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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.115, #1-3)

Origins of and changes in the symposium Series on Biotechnology for Fuels and Chemicals by Charles D. Scott (pp. 765-769).

More than two decades ago, a group of research engineers and applied scientists with interest in energy applications of bioprocessing initiated an annual symposium series ultimately entitled “Biotechnology for Fuels and Chemicals.” The Department of Energy, several of the national laboratories, and various industrial firms have supported these symposia that are now held alternately in the mountains of Tennessee and Colorado. There has been wide acceptance of these meetings, with participants from the government, academia, and the commercial sector, and more than 20 different nations have been represented. The peer-reviewed proceedings have been published and are an important source for innovative bioprocessing research.

Keywords: Symposium; biotechnology; fuels; chemicals; bioprocessing

Optimization of enzymatic production of biodiesel from castor oil in organic solvent medium by Débora de Oliveira; Marco Di Luccio; Carina Faccio; Clarissa Dalla Rosa; João Paulo Bender; Nádia Lipke; Silvana Menoncin; Cristiana Amroginski; José Vladimir de Oliveira (pp. 771-780).

We studied the production of fatty acid ethyl esters from castor oil using n-hexane as solvent and two commercial lipases, Novozym 435 and Lipozyme IM, as catalysts. For this purpose, a Taguchi experimental design was adopted considering the following variables: temperature (35–65°C), water (0–10 wt/wt%), and enzyme (5–20 wt/wt%) concentrations and oil-to-ethanol molar ratio (1∶3 to 1∶10). An empirical model was then built so as to assess the main and cross-variable effects on the reaction conversion and also to maximize biodiesel production for each enzyme. For the system containing Novozym 435 as tatalyst the maximum conversion obtained was 81.4% at 65°C, enzyme concentration of 20 wt/wt%, water concentration of 0 wt/wt%, and oil-to-ethanol molar ratio of 1∶10. When the catalyst was Lipozyme IM, a conversion as high as 98% was obtained at 65°C, enzyme concentration of 20 wt/wt%, water concentration of 0 wt/wt%, and oil-to-ethanol molar ratio of 1∶3.

Keywords: Alcoholysis; vegetable oils; lipases; reaction kinetics; biodiesel

Two-step preparation for catalyst-free biodiesel fuel production by Dadan Kusdiana; Shiro Saka (pp. 781-791).

Biodiesel fuel was prepared by a two-step reaction: hydrolysis and methyl esterification. Hydrolysis was carried out at a subcritical state of water to obtain fatty acids from triglycerides of rapeseed oil, while the methyl esterification of the hydrolyzed products of triglycerides was treated near the supercritical methanol condition to achieve fatty acid methyl esters. Consequently, the two-step preparation was found to convert rapessed oil to fatty acid methyl esters in considerably shorter reaction time and milder reaction condition than the direct supercritical methanol treatment. The optimum reaction condition in this two-step preparation was 270°C and 20 min for hydrolysis and methyl esterification, respectively. Variables affecting the yields in hydrolysis and methyl esterification are discussed.

Keywords: Supercritcal methanol; biodiesel; methyl esters; transesterification; methyl esterification

Biodiesel fuel from vegetable oil by various supercritical alcohols by Yuichiro Warabi; Dadan Kusdiana; Shiro Saka (pp. 793-801).

Biodiesel was prepared in various supercritical alcohol treatments with methanol, ethanol, 1-propanol, 1-butanol, or 1-octanol to study transesterification of rapeseed oil and alkyl esterification of fatty acid at temperatures of 300 and 350°C. The results showed that in transes terification, the reactivity was greatly correlated to the alcohol: the longer the alkyl chain of alcohol, the longer the reaction treatment. In alkyl esterification of fatty acids, the conversion did not depend on the alcohol type because they had a similar reactivity. Therefore, the selection of alcohol in biodiesel production should be based on consideration of its performance of properties and economics.

Keywords: Supercritical alcohol; biodiesel; fatty acids alkyl ester; transesterification; alkyl esterification

Effects of trace contaminants on catalytic processing of biomass-derived feedstocks by Douglas C. Elliott; Keith L. Peterson; Danielle S. Muzatko; Eric V. Alderson; Todd R. Hart; Gary G. Neuenschwander (pp. 807-825).

