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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.98-100, #1-9)

Two-step steam pretreatment of softwood with SO₂ impregnation for ethanol production by Johanna Söderström; Linda Pilcher; Mats Galbe; Guido Zacchi (pp. 5-21).

Two-step steam pretreatment of softwood was investigated with the aim of improving the enzymatic digestibility for ethanol production. In the first step, softwood was impregnated with SO₂ and steam pretreated at different severities. The first step was performed at low severity to hydrolyze the hemicellulose and release the sugars into the solution. The combination of time and temperature that yielded the highest amount of hemicellulosic sugars in the solution was determined. In the second step, the washed solid material from the optimized first step was impregnated once more with SO₂ and steam pretreated under more severe conditions to enhance the enzymatic digestibility. The investigated temperature range was between 180 and 220°C, and the residence times were 2, 5 and 10 min. The effectiveness of pretreatment was assessed by both enzymatic hydrolysis of the solids and simultaneous saccharification and fermentation (SSF) of the whole slurry after the second pretreatment step, in the presence of antibiotics. For each pretreatment combination, the liquid fraction was fermented to determine any inhibiting effects. At low severity in the second pretreatment step, a high conversion of cellulose was obtained in the enzymatic hydrolysis step, and at a high severity a high conversion of cellulose was obtained in the second pretreatment step. This resulted in an overall yield of sugars that was nearly constant over a wide range of severity. Compared with the one-step steam pretreatment, the two-step steam pretreatment resulted in a higher yield of sugar and in a slightly higher yield of ethanol. The overall sugar yield, when assessed by enzymatic hydrolysis, reached 80%. In the SSF configuration, an overall ethanol yield of 69% was attained.

Keywords: Enzymatic hydrolysis; spruce; simultaneous saccharification and fermentation

Predicting digestibility of ammonia fiber explosion (AFEX)-treated rice straw by Lisa E. Gollapalli; Bruce E. Dale; Douglas M. Rivers (pp. 23-35).

The enzymatic digestibility of ammonia fiber explosion (AFEX)-treated rice straw was modeled by statistically correlating the variability of samples to differences in treatment using several different analytical techniques. Lignin content and crystallinity index of cellulose affect enzymatic hydrolysis the most. X-ray diffraction was used to measure the crystallinity index (CrI), while fluorescence and diffuse reflectance infrared (DRIFT) spectroscopy measured the lignin content of the samples. Multivariate analysis was applied to correlate the enzymatic hydrolysis results of the various samples with X-ray diffraction and spectroscopic data. Principal component analysis (PCA) and multilinear regression (MLR) techniques did not accurately predict the digestibility of the rice straw samples. The best correlation (R value of 0.775) was found between the treatment conditions of the AFEX process and the concentration of xylose at 24 h after enzymatic hydrolysis.

Keywords: Lignocellulose; enzymatic digestibility; lignin; crystallinity; AFEX; correlation; pretreatment; X-ray diffraction; fluorescence; DRIFT

Improvement of photosynthetic CO₂ fixation at high light intensity through reduction of chlorophyll antenna size by James W. Lee; Laurens Mets; Elias Greenbaum (pp. 37-48).

At elevated light intensities (greater than ∼200 µE/[m²·s]), the kinetic imbalance between the rate of photon excitation and thermally activated electron transport results in saturation of the rate of photosynthesis. Since maximum terrestrial solar radiation can reach 200 µE/(m²·s), a significant opportunity exists to improve photosynthetic efficiency at elevated light intensities by achieving a kinetic balance between photon excitation and electron transport, especially in designed large-scale photosynthetic reactors in which a low-cost and efficient biomass production system is desired. One such strategy is a reduction in chlorophyll (chl) antenna size in relation to the reaction center that it serves. In this article, we report recent progress in this area of research. Light-saturation studies for CO₂ fixation were performed on an antenna-deficient mutant of Chlamydomonas (DS521) and the wild type (DES15) with 700 ppm of CO₂ in air. The light-saturated rate for CO₂ assimilation in the mutant DS521 was about two times higher (187 µmol/[h·mg of chl]) than that of the wild type, DES15 (95 µmol/[h·mg of chl]). Significantly, a partial linearization of the light-saturation curve was also observed. These results confirmed that DS521 has a smaller relative chl antenna size and demonstrated that reduction of relative antenna size can improve the overall efficiency of photon utilization at higher light intensities. The antenna-deficient mutant DS521 can provide significant resistance to photoinhibition, in addition to improvement in the overall efficiency of CO₂ fixation at high light. The experimental data reported herein support the idea that reduction in chl antenna size could have significant implications for both fundamental understanding of photosynthesis and potential application to improve photosynthetic CO₂ fixation efficiency.

Keywords: Photosynthesis; photosynthetic efficiency; chlorophyll antenna site; CO₂ fixation; photoinhibition resistance; Chlamydomonas mutant

Dilute-acid hydrolysis of sugarcane bagasse at varying conditions by Markus Neureiter; Herbert Danner; Christiane Thomasser; Bamusi Saidi; Rudolf Braun (pp. 49-58).

Sugarcane bagasse, a byproduct of the cane sugar industry, is an abundant source of hemicellulose that could be hydrolyzed to yield a fermentation feedstock for the production of fuel ethanol and chemicals. The effects of sulfuric acid concentration, temperature, time, and dry matter concentration on hemicellulose hydrolysis were studied with a 20-L batch hydrolysis reactor using a statistical experimental design. Even at less severe conditions considerable amounts (>29%) of the hemicellulose fraction could be extracted. The percentage of soluble oligosaccharides becomes very low in experiments with high yields in monosaccharides, which indicates that the cellulose fraction is only slightly affected. For the sugar yields, acid concentration appears to be the most important parameter, while for the formation of sugar degradation products, temperature shows the highest impact. It could be demonstrated that the dry matter concentration in the reaction slurry has a negative effect on the xylose yield that can be compensated by higher concentrations of sulfuric acid owing to a positive interaction between acid concentration and dry matter contents.

Keywords: Sugarcane bagasse; hemicellulose; dilute-acid hydrolysis

SO₂-catalyzed steam explosion of corn fiber for ethanol production by Renata Bura; Shawn D. Mansfield; John N. Saddler; Rodney J. Bothast (pp. 59-72).

Corn fiber, a by-product of the corn wet-milling industry, represents a renewable resource that is readily available in significant quantities and could potentially serve as a low-cost feedstock for the production of fuel-grade alcohol. In this study, we used a batch reactor to steam explode corn fiber at various degrees of severity to evaluate the potential of using this feedstock in the bioconversion process. The results indicated that maximum sugar yields (soluble and following enzymatic hydrolysis) were recovered from corn fiber that was pretreated at 190°C for 5 min with 6% SO₂. Sequential SO₂-catalyzed steam explosion and enzymatic hydrolysis resulted in very high conversion (81%) of all polysaccharides in the corn fiber to monomeric sugars. Subsequently, Saccharomyces cerevisiae was able to convert the resultant corn fiber hydrolysates to ethanol very efficiently, yielding 90–96% of theoretical conversion during the fermentation process.

Keywords: Corn fiber; steam pretreatment; enzymatic hydrolysis; fermentation; ethanol

Chemical pretreatments of corn stover for enhancing enzymatic digestibility by Enikö Varga; Zsolt Szengyel; Kati Réczey (pp. 73-87).

Corn stover, the most abundant agricultural residue in Hungary, is a potential raw material for the production of fuel ethanol as a result of its high content of carbohydrates, but a pretreatment is required for its efficient hydrolysis. In this article, we describe the results using various chemicals such as dilute H₂SO₄, HCl, and NaOH separately as well as consecutively under relative mild conditions (120°C, 1h). Pretreatment with 5% H₂SO₄ or 5% HCl solubilized 85% of the hemicellulose fraction, but the enzymatic conversion of pretreated materials increased only two times compared to the untreated corn stover. Applying acidic pretreatment following a 1-d soaking in base achieved enzymatic conversion that was nearly the theoretical maximum (95.7%). Pretreatment with 10% NaOH decreased the lignin fraction >95%, increased the enzymatic conversion more than four times, and gave a 79.4% enzymatic conversion. However, by increasing the reaction time, the enzymatic degradability could also be increased significantly, using a less concentrated base. When the time of pretreatment was increased three times (0.5% NaOH at 120°C), the amount of total released sugars was 47.9 g from 100 g (dry matter) of untreated corn stover.

Keywords: Biomass pretreatment; corn stover; enzymatic hydrolysis

Technoeconomic study on steam explosion application in biomass processing by Francesco Zimbardi; Esmeralda Ricci; Giacobbe Braccio (pp. 89-99).

This work is based on the data collected during trials of a continuous steam explosion (SE) plant, with a treatment capacity of about 350 kg/h, including the biomass fractionation section. The energy and water consumption, equipment cost, and manpower needed to run this plant have been used as the base case for a techno-economic evaluation of productive plants. Three processing plant configurations have been considered: (I) SE pretreatment only; (II) SE followed by the hemicellulose extraction; (III) SE followed by the sequential hemicellulose and lignin extractions. The biomass treatment cost has been evaluated as a function of the plant scale. For each configuration, variable and fixed cost breakdown has been detailed in the case of a 50,000 t/y plant.

Keywords: Steam explosion; economic evaluation; scale up; biomass processing

Modeling of 2,4-dichlorophenoxyacetic acid controlled-release kinetics from lignin-based formulations by Félix M. Pereira; Adilson R. Gonçalves; André Ferraz; Flávio T. Silva; Samuel C. Oliveira (pp. 101-107).

The second Fick’s law of diffusion, considering boundary conditions that at both slab faces the concentration of herbicide is equal to zero (sink conditions), has been adequate to describe our kinetic data obtained from experiments on 2,4-dichlorophenoxyacetic acid, (2,4-D) released from lignin-based formulations in a water static bath system. However, the same model proved to be invalid in describing the experimental data obtained with ametryn (2-ethylamino-4-isopropylamino-6-methylthio-1,3,5-triazine) and diuron (3-[3,4-dichlorophenyl]-1,1-dimethylurea) formulations in a water dynamic bath system. For ametryn and diuron formulations, because of the lower aqueous solubility of these herbicides, it was necessary to model a stagnant film at the formulation surface to describe better the release kinetics because the model incorporating sink conditions is insufficient. This study presents a new mathematical modeling of experimental data obtained with 2,4-D formulations in a water static bath system. The new model incorporates a stagnant film as the boundary condition at the formulation surface, and its diffusion coefficient value is more precise than the one estimated by the model employing sink conditions.

Keywords: Mathematical modeling; controlled release; lignin; herbicide; 2,4-dichlorophenoxyacetic acid; diffusion

Comparison of aspen wood hydrolysates produced by pretreatment with liquid hot water and carbonic acid by Robert C. McWilliams; G. Peter van Walsum (pp. 109-121).

Hydrolysates produced by the pretreatment of aspen wood with liquid hot water were compared with hydrolysates produced using carbonic acid pretreatment. Pretreatment temperatures tested ranged from 180° to 220°C; reaction times were varied between 2.5 and 30.5 min. Under most conditions tested, it was found that the presence of carbonic acid had no discernible effect on the amount of xylose released or concentration of furan compounds, as indicated by ultraviolet-visual absorbance between 270 and 280 nm. Thus, there appears to be little difference in the severity of the pretreatment conditions with or without the presence of carbonic acid. The presence of carbonic acid did, however, result in a hydrolysate with a higher final pH. It is hypothesized that the presence of the carbonic acid during the reaction may have the effect of reducing the accumulation of organic acids in the hydrolysate.

Keywords: Carbonic acid; pretreatment; liquid hot water pretreatment; autohydrolysis; aspen wood; pH

Optimizing ammonia processing conditions to enhance susceptibility of legumes to fiber hydrolysis by A. Ferrer; F. M. Byers; B. Sulbarán-de-Ferrer; B. E. Dale; C. Aiello (pp. 123-134).

An ammonia process was applied at several ammonia loadings, moisture contents, temperatures, and dwell times. A cellulase loading of 5 FPU /g dry matter and a 24 h incubation time were used to produce the sugars, which were measured as reducing sugars and by HPLC. Optimal processing conditions caused a 76% of theoretical yield (2.9-fold above untreated). Cellulose and hemicellulose conversions were 68 and 85% (vs 38 and 34% in untreated, respectively). The short hydrolysis time and relatively low enzyme loading suggests great potential to produce sugars from alfalfa.

Keywords: Ammonia; alfalfa; enzymatic hydrolysis; sugars

Optimizing ammonia processing conditions to enhance susceptibility of legumes to fiber hydrolysis by A. Ferrer; F. M. Byers; B. Sulbarán-de-Ferrer; B. E. Dale; C. Aiello (pp. 135-146).

A warm-season legume, Florigraze rhizoma peanut (FRP), was used as the source of fiber to produce sugars. FRP was subjected to several ammonia-processing conditions using temperature, biomass moisture content, and ammonia loading as process variables during a 5-min treatment. A cellulase loading of 2 FPU/g DM and 24 h incubation were used to produce the sugars. Total sugar yield was 3.34-fold higher in the optimal treatment (1.5 g ammonia/g DM-60%-90°C) compared to untreated and was 65.3% of theoretical. Cellulose and hemicellulose conversions increased from 30 and 15.5% in untreated FRP to 78 and 34% in treated FRP.

Keywords: Ammonia; florigraze rhizoma peanut; enzymatic hydrolysis; sugars

Effects of operating parameters on countercurrent extraction of hemicellulosic sugars from pretreated softwood by Kyoung Heon Kim; Melvin P. Tucker; Quang A. Nguyen (pp. 147-159).

