Biochemical and some thermochemical routes to producing advanced biofuels require the fractionation of lignocellulosic biomass into a sugar-rich stream. This requires the depolymerization of plant cell wall polysaccharides and is generally regarded as the major hurdle for cost-effective advanced biofuel production. To achieve the saccharification of cellulosics, a combination of pretreatment to enhance the reactivity of cellulose and catalysts (enzymes, etc.) are required. This task will describe advances in understanding how components of cellulosic biomass inhibit and deactivate cellulase enzymes. In addition, results will be presented from a novel approach using enzyme-mimicking acid catalysts to release and even convert cellulosic sugars to advanced biofuels and value-added compounds. These reseult suggest that technology approaches to control the ionic strength, ion character, and pH of aqueous solutions can control the selectivity of saccharification to favor sugar formation over undesired degradation products. A similar approach has application in starch conversion toward value-added chemicals.

Corn biorefineries with diversified product portfolios offer great potential for corn growers and sugar producers by providing new, high margin market opportunities to capture added value and a higher return on investment. Conventional dry grind utilizes starch to produce ethanol, while leaving all other components (germ, pericarp) unutilized and mixed together as in distillers' grains. Wet mill processes involve steeping at elevated temperatures. In this study, we present a new approach for disassembly rather than destruction of corn kernels into its components (starch, pericarp, and germ) by enzyme catalysis at at temperatures of 50 to 60 C. The enzymes are formulated to separate pericarp from endosperm while leaving germ floating on the reaction solution at the end of the process. The process involves no mechanical grinding and no chemical steeping of corn kernels prior to the enzymatic deconstruction and can be easily adapted to a conventional dry grind process. Fractionation of pericarp and germ, followed by washing will generate a starch stream which is subsequently hydrolyzed to glucose by amylases. The enzymes are specifically formulated for this task by screening numerous commercially available enzymes that will disassemble corn kernels. To facilitate the enzyme penetration, the tip caps of kernels are removed. This process provides an alternative approach to fractionate corn kernels into components that are suitable for production of chemical building blocks for polymers, chemicals, and liquid fuels.

Pretreatment is an important cost-driver of lignocellulose conversion to ethanol and a critical step prior to enzyme hydrolysis. It disrupts the plant cell wall network and partially separates the major polymer components (lignin, cellulose and hemicellulose). However, pretreatment of lignocellulosic materials may also result in the release of inhibitors and deactivators of the enzymatic hydrolysis of cellulose. Development of enzyme processes for hydrolysis of cellulose to glucose must reduce inhibition and deactivation effects in order to enhance hydrolysis and reduce enzyme usage. Here we report the identification of phenols with major inhibition and/or deactivation effect on enzymes used for conversion of cellulose to ethanol. The strength of the inhibition or deactivation effect depended on the type of enzyme, the microorganism from which the enzyme was derived, and the type of phenolic compounds present. The effects of inhibitors on enzyme hydrolysis of pretreated lignocellulosic materials are presented

Hydrothermal pretreatment of lignocellulosic materials generates a liquid stream rich in pentose sugar oligomers. Cost-effective hydrolysis and utilization of these soluble sugar oligomers is an integral process of biofuel production. We report integrated rate equations for hydrolysis of xylo-oligomers derived from pretreated hardwood by dicarboxylic maleic and oxalic acids. The highest xylose yield observed with dicarboxylic acids was 96%, and compared to sulfuric acid, was 5–15% higher with less xylose degradation. Dicarboxylic acids showed an inverse correlation between xylose degradation rates and acid loadings unlike sulfuric acid for which less acid results in less xylose degradation to aldehydes and humic substances. A combination of high acid and low-temperature leads to xylose yield improvement. Hydrolysis time course data at three different acid concentrations and three temperatures between 140 and 180°C were used to develop a reaction model for the hydrolysis of xylo-oligosaccharides to xylose by dicarboxylic acids.

