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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.130, #1-3)

Introduction to the Proceedings of the Twenty-Seventh Symposium on Biotechnology for Fuels and Chemicals by James D. McMillan (pp. iii-viii).

Introduction to session 2 by Tsobanakis Paris (pp. 347-348).

Existing biorefinery operations that benefit from fractal-based process intensification by Vadim Kochergin; Mike Kearney (pp. 349-360).

Ion exchange, adsorption, and chromatography are examples of separation processes frequently used in today's biorefineries. The particular tasks for which these technologies can be successfully applied are highly influenced by capital cost and efficiency. There exists a potential for significantly increasing the efficiency of these processes whereas simultaneously decreasing their size and capital cost. This potential for process intensification can be realized with the use of engineered fractal equipment. The cost savings potential and the possibilities for broadening the use of fractal-based separation technologies in future biorefinery concepts is illustrated by examples of full-scale implementation in the sugar and sweetener industries.

Keywords: Biorefinery; fractal; liquid separations; chromatography; ion exchange

The importance of utility systems in today's biorefineries and a vision for tomorrow by Tim Eggeman; Dan Verser (pp. 361-381).

Heat and power systems commonly found in today's corn processing facilities, sugar mills, and pulp and paper mills will be reviewed. We will also examine concepts for biorefineries of the future. We will show that energy ratio, defined as the ratio of renewable energy produced divided by the fossil energy input, can vary widely from near unity to values greater than 12. Renewable-based utility systems combined with low-fossil input agricultural systems lead to high-energy ratios.

Keywords: Ethanol; indirect ethanol process; green power

Extraction of hyperoside and quercitrin from mimosa (Albizia julibrissin) foliage by Adam K. Ekenseair; Lijan Duan; Danielle Julie Carrier; David I. Bransby; Edgar C. Clausen (pp. 382-391).

Mimosa, an excellent energy crop candidate because of its high growth yield, also contains, on a dry basis, 0.83% hyperoside and 0.90% quercitrin. Hyperoside has been documented as having anti-inflammatory and diurectic properties, whereas quercitrin may play a role in intestinal repair following chronic mucosal injury. Thus, mimosa might first be extracted for important antioxidant compounds and then used as a feedstock for energy production. This article presents results from studies aimed at determining the effect of three extraction parameters (temperature, solvent composition, and time) on the yield of these important quercetin compounds. Conditions are sought which maximize yield and concentration, whereas complementing subsequent biomass pretreatment, hydrolysis and fermentation.

Keywords: Biomass pretreatment; energy crops; hyperoside; mimosa quercitrin; value-added compounds

Foam control in fermentation bioprocess by A. Etoc; F. Delvigne; J. P. Lecomte; P. Thonart (pp. 392-404).

In this article, we describe the development of a simple laboratory test for the effective screening of foam control agents on a selected fermentation system, the mass production of Yarrowia lipolytica. Aeration testing is based on sparging air in the foaming medium allowing partial reproduction of the gas-liquid hydrodynamic encountered in bioreactors. “Dynamic sparge test”, for which measurements are made during foam formation, was used to compare the capacity of three antifoams, based on different technologies, to control the foam produced in the fermentation broth. The selected foam control agents were: (1) an organic antifoam (TEGO AFKS911), (2) a siliconebased emulsion containing in situ treated silica (DC-1520) and (3) a silicone/organic blend silica-free formulation. The testing results demonstrated dramatic differences among them and showed that the capacity of TEGO AFKS911 and DC-1520 to control the foam generated in the fermentation broth decreases as a function of fermentation time. This occurred to a much lesser extent for the silicone/organic blend formulation. These results were correlated with the change of the foam nature and the increase of foam stability of the fermentation broth with culture time. The increase in protein content as a function of growth time was correlated with an increase in foam stability and antifoam consumption. A “synthetic fermentation broth” was also developed, by adding both proteins and microorganism to the culture medium. This allowed us to mimic the fermentation broth, shown by the similar antifoams behaviour, and is therefore a simple methodology useful for the selection of appropriate antifoams.

Keywords: Antifoam; aeration; bioreactor; silicone

Optimization of biodiesel production from castor oil by Nivea de Lima Da Silva; Maria Regina Wolf Maciel; César Benedito Batistella; Rubens Maciel Filho (pp. 405-414).