In a previous study using a continuous countercurrent screw extractor for two-stage dilute-acid hydrolysis, which was focused on the effects of liquid-to-insoluble solids (L/IS) ratio, we demonstrated that by using low volumes of wash water soluble sugars can be recovered from first-stage pretreated softwood at high yields and also at high sugar concentrations. In this study, we investigated the effects of important operating parameters other than the L/IS ratio, such as the feed rates of water and pretreated biomass and the extractor inclined angle, on the performance of the extractor using first-stage pretreated softwood. As biomass and water feed rates increased at the same L/IS ratio, the recovery yield of soluble sugars decreased, probably owing to a reduced solids residence time in the extractor, which is related to the solid/liquid contact time. The sugar recovery yield was higher at a higher extractor inclined angle. This may be attributed to the effects of increased back mixing and a longer residence time for solids at a higher extractor angle. Countercurrent extraction was also carried out with other pretreated biomass having smaller particle sizes and poor drainage rates. The countercurrent screw extractor was found to be unsuitable for these fine materials due to the slow liquid drainage rate and filter-clogging problems. In a test for stability of soluble sugars in first-stage softwood hydrolysate, irrespective of the storage temperature and storage form, the sugar concentration slowly decreased with storage time. However, storage in slurry form showed higher sugar stability compared with that in liquor form at the same conditions.

Keywords: Continuous countercurrent extraction; hemicellulosic sugars; softwood; pretreatment; two-stage dilute-acid hydrolysis

Production of recombinant bleaching enzymes from thermophilic microorganisms in fungal hosts by Peter L. Bergquist; V. S. Junior Te’o; Moreland D. Gibbs; Angela C. E. Cziferszky; Fabricia P. De Faria; Maristela O. Azevedo; K. M. Helena Nevalainen (pp. 165-176).

Cost-effective production of enzymes for industrial processes makes the appropriate selection of the host-vector expression system critical. We have developed two systems for the bulk production of bleaching enzymes from thermophiles. Kluyveromyces lactis has been developed as a secretion host employing expression vectors based on the 2μ-like plasmid pKD1 of Kluyveromyces drosophilarium. Our second system involves the filamentous fungus Trichoderma reesei. Fusion and nonfusion vectors have been constructed using the strong cellobiohydrolase 1 (cbh1) promoter. The KEX2 protease cleavage site and a 6 × HIS-tag have been incorporated to facilitate both cleavage and purification of the mature foreign proteins.

Keywords: Trichoderma reesei ; Kluyveromyces lactis ; thermophilic xylanases; heterologous expression; gene redesign

Hydrogen production by the thermophilic bacterium Thermotoga neapolitana by Suellen A. Van Ooteghem; Stephen K. Beer; Paul C. Yue (pp. 177-189).

Virtually all members of the order Thermotogales have demonstrated the ability to produce hydrogen; however, some members of this order produce considerably greater quantities than others. With one representative of this order, Thermotoga neapolitana, we have consistently obtained accumulation of 25–30% hydrogen with 12–15% carbon dioxide as the only other prominent product in the batch reaction. In contradistinction to information widely disseminated in the literature, we have also found that most members of this order tolerate and appear to utilize the moderate amounts of oxygen present in the gaseous phase of batch reactors (6–12%), with no apparent decrease in hydrogen production. Hydrogen accumulation has been widely reported to inhibit growth of Thermotogales. While this may be true at very high hydrogen tensions, we have observed log phase bacterial morphology (rods) even in the presence of 25–35% hydrogen concentrations. To maximize hydrogen production and minimize production of hydrogen sulfide, inorganic sulfur donors are avoided and the cysteine concentration in the medium is increased. We and others have demonstrated that different members of the order Thermotogales utilize a wide variety of feedstocks, including complex carbohydrates and proteins. Thus, it appears that organisms within this order have the potential to utilize a variety of organic wastes and to cost-effectively generate hydrogen.

Keywords: Thermotogales ; Thermotoga neapolitana ; hydrogen generation; microaerophiles

4-Chlorophenol degradation by chloroperoxidase from Caldariomyces fumago by Camilo E. La Rotta H.; Elba P.S.Bon (pp. 191-203).

This study investigated the degradation of 4-chlorophenol (4-CP) by Caldariomyces fumago chloroperoxidase (CPO). Enzymatic oxidations were studied in reaction mixtures at pH 3.0, 4.0, and 6.0 in the presence and absence of Cl⁻ containing 3.5 IU of CPO and 4-CP and hydrogen peroxide concentrations within the range of 0.5–50 and 0.005–50 mM, respectively. Distinct patterns of products regarding color, concentration, and solubility were observed. Reaction mixtures at pH 6.0 containing 3.5 IU of CPO and 5.0 mM 4-CP and H₂O₂ (1:1 stoichiometry) showed the highest 4-CP removal of 95% and the highest formation of a dark precipitate.

Keywords: Caldariomyces fumago ; chloroperoxidase; 4-chlorophenol degradation; polymerization reactions; industrial effluents treatment

Effect of k L a on the production of glucose 6-phosphate dehydrogenase from Saccharomyces cerevisiae grown by fermentation process by Daniel Pereira Silva; Adalberto Pessoa Jr.; Ines C. Roberto; Michele Vitolo (pp. 205-213).

In a 5-L fermentor (NBS-MF 105), Saccharomyces cerevisiae (0.7 g/L) was inoculated into a liquid medium (pH 4.0) containing 17 g/L of glucose, 2.55 g/L of yeast extract, 4.25 g/L of peptone, 2.04 g/L of Na₂HPO₄·12H₂O, 4.34 g/L of (NH₄)₂SO₄ and 0.064 g/L of MgSO₄·7H₂O and aerobically cultivated at 35°C for 22 h. Agitation and aeration were adjusted to attain initial k L a values of 15, 60, 135, and 230 h⁻¹. The glucose 6-phosphate dehydrogenase (G6PDH) productivity (Pr _G6PDH) obtained for kLa values of 15, 60, 135, and 230 h⁻¹ was 10.6, 31.8, 30.3, and 23.3 U/([L·h]), respectively, whereas the cell productivity (Pr _x) for the same k L a values were 0.24, 0.69, 0.69, and 0.49 g/[L·h], respectively. Thus, both events are coupled and depend on the dissolved oxygen in the medium.

Keywords: Glucose 6-phosphate dehydrogenase; Saccharomyces cerevisiae ; fermentation; volumetric coefficient of oxygen transfer

Steady-state measurements of lactic acid production in a wild-type and a putative d-lactic acid dehydrogenase-negative mutant of zymomonas mobilis by Hugh G. Lawford; Joyce D. Rousseau (pp. 215-228).

This work represents a continuation of our investigation into environmental conditions that promote lactic acid synthesis by Zymomonas mobilis. The characteristic near theoretical yield of ethanol from glucose by Z. mobilis can be compromised by the synthesis of d- and l-lactic acid. The production of lactic acid is exacerbated by the following conditions: pH 6.0, yeast extract, and reduced growth rate. At a specific growth rate of 0.048/h, the average yield of dl-lactate from glucose in a yeast extract-based medium at pH 6.0 was 0.15 g/g. This represents a reduction in ethanol yield of about 10% relative to the yield at a growth rate of 0.15/h. Very little lactic acid was produced at pH 5.0 or using a defined salts medium (without yeast extract) Under permissive and comparable culture conditions, a tetracycline-resistant, d-ldh negative mutant produced about 50% less lactic acid than its parent strain Zm ATCC 39676. d-lactic acid was detected in the cell-free spent fermentation medium of the mutant, but this could be owing to the presence of a racemase enzyme. Under the steady-state growth conditions provided by the chemostat, the specific rate of glucose consumption was altered at a constant growth rate of 0.075/h. Shifting from glucose-limited to nitrogen-limited growth, or increasing the temperature, caused an increase in the specific rate of glucose catabolism. There was good correlation between an increase in glycolytic flux and a decrease in lactic acid yield from glucose. This study points to a mechanistic link between the glycolytic flux and the control of end-product glucose metabolism. Implications of reduced glycolytic flux in pentose-fermenting recombinant Z. mobilis strains, relative to increased byproduct synthesis, is discussed.

Keywords: Zymomonas mobilis ; lactic acid; end-product selectivity; d-lactate dehydrogenase; glycolytic flux; steady state

Enhanced formation of extracellular laccase activity by the white-rot fungus Trametes multicolor by Johann Hess; Christian Leitner; Christiane Galhaup; Klaus D. Kulbe; Barbara Hinterstoisser; Martin Steinwender; Dietmar Haltrich (pp. 229-241).

The white-rot fungus Trametes multicolor MB 49 has been identified as an excellent producer of the industrially important enzyme laccase. The formation of extracellular laccase could be considerably stimulated by the addition of Cu(II) to a simple, glycerol-based culture medium. In this study, optimal concentrations of copper were found to be 0.5–1 mM, which were added during the growth phase of the fungus. Other medium components important for laccase production are the carbon and nitrogen sources employed. When using an optimized medium containing glycerol (40 g/L), peptone from meat (15 g/L), and MgSO₄·7H₂O and stimulating enzyme formation by the addition of 1.0 mM Cu, maximal laccase activities obtained in shake-flask cultures were approx 85 U/mL. These results, however, could not be scaled up to a laboratory fermentor cultivation. Laccase production by T. multicolor decreased considerably when the fungus was grown in a stirred-tank reactor, presumably because of damage of the mycelia caused by shear stress and/or changes in the morphology of the fungus.

Keywords: Trametes multicolor ; laccase; polyphenol oxidase; culture medium development; morphology

Presence and physiologic regulation of alcohol oxidase activity in an indigenous fungus isolated from petroleum-contaminated soils by Yolanda Alvarado-Caudillo; José Carlos Bravo Torres; Vanesa Zazueta Novoa; Hortencia Silva Jiménez; J. Carlos Torres-Guzmán; J. Félix Gutiérrez-Corona; Roberto Zazueta-Sandoval (pp. 243-255).

A soluble alcohol oxidase (AO) activity was detected in the mycelium of a filamentous fungus strain named YR-1, isolated from petroleum-contaminated soils. AO activity from aerobically grown mycelium was detected in growth media containing the hydrocarbons decane or hexadecane; the enzyme activity exhibited optimum pH for the oxidation of different alcohols (methanol, ethanol, and hexadecanol) similar to that of the corresponding aldehyde. Zymogram analysis conducted with purified fractions from aerobic mycelium of YR-1 strain extracts indicated the existence of two AO enzymes (AO-1 and AO-2). Purified samples of both enzymes analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis indicated the presence of three protein bands with molecular sizes 20, 38, and 46 kDa that could be part of the native enzyme. In samples of both enzymes, the 46-kDa protein gave a positive reaction in immunodetection experiments with antibodies directed against AO from Hansenula polymorpha. The purified AO-2 enzyme oxidized different alcohols, although higher activity was displayed with hexadecanol. K m values obtained for methanol and hexa-decanol indicated a higher affinity for the latter. Analysis of the aminoter-minal sequence of the 46-kDa protein of AO-2 enzyme indicated significant similarity to enzymes involved in the metabolism of biphenyl polychloride compounds.

Keywords: Alcohol oxidase; filamentous fungi; hydrocarbon biodegradation; petroleum contamination; Alcohol oxidase; filamentous fungi; hydrocarbon biodegradation; petroleum contamination

Rapid selection system of strains with higher avicel degrading ability in a cellulolytic fungus, Trichoderma by Hideo Toyama; Naotsugu Yamagishi; Nobuo Toyama (pp. 257-263).

We have attempted to develop an active selection system for strains that have a higher potential for Avicel degradation using haploidized conidia from colchicine-treated Trichoderma reesei Rut C-30 as a model strain. Avicel, absorbent cotton, and wood powder were used as substrates for selection. It appeared that the strains that degrade Avicel actively could be effectively selected when the solid medium containing the selection substrate and the liquid medium containing Avicel were used.

Keywords: Trichoderma ; cellulase; colchicine; Avicel; cellulose

Effect of pH on the stability of hexokinase and glucose 6-phosphate dehydrogenase by Maria Aparecida Souza; Marcela Zanella Ribeiro; Daniel Pereira Silva; Adalberto Pessoa Jr.; Michele Vitolo (pp. 265-272).

Hexokinase (HK) and glucose 6-phosphate dehydrogenase (G6PDH) are important enzymes used in biochemical studies and in analytical methods. The stability of the enzymes can be affected by several variables, pH being one of them. The effect of pH on the stability of HK and G6PDH was evaluated in this work. Baker’s yeast cells were suspended in 50 mM Tris-HCl buffer (pH 7.5) containing 5.0 mM MgCl2, and submitted to disruption by agitation with glass beads and in the presence of protease inhibitors. The cell-free extract was obtained by centrifugation (2880g; 10 min), followed by dilution into the buffers: 0.1 M acetate-acetic acid (pH: 4.0, 4.5, 5.0, or 5.5), 0.1 M phosphate buffer (pH: 6.0, 6.5, or 7.0), and 0.1 M Tris-HCl buffer (pH: 7.5, 8.0, 8.5, 9.0 or 9.5). The residual activity of HK and G6PDH, expressed as µmol of NADPH formed per min, were measured through a period of buffer-enzyme contact from 0 to 51 h at 4°C. It was observed that up to 4 h both enzymes were stable in all buffers used. However, after 51 h HK was stable at pH 6.0 and 7.5, whereas G6PDH was stable at pH 7.0, 9.5, and between 4.5 and 5.5.

Keywords: pH; hexokinase; glucose 6-phosphate dehydrogenase

Exploration of cellulose surface-binding properties of Acidothermus cellulolyticus Cel5A by site-specific mutagenesis by Suzanne L. McCarter; William S. Adney; Todd B. Vinzant; Edward Jennings; Fannie Posey Eddy; Stephen R. Decker; John O. Baker; Joshua Sakon; Michael E. Himmel (pp. 273-287).

