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Microalgae are attractive feedstocks for biofuel production and are especially suitable for thermochemical conversion due to the presence of thermally labile constituents-lipids, starch and protein. However, the thermal degradation of starch and proteins produces water as well as other O- and N-compounds that are mixed-in with energy-dense lipid pyrolysis products. To produce hydrocarbon-rich products from microalgae biomass, we assessed in situ and ex situ catalytic pyrolysis of a lipid-rich Chlorella sp. in the presence of the HZSM-5 zeolite catalyst over a temperature range of 450-550°C. Results show that product yields and compositions were similar under both in situ and ex situ conditions with benzene, toluene and xylene produced as the primary aromatic products. Yields of aromatics increased with increasing temperature and the highest aromatic yield (36.4% g aromatics/g ash-free microalgae) and selectivity (87% g aromatics/g bio-oil) was obtained at 550°C. Also, at this temperature, oxygenates and nitrogenous compounds were not detected among the liquid products during ex situ catalytic pyrolysis. We also assessed the feasibility of a two-step fractional pyrolysis approach integrated with vapor phase catalytic upgrading. In these experiments, the biomass was first pyrolyzed at 320°C to degrade and volatilize starch, protein and free fatty acids. Then, the residual biomass was pyrolyzed again at 450°C to recover products from triglyceride decomposition. The volatiles from each fraction were passed through an ex situ catalyst bed. Results showed that net product yields from the 2-step process were similar to the single step ex situ catalytic pyrolysis at 450°C indicating that tailored vapor phase upgrading can be applied to allow separate recovery of products from the chemically distinct biomass components-(1) lower calorific value starch and proteins and (2) energy-dense lipids.

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Laboratory analytical techniques employed for triglyceride quantification in oleaginous biomass (e.g., microalgae and oilseeds) involve multiple steps and typically require use of volatile organic solvents. Here we describe a single-step approach for measurement of triglycerides using thermogravimetry (TG). We have observed that triglycerides undergo complete volatilization over a narrow temperature interval of 370-450 °C, with negligible solid residue under inert atmosphere, whereas other constituents of oleaginous biomass (such as proteins and carbohydrates) primarily degrade below 350 °C. As a result, triglyceride content of biomass can be estimated using TG by determining the mass loss of the sample in the temperature interval of 370-450 °C.

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Fatty nitriles are widely used as intermediate molecules in the pharmaceutical and polymer industries. In addition, hydrogenation of fatty nitriles produces fatty amines that are common surfactants. In the conventional fatty nitrile process, triglycerides are first hydrolyzed and the resulting fatty acids are catalytically reacted with NH3 in a liquid-phase reaction. In this study, we report a simpler one-step fatty nitrile production method that involves a direct vapor-phase reaction of triglycerides with NH3 in the presence of heterogeneous solid acid catalysts. The reactions were performed in a tubular reactor maintained at 400 °C into which triglycerides were injected through an atomizer to allow rapid volatilization and reaction; NH3 was fed as a gas. Several metal oxide catalysts were tested, and reactions in the presence of V2O5 resulted in near-theoretical fatty nitrile yields (84 wt % relative to the feed mass). In general, catalysts with higher acidity such as V2O5, Fe2O3, and ZnO showed higher fatty nitrile yields compared to low acidity catalysts such as ZrO, Al2O3, and CuO. Energy balance calculations indicate that the one-step reaction described here would require significantly lower energy than the conventional process primarily because of the elimination of the energy-intense triglyceride hydrolysis.

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A system that incorporates a packed bed reactor for isomerization of xylose and a hollow fiber membrane fermentor (HFMF) for sugar fermentation by yeast was developed for facile recovery of the xylose isomerase enzyme pellets and reuse of the cartridge loaded with yeast. Fermentation of pre-isomerized poplar hydrolysate produced using ionic liquid pretreatment in HFMF resulted in ethanol yields equivalent to that of model sugar mixtures of xylose and glucose. By recirculating model sugar mixtures containing partially isomerized xylose through the packed bed and the HFMF connected in series, 39 g/l ethanol was produced within 10h with 86.4% xylose utilization. The modular nature of this configuration has the potential for easy scale-up of the simultaneous isomerization and fermentation process without significant capital costs.

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Pyrolysis experiments were performed with algal and lignocellulosic feedstocks under similar reactor conditions for comparison of product (bio-oil, gas and bio-char) yields and composition. In spite of major differences in component bio-polymers, feedstock properties relevant to thermo-chemical conversions, such as overall C, H and O-content, C/O and H/C molar ratio as well as calorific values, were found to be similar for algae and lignocellulosic material. Bio-oil yields from algae and some lignocellulosic materials were similar; however, algal bio-oils were compositionally different and contained several N-compounds (most likely from protein degradation). Algal bio-char also had a significantly higher N-content. Overall, our results suggest that it is feasible to convert algal cultures deficient in lipids, such as nuisance algae obtained from natural blooms, into liquid fuels by thermochemical methods. As such, pyrolysis technologies being developed for lignocellulosic biomass may be directly applicable to algal feedstocks as well.