The transesterification of castor oil with ethanol in the presence of sodium ethoxide as catalyst is an exceptional option for the Brazilian biodiesel production, because the castor nut is quite available in the country. Chemically, its oil contains about 90% of ricinoleic acid that gives to the oil some beneficial characteristics such as its alcohol solubility at 30°C. The transesterification variables studied in this work were reaction temperature, catalyst concentration and alcohol oil molar ratio. Through a star configuration experimental design with central points, this study shows that it is possible to achieve the same conversion of esters carrying out the transesterification reaction with a smaller alcohol quantity, and a new methodology was developed to obtain high purity biodiesel.

Keywords: Biodiesel; castor oil; ethanolysis; transesterification; alkaline catalyst

Maniplating the phenolic acid content and digestibility of italian ryegrass (Lolium multiflorum)b y vacuolar-targeted epession of a fungal ferulic acid esterase by Marcia M. de O. Buanafina; Tim Langdon; Barbara Hauck; Sue J D Alton; Phil Morris (pp. 416-426).

In grass cell walls, ferulic acid esters linked to arabinosyl residues in arabinoxylans play a key role in crosslinking hemicellulose. Although such crosslinks have a number of important roles in the cell wall, they also hinder the rate and extent of cell wall degradation by ruminant microbes and by fungal glycohydrolyase enzymes. Ferulic acid esterase (FAE) can release both monomeric and dimeric ferulic acids from arabinoxylans making the cell wall more susceptible to further enzymatic attack. Transgenic plants of Lolium multiflorum expressing a ferulic acid esterase gene from Aspergillus niger, targeted to the vacuole under a constitutive rice actin promoter, have been produced following microprojectile bombardment of embryogenic cell cultures. The level of FAE activity was found to vary with leaf age and was highest in young leaves. FAE expression resulted in the release of monomeric and dimeric ferulic acids from cell walls on cell death and this was enhanced severalfold by the addition of exogenous β-1,4-endoxylanase. We also show that a number of plants expressing FAE had reduced levels of cell wall esterified monomeric and dimeric ferulates and increased in vitro dry-matter digestibility compared with nontransformed plants.

Keywords: Constitutive vacuolar-targeted expression; digestibility; ferulic acid esterase; L. multiflorum ; transgenic grasses

Variation of S/G ratio and lignin content in a Populus family influences the release of xylose by dilute acid hydrolysis by Brian H. Davison; Sadie R. Drescher; Gerald A. Tuskan; Mark F. Davis; Nhuan P. Nghiem (pp. 427-435).

Wood samples from second generation Populus cross were shown to have different lignin contents and S/G ratios (S: syringyl-like lignin structures; G: guaiacyl-like lignin structures). The lignin contents varied from 22.7% to 25.8% and the S/G ratio from 1.8 to 2.3. Selected samples spanning these ranges were hydrolyzed with dilute (1%) sulfuric acid to release fermentable sugars. The conditions were chosen for partial hydrolysis of the hemicellulosic fraction to maximize the expression of variation among samples. The results indicated that both lignin contents and S/G ratio significantly affected the yield of xylose. For example, the xylose yield of the 25.8% lignin and 2.3 S/G (hihg lignin, high S/G) sample produced 30% of the theoretical yield, whereas the xylose yield of the 22.7% lignin and 1.8 S/G (low lignin, low S/G) was 55% of the theoretical value. These results indicate that lignin content and composition among genetic variants within a single species can influence the hydrolyzability of the biomass.

Keywords: Cell wall chemistry; genetic variation; hybrid poplar; hydrolysis; lignin

Enhanced secondary metabolite biosynthesis by elicitation in transformed plant root system by Gwi-T Aek Jeong; Don-H ee Park (pp. 436-446).