Understanding the interactions between cellulases and cellulosic substrates is critical to the development of an efficient artificial cellulase system for conversion of biomass to sugars. We directed specific mutations to the interactive surface of the Acidothermus cellulolyticus EI endoglucanase catalytic domain. The cellulose-binding domain is not translated in these mutants. Amino acid mutations were designed either to change the surface charge of the protein or to modify the potential for hydrogen bonding with cellulose. The relationship between cellulase-to-cellulose (Avicel PH101) binding and hydrolysis activity was determined for various groupings of mutations. While a significant increase in hydrolysis activity was not observed, certain clusters of residues did significantly alter substrate binding and some interesting correlations emerged. In the future, these observations may be used to aid the design of endoglucanases with improved performance on pretreated biomass.

Keywords: Cellulase; endoglucanase; site-directed mutagenesis; Acidothermus cellulolyticus Cel5A

Use of steam explosion liquor from sugar cane bagasse for lignin peroxidase production by Phanerochaete chrysosporium by Maria Antonieta Ferrara; Elba P. S. Bon; Julio Silva Araujo Neto (pp. 289-300).

The possibility of using two by-products of the sugar cane industry, molasses and bagasse steam explosion liquor (SEL), for lignin peroxidase (LiP) production by Phanerochaete chrysosporium was investigated. For comparison, the fungus was initially cultivated in synthetic media containing either glucose, sucrose, xylose, or xylan as sole carbon sources. The effect of veratryl alcohol (VA) was also investigated in relation to the enzyme activity levels. Results showed that sucrose was not metabolized by this fungus, which precluded the use of molasses as a carbon source. Glucose, xylose, and xylan promoted equivalent cell growth. Enzyme levels in the absence of VA were lower than 28 UI/L and in the presence of VA reached 109 IU/L with glucose and 85 IU/L with xylose or xylan. SEL was adequate for P. chrysosporium LiP production as LiP activity reached 90 IU/L. When VA was added to this medium, enzyme concentration increased to 155 IU/L.

Keywords: Phanerochaete chrysosporium ; lignin peroxidase; steam explosion liquor; sugar cane bagasse

Isolation and characterization of thermophilic benzothiophene-degrading Mycobacterium sp. by Yuwadee Watanapokasin; Siriporn Nuchfoang; Sirinun Nilwarangkoon; Somsak Sarangbin; Toshihide Kakizono (pp. 301-309).

A bacterial strain, SWU-4, capable of using benzothiophene (BT) as a sole carbon and energy source was isolated from a petroleum-contaminated site in Thailand and identified by 16S rRNA gene sequence analysis to be in the genus of Mycobacterium. The strain was Gram-positive, nonspore former, and grew at 50° C. Colonies of the strain on nutrient agar were rod-shaped, smooth with a convex surface, slightly mucoid, and yellow pigmented. The thermophilic Mycobacterium sp. strain SWU-4 rapidly degraded 2% (w/v) BT at 50°C. Interestingly, this strain was able to degrade a wide variety of organosulfur compounds including thiophene, bromo(α)thiophene, and 3-methylthiophene in liquid minimum medium at 50°C, which will be beneficial for industrial applications.

Keywords: Thermophilic Mycobacterium sp.; organosulfur-degradation; 16S rDNA

A technique for mycelial development of ectomycorrhizal fungi on agar media by Álvaro Alberto De Araújo; Sevastianos Roussos (pp. 311-318).

A technique was established to study ectomycorrhizal fungi on agar media. Petri dishes, 60 mm in diameter, containing 10 mL of culture medium covered with a cellophane disk were used for easy collection of the mycelium after growth. For analysis of fungal biomass production, a sterilized cellophane sheet was placed on the medium’s surface. Inoculation was achieved by placing a mycelial block onto the center of the cellophane sheet and then incubating at 25°C in the dark. Colony radial growth was measured and biomass dry wt was determined. Fresh mycelia were homogenized with 10 mL of acetate buffer (pH 5.5) for enzyme analysis. A crude extract was obtained by adding all culture medium to 90 mL of distilled water and homogenizing in a Potter. Reducing sugars, enzyme concentration, and pH were determined. Three fungal strains, Suillus collinitus, Pisosithus arrhizus, and Hebeloma cylindrosporum, were grown in different culture media (potato dextrose agar or Pintro’s medium). Parameters measured over time included glucose concentration, phosphatase activity, biomass, and pH.

Keywords: Ectomycorrhizal fungi; physiology; apical growth; Pisolithus ; Suillus ; Hebeloma ; biomass; phosphatase; agar media

Screening of soil bacteria for production of biocleaner by Haruko Takeyama; Masumi Wada; Tadashi Matsunaga (pp. 319-326).

Soil bacteria were studied for the production of biodegradable cleaning agents. Among 86 bacterial strains resistant to liquid paraffin, 58 showed hemolytic activity. These strains were cultured, and the supernatant of culture broths was evaluated for cleaning activity against a dirty porcelain tile. Potent activity was exhibited in 18 strains. The lowest value of surface tension was obtained from Bacillus sp. NKB03 suggesting the presence of a biosurfactant. Aeromonas sp. NKB26c and Bacillus cereus NKB46b exhibited enzymatic cleaning activity. A cleaning efficiency of 82% was achieved when using a mixture of supernatants from culture broths of Bacillus sp. NKB03 and Aeromonas sp. NKB26c in synthetic minimal media. The cleaning efficiency using this mixture was higher than that of sodium dodecyl sulfate. These results suggest that a mixture of supernatants from culture broths of Bacillus sp. NKB03 and Aeromonas sp. NKB26c has potential for commercial use as a biocleaner.

Keywords: Biocleaner; NIHImage analysis; soil bacteria; biosurfactant; hemolytic activity

Reduction of furfural to furfuryl alcohol by ethanologenic strains of bacteria and its effect on ethanol production from xylose by Tony Gutiérrez; Marian L. Buszko; Lonnie O. Ingram; James F. Preston (pp. 327-340).

The ethanologenic bacteria Escherichia coli strains KO11 and LYO1, and Klebsiella oxytoca strain P2, were investigated for their ability to metabolize furfural. Using high performance liquid chromatography and ¹³C-nuclear magnetic resonance spectroscopy, furfural was found to be completely biotransformed into furfuryl alcohol by each of the three strains with tryptone and yeast extract as sole carbon sources. This reduction appears to be constitutive with NAD(P)H acting as electron donor. Glucose was shown to be an effective source of reducing power. Succinate inhibited furfural reduction, indicating that flavins are unlikely participants in this process. Furfural at concentrations >10 mM decreased the rate of ethanol formation but did not affect the final yield. Insight into the biochemical nature of this furfural reduction process may help efforts to mitigate furfural toxicity during ethanol production by ethanologenic bacteria.

Keywords: Furfural detoxification; furfurylalcohol; ethanol fermentation; Escherichia coli ; Klebsiella oxytoca

Characterization of heterologous and native enzyme activity profiles in metabolically engineered Zymomonas mobilis strains during batch fermentation of glucose and xylose mixtures by Qiang Gao; Min Zhang; James D. McMillan; Dhinakar S. Kompala (pp. 341-355).

Zymomonas mobilis has been metabolically engineered to broaden its substrate utilization range to include d-xylose and l-arabinose. Both genomically integrated and plasmid-bearing Z. mobilis strains that are capable of fermenting the pentose d-xylose have been created by incorporating four genes: two genes encoding xylose utilization metabolic enzymes (xylA/xylB) and two genes encoding pentose phosphate pathway enzymes (talB/tktA). We have characterized the activities of the four newly introduced enzymes for xylose metabolism, along with those of three native glycolytic enzymes, in two different xylose-fermenting Z. mobilis strains. These strains were grown on glucose-xylose mixtures in computer-controlled fermentors. Samples were collected and analyzed to determine extracellular metabolite concentrations as well as the activities of several intracellular enzymes in the xylose and glucose uptake and catabolism pathways. These measurements provide new insights on the possible bottlenecks in the engineered metabolic pathways and suggest methods for further improving the efficiency of xylose fermentation.

Keywords: Recombinant Zymomonas mobilis ; xylose fermentation; enzyme analysis; ethanol; byproducts

Action of white-rot fungus Panus tigrinus on sugarcane bagasse by Sirlene M. Costa; Adilson R. Gonçalves; Elisa Esposito (pp. 357-364).

Biological pretreatments with three selected strains of Panus tigrinus were used for delignification of sugarcane bagasse. The fungi with potential for delignification were analyzed by determining the chemical composition of the decayed bagasse samples, and the selectivity in terms of weight loss of the different components was evaluated. All the strains grow abundantly on bagasse as unique carbon source. After determining the chemical composition of degraded bagasse, P. tigrinus FTPT-4745 was selected as the most efficient strain on a 6-g scale, since the carbohydrates were preserved. P. tigrinus FTPT-4741 and FTPT-4742 were the most efficient strains on a large scale (100 g).

Keywords: Panus tigrinus ; selective biodegradation; sugarcane bagasse; white-rot fungus

Production of chelating agents through the enzymatic oxidation of acetosolv sugarcane bagasse lignin by Adilson R. Gonçalves; Mauro A. Soto-Oviedo (pp. 365-371).

Oxidation of lignin obtained from Acetosolv pulping of sugarcane bagasse was performed by polyphenoloxidase (PPO) using glycerol or polyethyleneglycol to increase the number of carbonyl and hydroxyl groups in lignin, and to improve its chelating capacity. Increase in the absorption in UV-spectrum related to α-carbonylphenolic and α,β-unsaturated structures was observed in all the experiments. The chelating properties of the original and oxidized lignins were compared by monitoring the amount of Cu²⁺ bound to lignin by gel permeation chromatography. The chelating capacity of original Acetosolv lignin was 354 mg Cu²⁺ /g lignin. On the other hand, lignin oxidized with PPO/O₂ showed an increase of 73% in chelating capacity in relation to the original lignin. The chelating capacity of lignin oxidized with PPO/O₂/glycerol was 110% higher than that of the original lignin. Glycerol stabilizes PPO, increasing its half-life. Average molecular weight $$left( {overline {MW} } ight)$$ , measured by size-exclusion chromatography, was smaller for the oxidized lignins than for the original Acetosolv lignin. This result suggests that quinones can eventually be formed through the action of PPO, but are not polymerized. The chelating capacity of oxidized lignins increases with the incorporation of vicinal hydroxyl groups.

Keywords: Oxidation of lignin; polyphenoloxidase; sugar cane bagasse; Acetosolv; chelating agents

Panus tigrinus strains used in delignification of sugarcane bagasse prior to kraft pulping by Adilson R. Gonçalves; Sirlene M. Costa; Elisa Esposito (pp. 373-382).

Three strains of the white-rot fungus Panus tigrinus (FTPT-4741, FTPT-4742, and FTPT-4745) were cultivated on sugarcane bagasse prior to kraft pulping. Pulp yields, kappa number, and viscosity of all pulps were determined and Fourier transform infrared (FTIR) spectra from the samples were recorded. The growth of P. tigrinus strains in plastic bags increased the manganese peroxide and xylanase activities. Lignin peroxidase was not detected in the three systems (shaken and nonshaken flasks and plastic bags). FTIR spectra were reduced to their principal components, and a clear separation between FTPT-4742 and the control was observed. Strain FTPT-4745 decayed lignin more selectively in the three systems utilized. Yields of kraft pulping were low, ranging from 20 to 45% for the plastic bag samples and from 12 to 38% for the flask samples. Kappa numbers were 1–18 and viscosity ranged from 2.3 to 6.8 cP.

Keywords: Panus tigrinus strains; sugarcane bagasse; kraft pulping; biological pretreatment

Effect of single active-site cleft mutation on product specificity in a thermostable bacterial cellulase by Tauna R. Rignall; John O. Baker; Suzanne L. McCarter; William S. Adney; Todd B. Vinzant; Stephen R. Decker; Michael E. Himmel (pp. 383-394).

Mutation of a single active-site cleft tyrosyl residue to a glycyl residue significantly changes the mixture of products released from phosphoric acidswollen cellulose (PSC) by EIcd, the catalytic domain of the endoglucanase-I from Acidothermus cellulolyticus. The percentage of glucose in the product stream is almost 40% greater for the Y245G mutant (and for an additional double mutant, Y245G/Q204A) than for the wild type enzyme. Comparisons of results for digestion PSC and of pretreated yellow poplar suggest that the observed shifts in product specificity are connected to the hydrolysis of a more easily digestible fraction of both substrates. A model is presented that relates the changes in product specificity to a mutation-driven shift in indexing of the polymeric substrate along the extended binding-site cleft.

Keywords: Acidothermus cellulolyticus ; endoglucanase; product specificity; saccharification; cellulose

Foam fractionation of a dilute solution of bovine lactoferrin by James Noel; Ales Prokop; Robert D. Tanner (pp. 395-402).

Lactoferrin (Lf), a protein found in human and bovine milk, tears, blood, and other secretory fluids, has been used to prevent infection from potential microbial pathogens by its ability to bind with iron (Fe³⁺). Currently, bovine lactoferrin can be purified from milk using ion exchange resin, which is a costly procedure making lactoferrin expensive. The purpose of this work was to investigate a low-cost foam fractionation process as the first step in separating lactoferrin from milk.

Keywords: Lactoferrin; foam fractionation; milk; surface tension

Xylose reductase and xylitol dehydrogenase activities of Candida guilliermondii as a function of different treatments of sugarcane bagasse hemicellulosic hydrolysate employing experimental design by Lourdes A. Alves; Michele Vitolo; Maria das Graças A. Felipe; João Batista de Almeida e Silva (pp. 403-413).