Model compound testing was conducted in a batch reactor to evaluate the effects of trace contaminant components on catalytic hydrogenation of sugars. Trace components are potential catalyst poisons when processing biomass feedstocks to value-added chemical products. Trace components include inorganic elements such as alkali metals and alkaline earths, phosphorus, sulfur, aluminum, silicon, chloride, or transition metals. Protein components in biomass feedstocks can lead to formation of peptide fractions (from hydrolysis) or ammonium ions (from more severe breakdown), both of which might interfere with catalysis. The batch reactor tests were performed in a 300-mL stirred autoclave, with multiple liquid samples withdrawn over the period of the experiment. Evaluation of these test results suggest that most of the catalyst inhibition is related to nitrogen-containing components.

Keywords: Catalysts; trace minerals; protein; catalytic hydrogenation; hydrolysates

Characterization of surfactin from Bacillus subtilis for application as an agent for enhanced oil recovery by Kastli D. Schaller; Sandra L. Fox; Debby F. Bruhn; Karl S. Noah; Gregory A. Bala (pp. 827-836).

Surfactin produced by Bacillus subtilis (ATCC 21332) was used to examine the effect of altering salt concentration, pH, and temperature on surfactin activity (as measured by reductions in surface tension). These parameters are some of the conditions that define oil reservoir characteristics and can affect the application of surfactants. The Biotechnology for Oilfield Operations research program at the Idaho National Engineering and Environmental Laboratory (INEEL) has successfully produced surfactin from potato process effluents for possible use as an economical alternative to chemical surfactants for improved oil recovery. Surfactants enhance the recovery of oil through a reduction of the interfacial tension between the oil and water interfaces, or by mediating changes in the wettability index of the system. We investigated changes in surfactin activity under a range of conditions by measuring surface tension. Surface tension was determined using video image analysis of inverted pendant drops. Experimental variables included NaCl (0–10%), pH (3.0–10.0), and temperature (21–70°C). Each of these parameters, as well as selected combinations, resulted in discrete changes in surfactin activity. It is therefore important to consider the exploration of the studied surfactin as an enhanced oil recovery agent.

Keywords: Bacillus subtilis ; biosurfactant; surfactin; improved oil recovery; oil recovery agent

Effect of germ and fiber removal on production of ethanol from corn by Elankovan Ponnampalam; D. Bernie Steele; Deborah Burgdorf; Darold McCalla (pp. 837-842).

Ethanol fermentations were conducted using both whole corn, and corn with 100% of the germ, and a portion (∼74%) of the fiber removed. Ethanol production increased 11% in the germ and fiber-removed corn vs the whole corn. The protein content of distiller's dried grains and solubles increased from 30 to 36%, and phosphate levels were 60% lower in corn with germ and fiber removed vs whole corn. Removal of germ and fiber prior to fermentation allows higher starch loading and results in increased ethanol production. The integration of germ and fiber removal in the dry-grind ethanol industry could increase capacity and add valuable coproducts, resulting in increased productivity and profits.

Keywords: Ethanol; germ; fiber oil; distiller's dried grains and solubles; termentation

Production of fumaric acid using rice bran and subsequent conversion to succinic acid through a two-step process by Se-Kwon Moon; Young-Jung Wee; Jong-Sun Yun; Hwa-Won Ryu (pp. 843-855).

The fungal production of fumaric acid using rice bran and subsequent bacterial conversion of succinic acid using fungal culture broth were investigated. Since the rice bran contains abundant proteins, amino acids, vitamins, and minerals, it is suitable material that fungi use as a nitrogen source. The effective concentration of rice bran to produce fumaric acid was 5 g/L. A large amount of rice bran caused excessive fungal growth rather than enhance fumaric acid production. In addition, we could produce fumaric acid without the addition of zinc and iron. Fungal culture broth containing appro × 25 g/L of fumaric acid was directly employed for succinic acid conversion. The amount of glycerol and yeast extract required for succinic acid conversion was reduced to 70 and 30%, respectively, compared with the amounts cited in previous studies.

Keywords: Fumaric acid; succinic acid; two-step process; fungal culture broth; rice bran

Catalytic hydrogenation of glutamic acid by Johnathan E. Holladay; Todd A. Werpy; Danielle S. Muzatko (pp. 857-869).

Technology to convert biomass into chemical building blocks provides an opportunity to displace fossil fuels and increase the economic viability of biorefineries. Coupling fermentation capability with aqueous-phase catalysis provides novel routes to monomers and chemicals, including those not accessible from petrochemical routes. Glutamic acid provides a platform to numerous compounds through thermochemical approaches including hydrogenation, cyclization, decarboxylation, and deamination. Hydrogenation of amino acids also provides access to chiral compounds with high enantiopurity. This article detals aqueous-phase hydrogenation reactions that we have developed that lead to valuable chemical intermediates from glutamic acid. In addition, ¹³C nuclear magnetic resonance and matrix-assisted laser desorption ionization mass spectral data are presented that provide a mechanistic picture of the reactions. The results show that hydrogenation of glutamic acid has unique characteristics from other amino acids and that paradigms in the literature do not hold up for this transformation.