Lignin content, composition, distribution as well as cell wall thickness, structures, and type of tissue all have measurable effects on enzymatic hydrolysis of cellulose in lignocellulosic feedstocks. Our work combined compositional analysis, pretreatment, enzyme hydrolysis and SEM imaging for fractionated pith, rind, and leaf tissues from a hybrid stay-green corn, in order to identify the role of structural characteristics on enzyme hydrolysis of cell walls. Hydrolysis followed the sequence rind < leaves < pith, with 75% conversion to glucose achieved with 9 mg enzyme protein/g glucan or 3.6 mg protein/total solids and 90% with l08 mg protein/g glucan or 43.2 mg protein/total solids in 24 hours. Physical fractionation of corn stalks or other C4 grasses into soft and hard tissue types could reduce cost of cellulose conversion by enabling reduced enzyme loadings to hydrolyze soft tissue, and directing the hard tissue to other uses. The amount of lignin alone remaining after pretreatment of the different fractions is about the same, so differences in lignin content do not explain the differences in enzymatic hydrolysis. SEM images show sugar yields correlate with changes in plant cell wall structure both before and after liquid hot water pretreatment.

The commercialization of cellulosic ethanol has faced a number of different technical hurdles. One major challenge is the negative impact of inhibitors on the fermentative performance of industrial microorganisms. Most inhibition studies have focused on furan derivatives and weak acids; however, potential fermentation inhibitors also include cations and anions. Cations and anions are present in cellulosic biomass and are also used for pH adjustment prior to and during fermentation. To characterize the inhibitory effect of cations (potassium, sodium, ammonium) and anions (chloride and sulfate), a series of lab-scale fermentations were completed using S. cerevisiae 424A(LNH-ST), a recombinant yeast strain that can effectively co-ferment glucose and xylose. The concentration of the cations and anions tested ranged from 0.1M to 0.5M. Preliminary analysis of these fermentations showed xylose fermentation to be more sensitive to the presence of cations and anions than glucose fermentation. Results also found sodium to be the most inhibitory cation. To further explore the effect of sodium, a comprehensive analysis of intracellular metabolites involved in glycolysis and the pentose phosphate pathway was conducted. The Global Isotope-labeled Internal Standard (GILISA) MS quantization method was used for the identification and quantification of intracellular metabolites at key metabolic stages during fermentation.

Lignocellulose consists of various components which are released by pretreatment and the actions of cellulolytic enzymes. In the case of liquid hot water pretreatment (LHW) of lignocellulosic biomass, the preatreatment solubilizes oligomers and acetic acid from hemicellulose and phenolic compounds from both hemicellulose and lignin. The soluble compounds in the liquid fraction of LHW pretreated cellulosic biomass strongly inhibits the cellulolytic activities of enzymes. In this study, the inhibitory effects of the soluble components in the LHW pretreatment liquid were assessed using pretreated maple and corn stover as a source of inhibitors and Solka Floc as the reactant, Solka Floc at 1% solids loading was readily hydrolyzed at an enzyme loading of 15 FPU cellulase per g cellulose. However when inhibitors were introduced by adding pretreatment liquid to the Solka Floc and buffer, the glucose yield after 72 hrs was reduced by 50%. Among the soluble components in the pretreatment liquid, phenolic compounds were found to be the strongest inhibitors of cellulose hydrolysis. This was further verified by removal of phenolics from the pretreatment liquid which resulted in a significant yield improvement. The relationship between hydrolysis efficiency and the mass ratio of phenolic compounds to cellulase proteins was also measured. The mechanisms of cellulase inhibition/deactivation by sugar-oligomers and phenolics were further probed using individual inhibitor molecules. The combined effects were then studied through simultaneous saccharification and fermentation of Solka Floc and pretreated lignocellulosic substrates. The results show that phenolics are strong inhibitors whose effects may be moderated by washing them away from the lignocellulosic substrates.

Cost-effective and efficient ethanol production from lignocellulosic materials requires the fermentation of all sugars recovered from such materials including glucose, xylose, mannose, galactose, and L-arabinose. Wild-type strains of Saccharomyces cerevisia used in industrial ethanol production cannot ferment D-xylose and L-arabinose. Our genetically engineered recombinant S. cerevisiae yeast 424A(LNH-ST) has been made able to efficiently ferment xylose to ethanol, which was achieved by integrating multiple copies of three xylose-metabolizing genes. This study reports the efficient anaerobic fermentation of L-arabinose by the derivative of 424A(LNH-ST). The new strain was constructed by over-expression of two additional genes from fungi L-arabinose utilization pathways. The resulting new 424A(LNH-ST) strain exhibited production of ethanol from L-arabinose, and the yield was more than 40%. An efficient ethanol production, about 72.5% yield from five-sugar mixtures containing glucose, galactose, mannose, xylose, and arabinose was also achieved. This co-fermentation of five-sugar mixture is important and crucial for application in industrial economical ethanol production using lignocellulosic biomass as the feedstock .