The sugarcane bagasse hydrolysate, which is rich in xylose, can be used as culture medium for Candida guilliermondii in xylitol production. However, the hydrolysate obtained from bagasse by acid hydrolysis at 120°C for 20 min has by-products (acetic acid and furfural, among others), which are toxic to the yeast over certain concentrations. So, the hydrolysate must be pretreated before using in fermentation. The pretreatment variables considered were: adsorption time (15,37.5, and 60 min), type of acid used (H2So4 and H3Po4), hydrolysate concentration (original, twofold, and fourfold. concentrated), and active charcoal (0.5, 1.75 and 3.0%). The suitability of the pretreatment was followed by measuring the xylose reductase (XR) and xylitol dehydrogenase (XD) activity of yeast grown in each treated hydrolysate. The response surface methodology (2⁴ full factorial design with a centered face) indicated that the hydrolysate might be concentrated fourfold and the pH adjusted to 7.0 with CaO, followed by reduction to 5.5 with H₃PO₄. After that it was treated with active charcoal (3.0%) by 60 min. This pretreated hydrolysate attained the high XR/XD ratio of 4.5.

Keywords: Xylose reductase; xylitol dehydrogenase; sugarcane bagasse hydrolysate; treatment; Candida guilliermondii

Purification and characterization of a microbial dehydrogenase by Ghislain Baré; Thierry Swiatkowski; Ahmed Moukil; Charles Gerday; Philippe Thonart (pp. 415-428).

Pseudomonas fluorescens (strain BTP9) was found to have at least two NAD(P)-dependent vanillin dehydrogenases: one is induced by vanillin, and the other is constitutive. The constitutive enzyme was purified by ammonium sulfate fractionation, gel-filtration, and Q-Sepharose chromatography. The subunit Mr value was 55,000, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The native M r value estimated by gelfiltration chromatography gave a value of 210,000. The enzyme made use of NAD⁺ less effectively than NADP⁺. Benzaldehyde, 4-hydroxybenzaldehyde, hexanal, and acetaldehyde were not oxidized at detectable rates in the presence of NAD⁺ or NADP⁺. The ultraviolet absorption spectrum indicated that there is no cofactor or prosthetic group bound. The vanillin oxidation reaction was essentially irreversible. The pH optimum was 9.5 and the pI of the enzyme was 4.9. Enzyme activity was not affected when assayed in the presence of salts, except FeCl₂. The enzyme was inhibited by the thiol-blocking reagents 4-chloromercuribenzoate and N-ethylmaleimide. NAD⁺ and NADP⁺ protected the enzyme against such a type of inhibition along with vanillin to a lesser extent. The enzyme exhibited esterase activity with 4-nitrophenyl acetate as substrate and was activated by low concentrations of NAD⁺ or NADP⁺. We compared the properties of the enzyme with those of some well-characterized microbial benzaldehyde dehydrogenases.

Keywords: Vanillin; enzyme purification; vanillin dehydrogenase; Pseudomonas fluorescens ; benzaldehyde dehydrogenase

Performance testing of Zymomonas mobilis metabolically engineered for cofermentation of glucose, xylose, and arabinose by Hugh G. Lawford; Joyce D. Rousseau (pp. 429-448).

IOGEN Corporation of Ottawa, Canada, has recently built a 40t/d biomass-to-ethanol demonstration plant adjacent to its enzyme production facility. It has partnered with the University of Toronto to test the C6/C5 cofermenta-tion performance characteristics of the National Renewable Energy Labora-tory’s metabolically engineered Zymomonas mobilis using various biomass hydrolysates. IOGEN’s feedstocks are primarily agricultural wastes such as corn stover and wheat straw. Integrated recombinant Z. mobilis strain AX101 grows on d-xylose and/or l-arabinose as the sole carbon/energy sources and ferments these pentose sugars to ethanol in high yield. Strain AX101 lacks the tetracycline resistance gene that was a common feature of other recombinant Zm constructs. Genomic integration provides reliable cofermentation performance in the absence of antibiotics, another characteristic making strain AX101 attractive for industrial cellulosic ethanol production. In this work, IOGEN’s biomass hydrolysate was simulated by a pure sugar medium containing 6% (w/v) glucose, 3% xylose, and 0.35% arabinose. At a level of 3 g/L (dry solids), corn steep liquor with inorganic nitrogen (0.8 g/L of ammonium chloride or 1.2 g/L of diammonium phosphate) was a cost-effective nutritional supplement. In the absence of acetic acid, the maximum volumetric ethanol productivity of a continuous fermentation at pH 5.0 was 3.54g/L·h. During prolonged continuous fermentation, the efficiency of sugar-to-ethanol conversion (based on total sugar load) was maintained at >85%. At a level of 0.25% (w/v) acetic acid, the productivity decreased to 1.17 g/L·h at pH 5.5. Unlike integrated, xylose-utilizing rec Zm strain C25, strain AX101 produces less lactic acid as byproduct, owing to the fact that the Escherichia coli arabinose genes are inserted into a region of the host chromosome tentatively assigned to the gene for d-lactic acid dehydrogenase. In pH-controlled batch fermentations with sugar mixtures, the order of sugar exhaustion from the medium was glucose followed by xylose and arabinose. Both the total sugar load and the sugar ratio were shown to be important determinants for efficient cofermentation. Ethanol at a level of 3% (w/v) was implicated as both inhibitory to pentose fermentation and as a potentiator of acetic acid inhibition of pentose fermentation at pH 5.5. The effect of ethanol may have been underestimated in other assessments of acetic acid sensitivity. This work underscores the importance of employing similar assay conditions in making comparative assessments of biocatalyst fermentation performance.

Keywords: Genomic integration; recombinant Zymomonas AX101; arabinose; xylose; ethanol; biomass hydrolysate; acetic acid; corn steep liquor

Influence of oxygen availability on cell growth and xylitol production by Candida guilliermondii by Luis F. Figueiredo Faria; Maria Antonieta P. Gimenes; Ronaldo Nobrega; Nei Pereira Jr. (pp. 449-458).

Oxygen availability is the most important environmental parameter in the production of xylitol by yeasts, directly affecting yields and volumetric productivity. This work evaluated the cell behavior in fermentations carried out with different dissolved oxygen concentrations (0.5–30.0% of saturation), as well as a limited oxygen restriction (0% of saturation), at several oxygen volumetric transfer coefficients (12 ≤ k L a ≤ 70 h⁻¹). These experiments allowed us to establish the specific oxygen uptake rate limits to ensure high yields and volumetric productivity. When oxygen availability was limited, the specific oxygen uptake rate values were between 12 and 26 mg of O₂/of g cell·h, resulting in a yield of 0.71 g of xylitol/xylose consumed, and 0.85 g/[L·h] for the volumetric productivity. According to the results, the effective control of the specific oxygen uptake rate makes it possible to establish complete control over this fermentative process, for both cell growth and xylitol production.

Keywords: Xylitol; xylose; aeration; Candida guilliermondii ; oxygen availability

Influence of mixing regime on enzymatic saccharification of steam-exploded softwood chips by Ursula Mais; Ali R. Esteghlalian; John N. Saddler (pp. 463-472).

In an attempt to elucidate the effect of reduced mixing on the enzymatic hydrolysis of lignocellulosic feedstocks, a pretreated softwood substrate was hydrolyzed under various mixing regimes using a commercial cellulase mixture. The substrate was generated by SO₂-catalyzed steam explosion of Douglas fir wood chips followed by alkali-peroxide treatment to remove lignin. Three mixing regimes were tested; continuous mixing at low (25 rpm) and high (150 rpm) speeds, and mixing at low-speed interspersed with 5-min intervals of high-speed agitation at 150 rpm. At both substrate concentrations (7.5 and 10% [w/w]), the mixed-speed mixing was able to produce sufficiently high conversion rates and yields (93% after 96 h), close or slightly better than those obtained under vigorous mixing (150 rpm). The low-speed shaking produced appreciably lower conversion yields at both levels of substrate concentration. Therefore, the mixed-speed regime may be a viable process option, because it does not seem to have an adverse impact on the cellulose conversion yield and can be an effective means of reducing the mixing energy requirements of an enzymatic hydrolysis process.

Keywords: Enzymatic hydrolysis; mixing; softwoods; Douglas fir; bioconversion

Hydrolysis of lactose by β-glycosidase CelB from hyperthermophilic archaeon Pyrococcus furiosus by Barbara Splechtna; Inge Petzelbauer; Bernhard Kuhn; Klaus D. Kulbe; Bernd Nidetzky (pp. 473-488).

Recombinant β-glycosidase CelB from the hyperthermophilic archaeon Pyrococcus furiosus was produced through expression of the plasmid-encoded gene in Escherichia coli. Bioreactor cultivations of E. coli in the presence of the inductor isopropyl-1-thio-β-d-galactoside (0.1 mM) gave approx 100,000 U of enzyme activity/L of culture medium after 8 h of growth. A technicalgrade enzyme for the hydrolysis of lactose was prepared by precipitating the mesophilic protein at 80°C. A hollow-fiber membrane reactor was developed, and its performance during continuous processing of ultrahigh temperature-treated (UHT) skim milk at 70°C was analyzed regarding long-term stability, productivity, and diffusional limitation thereof. CelB was covalently attached onto Eupergit C in yields of 80%, and a packed-bed immobilized enzyme reactor was used for the continuous hydrolysis of lactose in UHT skim milk at 70°C. The packed-bed reactor was ≈10-fold more stable and gave about the same productivity at 80% substrate conversion as the hollow-fiber reactor at 60% substrate conversion. The marked difference in the stability of free and immobilized CelB seems to reflect mainly binding of the soluble enzyme to the membrane surface of the hollow-fiber module. Under these bound conditions, CelB is essentially inactive. CelB is essentially inactive. Microbial contamination of the reactors did not occur during reaction times of up to 39 d, given that UHT skim milk and not pasteurized skim milk was used as the substrate.

Keywords: Reaction engineering; diffusional reactor; extremozyme; lactose process

Use of immobilized Candida yeast cells for xylitol production from sugarcane bagasse hydrolysate by Walter Carvalho; Silvio S. Silva; Attilio Converti; Michele Vitolo; Maria G. A. Felipe; Ines C. Roberto; Messias B. Silva; Ismael M. Mancilha (pp. 489-496).

Candida guilliermondii cells were immobilized in Ca-alginate beads and used for xylitol production from concentrated sugarcane bagasse hydrolysate. A full factorial design was employed to determine whether variations in the immobilization conditions would have any effects on the beads, chemical stability and on the xylitol production rates. Duplicate fermentation runs were carried out in 125-mL Erlenmeyer flasks maintained in a rotatory shaker at 30°C and 200 rpm for 72h. Samples were periodically analyzed to monitor xylose and acetic acid consumption, xylitol production, free cell growth, and bead solubilization. Concentrations of sodium alginate at 20.0 g/L and calcium chloride at 11.0 g/L and bead curing time of 24 h represented the most appropriate immobilization conditions within the range of conditions tested.

Keywords: Xylitol; sugarcane bagasse hydrolysate; immobilized cells; immobilization conditions; factorial design

Purification and characterization of a laccase from the white-rot fungus Trametes multicolor by Christian Leitner; Johann Hess; Christiane Galhaup; Roland Ludwig; Bernd Nidetzky; Klaus D. Kulbe; Dietmar Haltrich (pp. 497-507).

The wood-degrading fungus Trametes multicolor secretes several laccase isoforms when grown on a simple medium containing copper in the millimolar range for stimulating laccase synthesis. The main isoenzyme laccase II was purified to apparent homogeneity from the culture supernatant by using anion-exchange chromatography and gel filtration. Laccase II is a monomeric glycoprotein with a molecular mass of 63 kDa as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis, contains 18% glycosylation, and has a pI of 3.0. It oxidizes a variety of phenolic substrates as well as ferrocyanide and iodide. The pH optimum depends on the substrate employed and shows a bell-shaped pH activity profile with an optimum of 4.0 to 5.0 for the phenolic substrates, while the nonphenolic substrates ferrocyanide and 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate) show a monotonic pH profile with a rate decreasing with increasing pH.

Keywords: Trametes multicolor ; basidiomycete; laccase; polyphenol oxidase; lignin degradation

Use of measurement uncertainty analysis to assess accuracy of carbon mass balance closure for a cellulase production process by Daniel J. Schell; Juan Carlos Sáez; Jenny Hamilton; Arun Tholudur; James D. McMillan (pp. 509-523).

Closing carbon mass balances is a critical and necessary step for verifying the performance of any conversion process. We developed a methodology for calculating carbon mass balance closures for a cellulase production process and then applied measurement uncertainty analysis to calculate 95% confidence limits to assess the accuracy of the results. Cellulase production experiments were conducted in 7-L fermentors using Trichoderma reesei grown on pure cellulose (Solka-floc), glucose, or lactose. All input and output carbon-containing streams were measured and carbon dioxide in the exhaust gas was quantified using a mass spectrometer. On Solka-floc, carbon mass balances ranged from 90 to 100% closure for the first 48 h but increased to 101 to 135% closure from 72 h to the end of the cultivation at 168 h. Carbon mass balance closures for soluble sugar substrates ranged from 92 to 127% over the entire course of the cultivations. The 95% confidence intervals (CIs) for carbon mass balance closure were typically ±11 to 12 percentage points after 48 h of cultivation. Many of the carbon mass balance results did not bracket 100% closure within the 95% CIs. These results suggest that measurement problems with the experimental or analytical methods may exist. This work shows that uncertainty analysis can be a useful diagnostic tool for identifying measurement problems in complex biochemical systems.