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In this paper, the feasibility of a technology for fermenting sugar mixtures representative of cellulosic biomass hydrolyzates with native industrial yeast strains is demonstrated. This paper explores the isomerization of xylose to xylulose using a bi-layered enzyme pellet system capable of sustaining a micro-environmental pH gradient. This ability allows for considerable flexibility in conducting the isomerization and fermentation steps. With this method, the isomerization and fermentation could be conducted sequentially, in fed-batch, or simultaneously to maximize utilization of both C5 and C6 sugars and ethanol yield. This system takes advantage of a pH-dependent complexation of xylulose with a supplemented additive to achieve up to 86% isomerization of xylose at fermentation conditions. Commercially-proven Saccharomyces cerevisiae strains from the corn-ethanol industry were used and shown to be very effective in implementation of the technology for ethanol production.

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Crystallizing solutions of proteins often contain various nonelectrolyte additives that arise from the purification process of proteins or from the reagents employed in the screening kits. Currently, limited knowledge exists about the influence of these additives on the mechanisms underlying the crystallization process, in particular on the nucleation stage of crystals. To address this need, we studied crystallization of two proteins, D-xylose isomerase and chicken egg-white lysozyme, in small batches and in the presence of two solubility-enhancing additives, acetonitrile and glycerol. We have also measured the nucleation rates of crystals of these proteins in the presence and in the absence of acetonitrile using the method of initial rates. With the addition of the solubility enhancers, both proteins exhibited an increase in crystal nucleation at any given supersaturation. Solubility enhancing additives appear to lower the energy barrier to nucleation by influencing the strength of attraction between the protein molecules. We have characterized the quality of D-xylose isomerase crystals by determining the crystal mosaicity, which showed considerable improvement for crystals grown in the presence of additives. When compared to the crystals of chicken egg-white lysozyme, D-xylose isomerase crystals required higher supersaturations to nucleate. We attribute this result to the large size of the D-xylose isomerase molecule, which influences the energy barrier to nucleation by increasing the surface area of the critical nucleus. Contrary to the common expectation that reagents that solubilize the protein may hinder the crystallization process, our results suggest that solubility enhancers, in fact, can have a beneficial effect on the nucleation and growth of crystals. These findings are of importance in formulating successful strategies toward crystallizing new proteins.

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The application of ionic liquids (ILs) as nonderivatizing solvents for the pretreatment and regeneration of cellulose is a growing area of research. Here we report the development of a rapid and simple method for the determination of residual ethanol content in two hydrophilic ILs, 1-butyl-3-methylimidazolium chloride and 1-ethyl-3-methylimidazolium acetate. The method utilizes headspace solid-phase microextraction coupled with gas chromatography at elevated extraction temperatures, resulting in rapid equilibration times. The effect of IL water content on the ethanol extraction efficiency is presented. Recovery experiments carried out in real samples gave recoveries ranging from 96.8 to 98.2%.

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Of the sugars recovered from lignocellulose, D-glucose can be readily converted into ethanol by baker's or brewer's yeast (Saccharomyces cerevisiae). However, xylose that is obtained by the hydrolysis of the hemicellulosic portion is not fermentable by the same species of yeasts. Xylose fermentation by native yeasts can be achieved via isomerization of xylose to its ketose isomer, xylulose. Isomerization with exogenous xylose isomerase (XI) occurs optimally at a pH of 7-8, whereas subsequent fermentation of xylulose to ethanol occurs at a pH of 4-5. We present a novel scheme for efficient isomerization of xylose to xylulose at conditions suitable for the fermentation by using an immobilized enzyme system capable of sustaining two different pH microenvironments in a single vessel. The proof-of-concept of the two-enzyme pellet is presented, showing conversion of xylose to xylulose even when the immobilized enzyme pellets are suspended in a bulk solution whose pH is sub-optimal for XI activity. The co-immobilized enzyme pellets may prove extremely valuable in effectively conducting "simultaneous isomerization and fermentation" (SIF) of xylose. To help further shift the equilibrium in favor of xylulose formation, sodium tetraborate (borax) was added to the isomerization solution. Binding of tetrahydroxyborate ions to xylulose effectively reduces the concentration of xylulose and leads to increased xylose isomerization. The formation of tetrahydroxyborate ions and the enhancement in xylulose production resulting from the complexation was studied at two different bulk pH values. The addition of 0.05 M borax to the isomerization solution containing our co-immobilized enzyme pellets resulted in xylose to xylulose conversion as high as 86% under pH conditions that are suboptimal for XI activity. These initial findings, which can be optimized for industrial conditions, have significant potential for increasing the yield of ethanol from xylose in an SIF approach.