Keywords: Glutamic acid; catalytic hydrogenation; pyroglutaminol; pyroglutamic acid; phosphoric acid; prolinol

Opportunities in the industrial biobased products industry by Tracy M. Carole; Joan Pellegrino; Mark D. Paster (pp. 871-885).

Approximately 89 million metric to forganic chemicals and lubricants, the majority of which are fossil based, are produced annually in the United States. The development of new industrial bioproducts, for production in stand-alone facilities or biorefineries, has the potential to reduce our dependence on imported oil and improve energy security. Advances in biotechnology are enabling the optimization of feedstock composition and agronomic characteristics and the development of new and improved fermentation organisms for conversion of biomass to new end products or intermediates. This article reviews recent biotechnology efforts to develop new industrial bioproducts and improve renewable feedstocks and key market opportunities.

Keywords: Bioproducts; thermoplastics; fermentation; solvents; platform chemicals

Continuous production of butanol by clostridium acetobutylicum immobilized in a fibrous bed bioreactor by Wei-Cho Huang; David E. Ramey; Shang-Tian Yang (pp. 887-898).

We explored the influence of dilution rate and pH in continuous cultures of Clostridium acetobutylicum. A 200-mL fibrous bed bioreactor was used to produce high cell density and butyrate concentrations at pH 5.4 and 35°C. By feeding glucose and butyrate as a cosubstrate, the fermentation was maintained in the solventogenesis phase, and the optimal butanol productivity of 4.6g/(L h) and a yield of 0.42 g/g were obtained at a dilution rate of 0.9h⁻¹ and pH 4.3. Compared to the conventional acetone-butanol-ethanol fermentation, the new fermentation process greatly improved butanol yield, making butanol production from corn an attractive alternative to ethanol fermentation.

Keywords: ABE fermentation; butanol; Clostridium acetobutylicum ; fibrous bed bioreactor; dilution rate

Lipopeptide surfactant production by Bacillus subtilis grown on low-cost raw materials by Fabíula A. S. L. Reis; Eliana Flavia C. Sérvulo; Francisca P. De França (pp. 899-912).

The production of biosurfactant by Bacillus subtilis ATCC 6633 was investigated using commercial sugar, sugarcane juice and cane molasses, sugarcane juice alcohol stillage, glycerol, mannitol, and soybean oil. Commercial sugar generated the minimum values of surface tension, with the best results (28.7 mN/m, (relative critical micelle concentration [CMC⁻¹] of 78.6) being achieved with 10 g of substrate/L in 48 h. At a pH between 7.0 and 8.0, a higher production of surface-active compounds and a greater emulsifier activity was also observed. Enrichment of the culture medium with trace minerals and EDTA showed maximum yields, whereas supplementation with yeast extract stimulated only cell growth. The kinetic studies revealed that biosurfactant production is a cell growth-associated process; surface tension, CMC, and emulsification index values of 29.6 dyn/cm, 82.3, and 57%, respectively, were achieved, thus indicating that it is feasible to produce biosurfactants from a renewable and low-cost carbon source.

Keywords: Biosurfactant; Bacillus subtilis ; lipopeptide; surfactin; raw materials; commercial sugar

Higher-alcohols biorefinery by Edwin S. Olson; Ramesh K. Sharma; Ted R. Aulich (pp. 913-932).

The concept of a biorefinery for higher-alcohol production is to integrate ethanol and methanol formation via fermentation and biomass gasification, respectively, with, conversion of these simple alcohol intermediates into higher alcohols via the Guerbet reaction. 1-Butanol results from the selfcondensation of ethanol in this multistep reaction occurring on a single catalytic bed. Combining methanol with ethanol gives a mixture of propanol, isobutanol, and 2-methyl-1-butanol. All of these higher alcohols are usefulas solvents, chemical intermediates, and fuel additives and, consequently, have higher market values than the simple alcohol intermediates. Several new catalysts for the condensation of ethanol and alcohol mixtures to higher alcohols were designed and tested under a variety of conditions. Reactions of methanol ethanol mixtures gave as high as 100% conversion of the ethanol to form high yields of isobutanol with smaller amounts of 1-propanol, the amounts in the mixture depending on the starting mixture. The most successful catalysts are multifunctional with basic and hydrogen transfer components.