Keywords: Cellulase; carbon mass balance; cellulose; uncertainty analysis; Trichoderma reesei

The effect of bioindicator preparation and storage on thermal resistance of Bacillus stearothermophilus spores by Thereza Christina Vessoni Penna; Marina Ishii; Irene Alexeevna Machoshvili; Marcelo Marques (pp. 525-538).

Paper strips inoculated with spores of Bacillus stearothermophilus ATCC 7953 were conventionally dried (lot 1) and lyophilized (lot 2); stored in defined environments of 32 and 86% relative humidity at 10, 25 and 33°C for 210 d; and submitted to moist heat treatments at 121°C. A significant decrease in thermal resistance from initial starting levels was found for lyophilized bioindicators stored at 86% relative humidity. The respective average D _121°C values were 1.55 ± 0.05 and 1.37 ± 0.10 min for lyophilized bioindicators stored at 32 and 86% relative humidity; and 1.65±0.15min and 1.57 ± 0.11 min for dried bioindicators stored in the same environments.

Keywords: Bacillus stearothermophilus ; decimal reduction time; biological indicators; thermal resistance; sterilization

The effect of composition of parenteral solution on the thermal resistance of Bacillus stearothermophilus and Bacillus subtilis spores by Thereza Christina Vessoni Penna; Marcelo Marques; Irena A. Machoshvili; Marina Ishii (pp. 539-551).

Large-volume parenteral solutions were submitted to heat treatments after being inoculated with Bacillus stearothermophilus ATCC 7953 (T r=121°C) and Bacillus subtilis ATCC 9372 (T r=104.5°C) spores. The average decimal reduction time for B. stearothermophilus ranged from a D _121°C value of 1.31 to 3.14 min, in glucophysiologic and Ringer’s solutions respectively. For B. subtilis, D _104.5°C value increased from 0.69 to 1.37 min, in Ringer’s (pH=5.91) and 50% glucose (pH 3.05) solutions respectively. The z value ranged from 7.95°C (20% mannitol solution) to 13.14°C (50% glucose solution), corresponding to an activation energy (Ea) of 81.48 and 49.30 kcal/mol, respectively.

Keywords: Parenteral solutions; moist heat sterilization; Bacillus stearothermophilus ; Bacillus subtilis ; decimal reduction time

Production of acetone butanol ethanol from degermed corn using Clostridium beijerinckii BA101 by Edhilvia J. Campos; Nasib Qureshi; Hans P. Blaschek (pp. 553-561).

In this article we report on acetone butanol ethanol (ABE) fermentation characteristics of degermed corn when using Clostridium beijerinckii BA101. Recent economic studies suggested that recovery of germ from corn and hence corn oil would help to make the ABE fermentation process more economical. C. beijerinckii BA101 ferments corn mash efficiently to produce ABE under appropriate nutritional and environmental conditions. Corn mash contains germ/corn oil that is, possibly, ancillary to the production of butanol during the ABE fermentation process. Since the presence of corn oil is not a critical factor in solvent fermentation, it can be removed and this will allow for byproduct credit. Batch fermentation of degermed corn resulted in 8.93 g/L of total ABE production as compared with 24.80 g/L of total ABE when supplemented with P2 medium nutrients. During the course of the germ separation process, corn steeping is required prior to grinding and removing the germ. It is likely that some nutrients from the corn are leached out during the steeping process. This may reduce the rate of fermentation and impact the final concentration of butanol/ABE that can be achieved. Fermentation of degermed corn with corn steep liquor resulted in the production of 19.28 g/L of ABE.

Keywords: Degermed corn; butanol; Clostridium beijerinckii BA101; corn steep liquor; acetone butanol ethanol; fermentation

Treatment with lignin residue by Linda Björklund; Simona Larsson; Leif J. Jönsson; Anders Reimann; Nils-Olof Nivebrant (pp. 563-575).

Acid hydrolysis of lignocellulose to hydrolysates intended for production of fuel ethanol results in the formation of byproducts in addition to fermentable sugars. Some of the byproducts, such as phenolic compounds and furan aldehydes, are inhibitory to the fermenting microorganism. Detoxification of the hydrolysates may be necessary for production of ethanol at a satisfactory rate and yield. The lignin residue obtained after hydrolysis is a material with hydrophobic properties that is produced in large amounts as a byproduct within an ethanol production process based on lignocellulosic raw materials. We have explored the possibility of using this lignin residue for detoxification of spruce dilute-acid hydrolysates prior to fermentation with Saccharomyces cerevisiae. Three dilute-acid hydrolysates of spruce were treated with lignin residue, which in all cases resulted in improved fermentability in terms of productivity and yield of ethanol. The effect was improved by washing the lignin before treatment, by using larger amounts of lignin in the treatment, and by performing the treatment at low temperature. Treatment with the lignin residue removed up to 53% of the phenolic compounds and up to 68% of the furan aldehydes in a spruce dilute-acid hydrolysate. A larger fraction of furfural was removed compared to the less hydrophobic 5-hydroxymethylfurfural.

Keywords: Ethanol; lignocellulose; inhibitors; detoxification; lignin; Saccharomyces cerevisiae

Utilization of xylitol dehydrogenase in a combined microbial/enzymatic process for production of xylitol from d-glucose by Gerhard Mayer; Klaus D. Kulbe; Bernd Nidetzky (pp. 577-589).

The production of xylitol from d-glucose occurs through a three-step process in which d-arabitol and d-xylulose are formed as the first and second intermediate product, respectively, and both are obtained via microbial bioconversion reactions. Catalytic hydrogenation of d-xylulose yields xylitol; however, it is contaminated with d-arabitol. The aim of this study was to increase the stereoselectivity of the d-xylulose reduction step by using enzymatic catalysis. Recombinant xylitol dehydrogenase from the yeast Galactocandida mastotermitis was employed to catalyze xylitol formation from d-xylulose in an NADH-dependent reaction, and coenzyme regeneration was achieved by means of formate dehydrogenase-catalyzed oxidation of formate into carbon dioxide. The xylitol yield from d-xylulose was close to 100%. Optimal productivity was found for initial coenzyme concentrations of between 0.5 and 0.75 mM. In the presence of 0.30 M (45 g/L) d-xylulose and 2000 U/L of both dehydrogenases, exhaustive substrate turnover was achieved typically in a 4-h reaction time. The enzymes were recovered after the reaction in yields of approx 90% by means of ultrafiltration and could be reused for up to six cycles of d-xylulose reduction. The advantages of incorporating the enzyme-catalyzed step in a process for producing xylitol from d-glucose are discussed, and strategies for downstream processing are proposed by which the observed coenzyme turnover number of approx 600 could be increased significantly.

Keywords: Xylitol; coenzyme regeneration; optimization; biocatalysis

Butanol production by Clostridium beijerinckii BA101 in an immobilized cell biofilm reactor by Jason Lienhardt; Justin Schripsema; Nasib Qureshi; Hans P. Blaschek (pp. 591-598).

Acetone butanol ethanol was produced in a continuous immobilized cell (biofilm) plug-flow reactor inoculated with Clostridium beijerinckii BA101. To achieve high reactor productivity, C. beijerinckii BA101 cells were immobilized by adsorption onto clay brick. The continuous plug-flow reactor offers high productivities owing to reduced butanol inhibition and increased cell concentration. Although high productivity was achieved, it was at the expense of low sugar utilization (30.3%). To increase sugar utilization, the reactor effluent was recycled. However, this approach is complicated by butanol toxicity. The effluent was recycled after removal of butanol by pervaporation to reduce butanol toxicity in the reactor. Recycling of butanolfree effluent resulted in a sugar utilization of 100.7% in addition to high productivity of 10.2g/(L·h) at a dilution rate of 1.5 h⁻¹. A dilution rate of 2.0h⁻¹ resulted in a reactor productivity of 16.2g/(L·h) and sugar utilization of 101.4%. It is anticipated that this reactor-recovery system would be economical for butanol production when using C. beijerinckii BA101.

Keywords: Clostridium beijerinckii BA101; butanol; immobilized cell biofilm reactor; pervaporative recovery; selectivity; flux

Pressure pulsation in solid-phase fermentation by Xiaomin Yang; Tom Huang; George T. Tsao (pp. 599-610).

Inadequate dissipation of heat generated by biologic activities has prevented the use of solid-phase fermentation in large-scale applications. This study deals with the cooling effects of pressure pulsation on solid, porous beds. Pressure pulsation also enhances evaporation of medium moisture, which is also described. Computer software has been developed for on-line control of heat accumulation and moisture loss involving automatic variation in pressure pulsation frequency and airflow direction as well as replenishment of water. Aspergillus niger NRRL3 was cultivated on a moist, solid medium made of wheat bran and ground corncobs to produce cellobiase. During 100h of fermentation, the maximum temperature inside the solid bed was kept under 40°C, and the medium water content was successfully maintained between 61 and 65%, which was optimal for cell growth. Cells grew heavily on the solid-phase substrate and distributed uniformly. With good on-line control of temperature and moisture, the 12-L fermentor provided a better environment for enzyme production than 250 mL flasks did.

Keywords: Pressure pulsation; solid-phase fermentation; Aspergillus niger ; cellobiase

Bioreactor design studies for a hydrogen-producing bacterium by Edward J. Wolfrum; Andrew S. Watt (pp. 611-625).

Carbon monoxide (CO) can be metabolized by a number of microorganisms along with water to produce hydrogen (H₂) and carbon dioxide. National Renewable Energy Laboratory researchers have isolated a number of bacteria that perform this so-called water-gas shift reaction at ambient temperatures. We performed experiments to measure the rate of CO conversion and H₂ production in a trickle-bed reactor (TBR). The liquid recirculation rate and the reactor support material both affected the mass transfer coefficient, which controls the overall performance of the reactor. A simple reactor model taken from the literature was used to quantitatively compare the performance of the TBR geometry at two different size scales. Good agreement between the two reactor scales was obtained.

Keywords: Synthesis gas; water-gas shift; mass transfer; Rubrivivax gelatinosus ; hydrogen; carbon monoxide

Development of novel microscale system as immobilized enzyme bioreactor by Francis Jones; Zonghuan Lu; Bill B. Elmore (pp. 627-640).

This study involves a novel method for immobilized enzyme catalysis. The focus of the work was to design and construct a microscale bioreactor using microfabrication techniques traditionally employed within the semiconductor industry. Enzymes have been immobilized on the microreactor walls by incorporating them directly into the wall material. Fabricated microchannels have cross-sectional dimensions on the order of hundreds of micrometers, constructed using polydimethylsiloxane cast on silicon/SU-8 molds. The resulting ratio of high surface area to volume creates an efficient, continuous-flow reaction system. Transverse features also containing enzymes were molded directly into the channels.

Keywords: Immobilized enzymes; microscale bioreactor; polydimethylsiloxane microreactor

Cellulase adsorption and an evaluation of enzyme recycle during hydrolysis of steam-exploded softwood residues by Yanpin Lu; Bin Yang; David Gregg; John N. Saddler; Shawn D. Mansfield (pp. 641-654).

The sugar yield and enzyme adsorption profile obtained during the hydrolysis of SO₂-catalyzed steam-exploded Douglas-fir and posttreated steamexploded Douglas-fir substrates were determined. After hot alkali peroxide posttreatment, the rates and yield of hydrolysis attained from the posttreated Douglas-fir were significantly higher, even at lower enzyme loadings, than those obtained with the corresponding steam-exploded Douglas-fir. The enzymatic adsorption profiles observed during hydrolysis of the two substrates were significantly different. Ultrafiltration was employed to recover enzyme in solution (supernatant) and reused in subsequent hydrolysis reactions with added, fresh substrate. These recycle findings suggested that the enzyme remained relatively active for three rounds of recycle. It is likely that enzyme recovery and reuse during the hydrolysis of posttreated softwood substrates could lead to reductions in the need for the addition of fresh enzyme during softwood-based bioconversion processes.

Keywords: Cellulases; Douglas-fir; ultrafiltration; adsorption; enzyme recycle; hydrolysis; bioconversion

Production of specific copolymers of polyhydroxyalkanoates from industrial waste by Phoeby A. L. Wong; Hong Chua; Waihung Lo; Hugh G. Lawford; Peter H. Yu (pp. 655-662).

Polyhydroxyalkanoates, biodegradable plastics with the desired physical and chemical properties of conventional synthetic plastics, are extensively investigated. In this study, specific bacterial strains produced specific copolymers from food waste. Copolymers of HB and HV (poly[3-hydroxybutyrate-co-3-hydroxyvalerate]) were obtained using various ratios of butyric acid (C₄) and valeric acid (C₅) as carbon sources. The C₄ to C₅ ratio affected the melting points of the copolymers. Melting and glass transition temperatures and many other thermal properties are important parameters relative to in-service polymer applications. Higher ratios of butyrate to valerate gave higher melting points. When a mixed culture of activated sludge was employed to produce copolymers using food wastes as nutrients, the obtained copolymers showed various monomer compositions. Copolymers with a higher portion of HV were obtained using soy waste; copolymers with less HV were obtained using malt wastes. Pure strains, (i.e., Alcaligenes latus DSM 1122, and DSM 1124, Staphylococcus spp., Klebsiella spp.) produced specific copolymers from food waste. Only Klebsiella spp. produced different copolymers; the ratios of HB:HV were 93:7 and 79:21 from malt waste and soy waste, respectively. The other strains produced polymers of 100% HB. Selecting industrial food wastes as carbon sources can further reduce the cost of producing copolymers.

Growth of lignocellulosic-fermenting fungi on different substrates under low oxygenation conditions by Erika C. Pavarina; Lucia R. Durrant (pp. 663-677).