Efficient conversion of hemicellulose-derived sugars to ethanol at high yields and titers are goals toward commercializing cellulosic ethanol production. S. cerevisiae 424A (LNH-ST) developed at Purdue University can efficiently ferment glucose and xylose. However, inhibitors present in cellulosic feedstocks (acetic acid) and the desired fermentation product (ethanol) reduce yeast growth rate and fermentation rates, especially during xylose fermentation. Through adaptation we have developed new strains with improved xylose fermentation compared to the original strain. The new strain has 500% higher ethanol volumetric productivity on xylose in the presence of higher ethanol concentrations (above 6%) than the original strain. An acetic acid-resistant yeast strain co-fermenting glucose and xylose in the presence of acetic acid (10 g/L) when compared to the original strain has 3 times the rate of xylose utilization (1.05 g/L/h from 0.32 g/L/h) and results in a higher final ethanol titer (76.3 g/L from 61.2 g/L). We present the results from a system biology approach to analyzing differences between our original strain and newly developed strains. We focus not only on expression profiling (transcriptomics), but also report changes in metabolic intermediates and fluxes, and lipid membrane composition to elucidate the basis for improved yeast performance. Efficient conversion of hemicellulose-derived sugars to ethanol at high yields and titers are goals toward commercializing cellulosic ethanol production. S. cerevisiae 424A (LNH-ST) developed at Purdue University can efficiently ferment glucose and xylose. However, inhibitors present in cellulosic feedstocks (acetic acid) and the desired fermentation product (ethanol) reduce yeast growth rate and fermentation rates, especially during xylose fermentation. Through adaptation we have developed new strains with improved xylose fermentation compared to the original strain. The new strain has 500% higher ethanol volumetric productivity on xylose in the presence of higher ethanol concentrations (above 6%) than the original strain. An acetic acid-resistant yeast strain co-fermenting glucose and xylose in the presence of acetic acid (10 g/L) when compared to the original strain has 3 times the rate of xylose utilization (1.05 g/L/h from 0.32 g/L/h) and results in a higher final ethanol titer (76.3 g/L from 61.2 g/L). We present the results from a system biology approach to analyzing differences between our original strain and newly developed strains. We focus not only on expression profiling (transcriptomics), but also report changes in metabolic intermediates and fluxes, and lipid membrane composition to elucidate the basis for improved yeast performance. Efficient conversion of hemicellulose-derived sugars to ethanol at high yields and titers are goals toward commercializing cellulosic ethanol production. S. cerevisiae 424A(LNH-ST) developed at Purdue University can efficiently ferment glucose and xylose. However, inhibitors present in cellulosic feedstocks (acetic acid) and the desired fermentation product (ethanol) reduce yeast growth rate and fermentation rates, especially during xylose fermentation. Through adaptation we have developed new strains with improved xylose fermentation compared to the original strain. The new strain has 500% higher ethanol volumetric productivity on xylose in the presence of higher ethanol concentrations (above 6%) than the original strain. An acetic acid-resistant yeast strain co-fermenting glucose and xylose in the presence of acetic acid (10 g/L) when compared to the original strain has 3 times the rate of xylose utilization. (1.05 g/L/h from 0.32 g/.L/h) and results in a higher final ethanol titer (76.3 g/L from 61.2 g/L). We present the results from a system biology approach to analyzing differences between our original strain and newly developed strains. We focus not only on expression profiling (transcriptomics), but also report changes in metabolic intermediates and fluxes, and lipid membrane composition to elucidate the basis for improved yeast performance.

Both corn grain and grain stover have been examined and utilized as biofuel feedstocks. Maize silage (wet stored, partially fermented maize stover plus immature grain) is an alternative that combines starch and cellulosic processing in a single feedstock. The commercial brown midrib (BMR marketed by Mycogen, wholly owned subsidiary of Dow AgroSciences) has lowered expression of caffeic acid O-methyl transferase, a key enzyme in the biosynthesis of S monolignols. We carried out a compositional analysis for two commercial varieties of maize silage (regular and brown midrib) for starch, cellulose, hemicellulose, and lignin content. Our results show that for the commercial varieties, the lignin content (Klason lignin plus acid soluble lignin) is indistinguishable. However, the BMR silage exhibits significantly higher cellulose enzymatic digestibility. Liquid hot-water pretreatment was optimized for each silage variant. Optimal pretreatment conditions were similar between BMR and regular silage, which was less severe than required for dry stover from similar maize varieties. Simultaneous saccharifications and fermentations were subsequently performed on pretreated whole silage and ground silage at 25% (w/v) total solids using Celluclast 1.5L and Novozyme 188 and the glucose/xylose co-fermenting yeast S. cerevisiae 424A(LNH-ST). The results show that the improved cellulose hydrolysis performance of BMR silage compared to regular silage is also seen in pretreated material, resulting in significantly higher yields of ethanol after SSF.