Keywords: Biorefinery, alcohols; Guterbet reaction; methanol-ethanol mixtures; catalyst

Fermentation of “Quick Fiber” produced from a modified corn-milling process into ethanol and recovery of corn fiber oil by Bruce S. Dien; Nick Nagle; Kevin B. Hicks; Vijay Singh; Robert A. Moreau; Melvin P. Tucker; Nancy N. Nichols; David B. Johnston; Michael A. Cotta; Quang Nguyen; Rodney J. Bothast (pp. 937-949).

Approximately 9% of the 9.7 billion bushels of corn harvested in the United States was used for fuel ethanol production in 2002, half of which was prepared for fermentation by dry grinding. The University of Illinois has developed a modified dry grind process that allows recovery of the fiber fractions prior to fermentation. We report here on conversion of this fiber (Quick Fiber [QF]) to ethanol. QF was analyzed and found to contain 32%wt glucans and 65%wt total carbohydrates. QF was pretreated with dilute acid and converted into ethanol using either ethanologenic Escherichia coli strain FBR5 or Saccharomyces cerevisiae. For the bacterial fermentation the liquid fraction was fermented, and for the yeast fermentation both liquid and solids were fermented. For the bacterial fermentation, the final ethanol concentration was 30 g/L, a yield of 0.44 g ethanol/g of sugar(s) initially present in the hydrolysate, which is 85% of the theoretical yield. The ethanol yield with yeast was 0.096 gal/bu of processed corn assuming a QF yield of 3.04 lb/bu. The residuals from the fermentations were also evaluated as a source of corn fiber oil, which has value as a nutraceutical. Corn fiber oil yields were 8.28%wt for solids recovered following prtetreatment.

Keywords: Bioethanol; corn fiber oil; Escherichia coli ; pentose fermentations; Saccharomyces cerevisiae ; β-glucosidase

Ammonia fiber explosion treatment of corn stover by Farzaneh Teymouri; Lizbeth Laureano-Pérez; Hasan Alizadeh; Bruce E. Dale (pp. 951-963).

Optimizing process conditions and parameters such as ammonia loading, moisture content of biomass, temperature, and residence time is necessary for maximum effectiveness of the ammonia fiber explosion process. Approximate optimal pretreatment conditions for corn stover were found to be temperature of 90°C, ammonia: dry corn stover mass ratio of 1∶1, moisture content of corn stover of 60% (dry weight basis), and residence time (holding at target temperature), of 5 min. Approximately 98% of the theoretical glucose yield was obtained during enzymatic hydrolysis of the optimal treated corn stover using 60 filter paper units (FPU) of cellulase enzyme/g of glucan (equal to 22 FPU/g of dry corn stover). The ethanol yield from this sample was increased up to 2.2 times over that of untreated sample. Lowering enzyme loading to 15 and 7.5 FPU/g of glucan did not significantly affect the glucose yield compared with 60 FPU, and any differences between effects at different enzyme levels decreased as the treatment temperature increased.

Keywords: Ammonia fiber explosion; corn stover; enzymatic hydrolysis; simultaneous saccharification and fermentation; moisture content; residence time

Initial evaluation of simple mass transfer models to describe hemicellulose hydrolysis in corn stover by Michael A. Brennan; Charles E. Wyman (pp. 965-976).

The uncatalyzed hydrolysis and removal of xylan from corn stover is markedly enhanced when operation is changed from batch to continuous flow through conditions, and the increase in hemicellulose removal with flow rate is inconsistent with predictions by widely used first-order kinetic models. Mass transfer or other physical effects could influence the hydrolysis rate, and two models reported in the literature for other applications were adapted to investigate whether incorporation of mass transfer into the kinetics could explain xylan removal in both batch and continuous flow through reactors on a more consistent basis. It was found that a simple leaching model and a pore diffusion/leaching model could describe batch and flow through data with accuracy similar to that of conventional batch models and could provide a more rational explanation for changes in performance with flow rate.

Keywords: Pretreatment; flowthrough; batch; mass transfer; hemicellulose hydrolysis

Impact of fluid velocity on hot water only pretreatment of corn stover in a flowthrough reactor by Chaogang Liu; Charles E. Wyman (pp. 977-987).

Flowthrough pretreatment with hot water only offers many promising features for advanced pretreatment of biomass, and a better understanding of the mechanisms responsible for flowthrough behavior could allow researchers to capitalize on key attributes while overcoming limitations. In this study, the effect of fluid velocity on the fate of total mass, hemicellulose, and lignin was evaluated for hot water only pretreatment of corn stover in tubular flow through reactors. Increasing fluid velocity significantly accelerated solubilization of total mass, hemicellulose, and lignin at early times. For example, when fluid velocity was increased from 2.8 to 10.7 cm/min, xylan removal increased from 60 to 82% for hot water only pretreatment of corn stover at 200°C after 8 min. At the same time, lignin removal increased from 30 to 46%. Dissolved hemicellulose was almost all in oligomeric form, and solubilization of hemicellulose was always accompanied by lignin release. The increase in removal of xylan and lignin with velocity, especially in the early reaction stage, suggests that chemical reaction is not the only factor controlling hemicellulose hydrolysis and that mass transfer and other physical effects may also play an important trole in hemicellulose and lignin degradation and removal.