Four soil fungi able to grow under low oxygenation conditions were selected and used in studies to determine the production of enzymes that promote the degradation of lignocellulosic materials. The capacity of these fungi to ferment such materials was also investigated. The fungi were grown in sugarcane bagasse and sawdust at final concentrations of 4 and 10%, as the carbon sources. The strains were cultivated under microaerophilic and combined conditions of oxygenation (aerobic followed by microaerophilic conditions). The results obtained with the basidiomycete specie, Trichocladium canadense, Geotrichum sp., and Fusarium sp. suggest that they prefer lower oxygen concentration for growth and enzyme production. Lignocellulolytic activities were detected in all strains but varied with the carbon source used. The highest levels of these activities were produced by the Basidiomycete specie and Fusarium sp. Ethanol and other nongaseous fermentation products were detected following high-performance liquid chromatography analysis using a supelcogel C-610H column, demonstrating the fermentative capability of these strains. In view of their ability to produce enzymes necessary for the breakdown of lignocellulosic materials and to utilize most of the degradation products for growth, these strains have a great potential for biotechnological application.

Keywords: Biodegradation; lignocellulosic fermentation fungi; microaerophilic

Inoculum studies in production of penicillin g acylase by Bacillus megaterium ATCC 14945 by Laura M. Pinotti; Rosineide G. Silva; Roberto C. Giordano; Raquel L. C. Giordano (pp. 679-686).

This article reports studies concerning the production of penicillin G acylase (PGA) by Bacillus megaterium. This enzyme has industrial use in the hydrolysis of penicillin G to obtain 6-aminopenicillanic acid, an essential intermediate for the production of semisynthetic β-lactam antibiotics. Although most microorganisms produce the enzyme intracellularly, B. megaterium provides extracellular PGA. The enzyme production by microorganisms involves several steps, resulting in a many operational variables to be studied. The study of the inoculum is an important step to be accomplished, before addressing other issues such as culture optimization and downstream processing. In this study, using a standard inoculum as reference, several runs were performed aiming at the definition of operational conditions in the PGA production. Cell concentration and PGA activity in the production medium were measured after 24, 48, and 72 h of the beginning of the production phase. This study encompasses the duration of the inoculum germination phase and the concentration of cells used to startup the germination. Based on these results, PGA productivity during the production phase was maximized. The selected values for these variables were 1.5 × 10⁷ spores/mL of germination medium, germination during 24 h, and 72 h for the production phase.

Keywords: Penicillin G acylase; Bacillus megaterium ; inoculum preparation

Production of 1,3-propanediol by Klebsiella pneumoniae by He Huang; Cheng S. Gong; George T. Tsao (pp. 687-698).

1,3-Propanediol (1,3-PD) has numerous applications from polymers to cosmetics, foods, lubricants, and medicines. Recently, there are strong industrial interests in a new kind of polyester, polytrimethylene terephthalate, with 1,3-PD as a monomer. This new polyester shows significant promise for use in carpeting and textiles. In this article we introduce a mild aerobic fermentation process using a strain screened from Klebsiella pneumoniae ATCC 25955, which is insensitive to oxygen, to produce 1,3-PD. We also describe a two-step fermentation process starting with glucose that was converted into glycerol with a glycerol-producing yeast, followed by K. pneumoniae that converts glycerol into 1,3-PD without intermediate isolation and purification of glycerol.

Keywords: 1,3-Propanediol; glycerol; Klebsiella pneumoniae ; yeast; fermentation

Comparison of the fermentability of enzymatic hydrolyzates of sugarcane bagasse pretreated by steam explosion using different impregnating agents by Carlos Martín; Mats Galbe; Nils-Olof Nilvebrant; Leif J. Jönsson (pp. 699-716).

Sugarcane bagasse is a potential lignocellulosic feedstock for ethanol production, since it is cheap, readily available, and has a high carbohydrate content. In this work, bagasse was subjected to steam explosion pretreatment with different impregnation conditions. Three parallel pretreatments were carried out, one without any impregnation, a second with sulfur dioxide, and a third with sulfuric acid as the impregnating agent. The pretreatments were performed at 205°C for 10 min. The pretreated material was then hydrolyzed using celluloytic enzymes. The chemical composition of the hydrolyzates was analyzed. The highest yields of xylose (16.2 g/100 g dry bagasse), arabinose (1.5 g/100 g), and total sugar (52.9 g/100 g) were obtained in the hydrolysis of the SO₂-impregnated bagasse. The H₂SO₄-impregnated bagasse gave the highest glucose yield (35.9 g/100 g) but the lowest total sugar yield (42.3 g/100 g) among the three methods. The low total sugar yield from the H₂SO₄-impregnated bagasse was largely due to by-product formation, as the dehydration of xylose to furfural. Sulfuric acid impregnation led to a three-fold increase in the concentration of the fermentation inhibitors furfural and 5-hydroxymethylfurfural (HMF) and a two-fold increase in the concentration of inhibitory aliphatic acids (formic, acetic, and levulinic acids) compared to the other two pretreatment methods. The total content of phenolic compounds was not strongly affected by the different pretreatment methods, but the quantities of separate phenolic compounds were widely different in the hydrolyzate from the H₂SO₄-impregnated bagasse compared with the other two hydrolyzates. No major differences in the content of inhibitors were observed in the hydrolyzates obtained from SO₂-impregnated and non-impregnated bagasse. The fermentability of all three hydrolyzates was tested with a xylose-utilizing Saccharomyces cerevisiae strain with and without nutrient supplementation. The hydrolyzates of SO₂-impregnated and nonimpregnated bagasse showed similar fermentability, whereas the hydrolyzate of H₂SO₄-impregnated bagasse fermented considerably poorer.

Keywords: Sugarcane bagasse; ethanol; pretreatment; S. cerevisiae

Ethanol production from olive oil extraction residue pretreated with hot water by Ignacio Ballesteros; José Miguel Oliva; Maria José Negro; Paloma Manzanares; Mercedes Ballesteros (pp. 717-732).

The olive pulp fraction contained in the residue generated in olive oil extraction by a two-step centrifugation process can be upgraded by using the cellulose fraction to produce ethanol and recovering high value phenols (tyrosol and hydroxytyrosol). Olive pulp was pretreated in a laboratory scale stirred autoclave at different temperatures (150–250°C). Pretreatment was evaluated regarding cellulose recovery, enzymatic hydrolysis effectiveness ethanol production by a simultaneous saccharification and fermentation process (SSF), and phenols recovery in the filtrate. The pretreatment of olive pulp using water at temperatures between 200°C and 250°C enhanced enzymatic hydrolysis. Maximum ethanol production (11.9 g/L) was obtained after pretreating pulp at 210°C in a SSF fed-batch procedure. Maximum hydroxytyrosol recovery was obtained in the liquid fraction when pretreated at 230°C.

Keywords: Olive oil extraction by-products; pretreatment; simultaneous saccharification and fermentation; ethanol; hydroxytyrosol

Modeling simultaneous saccharification and fermentation of softwood by Pär O. Pettersson; Robert Eklund; Guido Zacchi (pp. 733-746).

Simultaneous saccharification and fermentation (SSF) of wood has been modeled for the past 15–20 years, but the substrates used for model evaluation have so far not included pretreated softwood. In the present study, data from lab-scale batch SSF of SO₂-impregnated, steam-pretreated spruce chips were used to evaluate a model found in the literature. The model, which was somewhat modified, consists of a number of nonlinear, coupled ordinary differential equations, which were solved numerically. Some parameter values were fitted to data by use of least-squares minimization. A difficulty in parameter estimation was the lack of cellobiose measurements, something that was relieved by adding assumptions about parameter relations. The simulated concentration profiles agreed well with the measured concentrations of glucose and ethanol. It is therefore concluded that the basic model features apply to softwood SSF. The model predicts rate saturation with respect to enzyme concentration at concentrations above 60 FPU/g cellulose, although this was not observed in the experimental data, which only comprised enzyme concentrations up to 32 FPU/g cellulose.

Keywords: Simultaneous saccharification and fermentation; softwood; kinetic model

Production of coconut aroma by fungi cultivation in solid-state fermentation by Álvaro Alberto de Aráujo; Gláucia M. Pastore; Ralf G. Berger (pp. 747-751).

The production of 6-pentyl-α-pyrone (6-PP), an unsaturated d-lactone with a strong coconut-like aroma was studied and compared with liquid and solid substrates. A fungi strain that produces coconut aroma compound was selected. The liquid medium of the submerged culture was used to impregnate a solid support of sugarcane bagasse in SSF (Solid State Fermentation). This substrate was adequate for growth and aroma production; the concentration obtained using SSF was higher than using liquid fermentation process. In the present work, it is demonstrated that, by solid-state-fermentation process, it is possible to produce 6-PP. The amount of 6-PP produced using a solid state substrate, following a 5 d culture, was 3 mg/g dry matter. Therefore, the amount of 6-PP produced during solid-state-fermentation process is higher than that reported in literature for submerged process.

Keywords: Aroma production; coconutaroma; Trichoderma ; solid-state fermentation; 6-PP; substrates

Mass production of methane from food wastes with concomitant wastewater treatment by Jung K. Kim; Jae H. Cho; Jung S. Lee; Kyung S. Hahm; Don H. Park; Si W. Kim (pp. 753-764).

We developed a process for production of methane at a pilot scale. This process consists of three stages. The first stage is a semianaerobic hydrolysis/acidogenic step in which organic wastes are converted to various sugars, amino acids, and volatile fatty acids (VFAs). Operation temperature and pH were 45°C, and 5.0–5.5, respectively. Hydraulic retention time (HRT) was 2 d. To remove the putrid odor and to enhance the hydrolysis of organic wastes, a mixture of bacteria isolated from landfill soil was inoculated into the reactor. Total chemical oxygen demand (tCOD) and biological oxygen demand (BOD) were 36,000 mg/L and 40,000 mg/L, respectively. The second stage was an anaerobic acidogenic process, which can produce large amount of VFAs including acetate, propionate, butyrate, valerate, and caproate. Operation temperature and pH were 35°C, and 5.0–5.5, respectively. HRT was 2 d. The third stage was a strictly anaerobic methane fermentation step producing methane and carbon dioxide from VFAs. The working volume of upflow anaerobic sludge blanket (UASB) type reactor was 1200 L, and operation temperature and pH were 41°C, and 7.7–7.9, respectively. HRT was 12 d. Seventy two percent of methane at maximum was generated and the yield was 0.45–0.50 m³/kg VS of food wastes. Through the process, 88% of tCOD and 95% of BOD were removed. The wastewater was treated with the biological aerobic and anaerobic filters immobilized with heterotrophic and autotrophic nitrifying and denitrifying bacteria. Ninety percent of total nitrogen (T-N) was removed by this treatment. The residual T-N and total phosphorous (T-P) were removed by the algal periphyton treatment system. The final concentrations of nitrogen and phosphorous in the drain water were 53 and 7 mg/L, respectively.

Keywords: Anaerobic digestion; volatile fatty acids; methane; biological filter; algal periphyton treatment system

Thermophilic fermentation of hydrolysates by Christiane Thomasser; Herbert Danner; Markus Neureiter; Bamusi Saidi; Rudolf Braun (pp. 765-773).

Lignocellulosic biomass has great potential as a cheap feedstock in biological processes to produce biofuels or chemicals; however, dilute acid pretreatment at high temperatures produces undesirable compounds. Toxicity tests were done with inhibitors in standard media, to predict the growth-limiting effects on thermophilic strains. The 22 inhibitors included furfural, levulinic acid, acetic acid, and cinnamaldehyde. Neutralizing reagents and additional treatment steps have been tested.

Keywords: Softwood hydrolysates; inhibitors; thermophiles

Optimization of nisin production by Lactococcus lactis by Thereza Christina Vessoni Penna; Dante Augusto Moraes (pp. 775-789).

The production of nisin by batch culture of Lactococcus lactis ATCC 11454 in MRS broth (pH 6.5), as treated in 30 assays, that were set up by a fractional factorial design of two levels (2^[4–1]), was improved. The minimum and maximum concentrations of sucrose (5.0–12.5 g/L), asparagine (7.5–75 g/L), potassium phosphate (6.0–18.0 g/L), and Tween-80 (1.0–6.6 g/L) were added to MRS broth. The best nisin activities ranged from 1.5×10⁴ to 1.8×10⁴ arbitrary units (AU)/mL for the maximum levels of sucrose, asparagine, and monobasic potassium phosphate, and for the minimum concentration of Tween-80. The best following proportions between nutrients were adopted as optimum for maximum specific nisin productivity of about 6.0 mg/mg of dry cell weight (related to 2.5 mg of pure nisin preparation with a specific activity of 1.0×10⁵ AU/mL): C/N=0.17, C/P=0.69, N/P=4.17 (C=sucrose, N=asparagine, P=phosphate, T=Tween-80).

Keywords: Nisin; nutrient balance; Lactococcus lactis ; Lactobacillus sake ; batch fermentation

Intracellular release of recombinant green fluorescent protein (gfp uv) from Escherichia coli by Thereza Christina Vessoni Penna; Eb Chiarini; Irene Alexeevna Machoshvili; Marina Ishii; Adalberto Pessoa Jr. (pp. 791-802).

The recombinant green fluorescent protein (gfp uv) was expressed by Escherichia coli DH5-α cells transformed with the plasmid pGFPuv. The gfp uv was selectively permeabilized from the cells in buffer solution (25 mM Tris-HCl, pH 8.0), after freezing (−70°C for 15 h), by four freeze (−20°C)/thaw cycles interlaid by sonication. The average content of released gfp uv (experiment 2) was 7.76, 34.58, 39.38, 12.90, and 5.38%, for the initial freezing (−70°C) and the first, second, third and fourth freeze/thaw cycles, respectively. Superfusion on freezing was observed between −11°C and −14°C, after which it reached −20°C at 0.83°C/min.

