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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.114, #1-3)

Polykaryon formation using a swollen conidium of Trichoderma reesei by Hideo Toyama; Makiko Yano; Takeshi Hotta (pp. 325-334).

The cellulolytic fungus, Trichoderma has oval and mononucleate conidia. When these conidia are incubated in a liquid medium, they begin to swell and their shape becomes spherical followed by an increase in inner space. In such swollen conidia, it is possible to produce a larger autopolyploid nucleus using a mitotic arrester compared with the case of the original conidia. In this study, polykaryon formation was attempted using these swollen conidia. Dried mature green conidia of Trichoderma reesei QM6a (IFO 31326) were incubated in Mandel's medium in order to swell. The swollen conidia were treated with a mitotic arrester, colchicine, for autopolyploidization. After autopolyploidization, polykary on formation was carried out using the swollen conidia. After the treatment, multiple smaller nuclei whose diameter was almost the same as that of the original strain were generated from an autopolyploid nucleus in a swollen conidium. A cellulase hyperproducer without decrease in growth rate could be selected using such swollen conidia.

Keywords: Trichoderma reesei ; polykaryon; cellulase; colchicines; polyploid

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by metabolically engineered Escherichia coli strains by Si Jae Park; Sang Yup Lee (pp. 335-346).

Biosynthesis of polyhydroxyalkanoates (PHAs) consisting of 3-hydroxyalkanoates (3HAs) of 4 to 10 carbon atoms was examined in metabolically engineered Escherichia coli strains. When the fadA and/or fadB mutant E. coli strains harboring the plasmid containing the Pseudomonas sp. 61-3 phaC2 gene and the Ralstonia eutropha phaAB genes were cultured in Luria-Bertani (LB) medium supplemented with 2 g/L of sodium decanoate, all the recombinant E. coli strains synthesized PHAs consisting of C4, C6, C8, and C10 monomer units. The monomer composition of PHA was dependent on the E. coli strain used. When the fadA mutant E. coli was employed, PHA containing up to 63 mol% of 3-hydroyhexanoate was produced. In fadB and fadAB mutant E. coli strains, 3-hydroxybutyrate (3HB) was efficiently incorporated into PHA up to 86 mol%. Cultivation of recombinant fadA and/or fadB mutant E. coli strains in LB medium containing 10 g/L of sodium gluconate and 2 g/L of sodium decanoate resulted in the production of PHA copolymer containing a very high fraction of 3HB up to 95 mol%. Since the material properties of PHA copolymer consisting of a large fraction of 3HB and a small fraction of medium-chain-length 3HA are similar to those of low-density polyethylene, recombinant E. coli strains constructed in this study should be useful for the production of PHAs suitable for various commercial applications.

Keywords: Polyhydroxyalkanoates; Escherichia coli ; ß-oxidation; sodium decanoate; copolymer

Effect of corn stover concentration on rheological characteristics by Natalia V. Pimenova; Thomas R. Hanley (pp. 347-360).

Corn stover, a well-known example of lignocellulosic biomass, is a potential renewable feed for bioethanol production. Dilute sulfuric acid pretreatment removes hemicellulose and makes the cellulose more susceptible to bacterial digestion. The rheologic properties of corn stover pretreated in such a manner were studied. The Power Law parameters were sensitive to corn stover suspension concentration becoming more non-Newtonian with slope n, ranging from 0.92 to 0.05 between 5 and 30% solids. The Casson and the Power Law models described the experimental data with correlation coefficients ranging from 0.90 to 0.99 and 0.85 to 0.99, respectively. The yield stress predicted by direct data extrapolation and by the Herschel-Bulkley model was similar for each concentration of corn stover tested.

Keywords: Corn stover; rheological measurement; shear stress; shear rate; non-Newtonian fluids; Power Law parameters

Construction of recombinant Escherichia coli strais for production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) by Kin-Ho Law; Pui-Ling Chan; Wai-Sum Lau; Yin-Chung Cheng; Yun-Chung Leung; Wai-Hung Lo; Hugh Lawford; Hoi-Fu Yu (pp. 361-372).

Plastic wastes constitute a worldwide environmental problem, and the demand for biodegradable plastics has become high. One of the most important characteristics of microbial polyesters is that they are thermoplastic with environmentally degradable properties. In this study, pUC 19/PHA was cloned and transformed into three different Escherichia coli strains. Among the three strains that were successfully expressed in the production of polyhydroxyalkanoates (PHA), E. coli HMS174 had the highest yield in the production of poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (P[HB-HV]). The cell dry weight and PHA content of recombinant HMS174 reached as high as 10.27 g/L and 43% (w/w), respectively, in fed-batch fermentor culture. The copolymer of PHA, P(HB-HV), was found in the cells, and the biopolymers accumulated were identified and analyzed by gas chromatography, proton nuclear magnetic resonance spectroscopy, and differential scanning calorimetry. We demonstrated clearly that the E. coli host for PHA production has to be carefully selected to obtain a high yield. The results obtained indicated that a superior E. coli with high PHA production can be constructed with a desirable ratio of P(HB-HV), which has potential applications in industry and medicine.