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Efficient hydrolysis of cellulose-to-glucose is critically important in producing fuels and chemicals from renewable feedstocks. Cellulose hydrolysis in aqueous media suffers from slow reaction rates because cellulose is a water-insoluble crystalline biopolymer. The high-crystallinity of cellulose fibrils renders the internal surface of cellulose inaccessible to the hydrolyzing enzymes (cellulases) as well as water. Pretreatment methods, which increase the surface area accessible to water and cellulases are vital to improving the hydrolysis kinetics and conversion of cellulose to glucose. In a novel technique, the microcrystalline cellulose was first subjected to an ionic liquid (IL) treatment and then recovered as essentially amorphous or as a mixture of amorphous and partially crystalline cellulose by rapidly quenching the solution with an antisolvent. Because of their extremely low-volatility, ILs are expected to have minimal environmental impact. Two different ILs, 1-n-butyl-3-methylimidazolium chloride (BMIMC1) and 1-allyl-3-methylimidazolium chloride (AMIMC1) were investigated. Hydrolysis kinetics of the IL-treated cellulose is significantly enhanced. With appropriate selection of IL treatment conditions and enzymes, the initial hydrolysis rates for IL-treated cellulose were up to 90 times greater than those of untreated cellulose. We infer that this drastic improvement in the "overall hydrolysis rates" with IL-treated cellulose is mainly because of a significant enhancement in the kinetics of the "primary hydrolysis step" (conversion of solid cellulose to soluble oligomers), which is the rate-limiting step for untreated cellulose. Thus, with IL-treated cellulose, primary hydrolysis rates increase and become comparable with the rates of inherently faster "secondary hydrolysis" (conversion of soluble oligomers to glucose).

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We have used liquid waste obtained from a beer brewery process to produce ethanol. To increase the productivity, genetically modified organism, Escherichia coli KO11, was used for ethanol fermentation. Yeast was also used to produce ethanol from the same feed stock, and the ethanol production rates and resulting concentrations of sugars and ethanol were compared with those of KO11. In the experiments, first the raw wastewater was directly fermented using two strains with no saccharification enzymes added. Then, commercial enzymes, alpha-amylase, pectinase, or a combination of both, were used for simultaneous saccharification and fermentation, and the results were compared with those of the no-enzyme experiments for KO11 and yeast. Under the given conditions with or without the enzymes, yeast produced ethanol more rapidly than E. coli KO11, but the final ethanol concentrations were almost the same. For both yeast and KO11, the enzymes were observed to enhance the ethanol yields by 61-84% as compared to the fermentation without enzymes. The combination of the two enzymes increased ethanol production the most for the both strains. The advantages of using KO11 were not demonstrated clearly as compared to the yeast fermentation results.

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Hydrolysis of cellulose to glucose in aqueous media catalyzed by the cellulase enzyme system suffers from slow reaction rates due in large part to the highly crystalline structure of cellulose and inaccessibility of enzyme adsorption sites. In this study, an attempt was made to disrupt the cellulose structure using the ionic liquid (IL), 1-n-butyl-3-methylimidazolium chloride, in a cellulose regeneration strategy which accelerated the subsequent hydrolysis reaction. ILs are a new class of non-volatile solvents that exhibit unique solvating properties. They can be tuned to dissolve a wide variety of compounds including cellulose. Because of their extremely low volatility, ILs are expected to have minimal environmental impact on air quality compared to most other volatile solvent systems. The initial enzymatic hydrolysis rates were approximately 50-fold higher for regenerated cellulose as compared to untreated cellulose (Avicel PH-101) as measured by a soluble reducing sugar assay.

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Fundamental understanding of protein crystal nucleation facilitates crystallization of biological macromolecules for structure determination and control of crystal size distribution. In the studies presented here, nucleation kinetics of hen egg-white lysozyme crystals were measured at solution conditions that exhibited equal solubility by adjusting pH, temperature, or sodium chloride concentration. It was observed that solution conditions that lead to equal solubility resulted in equal nucleation rates and hence kinetic parameters. Since the solubility of globular proteins correlates with the osmotic second virial coefficient, B(22), an integral measure of the protein pair interaction potential, this observation indicates that the protein pair interaction plays a key role in determining nucleation kinetic parameters.

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