Bio-ethanol has gained much attention due to its economical and environmental benefits as a renewable fuel. Our lab had genetically engineered a yeast strain 424A(LNH-ST) that can co-ferment glucose and xylose, the two most abundant sugars in cellulosic biomass. However, several inhibitors such as acetic acid, furfural, and ethanol are created and accumulated during the process of cellulosic biomass pretreatment, hydrolysis, and/or during fermentation. Our previous work has shown that acetic acid under process relevant conditions do not significantly affect glucose fermentation. However, xylose utilization is significantly affected, especially at low pH environment (pH < 5.5) and high acetic acid concentration (> 10 g/L). An acetic acid-resistant yeast strain alternated from original 424A(LNH-ST) strain was developed by adaptation to acetic acid. Small-scale fermentation (100 ml YEP) containing 120 g glucose and 80 g xylose per L with 10 g acetic acid per L has shown more than triple the rate of xylose utilization (1.05 g/L/h from 0.32 g/L/h) and higher final ethanol titer (76.3 g/L from 61.2 g/L) by the new strain compared to the original strain. In this study, a system biology analysis including transcriptomic and metabolomic measurements were completed to understand gene expression and metabolic fluxes in this improved strain as compared to the original strain.

Lignocellulosic biomass is a promising renewable feedstock for the microbial production of chemicals and fuels, especially ethanol. Processing lignocellulose for biofuel production results in the release of the major fermentable sugars glucose and xylose. However, the primary processing steps required for this conversion also produce a range of compounds that can inhibit the subsequent microbial fermentation. One such inhibitory compound is acetic acid, liberated from hemicelluloses during the pretreatment of the biomass. We previously reported acetic acid to be inhibitory to cell growth, substrate consumption (especially xylose), and ethanol productivity, and stimulatory to the metabolic yield of ethanol. To further explore the effect of acetic acid on a cellular level, a genome-wide analysis of gene expression levels over the course of a batch co-fermentation of glucose and xylose was conducted using microarray technology. RNA samples were extracted for analysis from S. cerevisiae 424A(LNH-ST) at various time points throughout the co-fermentation of glucose and xylose with either 0 or 10 g/L acetic acid at a controlled pH of 5.5. In this poster, we report the results of this transciptomic analysis, focusing on genes that are identified as differentially expressed when cells are inhibited by acetic acid.

The processing characteristics of biofuel feedstocks are strongly affected by the quantity and quality of lignin in the cell wall structure. We present the effect of brown midrib mutations on rates and yields of cellulosic ethanol production from maize silage. Both raw silage and silage from commercial sources were pretreated using liquid hot water (160-180 C) and assessed by enzymatic hydrolysis and fermentation using the glucose/xylose fermenting Purdue recombinant S. cerevisiae 424A(LNH-ST). At 20% solids concentration (20 g/L), pretreated bmr silage achieved higher yields of sugars than non-bmr silage pretreated under the same conditions. At the optimal pretreatment conditions, bmr silage achieved 62% of theoretical yield of glucose after 24 hours of enzymatic hydrolysis (15 FPU cellulase per gram glucan) compared to 50% yield from non-bmr silage. Sugars from both silage varieties fermented to ethanol at high yields using the Purdue recombinant yeast strain.

Lignocellulosic biomass, primarily comprised of cellulose, hemicellulose, and lignin, is a promising renewable feedstock for the microbial production of chemicals, especially ethanol. The major fermentable sugars (hydrolysates) released from the processing of the lignocellulose are glucose and xylose. However, the primary processing steps required for this conversion also produce a range of compounds that can inhibit the subsequent microbial fermentation. One such inhibitory compound is acetic acid, liberated during the pretreatment of the biomass. In this poster, we report the effect of acetic acid on glucose/xylose co-fermentation by the genetically modified S. cerevisiae 424A(LNH-ST). The co-fermentation of glucose and xylose was performed under acetic acid conditions of 5, 10, 15 g/L over a pH range of 5 – 6. To maintain the pH at the specified initial value, the fermentations were carried out in a 1L New Brunswick BioFlow 110 benchtop fermentor equipped with a pH controller. Results showed that the fermentation of both sugars was affected by the presence of acetic acid. The inhibitory effect of acetic acid increased as the pH decreased. The results also indicate that the utilization of xylose is more influenced by acetic acid concentration and pH than the utilization of glucose.