Keywords: Escherichia coli ; Polyhydroxyalkanoates; fed-batch fermentation; nuclear magnetic resonance; differential scanning calorimetry

Biosynthesis of (R)-3-hydroxyalkanoic acids by metabolically engineered Escherichia coli by Si Jae Park; Sang Yup Lee; Young Lee (pp. 373-379).

An efficient system for the production of (R)-hydroxyalkanoicacids (RHAs) was developed in natural polyhydroxyalkanoate (PHA)-producing bacteria and recombinant Escherichia coli. Acidic alcoholysis of purified PHA and in vivo depolymerization of PHA accumulated in the cells allowed the production of RHAs. In recombinant E. coli, RHA production was achieved by removing CoA from (R)-3-hydroxyacyl-CoA and by in vivo depolymerization of PHA. When the recombinant E. coli harboring the Ralstonia eutropha PHA biosynthesis genes and the depolymerase gene was cultured in a complex or a chemically defined medium containing glucose, (R)-3-hydroxybutyric acid (R3HB) was produced as monomers and dimers. R3HB dimers could be efficiently converted to monomers by mild alkaline heat treatment. A stable recombinant E. coli strain in which the R. eutropha PHA biosynthesis genes were integrated into the chromosome disrupting the pta gene was constructed and examined for the production of R3HB. When the R. eutropha intracellular depolymerase gene was expressed by using a stable plasmid containing the hok/sok locus of plasmid R1, R3HB could be efficiently produced.

Keywords: (R)-Hydroxyalkanoic acids; polyhydroxyalkanoate; Escherichia coli ; poly-(R)-3-hydroxybutyrate; (R)-3-hydroxybutyric acid

Gibberellic acid production by free and immobilized cells in different culture systems by Enrique Durán-Páramo; Héctor Molina-Jiménez; Marco A. Brito-Arias; Fabián Robles-Martínez (pp. 381-388).

Gibberellic acid production was studied in different fermentation systems. Free and immobilized cells of Gibberella fujikuroi cultures in shakeflask, stirred and fixed-bed reactors were evaluated for the production of gibberellic acid (GA₃). Gibberellic acid production with free cells cultured in a stirred reactor reached 0.206 g/L and a yield of 0.078 g of GA₃/g biomass.

Keywords: Cell immobilization; gibberellic acid; Gibberella fujikuroi ; shake flasks; free cells; immobilized cells

Screening genus Penicillium for producers of cellulolytic and xylanolytic enzymes by Kristian B. R. Krogh; Astrid Mørkeberg; Henning Jørgensen; Jens C. Frisvad; Lisbeth Olsson (pp. 389-401).

For enzymatic hydrolysis of lignocellulosic material, cellulolytic enzymes from Trichoderma reesei are most commently used, but, there is a need for more efficient enzyme cocktails. In this study, the production of cellulolytic and xylanolytic enzymes was investigated in 12 filamento us fungi from genus Penicillium and compared with that of T. reesei. Either Solka-Floc cellulose or oat spelt xylan was used as carbon source in shake flask cultivations. All the fungi investigated showed coinduction of cellulolytic and xylanolytic enzymes during growth on cellulose as well as on xylan. The highest filter paper activity was measured after cultivation of Penicillium brasilianum IBT 20888 on cellulose.

Keywords: Cellulolytic enzymes; hemicellulolyticenzymes; enzymatic hydrolysis; coinduction

Production of ethanol from cellulosic biomass hydrolysates using genetically engineered saccharomyces yeast capable of cofermenting glucose and xylose by Miroslav Sedlak; Nancy W. Y. Ho (pp. 403-416).

Recent studies have proven ethanol to be the idael liquid fuel for transportation, and renewable ligno cellulosic materials to be the attractive feed stocks for ethanol fuel production by fermentation. The major fermentable sugars from hydrolysis of most cellulosic biomass are D-glucose and D-xylose. The naturally occurring Saccharomyces yeasts that are used by industry to produce ethanol from starches and cane sugar cannot metabolize xylose. Our group at Purdue University succeded in developing genetically engineered Saccharomyces yeasts capable of effectively cofermenting glucose and xylose to ethanol, which was accomplished by cloning three xylose-metabolizing genes into the yeast. In this study, we demonstrated that our stable recombinant Sacharomyces yeast, 424A (LNH-ST), which contains the cloned xylose-metabolizing genes stably integrated into the yeast chromosome in high copy numbers, can efficiently ferment glucose and xylose present in hydrolysates from different cellulosic biomass to ethanol.

Keywords: Ethanol; Saccharomyces yeasts; hydrolysate; corn stover; corn fiber; xylose; glucose; glycerol; xylitol

Secondary membranes for flux optimization in membrane filtration of biologic suspensions by Parag R. Nemade; Robert H. Davis (pp. 417-432).