Plants generally produce secondary metabolites in nature as a defense mechanism against pathogenic and insect attack. In this study, we applied several abiotic elicitors in order to enhance growth and ginseng saponin biosynthesis in the hairy roots of Panax ginseng. Generally, elicitor treatments were found to inhibit the growth of the hairy roots, although simultaneously enhancing ginseng saponin biosynthesis. Tannic acid profoundly inhibited the hairy root growth during growth period. Also, ginseng saponin content was not significantly different from that of the control. The addition of selenium at inoculum time did not significantly affect ginseng saponin biosynthesis. However, when 0.5 mM selenium was added as an elicitor after 21 d of culture, ginseng saponin content and productivity increased to about 1.31 and 1.33 times control levels, respectively. Also, the addition of 20μM NiSO₄ resulted in an increase in ginseng saponin content and productivity, to about 1.20 and 1.23 times control levels, respectively, and also did not inhibit the growth of the roots. Sodium chloride treatment inhibited hairy root growth, except at a concentration of 0.3% (w/v). Increases in the amounts of synthesized ginseng saponin were observed at all concentrations of added sodium chloride. At 0.1% (w/v) sodium chloride, ginseng saponin content and productivity were increased to approx, 1.15 and 1.13 times control values, respectively. These results suggest that processing time for the generation of ginseng saponin in a hairy root culture can be reduced via the application of an elicitor.

Keywords: Elicitor; selenium; nickel; ginseng saponin; hairy roots

Preliminary results on optimization of pilot scale pretreatment of wheat straw used in coproduction of bioethanol and electricity by Mette Hedegaard Thomsen; Anders Thygesen; Henning Jørgensen; Jan Larsen; Børge Holm Christensen; Anne Belinda Thomsen (pp. 448-460).

The overall objective in this European Union-project is to develop cost and energy effective production systems for coproduction of bioethanol and electricity based on integrated biomass utilization. A pilot plan reactor for hydrothermal pretreatment (including weak acid hydrolysis, wet oxidation, and steam pretreatment) with a capacity of 100 kg/h was constructed and tested for pretreatment of wheat straw for ethanol production. Highest hemicellulose (C5 sugar) recovery and extraction of hemicellulose sugars was obtained at 190°C whereas highest C6 sugar yield was obtained at 200°C. Lowest toxicity of hydrolysates was observed at 190°C; however, addition of H₂O₂ improved the fermentability and sugar recoveries at the higher temperatures. The estimated total ethanol production was 223 kg/t straw assuming utilisation of both C6 and C5 during fermentation, and 0.5 g ethanol/g sugar.

Keywords: Lignocellulose; hydrothermal; pretreatment; pilot plant; SSF; bioethanol

The combined effects of acetic acid, formic acid, and hydroquinone on Debaryomyces hansenii physiology by Luís C. Duarte; Florbela Carvalheiro; Joana Tadeu; Francisco M. Gírio (pp. 461-475).

The combined effects of inhibitors present in lignocellulosic hydrolysates was studied using a multivariate statistical approach. Acetic acid (0–6 g/L), formic acid (0–4.6 g/L) and hydroquinone (0–3 g/L) were tested as model inhibitors in synthetic media containing a mixture of glucose, xylose, and arabinose simulating concentrated hemicellulosic hydrolysates. Inhibitors were consumed sequentially (acetic acid, formic acid, and hydroquinone), alongside to the monosaccharides (glucose, xylose, and arabinose). Xylitol was always the main metabolic product. Additionally, glycerol, ethanol, and arabitol were also obtained.The inhibitory action of acetic acid on growth, on glucose consumption and on all product formation rates was found to be significant (p≤0.05), as well as formic acid inhibition on xylose consumption and biomass production. Hydroquinone negatively affected biomass productivity and yield, but it significantly increased xylose consumption and xylitol productivity. Hydroquinone interactions, either with acetic or formic acid or with both, are also statistically signficant. Hydroquinone seems to partially lessen the acetic acid and amplify formic acid effects. The results clearly indicate that the interaction effects play an important role on the xylitol bioprocess.

Keywords: Lignocellulosic byproducts; xylitol; interaction effects; Debaryomyces hansenii ; inhibition

Bioethanol from cellulose with supercritical water treatment followed by enzymatic hydrolysis by Toshiki Nakata; Hisashi Miyafuji; Shiro Saka (pp. 476-485).

The water-soluble portion and precipitates obtained by supercritical (SC) water treatment of microcrystalline cellulose (Avicel) were enzymatically hydrolyzed. Glucose could be produced easily from both substrates, compared with the Avicel. Therefore, SC water treatment was found to be effective for enhancing the productivity of glucose from cellulose by the enzymatic hydrolysis. It is also found that alkaline treatment or wood charcoal treatment reduced inhibitory effects by various decomposed compounds of cellulose on the enzymatic hydrolysis to achieve higher glucose yields. Furthermore, glucose obtained by SC water treatment followed by the enzymatic hydrolysis of cellulose could be converted to ethanol by fermentation without any inhibition.