The conversion of switchgrass to fermentable sugars and ethanol provides a cellulosic feedstock for production of fuel ethanol which may be grown on lands not suitable for food agriculture. Switchgrass itself consists of 33% cellulose, 25% hemicelluloses, 18% lignin, and 24% other. If switchgrass is processed without pretreatment, the maximal conversion achieved at an enzyme loading of 15 FPU/g glucan (5 FPU/g biomass) is less than 5%. When the switchgrass is pretreated in liquid hot water, the conversion increases by 25-fold, resulting in 80% glucose yield. The utilization of liquid hot water followed by enzyme hydrolysis and fermentation is described in this paper. The levels of enzyme loading and inhibition effects are briefly discussed as part of the overall CAFI research project.

The production of ethanol from cellulose for use as a liquid transportation fuel requires a combination of process engineering, microbiology, and accessibility to feedstock. The feedstock must be available to supply the plant 24 hours / day, 7 days per week. Siting of the plant is key to ensuring feedstock supply. Conversion of the feedstock to sugars and to ethanol requires pretreatment, hydrolysis, and fermentation. Pretreatment softens up the plant cell wall structure and enables enzymes to access the cellulose so that they may catalyze the formation of monosaccharides. The monsaccahrides, in turn, may be converted to ethanol through microbial fermentation by yeast or bacteria that have been engineered to convert both glucose and xylose to ethanol. During the bioconversion steps, cascading molecular control of enzyme activity occurs due to inhibitors that are formed during the pretreatment and/or hydrolysis steps. This paper discusses the role of process engineering in addressing issues of inhibition, solids loading, and fermentation, and gives a review of fundamental mechanisms and future research needs for converting renewable resources to biofuels in a cost effective manner.

Commercial cellulasse preparations effectively hydrolyze cellulose present in liquid hot water pretreated distillers grains (DG) to glucose. However, commercial xylanase preparations yield approximately 25% of the xylose and arabinose from the hemicellulose fraction of the same hydrolysate. Since hemicellulose accounts for nearly 40% of the carbohydrate content of DG, optimizing enzyme activities is required to maximize total fermentatble sugar yields from this biomass material. In this paper we report the effect of supplementing commercial xylanase with additional enzyme activities. The addition of these enzymes to commercial cellulase significantly increased the yields of arabinose and xylose to 78%. Also presented is the effect of high solids concentrations on yield and rate of xylose and arabinose liberation.

Furfural, the acid-catalyzed degradation product of pentoses, has been shown to decrease the fermentability and the ethanol yields from sugars derived from lignocellulose. This paper reports a systematic study of the effect of furfural on cell growth and fermentation of both glucose and xylose to ethanol by the recombinant yeast S. cerevisiae 424A(LNH-ST). Fermentations were run with furfural, HMF, or both in a control solution of YEP with glucose and xylose as co-substrates or xylose alone. Cell concentrations at the beginning of the fermentation varied between 0.1 and 9 g/L. Inhibitor concentrations were varied from 0 to 40 g/L. Batch fermentations were carried out for at least 48 hours in 300 mL sidearm flasks at 30 C and 200 rpm with periodic sampling for analysis by HPLC. Our results show that concentrations of either furfural below about 5 g/L cause negligible inhibition for yeast cells in early stationary phase while similar concentrations will lengthen the lag phase of lower innoculations of cells. Xylose fermentation to ethanol is more sensitive to furfural than glucose for fermentation to ethanol. These results are then compared to the fermentation of xylose obtained from pretreated corn stover and pretreated poplar hydrolyzates from the Biomass Refining Consortium for Applied Fundamentals and Innovation (CAFI) that contain varying concentrations of inhibitors.