Keywords: Pretreatment; hot water; hemicellulose hydrolysis; fluid velocity; corn stover

Combined steam pretreatment and enzymatic hydrolysis of starch-free wheat fibers by Beatriz Palmarola-Adrados; Mats Galbe; Guido Zacchi (pp. 989-1002).

Steam treatment of an industrial process stream, denoted starch-free wheat fiber, was investigated to improve the formation of monomeric sugars in subsequent enzymatic hydrolysis for further bioconversion into ethanol. The solid fraction in the process stream, derived from a combined starch and ethanol factory, was rich in arabinose (21.1%), xylose (30.1%), and glucose (18.6%), in the form of polysaccharides. Various conditions of steam pretreatment (170–220°C for 5–30 min) were evaluated, and their effect was assessed by enzymatic hydrolysis with 2 g of Celluclast + Ultraflo mixture/ 100 g of starch-free fiber (SFF) slurry at 5% dry matter (DM). The highest overall sugar yield for the combined steam pretreatment and enzymatic hydrolysis, 52g/100 g of DM of SFF, corresponding to 74% of the theoretical, was achieved with pretreatment at 190°C for 10 min followed by enzymatic hydrolysis.

Keywords: Wheat fibers; steam pretreatment; enzymatic hydrolysis; starch-free fiber; microwave oven

Application of xylanase from thermomyces lanuginosus IOC-4145 for enzymatic hydrolysis of corncob and sugarcane bagasse by Mônica Caramez Triches Damaso; Aline Machado de Castro; Raquel Machado Castro; Carolina Maria M. C. Andrade; Nei Pereira Jr. (pp. 1003-1012).

Xylanases have significant current and potential uses for several industries including paper and pulp, food, and biofuel. For the biofuel industry, xylanases can be used to aid in the conversion of lignocellulose to fermentable sugars (e.g., xylose). We investigated the thermophilic fungus Thermomyces lanuginosus was yielded for xylanase production and found that the highest activity (850 U/mL) was yielded after 96 h of semisolid fermentation. The enzyme was used for hydrolyzing agricultural residues with and without pretreatment. Such residues were characterized in relation to the maximum xylose content by total acid hydrolysis. The highest xylose yields realized by enzymatic hydrolysis were 24 and 52%, achieved by using 3000 U/g (dried material) of sugarcane bagasse and corncob, respectively, which received both alkali and thermal pretreatment.

Keywords: Xylanase; Thermomyces lanuginosus ; agricultural residues; enzymatic hydrolysis; corncob; sugarcane bagasse

Predicted effects of mineral neutralization and bisulfate formation on hydrogen ion concentration for dilute sulfuric acid pretreatment by Todd A. Lloyd; Charles E. Wyman (pp. 1013-1022).

Dilute acid and water only hemicellulose hydrolysis are being examined as part of a multiin stitutional cooperative effort to evaluate the performance of leading cellulosic biomass pretreatment technologies on a common basis. Cellulosic biomass, such as agricultural residues and forest wastes, canhave a significant mineral content. It has been shown that these minerals neutralize some of the acid during dilute acid pretreatment, reducing its effectiveness, and the higher solids loadings desired to minimize costs will require increased acid use to compensate. However, for sulfuric acid in particular, an equilibrium shift to formation of bisulfate during neutralization can further reduce hydrogen ion concentrations and compound the effect of neutralization. Because the equilibrium shift has a more pronounced effect at lower acid concentrations, additional acid is needed to compensate. Coupled with the effect of temperature on acid dissociation, these effects increase acid requirements to achieve a particular reaction rate unless minerals are removed prior to hydrolysis.

Keywords: Pretreatment; dilute acid; hemicellulosehydrolysis; bisulfate; neutralization

Enhancement of enzymatic digestibility of recycled newspaper by addition of surfactant in ammonia-hydrogen peroxide pretreatment by Sung Bae Kim; Jin Won Chun (pp. 1023-1031).