Keywords: Lignocellulosics; supercritical water; enzymatic hydrolysis; inhibitor; ethanol

Enhancement of the enzymatic digestibility of waste newspaper using tween by Sung Bae Kim; Hyun Joo Kim; Chang Joon Kim (pp. 486-495).

Methods of increasing the enzymatic digestibility of waste newspaper by adding Tween (TW)-20 and 80 surfactants were investigated. Tween-series surfactants were selected because these surfactants increase cellulase activity during enzymatic hydrolysis and do not inhibit cell growth in downstream fermentation processes. When surfactant was used in a pretreatment, a benefic effect was expected in the enzymatic hydrolysis stage owing to surfactant carry-over from the pretreatment stage immediately upstream of the hydrolysis. However, because it was necessary to wash the pretreated substrate with water to remove inhibitors produced during pretreatment, no added benefit was obtained. When surfactant was used in the pretreatment only, it was found that it had a marked effect on digestibility and that this effect was higher at lower enzyme loadings. Also, TW-80 was found to be more effective than TW-20, and the addition of enzyme and TW-80 to substrate at the beginning of enzyme reaction was found to most effectively increase digestibility. When TW-80 was added into either the pretreatment stage or the hydrolysis stage the digestibilities of untreated sample increased by approx 40%, whereas an increase of only 45% was observed when TW-80 was added to both stages. These results show that the addition of surfactant to either the pretreatment or the enzymatic hydrolysis stage is sufficient to increase digestibility.

Keywords: Pretreatment; newspaper; surfactant; hydrolysis; enzymatic digestibility

Ethanol production from steam-explosion pretreated wheat straw by Ignacio Ballesteros; Ma José Negro; José Miguel Oliva; Araceli Cabañas; Paloma Manzanares; Mercedes Ballesteros (pp. 496-508).

Bioconversion of cereal straw to bioethanol is becoming an attractive alternative to conventional fuel ethanol production from grains. In this work, the best operational conditions for steam-explosion pretreatment of wheat straw for ethanol production by a simultaneous saccharification and fermentation process were studied, using diluted acid [H₂SO₄ 0.9% (w/w)] and water as preimpregnation agents. Acid-or water-impregnated biomass was steam-exploded at different temperatures (160–200°C) and residence times (5, 10, and 20 min). Composition of solid and filtrate obtained after pretreatment, enzymatic digestibility and ethanol production of pretreated wheat straw at different experimental conditions was analyzed. The best pretreatment conditions to obtain high conversion yield to ethanol (approx 80% of theoretical) of cellulose-rich residue after steam-explosion were 190°C and 10 min or 200°C and 5 min, in acid-impregnated straw. However, 180°C for 10 min in acid-impregnated biomass provided the highest ethanol yield referred to raw material (140 L/t wheat straw), and sugars recovery yield in the filtrate (300 g/kg wheat straw).

Keywords: Wheat straw; ethanol; diluted acid pretreatment; steam-explosion

Catalyst transport in corn stover internodes by Sridhar Viamajala; Michael J. Selig; Todd B. Vinant; Melvin P. Tucker; Michael E. Himmel; James D. McMillan; Stephen R. Decker (pp. 509-527).

The transport of catalysts (chemicals and enzymes) within plant biomass is believed to be a major bottleneck during thermochemical pretreatment and enzymatic conversion of lignocellulose. Subjecting biomass to size reduction and mechanical homogenization can reduce catalyst transport limitations; however, such processing adds complexity and cost to the over-all process. Using high-resolution light microscopy, we have monitored the transport of an aqueous solution of Direct Blue-I (DB-I) dye through intact corn internodes under a variety of impregnation conditions. DB-I is a hydrophilic anionic dye with affinity for cellulose. This model system has enabled us to visualize likely barriers and mechanisms of catalyst transport in corn stems. Microscopic images were compared with calculated degrees of saturation (i.e., volume fraction of internode void space occupied by dye solution) to correlate impregnation strategies with dye distribution and transport mechanisms. Results show the waxy rind exterior and air trapped within individual cells to be the major barriers to dye transport, whereas the vascular bundles, apoplastic continuum (i.e., the intercellular void space at cell junctions), and fissures formed during the drying process provided the most utilized pathways for transport. Although representing only 20–30% of the internode volume, complete saturation of the apoplast and vascular bundles by fluid allowed dye contact with a majority of the cells in the internode interior.