We have recently shown that modification of lignin subunit composition can significantly increase the yield of fermentable sugars obtained from enzymatic saccharification of maize stover. The brown midrib1 (bmi) and bm3 mutations each increase the yield of glucose per gram dry stover by 50% relative to the wild-type control (inbred A619). When combined in a near-isogenic bm1-bm3 double mutant, the two mutations act in an additive manner, resulting in a doubling of the yield of glucose. Even though there was no apparent increase in cellulose content, based on kinetic studies both the rate of hydrolysis and the overall yield of glucose increased as a result of the mutations. In order to be able to generalize our results, we are investigating if this increased yield is consistent in different genetic backgrounds. In addition, we are investigating what the basis is of the enhanced hydrolysis in these bm mutants by in situ visualization of cellulases. We have designed recombinant proteins consisting of the cellulose binding domain (CBD) isolated from Trichoderma reesei endoglucanases labeled with green-fluorescent protein (GFP) to study how changes in cell wall composition and architecture impact the distribution of cellulolytic enzymes. These analyses will be performed in intact plant tissue as well as in ground stover using UV fluorescence microscopy. The resulting information will be valuable for designing plant cell wall composition in such a way that agronomic properties and biomass conversion are optimally balanced.

Mid-severity dilute acid pretreatment liquor from Kramer corn stover pretreated in the Sunds reactor at NREL was analyzed, conditioned, and fermented by glucose/xylose co-fermenting yeast (S. cerevisiae 424A(LNH-ST). This yeast is currently being validated for large scale industrial cellulosic ethanol production. The pretreatment hydrolysate liquid contained 22.4 to 24.6 g/L glucose, 72.7 to 76.2 g/L xylose, 13 g/L acetic acid, 2.1 g/L furfural and 2.7 g/L HMF, and was conditioned by over-limiting contact with polymeric (XAD-4 resin), or a combination of the two steps before fermentation. The sugar compositions were similar to those for the untreated hydrolysate, although in all cases a significant fraction of the furfural was removed, and in the case of overliming, some HMF was also removed. XAD4 has been previously shown to selectively remove furfural and color from the aqueous sugar solutions. S. cerevisiae 424A(LNH-ST) completes the fermentation in 48 hours for media containing the same amounts of pure sugars as are found in the hydrolysates. However, high salt and acetic acid concentration in the dilute acid pretreatment liquor, and/or residual HMF, is known to decrease the fermentation rate, and this was found to be the cse here as well. When the different solutions were fermented by 424A(LNH-ST), glucose was consumed in 2 to 6 hours, but only 40% of the xylose was fermented to ethanol within 72 hours as compared to complete fermentation in 48 hours in the synthetic and other media. Research is continuing to optimize conditions and enhance rates and extents of ethanol fermentation from xylose in hydrolysates obtained from acid pretreated corn stover.

Cellulolytic enzymes consist of a catalytic domain, a linking peptide, and a binding domain. This poster describes research on carboxylic acids that have potential as the catalytic domain and planar cellulose adsorbing molecules for constructing organic catalysts that mimic the action of enzymes in hydrolyzing cellulose by adsorbing the acid catalyst near the cellulose substrate. Glucose degradation, unlike cellulose hydrolysis, was shown to be independent of hydrogen ion concentration for carboxylic acids. Maleic acid, a dicarboxylic acid, effectively hydrolyzes cellobiose, the repeat unit of cellulose, by the relatively well-understood mechanism of acid hydrolysis. However, unlike sulfuric acid, maleic acid does not catalyze glucose degradation. Consequently, overall yields of glucose from cellulose were shown to be higher for maleic acid, when compared to sulfuric acid at equivalent solution pH. A number of organic, planar, molecules were screened for adsorption to cellulose at temperatures ranging from 30 - 140 C using a chromatogrpahic method. Trypan blue was shown to strongly adsorb to cellulose at high temperatures and possesses moieties that offer possibilities for linking acid catalysts to this cellulose adsortive compound.

Cellulosic biomass is known to be an ideal raw material for the production of chemicals by microbial processes, particularly those produced in large volumes such as ethanol. However, cellulosic biomass contains large amounts of xylose in addition to glucose. The naturally-occurring Saccharomyces yeasts used for large-scale ethanol production from starch (glucose) cannot metabolize xylose. In recent years, we have been able to genetically engineer the Saccharomyces yeasts to effectively co-metabolize glucose and xylose both aerobically and anaerobically. This was accomplished by cloning and overexpressing three major xylose-metabolizing genes - xylose reductase, xylitol dehydrogenase, and xylulokinase genes (KDR). The resulting genetically engineered yeast can metabolize xylose aerobically and anaerobically as well as effectively co-ferment both glucose and xylose simultaneously to ethanol. First, these three genes were cloned on a high copy number plasmid. Subsequently, we developed an effective and reliable system for integrating multiple copies of multiple genes into the yeast chromosome, and made it possible to effectively integrate the three genes into the chromosomes of any Saccharomyces yeast. In this paper, we compare the ability of haploid, diploid and tetraploid S. cerevisiae with identical genetic background to co-ferment glucose and xylose when transformed with multiple copies of KDR, either on high-copy-number plasmid or integrated on the host chromosomes.