The effect of surfactant on enzymatic digestibility was investigated during the pretreatment stage. Newspaper was pretreated with an ammonia-hydrogen peroxide mixture on a shaking bath at 40°C and 130 strokes/min for 3 h. Two kinds of nonionic surfactants, NP series and Tween series, were utilized. The effect of hydrophile-lipophile balance (HLB) value of both series surfactants on digestibility was found to be negligible, even though de-inking efficiency was improved as HLB value was increased. The effect of surfactant loading on digestibility was small, below 0.5 wt%, and negligible above 0.5 wt% at 60 international filter paper units (IFPU). The percentage improvement in digestibility increased as enzyme loading decreased. Digestibility of NP-5-added sample relative to control sample, increased significantly at an enzyme loading <60 IFPU, i.e., 19 and 13% at 15 and 30 IFPU, respectively. Such an increase in digestibility was not explained clearly from the experimental results. It was also found that ink removal before enzymatic hydrolysis is very important to enhance digestibility.

Keywords: Pretreatment; newspaper; ammonia; hydrogen peroxide; enzymatic digestibility; surfactant

Study on methane fermentation and production of vitamin B₁₂ from alcohol waste slurry by Zhenya Zhang; Taisheng Quan; Pomin Li; Yansheng Zhang; Norio Sugiura; Takaaki Maekawa (pp. 1033-1039).

We studied biogas fermentation from alcohol waste fluid to evaluate the anaerobic digestion process and the production of vitamin B₁₂ as a byproduct. Anaerobic digestion using acclimated methanogens was performed using the continuously stirred tank reactor (CSTR) and fixed-bed reactor packed with rock wool as carrier material at 55°C. We also studied the effects of metal ions added to the culture broth on methane and vitamin B₁₂ formation. Vitamin B₁₂ production was 2.92 mg/L in the broth of the fixed-bed reactor, twice that of the CSTR. The optimum concentrations of trace metal ions added to the culture liquid for methane and vitamin B₁₂ production were 1.0 and 8 mL/L for the CSTR and fixed-bed reactor, respectively. Furthermore, an effective method for extracting and purifying vitamin B₁₂ from digested fluid was developed.

Keywords: Vitamin B₁₂ ; methane; acclimated methanogens; trace metal ions; rock wool

Comparison of two posthydrolysis processes of Brewery's spent grain autohydrolysis liquor to produce a pentose-containing culture medium by Luís C. Duarte; Florbela Carvalheiro; Sónia Lopes; Susana Marques; Juan Carlos Parajo; Francisco M. Gírio (pp. 1041-1058).

A readily fermentable pentose-containing hydrolysate was obtained from Brewery's spent grain by a two-step process consisting of an auto-hydrolysis (converting the hemicelluloses into oligosaccharides) followed by an enzymatic or sulfuric acid-catalyzed posthydrolysis (converting the oligosaccharides into monosaccharides). Enzymatic hydrolyses were performed with several commercial enzymes with xylanolytic and cellulolytic activities. Acid-catalyzed hydrolyses were carried out at 121°C under various sulfuric acid concentrations and reaction times, and the effects of treatments were interpreted by means of a corrected combined severity factor (CS ¹), which varied in the range of 0.80–2.01. Under the tested conditions, chemicalhydrolysis allowed higher pentose yields than enzymatic hydrolysis. Optimized conditions (defined by CS ¹=1.10) allowed both complete monosaccharide recovery and low content of inhibitors. Liquors subjected to posthydrolysis under optimal conditions were easily fermented by Debaryomyces hansenii CCMI 941 in semiaerobic shake-flask experiments, leading to xylitol and arabitol as major fermentation products. The bioconversion process was improved by hydrolysate concentration and supplementation of fermentation media with casamino acids.

Keywords: Arabitol; Debaryomyces hansenii ; hemicellulose hydrolysate; combined severity; xylitol

Optimization of Brewery's spent grain dilute-acid hydrolysis for the production of pentose-rich culture media by Florbela Carvalheiro; Luís C. Duarte; Raquel Medeiros; Francisco M. Gírio (pp. 1059-1072).

Dilute-acid hydrolysis of brewery's spent grain to obtain a pentose-rich fermentable hydrolysate was investigated. The influence of operational conditions on polysaccharide hydrolysis was assessed by the combined severity parameter (CS) in the range of 1.39–3.06. When the CS increased, the pentose sugars concentration increased to a maximum at a CS of 1.94, whereas the maximum glucose concentration was obtained for a CS of 2.65. The concentrations of furfural, hydroxymethylfurfural (HMF), as well as formic and levulinic acids and total phenolic compounds increased with severity. Optimum hydrolysis conditions were found at a CS of 1.94 with >95% of feedstock pentose sugars recovered in the monomeric form, together with a low content of furfural, HMF, acetic and formic acids, and total phenolic compounds. This hydrolysate containing glucose, xylose, and arabinose (ratio 10∶67∶32) was further supplemented with inorganic salts and vitamins and readily fermented by the yeast Debaryomyces hansenii CCMI 941 without any previous detoxification stage. The yeast was able to consume all sugars furfural, HMF, and acetic acid with high biomass yield, 0.68C-mol/C-mol, and productivity, 0.92 g/(L·h). Detoxification with activated charcoal resulted in a similar biomass yield and a slight increase in the volumetric productivity (11%).