We employ in situ deposited secondary membranes of yeast (SMYs) to optimize permeate flux during microfiltration and ultrafiltration of protein solutions. The deposited secondary membrane was periodically removed by backflushing, and a new cake layer was deposited at the start of the next cycle. The effects of backflushing time, backflushing strength, wall shear rate, and amount of secondary membrane deposited on the permeate flux were examined. Secondary membranes were found to increase the permeate fluxin microfiltration by severalfold. Protein transmission was also enhanced owing to the presence of the secondary membrane, and the amount of protein recovered was more than twice that obtained during filtration of protein-only solutions under othewise identical conditions. In ultrafiltration, the flux enhancement owing to the secondary membrane was only 50% or less. In addition, the flux for ultrafiltration was relatively insensitive to changes in the concentration of yeast used during deposition of SMY and to the backflushing strength used to periodically remove the secondary membrane.

Keywords: Secondary membrane; backflushing; microfiltration; ultrafiltration; direct visual observation; fouling

Enzymatic synthesis of monolaurin by Carla C. B. Pereira; Mônica A. P. da Silva; Marta A. P. Langone (pp. 433-445).

The aim of this study was to produce monolaurin utilizing a commercial immobilized lipase (Lipozyme IM-20; Novo Nordisk, Bagsvaerd, Denmark) through the direct esterification of lauric acid and glycerol in a solvent-free system. The influence of fatty acid/glycerol molar ratio, temperature, and Lipozyme (IM-20) concentration on the molar fraction of monolaurin were determined using an experimental design. The best conditions employed were 55°C, lauric acid/glycerol molar ratio of 1.0, and 3.0% (w/w) enzyme concentration. The final product, obtained after 6 h of reaction, was 45.5% monolaurin, 26.8% dilaurin, 3.1% trilaurin, and 24.6% lauric acid. The reusability of the enzyme was also studied.

Keywords: Monolaurin; Immobilized lipase; esterification; experimental design; solvent-free medium

Evaluation of recombinant green fluorescent protein, under various culture conditions and purification with HiTrap hydrophobic interaction chromatography resins by Thereza Christina Vessoni Penna; Marina Ishii; Adalberto Pessoa Junior; Laura de Oliveira Nascimento; Luciana Cambricoli de Souza; Olivia Cholewa (pp. 453-468).

To determine the influence of various culture conditions, transformed cells of Escherichia coli expressing recombinant green fluorescent protein (GFPuv) were grown in nine cultures with four variable conditions (storage of inoculated broth at 4°C prior to incubation, agitation speed, isopropyl-β-d-thiogalactopyranoside [IPTG] concentration, and induction time). The pelleted cells were resuspended in extraction buffer and subjected to the three-phase partitioning (TPP) extraction method. To determine the most appropriate purification resin, protein extracts were eluted through one of four types of HiTrap hydrophobic interaction chromatography (HIC) columns prepacked with methyl, butyl, octyl, or phenyl resins and analyzed further on a 12% sodium dodecylsulfatepolyacrylamidegel. With Coomassie staining, a single band between 27 (standard GFPuv) and 29 kDa (molecular weight standard) was visualized for every HIC column sample. TPP extraction with HIC elution provided about 90% of the GFPuv recovered and eight-fold GFPuv enrichment related to the specific mass. Rotary speed and IPTG concentration showed, respectively, greater negative and positive influences on GFPuv expression at the beginning of the logarithmic phase for the set culture conditions (37°C, 24-h incubation).

Keywords: Recombinant green fluorescent protein; GFPuv; hydrophobic interaction chromatography; sodium dodecylsulfate polyacrylamide gel electrophoresis; three-phase partitioning extraction

Thermal stability of recombinant green fluorescent protein (GFPuv) at various pH values by Thereza Christina Vessoni Penna; Marina Ishii; Adalberto Pessoa Junior; Olivia Cholewa (pp. 469-483).

The thermal stability of the recombinant green fluorescent protein (GFPuv) expressed by Escherichia coli cells and isolated by three-phase partitioning extraction with hydrophobic interaction chromatography was studied. The GFPuv (3.5–9.0 μg of GFPuv/mL) was exposed to various pH conditions (4.91–9.03) and temperatures (75–95°C) in the 10 mM buffers: acetate (pH 5.0–7.0), phosphate (pH 5.5–8.0), and Tris-HCl (pH 7.0–9.0). The extent of protein denaturation (loss of fluorescence intensity) was expressed in decimal reduction time (D-value), the time exposure required to reduce 90% of the initial fluorescence intensity of GFPuv. For pH 7.0 to 8.0, the thermostability of GFPuv was slightly greater in phosphate buffer than in Tris-HCl. At 85°C, the D-values (pH 7.1–7.5) ranged from 7.24 (Tris-HCl) to 13.88 min (phosphate) The stability of GFPuv in Tris-HCl (pH>8.0) was constant at 90 and 95°C, and the D-values were 7.93 (pH 8.38–8.92) and 6.0 min (pH 8.05–8.97), respectively. The thermostability of GFPuv provides the basis for its potential utility as a fluorescent biologic indicator to assay the efficacy of moist-heat treatments at temperatures lower than 100°C.

