Alfalfa stems, reed canarygrass, and switchgrass; perennial herbaceous species that have potential as biomass energy crops in temperate regions; were evaluated for their bioconversion potential as energy crops. Each forage species was harvested at two or three maturity stages and analyzed for carbohydrates, lignin, protein, lipid, organic acids, and mineral composition. The biomass samples were also evaluated for sugar yields following pretreatment with dilute sulfuric followed by enzymatic saccharification using a commercial cellulase preparation. Total carbohydrate content of the plants varied from 518 to 655 g kg À1 dry matter (DM) and cellulose concentration from 209 to 322 g kg À1 DM. Carbohydrate and lignin contents were lower for samples from early maturity samples compared to samples from late maturity harvests. Several important trends were observed in regards to the efficiency of sugar recovery following treatments with dilute acid and cellulase. First, a significant amount of the available carbohydrates were in the form of soluble sugars and storage carbohydrates (4.3-16.3% wt/wt). Recovery of soluble sugars following dilute acid pretreatment was problematic, especially that of fructose. Fructose was found to be extremely labile to the dilute acid pretreatments. Second, the efficiency at which available glucose was recovered was inversely correlated to maturity and lignin content. However, total glucose yields were higher for the later maturities because of higher cellulose contents compared to the earlier maturity samples. Finally, cell wall polysaccharides, as determined by the widely applied detergent fiber system were found to be inaccurate. The detergent fiber method consistently overestimated cellulose and hemicellulose and underestimated lignin by substantial amounts.

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Rice hulls, a complex lignocellulosic material with high lignin (15.38 +/- 0.2%) and ash (18.71 +/- 0.01%) content, contain 35.62 +/- 0.12% cellulose and 11.96 +/- 0.73% hemicellulose and has the potential to serve as a low-cost feedstock for production of ethanol. Dilute H2SO4 pretreatments at varied temperature (120-190 degrees C) and enzymatic saccharification (45 degrees C, pH 5.0) were evaluated for conversion of rice hull cellulose and hemicellulose to monomeric sugars. The maximum yield of monomeric sugars from rice hulls (15%, w/v) by dilute H2SO4 (1.0%, v/v) pretreatment and enzymatic saccharification (45 degrees C, pH 5.0, 72 h) using cellulase, beta-glucosidase, xylanase, esterase, and Tween 20 was 287 +/- 3 mg/g (60% yield based on total carbohydrate content). Under this condition, no furfural and hydroxymethyl furfural were produced. The yield of ethanol per L by the mixed sugar utilizing recombinant Escherichia colistrain FBR 5 from rice hull hydrolyzate containing 43.6 +/- 3.0 g fermentable sugars (glucose, 18.2 +/- 1.4 g; xylose, 21.4 +/- 1.1 g; arabinose, 2.4 +/- 0.3 g; galactose, 1.6 +/- 0.2 g) was 18.7 +/- 0.6 g (0.43 +/- 0.02 g/g sugars obtained; 0.13 +/- 0.01 g/g rice hulls) at pH 6.5 and 35 degrees C. Detoxification of the acid- and enzyme-treated rice hull hydrolyzate by overliming (pH 10.5, 90 degrees C, 30 min) reduced the time required for maximum ethanol production (17 +/- 0.2 g from 42.0 +/- 0.7 g sugars per L) by the E. coli strain from 64 to 39 h in the case of separate hydrolysis and fermentation and increased the maximum ethanol yield (per L) from 7.1 +/- 2.3 g in 140 h to 9.1 +/- 0.7 g in 112 h in the case of simultaneous saccharification and fermentation.

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To maximize the production of carboxylic acids with open cultures of microbial consortia (reactor microbiomes), we performed experiments to understand which factors affect the community dynamics and performance parameters. We operated six thermophilic (55 °C) bioreactors to test how the factors: (i) biomass pretreatment; (ii) bioreactor operating conditions; and (iii) bioreactor history (after perturbations during the operating period) affected total fermentation product and n-butyrate performance parameters with corn fiber as the cellulosic biomass waste. We observed a maximum total fermentation product yield of 39%, a n-butyrate yield of 23% (both on a COD basis), a maximum total fermentation production rate of 0.74 g COD l(-1) d(-1) and n-butyrate production rate of 0.47 g COD l(-1) d(-1) in bioreactors that were fed with dilute-acid pretreated corn fiber at a pH of 5.5. Pyrosequencing of 16S rRNA genes with constrained ordination and other statistical methods showed that changes in operating conditions to enable dilution of toxic carboxylic acid products, which lead to these maximum performance parameters, also altered the composition of the microbiome, and that the microbiome, in turn, affected the performance. Operating conditions are an important factor (tool for operators) to shape reactor microbiomes, but other factors, such as substrate composition after biomass pretreatment and bioreactor history are also important. Further optimization of operating conditions must relieve the toxicity of carboxylic acids at acidic bioreactor pH levels even more, and this can, for example, be accomplished by extracting the product from the bioreactor solutions.

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Corn hybrids were compared to determine the fate of recombinant Bt protein (CRY1Ab from Bacillus thuringiensis) in coproducts from dry grind and wet‐milled corn during production of fuel ethanol. Two pairs of Bt and non‐Bt hybrids were wet milled, and each fraction was examined for the presence of the Bt protein. Bt protein was found in the germ, gluten, and fiber fractions of Bt hybrids. In addition, one set of Bt and non‐Bt hybrids were treated by the dry‐grind ethanol process and Bt protein was monitored during each step of the process. The Bt protein was not detected after liquefaction. Subsequent experiments determined that the Bt protein is rapidly denatured at liquefaction temperatures. Finally, five hybrids were compared for ethanol yield after dry grinding. Analysis of fermentation data with an F‐test revealed the percent of total starch available for conversion into ethanol varied significantly among the hybrids (P < 0.002), indicating ethanol yield is not exclusively dependent on starch content. No difference, however, was observed between Bt and non‐Bt corn hybrids for either ethanol productivity or yield.