Keywords: Biomass conversion; internode transport; dilute acid pretreatment; direct blue-I; biomass recalcitrance

Evaluation of cellulase preparations for hydrolysis of hardwood substrates by Alex Berlin; Neil Gilkes; Douglas Kilburn; Vera Maximenko; Renata Bura; Alexander Markov; Anton Skomarovsky; Alexander Gusakov; Arkady Sinitsyn; Oleg Okunev; Irina Solovieva; John N. Saddler (pp. 528-545).

Seven cellulase preparations from Penicillium and Trichoderma spp. were evaluated for their ability to hydrolyze the cellulose fraction of hardwoods (yellow poplar and red maple) pretreated by organosolv extraction, as well as model cellulosic substrates such as filter paper. There was no significant correlation among hydrolytic performance on pretreated hardwood, based on glucose release, and filter paper activity. However, performance on pretreated hardwood showed significant correlations to the levels of endogenous β-glucosidase and xylanase activities in the cellulase preparation. Accordingly, differences in performance were reduced or eliminated following supplementation with a crude β-glucosidase preparation containing both activities. These results complement a previous investigation using softwoods pretreated by either organosolv extraction or steam explosion. Cellulase preparations that performed best on hardwood also showed superior performance on the softwood substrates.

Keywords: Cellulase; xylanase; hemicellulose; lignocellulose; bioconversion

Steam pretreatment of acid-sprayed and acid-soaked barley straw for production of ethanol by Marie Linde; Mast Galb; Guido Zacchi (pp. 546-562).

Barley is an abundant crop in Europe, which makes its straw residues an interesting cellulose source for ethanol production. Steam pretreatment of the straw followed by enzymatic hydrolysis converts the cellulose to fermentable sugars. Prior to pretreatment the material is impregnated with a catalyst, for example, H₂SO₄, to enhance enzymatic digestibility of the pretreated straw. Different impregnation techniques can be applied. In this study, soaking and spraying were investigated and compared at the same pretreatment condition in terms of overall yield of glucose and xylose. The overall yield includes the soluble sugars in the liquid from pretreatment, including soluble oligomers, and monomer sugars obtained in the enzymatic hydrolysis. The yields obtained differed for the impregnation techniques. Acid-soaked barley straw gave the highest overall yield of glucose, regardless of impregnation time (10 or 30 min) or acid concentration (0.2 or 1.0 wt%). For xylose, soaking gave the highest overall yield at 0.2 wt% H₂SO₄. An increase in acid concentration resulted in a decrease in xylose yield for both acid-soaked and acid-sprayed barley straw. Optimization of the pretreatment conditions for acid-sprayed barley straw was performed to obtain yields using spraying that were as high as those with soaking. For acid-sprayed barley straw the optimum pretreatment condition for glucose, 1.0 wt% H₂SO₄ and 220°C for 5 min, gave an overall glucose yield of 92% of theoretical based on the composition of the raw material. Pretreatment with 0.2wt% H₂SO₄ at 190°C for 5 min resulted in the highest overall xylose yield, 67% of theoretical based on the composition of the raw material.

Keywords: Barley straw; pretreatment; enzymatic hydrolysis; H₂SO₄ ; ethanol

Reaction kinetics of stover liquefaction in recycled stover polyol by Fei Yu; Roger Ruan; Xiangyang Lin; Yuhuan Liu; Rong Fu; Yuhong Li; Paul Chen; Yinyu Gao (pp. 563-573).