Keywords: gfp uv ; Escherichia coli ; physical permeabilization; superfusion phenomenon; freeze/thaw/sonication procedures

Development of continuous surfactin production from potato process effluent by Bacillus subtilis in an airlift reactor by Karl S. Noah; Sandra L. Fox; Debby F. Bruhn; David N. Thompson; Gregory A. Bala (pp. 803-813).

The biosurfactant surfactin has the potential to aid in the recovery of subsurface organic contaminants (environmental remediation) or crude oils (oil recovery). However, high medium and purification costs limit its use in these high-volume applications. In previous work, we showed that surfactin can be produced from an inexpensive low-solids (LS) potato process effluent with minimal amendments or pretreatments. Previous research has also shown that 95% or more of the surfactin in Bacillus subtilis cultures can be recovered by foam fractionation. In this work, we present the results of research to integrate surfactin production with foam fractionation. Experiments were performed in an airlift reactor, with continuous collection of the foam through a tube at the top of the column. Preliminary results using both purified potato starch and unamended low-solids potato process effluent as substrates for surfactin production indicate that the process is oxygen limited and that recalcitrant indigenous bacteria in the potato process effluent may hamper continuous surfactin production.

Keywords: Bacillus subtilis ; biosurfactant; surfactin; alternate feedstock; enhanced oil recovery

Enhancing the enzymatic hydrolysis of cellulosic materials using simultaneous ball milling by Ursula Mais; Ali R. Esteghlalian; John N. Saddler; Shawn D. Mansfield (pp. 815-832).

One of the limiting factors restricting the effective and efficient bioconversion of softwood-derived lignocellulosic residues is the recalcitrance of the substrate following pretreatment. Consequently, the ensuing enzymatic process requires relatively high enzyme loadings to produce monomeric carbohydrates that are readily fermentable by ethanologenic microorganisms. In an attempt to circumvent the need for larger enzyme loadings, a simultaneous physical and enzymatic hydrolysis treatment was evaluated. A ball-mill reactor was used as the digestion vessel, and the extent and rate of hydrolysis were monitored. Concurrently, enzyme adsorption profiles and the rate of conversion during the course of hydrolysis were monitored. α-Cellulose, employed as a model substrate, and SO₂-impregnated steam-exploded Douglas-fir wood chips were assessed as the cellulosic substrates. The softwood-derived substrate was further posttreated with water and hot alkaline hydrogen peroxide to remove >90% of the original lignin. Experiments at different reaction conditions were evaluated, including substrate concentration, enzyme loading, reaction volumes, and number of ball beads employed during mechanical milling. It was apparent that the best conditions for the enzymatic hydrolysis of α-cellulose were attained using a higher number of beads, while the presence of air-liquid interface did not seem to affect the rate of saccharification. Similarly, when employing the lignocellulosic substrate, up to 100% hydrolysis could be achieved with a minimum enzyme loading (10 filter paper units/g of cellulose), at lower substrate concentrations and with a greater number of reaction beads during milling. It was apparent that the combined strategy of simultaneous ball milling and enzymatic hydrolysis could improve the rate of saccharification and/or reduce the enzyme loading required to attain total hydrolysis of the carbohydrate moieties.

Keywords: Cellulose hydrolysis; cellulase; enzyme adsorption; ball mill reactor; softwood; enzymatic hydrolysis; steam explosion; bioconversion

Cadmium recovery by a sulfate-reducing magnetotactic bacterium, Desulfovibrio magneticus RS-1, using magnetic separation by Atsushi Arakaki; Haruko Takeyama; Tsuyoshi Tanaka; Tadashi Matsunaga (pp. 833-840).

Cadmium recovery by a sulfate-reducing magnetotactic bacterium, Desulfovibrio magneticus strain RS-1, was investigated. D. magneticus precipitated >95% of cadmium at an initial concentration of 1.3 ppm in the growth medium. Electron microscopic analysis revealed that D. magneticus formed electron-dense particles on its surface when cultivated in the presence of cadmium ions (Cd²⁺). Sulfide was also found in the precipitate, and the composition ratio of sulfide/cadmium was 0.7. Sixty percent of viable RS-1 cells was recovered by a simple magnetic separation revealing the removal of 58% cadmium from the culture medium.

Keywords: Desulfovibrio magneticus ; sulfate-reducing magnetotactic bacterium; cadmium, magnetic separation; bioremediation

The influence of vegetable oils on biosurfactant production by Serratia marcescens by Cristina Ferraz; Álvaro A. De Araújo; Gláucia M. Pastore (pp. 841-847).

The production of biosurfactant, a surface-active compound, by two Serratia marcescens strains was tested on minimal culture medium supplemented with vegetable oils, considering that it is well known that these compounds stimulate biosurfactant production. The vegetable oils tested included soybean, olive, castor, sunflower, and coconut fat. The results showed a decrease in surface tension of the culture medium without oil from 64.54 to 29.57, with a critical micelle dilution (CMD⁻¹) and CMD⁻² of 41.77 and 68.92 mN/m, respectively. Sunflower oil gave the best results (29.75 mN/m) with a CMD⁻¹ and CMD⁻² of 36.69 and 51.41 mN/m, respectively. Sunflower oil contains about 60% of linoleic acid. The addition of linoleic acid decreased the surface tension from 53.70 to 28.39, with a CMD⁻¹ of 29.72 and CMD⁻² of 37.97, suggesting that this fatty acid stimulates the biosurfactant production by the LB006 strain. In addition, the crude precipitate surfactant reduced the surface tension of water from 72.00 to 28.70 mN/m. These results suggest that the sunflower oil’s linoleic acid was responsible for the increase in biosurfactant production by the LB006 strain.

Keywords: Biosurfactant; Serratia marcescens ; fermentation; surface tension; vegetable oils

Recirculation of process streams in fuel ethanol production from softwood based on simultaneous saccharification and fermentation by Malek Alkasrawi; Mats Galbe; Guido Zacchi (pp. 849-861).

The effect of process stream recirculation on ethanol production from steam- pretreated softwood based on simultaneous saccharification and fermentation (SSF) was investigated for two process configurations. In the first configuration, a part of the stillage stream after distillation was recycled and, in the second configuration, the liquid after SSF was recycled. The aim was to minimize the energy consumption in the distillation of the fermentation broth and in the evaporation of the stillage, as well as the use of fresh water. However, recirculation leads to an increased concentration of nonvolatiles in the first configuration, and of both volatiles and nonvolatiles in the second configuration. These substances might be inhibitory to the enzymes and the yeast in SSF. When 60% of the fresh water was replaced by stillage, the ethanol yield and the productivity were the same as for the configuration without recirculation. The ethanol production cost was reduced by 17%. In the second configuration, up to 40% of the fresh water could be replaced without affecting the final ethanol yield, although the initial ethanol productivity decreased. The ethanol production cost was reduced by 12%. At higher degrees of recirculation, fermentation was clearly inhibited, resulting in a decrease in ethanol yield while hydrolysis seemed unaffected.

Keywords: Recirculation; ethanol; simultaneous saccharification and fermentation; inhibition; softwood

Intensification of lipase performance for long-term operation by immobilization on controlled pore silica in presence of polyethylene glycol by Cleide M. F. Soares; Heizir F. De Castro; M. Helena A. Santana; Gisella M. Zanin (pp. 863-874).

In agreement with previous studies, promising results were obtained when lipase was immobilized on controlled pore silica (CPS) in the presence of polyethylene glycol (PEG 1500). This methodology rendered immobilized derivatives with higher operational stability than those lacking PEG 1500. This article extends the scope of this approach by evaluating the combined effects of PEG concentration and lipase loading employing a multivariate statistical approach. A 2² factorial design with center point was adopted for a full understanding of these effects and their interactions. Conditions that maximize the immobilization yield were different from those attained for the biocatalyst’s operational stability. Possible reasons for the increase in both activity and stability of lipase immobilized on CPS in the presence of PEG 1500 are discussed in light of the influence of surface hydrophilic/hydrophobic balance.

Keywords: Immobilization; lipase; controlled pore silica; stability; experimental design

Xylose reductase activity of Candida guilliermondii during xylitol production by fed-batch fermentation by Denise C. G. A. Rodrigues; Silvio S. Da Silva; J. B. Almeida E Silva; Michele Vitolo (pp. 875-883).

Xylose reductase activity of Candida guilliermondii FTI 20037 was evaluated during xylitol production by fed-batch fermentation of sugarcane bagasse hydrolysate. A 2^4-1 fractional factorial design was used to select process variables. The xylose concentrations in the feeding solution (S F) and in the fermentor (S ₀), the pH, and the aeration rate were selected for optimization of this process, which will be undertaken in the near future. The best experimental result was achieved at S F=45 g/L, S ₀=40 g/L, pH controlled at 6.0, and aeration rate of 1.2 vvm. Under these conditions, the xylose reductase activity was 0.81 U/mg of protein and xylitol production was 26.3 g/L, corresponding to a volumetric productivity of 0.55 g/(L·h) and a xylose xylitol yield factor of 0.68 g/g.

Keywords: Xylitol; sugar cane bagasse; Candida guilliermondii ; xylose reductase; fed batch

Cofermentation of glucose, xylose, and arabinose by genomic DNA-integrated xylose/arabinose fermenting strain of Zymomonas mobilis AX101 by Ali Mohagheghi; Kent Evans; Yat-Chen Chou; Min Zhang (pp. 885-898).

Cofermentation of glucose, xylose, and arabinose is critical for complete bioconversion of lignocellulosic biomass, such as agricultural residues and herbaceous energy crops, to ethanol. We have previously developed a plasmid-bearing strain of Zymomonas mobilis (206C[pZB301]) capable of cofermenting glucose, xylose, and arabinose to ethanol. To enhance its genetic stability, several genomic DNA-integrated strains of Z. mobilis have been developed through the insertion of all seven genes necessay for xylose and arabinose fermentation into the Zymomonas genome. From all the integrants developed, four were selected for further evaluation. The integrants were tested for stability by repeated transfer in a nonselective medium (containing only glucose). Based on the stability test, one of the integrants (AX101) was selected for further evaluation. A series of batch and continuous fermentations was designed to evaluate the cofermentation of glucose, xylose, and l-arabinose with the strain AX101. The pH range of study was 4.5, 5.0, and 5.5 at 30°C. The cofermentation process yield was about 84%, which is about the same as that of plasmid-bearing strain 206C(pZB301). Although cofermentation of all three sugars was achieved, there was a preferential order of sugar utilization: glucose first, then xylose, and arabinose last.

Keywords: Recombinant Zymomonas ; genomic integration; cofermentation; continuous fermentation; xylose; arabinose; ethanol productivity; acetic acid

Performance of immobilized Zymomonas mobilis 31821 (pZB5) on actual hydrolysates produced by arkenol technology by Tomiaki Yamada; Michael A. Fatigati; Min Zhang (pp. 899-907).

By applying the Arkenol process using highly concentrated sulfuric acid, various biomass feedstocks, including cedar tree, rice straw, newspaper, and bagasse, were successfully processed and converted into glucose and xylose for fermentation usage in a flash fermentation reactor in which the performance of National Renewable Energy Laboratory’s patented rec-Zymomonas mobilis 31821 (pZB5) after immobilization was investigated. The immobilization medium is a photocrosslinked resin made from polyethylene glycols or polypropylene glycols. Recombinant or rec-Z. mobilis used in the study has been shown to efficiently ferment glucose and xylose at a relatively high concentration (12–15%), that is a typical hydrolysate produced from cellulosic feedstocks. The application of immobilized rec-Z. mobilis and flash fermentation technology, together with concentrated acid technology producing a high concentration sugar solution, promises to speed the development of the cellulose-to-ethanol industry.

Enhancement of oxygen transfer by pressure pulsation in aqueous glycerol fermentation by Wei-Cho Huang; Cheng S. Gong; George T. Tsao (pp. 909-920).

Aeration plays an important role in the production of glycerol by fermentation with yeast. Effective aeration depends on a number of factors, such as amount of air, fineness of air dispersion, rate of agitation, and time of gasliquid contact. This investigation dealt with the effect of periodic variation in gas pressure on oxygen transfer measured by sulfite oxidation and glycerol fermentation in stirred tanks. The oxygen transfer rate measured with the sulfite oxidation method was improved by 20–30% under the condition of pressure pulsation (PP) at 30°C. The yield and productivity of glycerol were increased by about 26 and 6.8%, respectively, in 48 h by employing a glucose concentration of 250 g/L with PP at 30°C.

Keywords: Glycerol fermentation; osmotolerant yeast; oxygen transfer; pressure pulsation; sulfite oxidation

Cellulase production on bagasse pretreated with hot water by Mary Bigelow; Charles E. Wyman (pp. 921-934).

Because pretreatment of biomass with hot water only in differential flow systems offers very digestible cellulose and potentially less inhibition by liquid hydrolysate, solids and liquid hydrolysate from bagasse pretreated with hot water were fed to a batch cellulase production system using the Rut C30 strain of Trichoderma reesei to determine the suitability of these substrates for cellulase production. The organism was found to be sensitive to inhibitors in the liquid hydrolysate but could be adapted to improve its tolerance. In addition, filtering of the material reduced inhibitory effects. The organism was also sensitive to some component in the solids, and they had to be washed heavily to achieve good growth and cellulase production rates. Even then, a lag was found before enzyme production would commence on pretreated solids whereas no such lag was experienced with Solka Floc. However, once enzyme production began, as high and even somewhat greater cellulase productivities were realized with washed pretreated solids. Adding lignin to Solka Floc delayed enzyme production, suggesting that lignin or other materials in the lignin solids could cause the lag observed for pretreated bagasse, but more studies are needed to resolve the actual reason for this delay.