Keywords: Green fluorescent protein; thermal stability; decimal reduction time; three-phase partitioning; fluorescence intensity; acetate; phosphate

Evaluation of optimization techniques for an extractive alcoholic fermentation process by Aline C. da Costa; Rubens Maciel Filho (pp. 485-496).

The mathematical optimization of a continuous alcoholic fermentation process combined with a flash column under vacuum was studied. The objective was to maximize % yield and productivity in the fermentor. The results using surface response analysis combined with modeling and simulation were compared withy those obtained when the problem was written as a nonlinear programming problem and was solved with a successive quadratic programming (SQP) technique. Two process models were evaluated when the process was optimized using the SQP technique. The first one is a deterministic model, whose kinetic parameters were experimentally determined as functions of the temperature, and the second is a statistical model obtained using the factorial design technique combined with simulation. Although the best result was the one obtained using the rigorous model, the values for productivity and % yield obtained using the simplified model are acceptable, and these models can be used when the development of a rigorous model is excessively difficult, slow, or expensive.

Keywords: Extractive alcoholic fermentation; optimization; successive quadratic programming; factorial design; response surface methodology; productivity

Yields from glucose, xylose, and paper sludge hydrolysate during hydrogen production by the extreme thermophile Caldicellulosiruptor saccharolyticus by Zsófia Kádár; Truus de Vrije; Giel E. van Noorden; Miriam A. W. Budde; Zsolt Szengyel; Kati Réczey; Pieternel A. M. Claassen (pp. 497-508).

This study addressed the utilization of an industrial waste stream, paper sludge, as a renewable cheap feedstock for the fermentative production of hydrogen by the extreme thermophile Caldicellulosiruptor saccharolyticus. Hydrogen, acetate, and lactate were produced in medium in which paper sludge hydrolysate was added as the sole carbon and energy source and in control medium with the same concentration of analytical grade glucose and xylose. The hydrogen yield was dependent on lactate formation and varied between 50 and 94% of the theoretical maximum. The carbon balance in the medium with glucose and xylose was virtually 100%. The carbon balance was not complete in the paper sludge medium because the measurement of biomass was impaired owing to interfering components in the paper sludge hydrolysate. Nevertheless, >85% of the carbon could be accounted for in the products acetate and lactate. The maximal volumetric hydrogen production rate was 5 to 6 mmol/(L·h), which was lower than the production rate in media with glucose, xylose, or a combination of these sugars (9–11 mmol/[L·h]). The reduced hydrogen production rate suggests the presence of inhibiting components in paper sludge hydrolysate.

Keywords: Hydrogen production; paper sludge hydrolysate; extreme thermophile; Caldicellulosiruptor saccharolyticus, glucose; xylose; carbon balances

Optimization of steam pretreatment of corn stover to enhance enzymatic digestibility by Enikő Varga; Kati Réczey; Guido Zacchi (pp. 509-523).

Among the available agricultural byproducts, corn stover, with its yearly production of 10 million t (dry basis), is the most abundant promising raw material for fuel ethanol production in Hungary. In the United States, more than 216 million to fcorn stover is produced annually, of which a portion also could possibly be collected for conversion to ethanol. However, a network of lignin and hemicellulose protects cellulose, which is the major source of fermentable sugars in corn stover (approx 40% of the dry matter [DM]). Steam pretreatment removes the major part of the hemicellulose from the solid material and makes the cellulose more susceptible to enzymatic digestion. We studied 12 different combinations of reaction temperature, time, and pH during steam pretreatment. The best conditions (200°C, 5 min, 2% H₂SO₄) increased the enzymatic conversion (from cellulose to glucose) of corn stover more then four times, compared to untreated material. However, steam pretreatment at 190°C for 5 min with 2% sulfuric acid resulted in the highest overall yield of sugars, 56.1 g from 100 g of untreated material (DM), corresponding to 73% of the theoretical. The liquor following steam explosion was fermented using Saccharomyces cerevisiae to investigate the inhibitory effect of the pretreatment. The achieved ethanol yield was slightly higher than that obtained with a reference sugar solution. This demonstrates that baker's yeast could adapt to the pretreated liquor and ferment the glucose to ethanol efficiently.

Keywords: Corn stover; pretreatment; steam explosion; hydrolysis; bioethanol

Selection of anion exchangers for detoxification of dilute-acid hydrolysates from spruce by Ilona Sárvári Horváth; Anders Sjöde; Nils-Olof Nilvebrant; Andrei Zagorodni; Leif J. Jönsson (pp. 525-538).