Keywords: Brewery's spent grain; dilute-acid hydrolysis; pentose-rich media; Debaryomyces hansenii

Comparison of microbial inhibition and enzymatic hydrolysis rates of liquid and solid fractions produced from pretreatment of biomass with carbonic acid and liquid hot water by Damo M. Yourchisin; G. Peter Van Walsum (pp. 1073-1086).

This research quantified the enzymatic digestibility of the solid component and the microbial inhibition of the liquid component of pretreated aspen wood and cornstover hydrolysates. Products of liquid hot water and carbonic acid pretreatment were compared. Pretreatment temperatures tested ranged from 180 to 220°C, and reaction times were varied between 4 and 64 min. Both microbial inhibition rates and enzymatic hydrolysis rates showed no difference between pretreatments containing carbonic acid and those not containing no carbonic acid. Microbial inhibition increased as the reaction severity increased, but only above a midpoint severity parameter of 200°C for 16 min. Both the rates and yields of enzymatic hydrolysis displayed an increase from the lowest tested reaction severity to the highest tested reaction severity.

Keywords: Carbonic acid; pretreatment; hydrolysis; corn stover; aspen wood

Modeling of carbonic acid pretreatment process using ASPEN-plus^Ŗ by Kemantha Jayawardhana; G. Peter Van Walsum (pp. 1087-1102).

ASPEN-Plus^Ŗ process modeling software is used to model carbonic acid pretreatment of biomass. ASPEN-Plus was used because of the thorough treatment of thermodynamic interactions and its status as a widely accepted process simulator. Because most of the physical property data for many of the key components used in the simulation of pretreatment processes are not available in the standard ASPEN-Plus property databases, values from an in-house database (INHSPCD) developed by the National Renewable Energy Laboratory were used. The standard non-random-two-liquid (NRTL) or renon route was used as the main property method because of the need to distill ethanol and to handle dissolved gases. The pretreatment reactor was modeled as a “black box” stoichiometric reactor owing to the unavailability of reaction kinetics. The ASPEN-Plus model was used to calculate the process equipment costs, power requirements, and heating and cooling loads. Equipment costs were derived from published modeling studies. Wall thickness calculations were used to predict construction costs for the high-pressure pretreatment reactor. Published laboratory data were used to determine a suitable severity range for the operation of the carbonic acid reactor. The results indicate that combined capital and operating costs of the carbonic acid system are slightly higher than on H₂SO₄-based system and highly sensitive to reactor pressure and solids concentration.

Keywords: Acid pretreatment; carbonic acid; ASPEN-Plus model; alcohol fuels; biomass

Enhanced enzymatic hydrolysis of steam-exploded douglas fir wood by alkali-oxygen post-treatment by Xuejun Pan; Xiao Zhang; David J. Gregg; John N. Saddler (pp. 1103-1114).

Good enzymatic hydrolysis of steam-exploded Douglas fir wood (SEDW) cannot be achieved owing to the very high lignin content (>40%) that remains associated with this substrate. Thus, in this study, we investigated the use of alkali-oxygen treatment as a posttreatment to delignify SEDW and also considered the enzymatic hydrolyzability of the delignified SEDW. The results showed that under optimized conditions of 15% NaOH, 5% consistency, 110°C, and 3h, approx84% of the lignin in SEDW could be removed. The resulting delignified SEDW had good hydrolyzability, and cellulose-to-glucose conversion yields of over 90 and 100% could be achieved within 48 h with 20 and 40 filter paper units/g of cellulose enzyme loadings, respectively. It was also indicated that severe conditions, such as high NaOH concentration and high temperature, should not be utilized in oxygen delignification of SEDW in order to avoid extensive condensation of lignin and significant degradation of cellulose.

Keywords: Steam explosion; Douglas fir; oxygen delignification; enzymatic hydrolysis; lignin

Effects of sugar inhibition on cellulases and β-glucosidase during enzymatic hydrolysis of softwood substrates by Zhizhuang Xiao; Xiao Zhang; David J. Gregg; John N. Saddler (pp. 1115-1126).