The purpose of this research was to study the kinetics of liquefaction of crop residues. The liquefaction of corn stover in the presence of ethylene glycol and ethylene carbonate using sulfuric acid as a catalyst was studied. It was found that the liquefaction yield was a function of ratio of solvent to corn stover, temperature, residence time, and amount of catalyst. Liquefaction of corn stover was conducted over a range of conditions encompassing residence times of 0–2.5 h, temperatures of 150–170°C, sulfuric acid concentrations of 2–4% (w/w), and liquefaction reagent/corn stover ratio of 1–3. The liquefaction rate constants for individual sets of conditions were examined using a first-order reaction model. Rate constant increased with the increasing of liquefaction temperature, catalyst content, and liquefaction reagent/corn stover ratio. Reuse of liquefied biomass as liquefying agent was also evaluated. When using recycled liquefied biomass instead of fresh liquefaction reagent, the conversion is reduced. It appeared that 82% of liquefaction yield was achieved after two times of reuse.

Keywords: Corn stover; ethylene carbonate; ethylene glycol; liquefaction; polyol

Liquefaction of corn stover and preparation of polyester from the liquefied polyol by Fei Yu; Yuhuan Liu; Xuejun Pan; Xiangyang Lin; Chengmei Liu; Paul Chen; Roger Ruan (pp. 574-585).

This research investigated a novel process to prepare polyester from corn stover through liquefaction and crosslinking processes. First, corn stover was liquefied in organic solvents (90 wt% ethylene glycol and 10 wt% ethylene carbonate) with catalysts at moderate temperature under atmospheric pressure. The effect of liquefaction temperature, biomass content, and type of catalyst, such H₂SO₄, HCl, H₃PO₄, and ZnCl₂, was evaluated. Higher liquefaction yield was achieved in 2 wt% sulfuric acid, 1/4 (w/w) stover to liquefying reagent ratio; 160°C temperature, in 2h. The liquefied corn stover was rich in polyols, which can be directly used as feedstock for making polymers without further separation or purification. Second, polyester was made from the liquefied corn stover by crosslinking with multifunctional carboxylic acids and/or cyclic acid anhydrides. The tensile strength of polyester is about 5 MPa and the elongation is around 35%. The polyester is stable in cold water and organic solvents and readily biodegradable as indicated by 82% weight loss when buried in damp soil for 10 mo. The results indicate that this novel polyester could be used for the biodegradable garden mulch film production.

Keywords: Biodegradability; liquefaction; polyester; strength; solubility

Enzymatic production of xylooligosaccharides from corn stover and corn cobs treated with aqueous ammonia by Yongming Zhu; Tae Hyun Kim; Y. Y. Lee; Rongfu Chen; Richard T. Elander (pp. 586-598).

A novel method of producing food-grade xylooligosaccharides from corn stover and corn cobs was investigated. The process starts with pretreatment of feedstock in aqueous ammonia, which results delignified and xylan-rich substrate. The pretreated substrates are subjected to enzymatic hydrolysis of xylan using endoxylanase for production of xylooligosaccharides. The conventional enzyme-based method involves extraction of xylan with a strong alkaline solution to form a liquid intermediate containing soluble xylan. This intermediate is heavily contaminated with various extraneous components. A costly purification step is therefore required before enzymatic hydrolysis. In the present method, xylan is obtained in solid form after pretreatment. Water-washing is all that is required for enzymatic hydrolysis of this material. The complex step of purifying soluble xylan from contaminant is essentially eliminated.Refining of xylooligosaccharides to food-grade is accomplished by charcoal adsorption followed by ethanol elution. Xylanlytic hydrolysis of the pretreated corn stover yielded glucan-rich residue that is easily digestible by cellulase enzyme. The digestibility of the residue reached 86% with enzyme loading of 10 filter paper units/g-glucan. As a feedstock for xylooligosaccharides production, corn cobs are superior to corn stover because of high xylan content and high packing density. The high packing density of corn cobs reduces water input and eventually raises the product concentration.

Keywords: Corn stover; corn cobs; xylooligosaccharides; xylan; aqueous ammonia; pretreatment; endoxylanase

Optimal conditions for alkaline detoxification of dilute-acid lignocellulose hydrolysates by Björn Alriksson; Anders Sjöde; Nils-Olof Nilvebrant; Leif J. Jönsson (pp. 599-611).