Six anion-exchange resins with different properties were compared with respect to detoxification of a dilute-acid hydrolysate of spruce prior to ethanolic fermentation with Saccharomyces cerevisiae. The six resins encompassed strong and weak functional groups as well as styrene-, phenol-, and acrylic-based matrices. In an analytical experimental series, fractions from columns packed with the different resins were analyzed regarding pH, glucose, furfural, hydroxymethylfurfural, phenolic compounds, levulinic acid, acetic acid, formic acid, and sulfate. An initial adsorption of glucose occurred in the strong alkaline environment and led to glucose accumulation at a later stage. Acetic and levulinic acid passed through the column before formic acid, whereas sulfate had the strongest affinity. In a preparative experimental series, one fraction from each of six columns packed with the different resins was collected for assay of the fermentability and analysis of glucose, mannose, and fermentation inhibitors. The fractions collected from strong anion-exchange resins with styrene-based matrices displayed the best fermentability: a sevenfold enhancement of ethanol productivity compared with untreated hydrolysate. Fractions from a strong anion exchanger with acrylic-based matrix and a weak exchanger with phenol-based resin displayed an intermediate improvement in fermentability, a four- to fivefold increase in ethanol productivity. The fractions from two weak exchangers with styrene- and acrylic-based matrices displayed a twofold increase in ethanol productivity. Phenolic compounds were more efficiently removed by resins with styrene-and phenol-based matrices than by resins with acrylic-based matrices.

Keywords: Saccharomyces cerevisiae ; anion-exchange resins; hydrolysate; fermentability

Ethanol production in immobilized-cell bioreactors from mixed sugar syrups and enzymatic hydrolysates of steam-exploded biomass by Isabella De Bari; Daniela Cuna; Francesco Nanna; Giacobbe Braccio (pp. 539-557).

We investigated ethanol production from mixed sugar syrups. Hydrolysates were prepared from enzymatic saccharification of steam-pretreated aspen chips. Syrups containing 45 g/L of glucose and 12 g/L of xylose were detoxified through two ion-exchange resins and then fermented with Pichia stipitis and Saccharomyces cerevisiae immobilized in Ca-alginate gel beads. Combinations of different gel fractions in the fermentation volume, amount of yeast cells, and ratios of P. stipitis vs S. cerevisiae within each bead were compared. In the best conditions, by using a total beads volume corresponding to 25% of the working volume, we obtained a yield of 0.39 g_ethanol/g_{initial sugars}. This amount of gel entrapped an initial cell concentration of 6×10¹²cells/L with ratio of S. cerevisiae/P. stipitis of 0.25 g/g. Modified stirredtank reactors were obtained either by adding marbles or by inserting a perforated metal cylinder, which reduced considerably the rupture of beads while visibly improving oxygenation of the medium.

Keywords: Biomass; ethanol; coimmobilization; Ca-alginate; Pichia stipitis ; steam explosion

Silymarin extraction from milk thistle using hot water by Lijun Duan; Danielle Julie Carrier; Edgar C. Clausen (pp. 559-568).

Hot water is attracting attention as an extraction solvent in the recovery of compounds from plant material as the search for milder and “greener” solvents intensifies. The use of hot water as an extraction solvent for milk thistle at temperatures above 100°C was explored. The maximum extraction yield of each of the silymarin compounds and taxifolin did not increase with temperature, most likely because significant compound degradation occurred. However, the time required for the yields of the compounds to reach their maxima was reduced from 200 to 55 min when the extraction temperature was increased from 100 to 140°C. Severe degradation of unprotected (plant matrix not present) silymarin compounds was observed and first-order degradation kinetics were obtained at 140°C.

Keywords: Milk thistle; extraction; silymarin; hot water; silybinin; taxifolin; silychristin

Extraction of antioxidant compounds from energy crops by Ching S. Lau; Danielle Julie Carrier; Luke R. Howard; Jackson O. Lay Jr.; Jean A. Archambault; Edgar C. Clausen (pp. 569-583).

Energy crops offer enormous opportunities for increasing the sustain ability of agriculture and energy production in the United States. Nevertheless, opportunities for sustaining biomass energy production may well hinge on producing energy and extracting high-value products from the same crop. Seven potential energy crops (mimosa, sericea, kudzu, arunzo, switchgrass, velvetbean, and castor) were extracted and assayed for the presence of potentially high-value antioxidant compounds. Of these crops, mimosa and sericea had the highest antioxidant potential and were selected for further study. High-performance liquid chromatography (ultraviolet) and liquid chromatography/mass spectrometry techniques were then utilized to help identify the compounds with high antioxidant potential using extract fractionation, and total phenolics and oxygen radical absorbance capability assays as a guide. These analyses indicate that methanol extracts of mimosa foliage most likely contain quercetin, a flavonol that has been associated with cardioprotection. Future work will concentrate on quantifying the quercetin content of mimosa (likely parts-per-million levels), as well as identifying and quantifying other antioxidants found in energy crops.

Keywords: Mimosa; sericea; energy crops; quercetin; antioxidant

Cellulase retention and sugar removal by membrane ultrafiltration during lignocellulosic biomass hydrolysis by Jeffrey S. Knutsen; Robert H. Davis (pp. 585-599).