A quantitative approach was taken to determine the inhibition effects of glucose and other sugar monomers during cellulase and β-Glucosidase hydrolysis of two types of cellulosic material: Avicel and acetic acid-pretreated softwood. The increased glucose content in the hydrolysate resulted in a dramatic increase in the degrees of inhibition on both β-Glucosidase and cellulase activities. Supplementation of mannose, xylose, and galactose during cellobiose hydrolysis did not show any inhibitory effects on β-Glucosidase activity. However, these sugars were shown to have significant inhibitory effects on cellulase activity during cellulose hydrolysis. Our study suggests that high-substrate consistency hydrolysis with supplementation of hemicellulose is likely to be a practical solution to minimizing end-product inhibition effects while producing hydrolysate with high glucose concentration.

Keywords: β-Glucosidase; cellulase; degree of inhibition; softwood; glucose; hydrolysate

Kinetics of glucose decomposition during dilute-acid hydrolysis of lignocellulosic biomass by Qian Xiang; Yong Y. Lee; Robert W. Torget (pp. 1127-1138).

Recent research work in-house both at Auburn University and National Renewable Energy Laboratory has demonstrated that extremely low concentrations of acid (e.g., 0.05–0.2 wt% sulfuric acid) and high temperatures (e.g., 200–230°C) are reaction conditions that can be effectively applied for hydrolysis of the cellulosic component of biomass. These conditions are far from those of the conventional dilute-acid hydrolysis processes, and the kinetic data for glucose decomposition are not currently available. We investigated the kinetics of glucose decomposition covering pH values of 1.5–2.2 and temperatures of 180–230°C using glass ampoule reactors. The primary factors controlling glucose decomposition are the reaction medium, acid concentration, and temperature. Based on the experimental data, a kinetic model was developed and the best-fit kinetic parameters were determined. However, a consistent discrepancy in the rate of glucose disappearance was found between that of the model based on pure glucose data and that observed during the actual process of lignocellulosic biomass hydrolysis. This was taken as an indication that glucose recombines with acid-soluble lignin during the hydrolysis process, and this conclusion was incorporated accordingly into the overall model of glucose decomposition.

Keywords: Reaction kinetics; glucose decomposition; dilute acid hydrolysis; kinetic modeling; acid-soluble lignin; acid-base catalysis rules

Conversion of distiller's grain into fuel alcohol and a higher-value animal feed by dilute-acid pretreatment by Melvin P. Tucker; Nicholas J. Nagle; Edward W. Jennings; Kelly N. Ibsen; Andy Aden; Quang A. Nguyen; Kyoung H. Kim; Sally L. Noll (pp. 1139-1159).

Over the past three decades ethanol production in the United States has increased more than 10-fold, to approx 2.9 billion gal/yr (mid-2003), with ethanol production expected to reach 5 billion gal/yr by 2005. The simultaneous coproduction of 7 million t/yr of distiller's grain (DG) may potentially drive down the price of DG as a cattle feed supplement. The sale of residual DG for animal feed is an important part of corn dry-grind ethanol production economics; therefore, dry-grind ethanol producers are seeking ways to improve the quality of DG to increase market penetration and help stabilize prices. One possible improvement is to increase the protein content of DG by converting the residual starch and fiber into ethanol. We have developed methods for steam explosion, SO₂, and dilute-sulfuric acid pretreatment of DG for evaluation as a feedstock for ethanol production. The highest soluble sugar yields (∼77% of available carbohydrate) were obtained by pretreatment of DG at 140°C for 20 min with 3.27 wt% H₂SO₄. Fermentation protocols for pretreated DG were developed at the bench scale and scaled to a working volume of 809 L for production of hydrolyzed distiller's grain (HDG) for feeding trials. The pretreated DG was fermented with Saccharomyces cerevisiae D₅A, with ethanol yields of 73% of theoretical from available glucans. The HDG was air-dried and used for turkey-feeding trials. The inclusion of HDG into turkey poult (as a model non-ruminant animal) diets at 5 and 10% levels, replacing corn and soybean meal, showed weight gains in the birds similar to controls, whereas 15 and 20% inclusion levels showed slight decreases (−6%) in weight gain. At the conclusion of the trial, no negative effects on internal organs or morphology, and no mortality among the poults, was found. The high protein levels (58–61%) available in HDG show promising economics for incorporation of this process into corn dry-grind ethanol plants.

Keywords: Distiller's grain; corn dry-grind; pretreatment; enzymatic hydrolysis; ethanol; animal feed

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.115, #1-3)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.115, #1-3)