Extraction of fermentable substrates from biopolymers is a form of primary separatin. Pretreatment of corn fiber by pressure cooking a 15 g/L fiber slurry in water at controlled pH produces soluble oligosaccharides. Our quest for catalysts that mimic the selectivity of cellulolytic enzymes, but at a lower cost, led us to rediscover the utility of a packed bed of strong cation exchange resin for saccharification of these oligosaccharides. The combination of controlled residence time, high ratio of diffisivity of monosaccharides to oligosaccharide, pore structure of the resin, and reactivity of glycosidic bonds in dissolved oligosaccharides, enables hydrolysis to be achieved in a flow reactor while minimizing formation of aldehydes and fermentation inhibitors. We report hydrolysis and diffusional effects for Amberlyst 35W over a temperature range of 100 to 130 C, as well as approaches that minimize fouling of the catalyst by proteins, phenolics and minerals. Conversions of over 80% are achieved.

The naturally occurring Saccharomyces yeasts, particularly those capable of effectively fermenting glucose to ethanol, are unable to metabolize xylose aerobically or anaerobically. We succeeded in developing genetically engineered yeasts that effectively utilize xylose aerobically for growth, as well as effectively co-ferment glucose and xylose to ethanol. However, our genetically engineered yeasts still utilize glucose much faster than xylose. One reason is that the Saccharomyces yeasts deo not contain specific transporters for xylose but instead rely on glucose transporters to transport xylose. Unfortunately, the glucose transporters greatly favor glucose over xylose. Saccharomyces yeasts have at least 7 major glucose transporters (Hxt1-7) with varying affinities for glucose. We studied the affinity of each yeast Hxt transporters for xylose and found that Hxt 4 is one of the transporters with moderate affinity to glucose and xylose. We believe that converting such an Hxt transporter to solely transport xylose could lead to the development of yeast that ferments xylose more efficiently. It was reported that Phe431 is crucial for yeaqst Hxt 2 to transport glucose. In this presentation, we report our recent finding on the role of Phe431 in Hxt 4 for transporting glucose and xylose.

Ethanol production utilizing five and six carbon sugars recovered from corn stover hydrolysate has been documented. Hot water pretreatment of corn stover has been shown to assist in the enzxymatic hydrolysis of the biomass to fermentable sugars. Corn stover contains carbon sources other than carbohydrates including lignin (17-18% dry mass) and crude fat (1-2% dry mass). The first objective of this study was to investigate the coproducts generated by modification of the hot water pretreatment method by the addition of varying concentrations of ethanol. Sample from this study were analyzed by GC/MS and contained free fatty-acids (Palmitic and Linoleic acids) and lignin derivatives (coniferyl alcohol, vanillin, etc.) that are soluble in ethanol-water mixtures. Phase two of this study involved passing the pretreatment liquid stream through a tubular reactor containing Amberlyst 35 catalyst. This catalyst is sulfonic acid-based and has an ion exchange capacity of 5.48 meq/gram. Analysis of this liquid stream by GC/MS found ethyl esters of Palmitic acid, Linoleic acid, Oleic acid and Steric acid which are components of bio-diesel. Phenolic compounds identified included 2 ethyl phenol and ethyl 3-(4-hydroxyphenol)-propenate. Solids remaining following pretreatment were hydrolyzed by enzyme with minimal difference in results as compared to water only pretreatment at up to 50% ethanol.

We should strive to make the cost for the production of cellulosic ethanol as low as possible. One way to reduce the overall cost for the production of cellulosic ethanol is to produc ehigh valued co-products or by-products during the production of ethanol. One class of co-products could be various industrial enzymes that are high priced products. One important industrial enzyme is phytase, which is used as a supplement in animal feed to improve phosphorus nutrition and to reduce phosphorus pollution of animal excreta. Saccharomyces yeast has the GRAS status and has been used for the preparation of food and drinks for human consumption for thousands of years. Thus, it can be used for the production of any enzyme or special protein including those for human and animal consumption. In this presentation we focus on the expression and secretion of a bacterial phytase in our glucose/xylose co-fermenting Saccharomyces yeast.