Technologies suitable for the separation and reuse of cellulase enzymes during the enzymatic saccharification of pretreated corn stover are investigated to examine the economic and technical viability of processes that promote cellulase reuse while removing inhibitory reaction products such as glucose and cellobiose. The simplest and most suitable separation is a filter with relatively large pores on the order of 20–25 mm that retains residual corn stover solids while passing reaction products such as glucose and cellobiose to form a sugar stream for a variety of end uses. Such a simple separation is effective because cellulase remains bound to the residual solids. Ultrafiltration using 50-kDa polyethersulfone membranes to recover cellulase enzymes in solution was shown not to enhance further the saccharification rate or overall conversion. Instead, it appears that the necessary cellulase enzymes, including β-glucosidase, are tightly bound to the substrate; when fresh corn stover is contacted with highly washed residual solids, without the addition of fresh enzymes, glucose is generated at a high rate. When filtration was applied multiple times, the concentration of inhibitory reaction products such as glucose and cellobiose was reduced from 70 to 10 g/L. However, an enhanced saccharification performance was not observed, most likely because the concentration of the inhibitory products remained too high. Further reduction in the product concentration was not investigated, because it would make the reaction unnecessarily complex and result in a product stream that is much too dilute to be useful. Finally, an economic analysis shows that reuse of cellulase can reduce glucose production costs, especially when the enzyme price is high. The most economic performance is shown to occur when the cellulase enzyme is reused and a small amount of fresh enzyme is added after each separation step to replace lost or deactivated enzyme.

Keywords: Saccharification; corn stover; cellulase; glucose; ultrafiltration; vacuum filtration

Controlled fed-batch fermentations of dilute-acid hydrolysate in pilot development unit scale by Andreas Rudolf; Mats Galbe; Gunnar Lidén (pp. 601-617).

Inhibitors formed during wood hydrolysis constitute a major problem in fermenting dilute-acid hydrolysates. By applying a fed-batch technique, the levels of inhibitory compounds may be held low, enabling high ethanol productivity. In this study, a previously developed fed-batch strategy was modified and implemented for use in pilot development unit (PDU) scale. The rate of total gas formation, measured with a mass flow meter, was used as input variable in the control algorithm. The feed rate in the PDU-scale experiments could be properly controlled based on the gas evolution from the reactor. In fed-batch experiments utilizing TMB 3000, an inhibitor-tolerant strain of Saccharomyces cerevisiae, close to 100% of the hexoses in the hydrolysate was converted.

Keywords: Pilot development unit scale; dilute-acid hydrolysate; fedbatch fermentation; feed rate; carbon dioxide evolution rate

Degeneration of β-glucosidase activity in a foam fractionation process by Vorakan Burapatana; Ales Prokop; Robert D. Tanner (pp. 619-625).

Foam fractionation is a promising technique for concentrating proteins because of its simplicity and low operating cost. One such protein that can be foamed is the enzyme cellulase. The use of inexpensively purified cellulase may be a key step in the economical production of ethanol from biomass. We conducted foam fractionation experiments at total reflux using the cellulase component β-glucosidase to study how continuous shear affects β-glucosidase in a foam such as a fermentation or foam fractionation process. The experiments were conducted at pH 2.4, 5.4, and 11.6 and airflow rates of 3, 6, 15, 20, and 32 cc/min to determine how β-glucosidase activity changes in time at these different conditions. This is apparently a novel and simple way of testing for changes in enzyme activity within a protein foam. The activity did not degenerate during 5 min of reflux at pH 5.4 at an airflow rate of 10 cc/ min. It was established that at 10 min of refluxing, the β-glucosidase denatured more as the flow rate increased. At pH 2.4 and a flow rate of 10 cc/min, the activity remained constant for at least 15 min.

Keywords: β-Glucosidase; foam fractionation; cellulase; reflux time; airflow rate

Simultaneous production of nisin and lactic acid from cheese whey by Chuanbin Liu; Yan Liu; Wei Liao; Zhiyou Wen; Shulin Chen (pp. 627-638).

A biorefinery process that utilizes cheese whey as substrate to simultaneously produce nisin, a natural food preservative, and lactic acid, a raw material for biopolymer production, was studied. The conditions for nisin biosynthesis and lactic acid coproduction by Lactococcus lactis subsp. lactis (ATCC 11454) in a whey-based medium were optimized using statistically based experimental designs. A Plackett-Burman design was applied to screen seven parameters for significant factors for the production of nisin and lactic acid. Nutrient supplements, including yeast extract, MgSO₄, and KH₂PO₄, were found to be the significant factors affecting nisin and lactic acid formation. As a follow-up, a central-composite design was applied to optimize these factors. Second-order polynomial models were developed to quantify the relationship between nisin and lactic acid production and the variables. The optimal values of these variables were also determined. Finally, a verification experiment was performed to confirm the optimal values that were predicted by the models. The experimented results agreed well with the model prediction, giving a similar production of 19.3 g/L of lactic acid and 92.9 mg/L of nisin.

Keywords: Nisin; whey; fermentation; optimization; experimental design

Effect of process parameters on production of a biopolymer by Rhizobium sp. by Flávia Pereira Duta; Francisca Pessôa De França; Eliana Flávia Camporese Sérvulo; Léa Maria De Almeida Lopes; Antonio Carlos Augusto Da Costa; Ana Barros (pp. 639-652).