Alkaline detoxification strongly improves the fermentability of dilute-acid hydrolysates in the production of bioethanol from lignocellulose with Saccharomyces cerevisiae. New experiments were performed with NH₄OH and NaOH to define optimal conditions for detoxification and make a comparison with Ca(OH)₂ treatment feasible. As too harsh conditions lead to sugar degradation, the detoxification treatments were evaluated through the balanced ethanol yield, which takes both the ethanol production and the loss of fermentable sugars into account. The optimization treatments were performed as factorial experiments with 3-h duration and varying pH and temperature. Optimal conditions were found roughly in an area around pH 9.0/60°C for NH₄OH treatment and in a narrow area stretching from pH 9.0/80°C to pH 12.0/30°C for NaOH treatment. By optimizing treatment with NH₄OH, NaOH, and Ca(OH)₂, it was possible to find conditions that resulted in a fermentability that was equal or better than that of a reference fermentation of a synthetic sugar solution without inhibitors, regardless of the type of alkali used. The considerable difference in the amount of precipitate generated after treatment with different types of alkali appears critical for industrial implementation.

Keywords: Ethanol; lignocellulose; detoxification; alkali; inhibitor

Reintroduced solids increase inhibitor levels in a pretreated corn stover hydrolysate by R. Eric Berson; John S. Young; Thomas R. Hanley (pp. 612-620).

Following detoxification of the liquid hydrolysate produced in a corn stover pretreatment process, inhibitor levels are seen to increase with the re-addition of solids for the ensuing hydrolysis and fermentation processes. The solids that were separated from the slurry before detoxification of the liquor contain approx 60% (w/w) moisture, and contamination occurs owing to the diffusion of inhibitors from the moisture entrained in the porous structure of the corn stover solids into the bulk fluid. This evidence suggests the need for additional separation and detoxification steps to purge residual inhibitors entrained in the moisture in the solids. An overliming process to remove furans from the hydrolysate failed to reduce total organic acids concentration, so acids were removed by treatment with an activated carbon powder. Smaller carbon doses proved more efficient in removing organic acids in terms of grams of acid removed per gram of carbon powder. Sugar adsorption by the activated carbon powder was minimal.

Keywords: Activated carbon; detoxification; organic acids; overliming; pretreated corn stover hydrolyzate

Modeling of a continuous pretreatment reactor using computational fluid dynamics by R. Eric Berson; Rajesh K. Dasari; Thomas R. Hanley (pp. 621-630).

Computational fluid dynamic simulations are employed to predict flow characteristics in a continuous auger driven reactor designed for the dilure acid pretreatment of biomass. Slurry containing a high concentration of biomass solids exhibits a high viscosity, which poses unique mixing issues within the reactor. The viscosity increases significantly with a small increase in solids concentration and also varies with temperature. A well-mixed slurry is desirable to evenly distribute acid on biomass, prevent buildup on the walls of the reactor, and provides an uniform final product. Simulations provide flow patterns obtained over a wide range of viscosities and pressure distributions, which may affect reaction rates. Results provide a tool for analyzing sources of inconsistencies in product quality and insight into future design and operating parameters.

Keywords: Auger; biomass; CFD simulations; pretreatment reactor; screw conveyor

Ethanol production from pretreated olive tree wood and sunflower stalks by an SSF process by Encarnación Ruiz; Cristóbal Cara; Mercedes Ballesteros; Paloma Manzanares; Ignacio Ballesteros; Eulogio Castro (pp. 631-643).

Olive tree wood and sunflower stalks are agricultural residues largely available at low cost in Mediterranean countries. As renewable lignocellulosic materials, their bioconversion may allow both obtaining a value-added product, for fuel ethanol, and facilitating their elimination. In this work, the ethanol production from olive tree wood and sunflower stalks by a simultaneous saccharification and fermentation (SSF) process is studied. As a pretreatment, steam explosion at different temperatures was applied. The water insoluble fractions of steam-pretreated sunflower stalks and steamed, delignified olive tree wood were used as substrates at 10% w/v concentration for an SSF process by a cellulolytic commercial complex and Saccharomyces cerevisiae. After 72-h fermentation, ethanol concentrations up to 30 g/L were obtained in delignified steam-pretreated olive tree wood at 230°C and 5 min. Sunflower stalks pretretated at 220°C and 5 min gave maximum ethanol concentrations of 21 g/L in SSF experiments.

Keywords: Ethanol; olive tree wood; sunflower stalks; SSF; pretreatment

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.130, #1-3)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.130, #1-3)