, most importantly, increases the ethanol yield by up to 38% using conventional biomass fermentation processes. The down-regulated lines require less severe pretreatment and 300-400% lower cellulase dosages for equivalent product yields using simultaneous saccharification and fermentation with yeast. Furthermore, fermentation of diluted acid-pretreated transgenic switchgrass using Clostridium thermocellum with no added enzymes showed better product yields than obtained with unmodified switchgrass. Therefore, this apparent reduction in the recalcitrance of transgenic switchgrass has the potential to lower processing costs for biomass fermentation-derived fuels and chemicals significantly. Alternatively, such modified transgenic switchgrass lines should yield significantly more fermentation chemicals per hectare under identical process conditions.ignocellulosic biomass is an abundant, domestic, renewable feedstock source that can be converted to liquid transportation fuels and other chemicals by fermentation. Cellulosic ethanol is a promising near-term technological option to reduce transportation sector greenhouse gas emissions (1). Because lignocellulosic biomass is made up of the complex structures of cellulose, hemicellulose, and lignin, such feedstock is highly recalcitrant to bioconversion of its carbohydrates into ethanol compared with starch (2, 3). Current biomass fermentation processes for fuels and chemicals have a relatively high cost primarily because of this recalcitrance, which in turn has limited commercialization of biomass ethanol (4). To achieve sustainable energy production, it is necessary to overcome the chemical and structural properties of biomass that inhibit its deconstruction in dedicated bioenergy crops (5).The conversion of lignocellulosic biomass to ethanol is a threestep process that involves pretreatment followed by polysaccharide hydrolysis to simple sugars followed by sugar fermentation to ethanol (6). The presence of lignin in cell walls negatively impacts these conversion steps (7,8). Examination of natural variation in alfalfa, switchgrass, canarygrass, and sorghum has shown that decreased lignin levels improve in vitro enzyme hydrolysis (9, 10). Lignin pathway modification in alfalfa generated transgenic lines with increased enzymati...

Effectively releasing the locked polysaccharides from recalcitrant lignocellulose to fermentable sugars is among the greatest technical and economic barriers to the realization of lignocellulose biorefineries because leading lignocellulose pre-treatment technologies suffer from low sugar yields, and/or severe reaction conditions, and/or high cellulase use, narrow substrate applicability, and high capital investment, etc. A new lignocellulose pre-treatment featuring modest reaction conditions (508C and atmospheric pressure) was demonstrated to fractionate lignocellulose to amorphous cellulose, hemicellulose, lignin, and acetic acid by using a non-volatile cellulose solvent (concentrated phosphoric acid), a highly volatile organic solvent (acetone), and water. The highest sugar yields after enzymatic hydrolysis were attributed to no sugar degradation during the fractionation and the highest enzymatic cellulose digestibility ($97% in 24 h) during the hydrolysis step at the enzyme loading of 15 filter paper units of cellulase and 60 IU of betaglucosidase per gram of glucan. Isolation of high-value lignocellulose components (lignin, acetic acid, and hemicellulose) would greatly increase potential revenues of a lignocellulose biorefinery.