The production of biopolymers by a Rhizobium strain was studied under batch and bioreactor conditions. The best viscosity levels were obtained under low mannitol concentrations as well as low agitation and aeration conditions. Infrared spectra indicated the presence of chemical groups characteristic of microbially produced biopolymers, including C=O and O-acetyl groups. Thermogravimetric analysis showed the characteristic degradation profiles of the exopolysaccharide produced (T _onset=290°C). The experimental design showed that a low substrate concentration (10.0 g/L), and low aeration (0.2 vvm) and agitation (200 rpm) levels should be used. The maximum yield of the process was a Yp/s (g/g) of 0.19±0.1, obtained under optimized conditions.

Keywords: Biopolymer; exopolysaccharides; Rhizobium sp; viscosity; conversion factor

Succinic acid adsorption from fermentation broth and regeneration by Brian H. Davison; Nhuan P. Nghiem; Gerald L. Richardson (pp. 653-669).

More than 25 sorbents were tested for uptake of succinic acid from aqueous solutions. The best resins were then tested for successive loading and regeneration using hotwater. The key desired properties for an ideal sorbent are high capacity, complete stable regenerability, and specificity for the product. The best resins have a stable capacity of about 0.06 g of succinic acid/g of resin at moderate concentrations (1–5 g/L) of succinic acid. Several sorbents were tested more exhaustively for uptake of succinic acid and for successive loading and regeneration using hot water. One resin, XUS 40285, has a good stable isotherm capacity, prefers succinate over glucose, and has good capacities at both acidic and neutral pH. Succinic acid was removed from simulated media containing salts, succinic acid, acetic acid, and sugar using a packed column of sorbent resin, XUS 40285. The fermentation byproduct, acetate, was completely separated from succinate. A simple hot water regeneration successfully concentrated succinate from 10 g/L (inlet) to 40–110 g/L in the effluent. If successful, this would lower separation costs by reducing the need for chemicals for the initial purification step. Despie promising initial results of good capacity (0.06 g of succinic/g of sorbent), 70% recovery using hot water, and a recovered concentration of >100 g/L, this regeneration was not stable over 10 cycles in the column. Alternative regeneration schemes using acid and base were examined. Two (XUS 40285 and XFS-40422) showed both good stable capacities for succinic acid over 10 cycles and >95% recovery in a batch operation using a modified extraction procedure combining acid and hot water washes. These resins showed comparable results with actual broth.

Keywords: Succinic acid; sorbent; adsorption; hot water regeneration; glucose; fermentation broth

A hollow-fiber membrane extraction process for recovery and separation of lactic acid from aqueous solution by Hanjing Huang; Shang-Tian Yang; David E. Ramey (pp. 671-688).

An energy-efficient hollow-fiber membrane extraction process was successfully developed to separate and recover lactic acid produced in fermentation. Although many fermentation processes have been developed for lactic acid production, and economical method for lactic acid recovery from the fermentation broth is still needed. Continuous extraction of lactic acid from a simulated aqueous stream was achieved by using Alamine 336 in 2-octanol contained in a hollow-fiber membrane extractor. In this process, the extractant was simultaneously regenerated by stripping with NaOH in a second membrane extractor, and the final product is a concentrated lactate salt solution. The extraction rate increased linearly with an increase in the Alamine 336 content in the solvent (from 5 to 40%). Increasing the concentration of the undissociated lactic acid in the feed solution by either increasing the lactate concentration (from 5 to 40 g/L) or decreasing the solution pH (from 5.0 to 4.0) also increased the extraction rate. Based on these observations, a reactive extraction model with a first-order reaction mechanism for both lactic acid and amine concentrations was proposed. The extraction rate also increased with an increase in the feed flow rate, but not the flow rates of solvent and the stripping solution, suggesting that the process was not limited by diffusion in the liquid films or membrane pores. A mathematical model considering both diffusion and chemical reaction in the extractor and back extractor was developed to simulate the process. The model fits the experimental data well and can be used in scale up design of the process.

Keywords: Reactive extraction; hollow-fiber membrane; lactic acid; mass transfer; Alamine 336

Evaluation of tocopherol recovery through simulation of molecular distillation process by E. B. Moraes; C. B. Batistella; M. E. Torres Alvarez; Rubens Maciel Filho; M. R. Wolf Maciel (pp. 689-711).

DISMOL simulator was used to determine the best possible operating conditions to guide, in future studies, experimental works. This simulator needs several physical-chemical properties and often it is very difficult to determine them because of the complexity of the involved components. Their determinations must be made through correlations and/or predictions, in order to characterize the system and calculate it. The first try is to have simulation results of a system that later can be validated with experimental data. To implement, in the simulator, the necessary parameters of complex systems is a difficult task. In this work, we aimed to determe these properties in order to evaluate the tocopherol (vitamin E) recovery using a DISMOL simulator. The raw material used was the crude deodorizer distillate of soya oil. With this procedure, it is possible to determine the best operating conditions for experimental works and to evaluated the process in the separation of new systems, analyzing the profiles obtained from these simulations for the falling film molecular distillator.