We are looking at c hanging lignin composition as a way to improve the efficiency of bio-fuel production from maize stover. In secondary cell walls, carbohydrates are intimately associated with the hydrophobic polymer lignin. We hypothesize that the enzymatic or chemical hydrolysis of cell wall carbohydrates is impeded by the presence of lignin. Changing the content and subunit composition of lignin is expected to alter the interaction between lignin and carbohydrates and therefore affect the yield of fermentable sugars, ideally in a positive manner. Preliminary experiments with a set of near-isogenic maize mutants with altered lignin composition revealed that (1) changes in lignin composition could increase the yield of fermentable sugars by as much as 35%, and (2) lignin composition is a more important determinant of the yield of fermentable sugars than lignin content. We are currently using a deconvolution strategy to define a relationship between lignin subunit composition and the efficiency of hydrolysis of cell wall carbohydrates. This involves the analysis of a set of single and double cell wall mutants in terms of bio-fuel production, but, given the importance of lignin in the overall viability of the plant, also in terms of agronomic performance. This approach is expected to lead to the development of high-efficiency biofuel crops that still perform well agronomically.

Recent studies have proven ethanol to be the ideal liquid fuel for transportation and renewable cellulosic biomass to be the attractive feedstocks for ethanol-fuel production by fermentation. The major fermentable sugars from hydrolysis of cellulosic biomass (such as rice stow, sugarcane bagasse, corn fiber, softwoods, hardwoods, and grasses) are D-glucose and D-xylose. The efficient fermentation of both glucose and xylose present in cellulosic biomass to ethanol is essential for these renewable resources to be used as feedstocks for bio-fuel production. The naturally-occurring Saccharomyces yeasts have proven to be safe, effective, and user-friendly microorganisms for the large-scale production of industrial ethanol from glucose-based feedstocks. However, these yeasts cannot metabolize xylose. Our group at Purdue University succeeded in the development of the genetically engineered Saccharomyces yeasts that can effectively co-ferment glucose and xylose to ethanol. This was accomplished by the cloning and over-expression of three major xylose-metabolizing genes; xylose reductase, xylitol dehydrogenase, and xylulokinase genes in yeast. In this presentation, we demonstrate that our stable recombinant Saccharomyces yeast can efficiently ferment glucose and xylose present in hydrolysates from different cellulosic biomass to ethanol.

A process was designed, based on experimental knowledge and industrial experience, to incorporate a corn fiber pretreatment/enzyme hydrolysis/ethanol fermentation system into an existing corn starch-fermenting ethanol plant. This process for corn residue pretreatment was incorporated into an existing corn starch-fermenting ethanol plant for a pilot-scale test of the design. The pretreatment process cnsists of several steps. The corn fiber enters a storage tank where it is mixed with stillage. The resulting slurr is pumped through two heat exchangers; the first heat exchanger transfers heat from the fiber stream leaving the pretreatment reactor to the fiber entering the pretreatment reactor, and the second heat exchanger transfers heat from steam to the fiber stream. The hot fiber stream passes through a snake-coil at 16 C for 20 minutes. It is during this time that the cellulose structure loses the crystalline structure. The fiber stream leaves the pretreatment reactor and exchanges heat with the incoming fiber stream. Finally, an economic analysis of the key process steps was conducted to generate a pro forma analysis for corn fiber/enzyme hydrolysis/ethanol fermentation.

The production of aldehydes that are microbial inhibitors may occur when hexoses and pentoses are exposed to temperatures above 150 C and acidic pH in water. These are common conditions encountered when biomass is pretreated. Concentrations of about 0.1% or higher of the degradation product, furfural, strongly inhibit fermentation as was confirmed for hydrolysate that contained 0.5% (w/o) furfural. This paper reports contacting of a polymeric adsorbent, XAD-4, with biomass hydrolysate that contains furfural. Liquid chromatographic analysis of the remaining effluent showed that furfural concentrations were less than 0.1 g/L in contrast to the initial concentrations, which were in the range jof 1 to 5 g/L. Fermentation of the resulting sugars with recombinant E. coli ethanologenic strain K011 confirmed that the concentration of furfural in the hydrolysate was at a low enough level that the inhibition effect was negligible. Fermentation of XAD-4 treated hydrolysate with E. coli K011 was near as rapid as the control medium, which was formulated with reagent grade sugars of the same concentration. Ethanol yields for both fermentations were 90% of theoretical. Modeling of the adsorptive properties of this styrene-based adsorbent indicates that it is suitable for on-off chromatography, and could be useful for removing small amounts of aldehydes that might otherwise inhibit fermentation.