BackgroundEconomic feasibility and sustainability of lignocellulosic ethanol production requires the development of robust microorganisms that can efficiently degrade and convert plant biomass to ethanol. The anaerobic thermophilic bacterium Clostridium thermocellum is a candidate microorganism as it is capable of hydrolyzing cellulose and fermenting the hydrolysis products to ethanol and other metabolites. C. thermocellum achieves efficient cellulose hydrolysis using multiprotein extracellular enzymatic complexes, termed cellulosomes.Methodology/Principal FindingsIn this study, we used quantitative proteomics (multidimensional LC-MS/MS and 15N-metabolic labeling) to measure relative changes in levels of cellulosomal subunit proteins (per CipA scaffoldin basis) when C. thermocellum ATCC 27405 was grown on a variety of carbon sources [dilute-acid pretreated switchgrass, cellobiose, amorphous cellulose, crystalline cellulose (Avicel) and combinations of crystalline cellulose with pectin or xylan or both]. Cellulosome samples isolated from cultures grown on these carbon sources were compared to 15N labeled cellulosome samples isolated from crystalline cellulose-grown cultures. In total from all samples, proteomic analysis identified 59 dockerin- and 8 cohesin-module containing components, including 16 previously undetected cellulosomal subunits. Many cellulosomal components showed differential protein abundance in the presence of non-cellulose substrates in the growth medium. Cellulosome samples from amorphous cellulose, cellobiose and pretreated switchgrass-grown cultures displayed the most distinct differences in composition as compared to cellulosome samples from crystalline cellulose-grown cultures. While Glycoside Hydrolase Family 9 enzymes showed increased levels in the presence of crystalline cellulose, and pretreated switchgrass, in particular, GH5 enzymes showed increased levels in response to the presence of cellulose in general, amorphous or crystalline.Conclusions/SignificanceOverall, the quantitative results suggest a coordinated substrate-specific regulation of cellulosomal subunit composition in C. thermocellum to better suit the organism's needs for growth under different conditions. To date, this study provides the most comprehensive comparison of cellulosomal compositional changes in C. thermocellum in response to different carbon sources. Such studies are vital to engineering a strain that is best suited to grow on specific substrates of interest and provide the building blocks for constructing designer cellulosomes with tailored enzyme composition for industrial ethanol production.

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BackgroundThe future hydrogen economy offers a compelling energy vision, but there are four main obstacles: hydrogen production, storage, and distribution, as well as fuel cells. Hydrogen production from inexpensive abundant renewable biomass can produce cheaper hydrogen, decrease reliance on fossil fuels, and achieve zero net greenhouse gas emissions, but current chemical and biological means suffer from low hydrogen yields and/or severe reaction conditions.Methodology/Principal FindingsHere we demonstrate a synthetic enzymatic pathway consisting of 13 enzymes for producing hydrogen from starch and water. The stoichiometric reaction is C6H10O5 (l)+7 H2O (l)→12 H2 (g)+6 CO2 (g). The overall process is spontaneous and unidirectional because of a negative Gibbs free energy and separation of the gaseous products with the aqueous reactants.ConclusionsEnzymatic hydrogen production from starch and water mediated by 13 enzymes occurred at 30°C as expected, and the hydrogen yields were much higher than the theoretical limit (4 H2/glucose) of anaerobic fermentations.SignificanceThe unique features, such as mild reaction conditions (30°C and atmospheric pressure), high hydrogen yields, likely low production costs ($∼2/kg H2), and a high energy-density carrier starch (14.8 H2-based mass%), provide great potential for mobile applications. With technology improvements and integration with fuel cells, this technology also solves the challenges associated with hydrogen storage, distribution, and infrastructure in the hydrogen economy.

We have engineered an industrial strain of the yeast, Candida tropicalis, for the efficient production of long-chain dicarboxylic acids, which are important raw materials for the chemical industry. By sequential disruption of the four genes encoding both isozymes of the acyl-CoA oxidase which catalyzes the first reaction in the beta-oxidation pathway, alkane and fatty acid substrates have been successfully redirected to the omega-oxidation pathway. Consequently, the conversion efficiency and chemical selectivity of their terminal oxidation to the corresponding dicarboxylic acids has been improved to 100 percent. The specific productivity of the bioconversion has been increased further by amplification of the cytochrome P450 monooxygenase and NADPH-cytochrome reductase genes encoding the rate-limiting omega-hydroxylase in the omega-oxidation pathway. The amplified strains demonstrated increased omega-hydroxylase activity and a 30% increase in productivity compared to the beta-oxidation-blocked strain in fermentations. The bioconversion is effective for the selective terminal oxidation of both saturated and unsaturated linear aliphatic substrates with chain-lengths ranging from 12 carbons to 22 carbons and also avoids the undesirable chain modifications associated with passage through the beta-oxidation pathway, such as unsaturation, hydroxylation, or chain shortening. It is now possible to efficiently produce a wide range of previously unavailable saturated and unsaturated dicarboxylic acids with a high degree of purity.

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