Keywords: Molecular distillation; vitamin E; deodorizer distillate of soya oil; DISMOL; property estimation

High-productivity continuous biofilm reactor for butanol production by Nasib Qureshi; Patrick Karcher; Michael Cotta; Hans P. Blaschek (pp. 713-721).

Corn steep liquor (CSL), a byproduct of the corn wet-milling process, was used in an immobilized cell continuous biofilm reactor to replace the expensive P2 medium ingredients. The use of CSL resulted in the production of 6.29 g/L of total acetone-butanol-ethanol (ABE) as compared with 6.86 g/L in a control experiment. These studies were performed at a dilution rate of 0.32 h⁻¹. The productivities in the control and CSL experiment were 2.19 and 2.01 g/(L·h), respectively. Although the use of CSL resulted in a 10% decrease in productivity, it is viewed that its application would be economical compared to P2 medium. Hence, CSL may be used to replace the P2 medium. It was also demonstrated that inclusion of butyrate into the feed was beneficial to the butanol fermentation. A control experiment produced 4.77 g/L of total ABE, and the experiment with supplemented sodium butyrate produced 5.70 g/L of total ABE. The butanol concentration increased from 3.14 to 4.04 g/L. Inclusion of acetate in the feed medium of the immobilized cell biofilm reactor was not found to be beneficial for the ABE fermentation, as reported for the batch ABE fermentation.

Keywords: Immobilized cell biofilm reactor; butanol; corn steep liquor; sodium butyrate; Clostridium beijerinckii BA101; sodium acetate

Measurement of rheology of distiller's grain slurries using a helical impeller viscometer by Tiffany L. Houchin; Thomas R. Hanley (pp. 723-732).

Current research is focused on developing a process to convert the cellulose and hemicellulose in distiller's grains into fermentable sugars, increasing both ethanol yield and the amount of protein in the remaining solid product. The rheologic properties of distiller's grain slurries were determined for concentrations of 21, 23, and 25%. Distiller's grain slurries are non-Newtonian, heterogeneous fluids subject to particle settling. Traditional methods of viscosity measurement, such as cone-and-plate and concentric cylinder viscometers, are not adequate for these fluids. A helical impeller viscometer was employed to measure impeller torque over a range of rotational speeds. Newtonian and non-Newtonian calibration fluids were utilized to obtain constants that relate shear stresses and shear rates to the experimental data. The Newtonian impeller constant, c, was 151; the non-Newtonian shear rate constant, k, was 10.30. Regression analysis of experimental data was utilized for comparison to power law, Herschel-Bulkley, and Casson viscosity models with regression coefficients exceeding 0.99 in all cases.

Keywords: Distiller's grain slurries; rheologic properties; wet grains; calibration fluids; helical impeller

Computational fluid dynamics simulation and redesign of a screw conveyor reactor by Yinkun Wan; Thomas R. Hanley (pp. 733-745).

National Renewable Energy Laboratory (NREL) designed a shrinking-bed reactor to maintain a constant bulk packing density of cellulosic biomass. The high solid-to-liquid ratio in the pretreatment process allows a high sugar yield and avoids the need to flush large volumes of solution through the reactor. To scale up the shrinking-bed reactor, NREL investigated a pilot-scale screw conveyor reactor in which an interrupted flight between screws was employed to mimic the “shrinking-bed” effect. In the experiments with the screw conveyor reactor, overmixing and uneven flow occurred. These phenomena produce negative effects on biomass hydrolysis. The flow behavior inside the reactor was analyzed to allow redesign of the screw to achieve adequate mixing and even flow. In the present study, computational fluid dynamics (CFD) was utilized to simulate the fluid flow in the porous media, and a new screw design was proposed. CFD analysis performed on the redesigned reactor indicated that an even flow pattern was achieved.

Keywords: Screw reactor; computational fluid dynamics; modeling; backflow; hydrolysis

Production of biodiesel fuel by transesterification of rapeseed oil by Gwi-Taek Jeong; Don-Hee Park; Choon-Hyoung Kang; Woo-Tai Lee; Chang-Shin Sunwoo; Chung-Han Yoon; Byung-Chul Choi; Hae-Sung Kim; Si-Wouk Kim; Un-Taek Lee (pp. 747-758).

Fatty acid methyl esters (FAMEs) show large potential applications as diesel substitutes, also known as biodiesel fuel. Biodiesel fuel as renewable energy is an alternative that can reduce energy dependence on petroleum as well as air pollution. Several processes for the production of biodiesel fuel have been developed. Transesterification processes under alkali catalysis with short-chain alcohols give high yields of methyl esters in short reaction times. We investigated transesterification of rapeseed oil to produce the FAMEs. Experimental reaction conditions were molar ratio of oil to alcohol, concentration of catalyst, type of catalyst, reaction time, and temperature. The conversion ratio of rapeseed oil was enhanced by the alcohol:oil mixing ratio and the reaction temperature.

Keywords: Biodiesel fuel; transesterification; rapeseed oil; molar ratio; fatty acid methyl ester

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.114, #1-3)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.114, #1-3)