Keywords: cellulase production; hot water pretreatment; Rut C30; Trichoderma reesei

Treatment of sewage sludge generated in municipal wastewater treatment plants by Célia R. G. Tavares; Cláudia T. Benatti; Benedito P. Dias Filho (pp. 935-945).

This study was designed to evaluate the performance of a cylindrical anaerobic digester in treating secondary sewage sludge. A series of three independent batch experiments was performed for a total operation time of 60 d. The system of anaerobic digestion showed stability conditions, with no noticeable scum or foaming problems. The chemical oxygen demand reduction reached 29, 21, and 45% in sludge and 95, 85, and 82% in supernatant for the three experiments, respectively. Total coliform bacteria levels in the digester ranged from 10⁴ to 10⁵ in influent sludge and from 10⁴ to 10³ in effluent sludge, with an average reduction of 90%. Fecal coliforms of the order of 10⁴ were enumerated in influent sludge and those of the order of 10⁰ were enumerated in effluent sludge, with an average reduction of 99.9%. The studied system had satisfactory results, showing that both organic matter and indicator bacteria levels substantially decrease when the sludge is submitted to anaerobic digestion.

Keywords: Sewage sludge; anaerobic digestion; stabilization; characterization; coliform bacteria

Influence of substrate and product concentrations on the production of cyclodextrins by CGTase of Bacillus firmus, strain no. 37 by Graciette Matioli; Gisella M. Zanin; Flávio F. De Moraes (pp. 947-961).

The influence of substrate or product level on the initial velocity of cyclodextrin (CD) production by cyclodextringlycosyltransferase from a Brazilian isolate of Bacillus firmus was studied. Our results indicate that the product γ-CD is a stronger inhibitor to the reaction than β-CD. Small saccharides could also inhibit CD production, although to a lesser extent than the products, and maltose was the strongest inhibitor among small saccharides. Increasing substrate concentration resulted in greater reduction on enzyme activity for the formation of β-CD than for γ-CD. We modeled the kinetics of CD production with a set of four reversible reactions including the cyclization/coupling reaction that forms/opens CDs, and three disproportionation reactions. Our model on the initial velocity data explained well the substrate inhibition phenomenon. Kinetic parameters were determined by fitting the initial velocity data into our model.

Keywords: Cyclodextrin; cyclodextringlycosyltransferase; inhibition; maltodextrin; kinetic parameters

Gas-phase enzymatic esterification on immobilized lipases in MCM-41 molecular sieves by Emerson Luis Pires; Everson Alves Miranda; Gustavo Paim Valença (pp. 963-976).

Several lipolytic enzymes were immobilized in the pores of MCM-41 and Al-MCM-41 molecular sieves and used as catalysts in the gas-phase esterification of acetic acid with ethanol. The entrapment of enzymes depended on the molecular sieve and the type of enzyme used. The order of enzymatic activity for enzymes entrapped in the pores of MCM-41 and Al-MCM-41 in the esterification reaction was OF (Rhizopus niveus lipases)Rhizopus oryzae lipases)Mucor javanicus lipases)Pseudomonas cepacia lipases)Pseudomonas fluorescens lipases). Experiments carried out between 298 and 318 K showed no effect of temperature on catalyst yield, suggesting that the enzymes were appropriately immobilized in the pores of the molecular sieves, thus avoiding possible processes such as denaturing or autolysis.

Keywords: Enzyme; lipase; molecular sieves; MCM-41; ethyl acetate; gas-phase

Esterification activity and stability of Candida rugosa lipase immobilized into chitosan by Ernandes B. Pereira; Heizir F. de Castro; Flávio F. De Moraes; Gisella M. Zanin (pp. 977-986).

Microbial lipase from Candida rugosa immobilized into porous chitosan beads was tested for esterification selectivity with butanol and different organic acids (C2–C12), and butyric acid and different aliphatic alcohols (C2–C10). After 24 h, the acids tested achieved conversions of about 40–45%. Acetic acid was the only exception, and in this case butanol was not consumed. Different alcohols led to butyric acid conversions >40%, except for ethanol, in which case butyric acid was converted only 26%. The system’s butanol and butyric acid were selected for a detailed study by employing an experimental design. The influence of temperature, initial catalyst concentration, and acid:alcohol molar ratio on the formation of butyl butyrate was simultaneously investigated, employing a 2³ full factorial design. The range studied was 37–50°C for temperature (X₁), 1.25–2.5% (w/v) for the catalyst concentration (X₂), and 1 and 2 for the acid:alcohol molar ratio (X₃). Catalyst concentration (X₂) was found to be the most significant factor and its influence was positive. Maximum ester yield (83%) could be obtained when working at the lowest level for temperature (37°C), highest level for lipase concentration (2.5% [w/v]), and center level of acid:alcohol molar ratio (1.5). The immobilized lipase was also used repeatedly in batch esterification reactions of butanol with butyric acid, revealing a half-life of 86 h.

Keywords: Lipase; immobilization; chitosan; esterification; activity; stability

Enzymatic synthesis of medium chain monoglycerides in a solvent-free system by Marta A. P. Langone; Melissa E. De Abreu; Michelle J. C. Rezende; Geraldo L. Sant’Anna Jr. (pp. 987-996).

The synthesis of monocaprin, monolaurin, and monomyristin in a solvent-free system was conducted by mixing a commercial immobilized lipase with the organic reactants (glycerol and fatty acids) in a 20-mL batch reactor with constant stirring. The effects of temperature, fatty acid/glycerol molar ratio, and enzyme concentration on the reaction conversion were determined. The addition of molecular sieves in the assays of monomyristin synthesis was also evaluated. The reactions were carried out for 5 to 6 h and the nonpolar phase was analyzed by gas chromatography. The best results in terms of selectivity and conversion (defined as the percentage of fatty acid consumed) were achieved when the stoichiometric amount of reagents (molar ratio=1) and 9% (w/w) commercial enzyme were used and the reaction was performed at 60°C. The addition of molecular sieves did not improve the synthesis of monomyristin. Conversions as high as 80%, with monoglycerides being the major products, were attained. After 5 h of reaction, the concentration of monoglyceride was about twice that of diglyceride, and only trace amounts of triglyceride were found. The results illustrate the technical possibility of producing medium chain monoglycerides in a solvent-free medium using a simple batch reactor.

Keywords: Medium chain monoglycerides; immobilized lipase; esterification

Process development for production of medium chain triglycerides using immobilized lipase in a solvent-free system by Marta A. P. Langone; Geraldo L. Sant’Anna Jr. (pp. 997-1008).

The synthesis of tricaprylin, tricaprin, trilaurin, and trimyristin in a solvent-free system was conducted by mixing a commercial immobilized lipase with the organic reagents (glycerol and fatty acid) in a 20-mL batch reactor with constant stirring. The effects of temperature, fatty acid/glycerol molar ratio, and enzyme concentration on the reaction conversion were determined. The reactions were carried out for 26 h and the nonpolar phase was analyzed by gas chromatography. Appreciable levels of medium chain triglycerides were achieved, except for tricaprylin. The higher selectivity values for the production of triglycerides were attained under the following conditions: a fatty acid/glycerol molar ratio of 5; enzyme concentration of 5 or 9% (w/w); and temperatures of 70°C (tricaprin), 80°C (trilaurin), and 90°C (trimyristin). After completion of the esterification reaction under these conditions, the recovery of the triglyceride and fatty acids, and the reusability of the enzyme were studied. The unreacted fatty acid and the produced triglyceride were satisfactorily recovered. The commercial immobilized lipase was used in 10 consecutive batch reactions at 80°C, with 100% selectivity in the trilaurin and trimyristin synthesis. The possibility of enzyme reuse and the recovery of residual fatty acid are relevant results that contribute to increasing the viability of the process.

Keywords: Medium chain triglycerides; immobilized lipase; esterification

Hybrid neural modeling of bioprocesses using functional link networks by Layse H. P. Harada; Aline C. da Costa; Rubens Maciel Filho (pp. 1009-1023).

The objective of this work was to develop a model for an extractive ethanol fermentation in a simple and rapid way. This model must be sufficiently reliable to be used for posterior optimization and control studies. A hybrid neural model was developed, combining mass and energy balances with neural networks, which describe the process kinetics. To determine the best model, two structures of neural networks were compared: the functional link networks and the feedforward neural networks. The two structures are shown to describe well the process kinetics, and the advantages of using the functional link networks are discussed.

Keywords: Extractive alcoholic fermentation; functional link networks; hybrid model; bioprocess; modeling

Evaluation of different carbon and nitrogen sources in production of rhamnolipids by a strain of Pseudomonas aeruginosa by Alexandre S. Santos; Ana Paula W. Sampaio; Gina S. Vasquez; Lídia M. Santa Anna; Nei Pereira Jr.; Denise M. G. Freire (pp. 1025-1035).

Culture conditions involving variations in carbon and nitrogen sources and different C:N ratios were examined with the aim of increasing productivity in the process of rhamnolipid synthesis by Pseudomonas aeruginosa. In addition to the differences in productivity, the use of different carbon sources resulted in several proportions related to the types of rhamnolipids synthesized (monorhamnolipids and dirhamnolipids). Furthermore, the variation in nutrients, mainly the nitrogen source, resulted in different amounts of virulence factors, as phenazines and extracellular proteins. The data point out a new concern in the choice of substrate to be used for rhamnolipid production by P. aeruginosa: toxic byproducts.

Keywords: Rhamnolipids; biosurfactant; virulence factors; Pseudomonas aeruginosa

Influence of a new axial impeller on K L a and xylanase production by Penicillium canescens 10-10c by Yasser Bakri; Philippe Jacques; Lin Kui Shi; Philippe Thonart (pp. 1037-1048).

The effects of a new axial impeller (HTPG4) on oxygen volumetric transfer coefficient, K L a, and xylanase production by Penicillium canescens 10-10c were studied and compared for dual-impeller systems, one with one DT4 impeller below and one HTPG4 above (DT4-HTPG4) and one with two DT4 (DT4-DT4) impellers, in a 5-L bioreactor. The volumetric coefficient of oxygen transfer was measured in culture medium using a gassing-out method at different gassing rates and agitation speeds. We observed that the DT4-HTPG4 combination provided better K L a performance than the DT4-DT4 combination. The two combinations were also tested for their influence on xylanase production by a filamentous microorganism; P. canescens 10-10c. These experiments demonstrated that the DT4-HTPG4 combination impeller enhanced enzyme production up to 23% compared with the DT4-DT4 combination at an aeration rate of 1 vvm and an agitation speed of 600 rpm. The main cause for this difference is thought to be a higher shear stress generated by the DT4-DT4 combination, which damages the mycelium of P. canescens and decreases xylanase production.

Keywords: K L a ; xylanase; Penicillium canescens ; down-pumping impeller; dual-impeller

Oxygen uptake rate in production of xylitol by Candida guilliermondii with different aeration rates and initial xylose concentrations by Maria Antonieta P. Gimenes; Luiz Cláudio S. Carlos; Luís F. F. Faria; Nei Pereira Jr. (pp. 1049-1059).

The global oxygen uptake rate (OUR) and specific oxygen uptake rates (SOUR) were determined for different values of the volumetric oxygen mass transfer coefficient (15, 43, and 108 h⁻¹), and for varying initial xylose concentrations (50, 100, 150, and 200 g/L) in shaking flasks. The initial cell concentration was 4.0 g/L, and there was only significant growth in the fermentation with the highest oxygen availability. In this condition, OUR increased proportionally to cell growth, reaching maximum values from 2.1 to 2.5 g of O₂/(L·h) in the stationary phase when the initial substrate concentration was raised from 50 to 200 g/L, respectively. SOUR showed different behavior, growing to a maximum value coinciding with the beginning of the exponential growth phase, after which point it decreased. The maximum SOUR values varied from 265 to 370 mg of O₂/(g of cell·h), indicating the interdependence of this parameter and the substrate concentration. Although the volumetric productivity dropped slightly from 1.55 to 1.18 g of xylitol/(L·h), the strain producing capacity (γ P/X) rose from 9 to 20.6 g/g when the initial substrate concentration was increased from 50 to 200 g/L. As for the xylitol yield over xylose consumed (γ P/S), there was no significant variation, resulting in a mean value of 0.76 g/g. The results are of interest in establishing a strategy for controlling the dynamic oxygen supply to maximize volumetric productivity.

Keywords: Xylitol; Candida guilliermondii ; xylose-fermenting yeasts; oxygen uptake

Sphaerotilus natans isolated from activated sludge and its production of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) by Kang Liu; Hong Chua; Wai-hung Lo; Hugh Lawford; Peter Hoi-Fu Yu (pp. 1061-1073).

Sphaerotilus natans is a sheathed bacterium existing in the activated sludge of wastewater treatment plants. IT is one of the filamentous bacteria causing the bulking and foaming difficulties of activated sludge. Isolating the strain and culturing it in an axenic environment could not only provide the metabolic knowledge of the strains that would be useful in the development of wastewater treatment methods, but also could enable us to gain an understanding of the mechanism by which poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (poly[3-HB-co-3-HV]) is produced by this strain. This article reports the screening and isolation of the strain from the activated sludge using the Nile blue staining method together with Fourier transform infrared analysis. We investigated the ability of the selected strain to produce poly(3-HB-co-3-HV) copolymer using glucose and peptone, or by adding valeric acid or sodium propionate as precursor. Proper precursor feeding could dramatically enhance its 3HV content in the copolymer P(3HB-co-3HV). By controlling the different feeding times in fed-batch fermentation, different desired copolymers were obtained with 15, 40, and 70% 3HV mole fraction of the copolymer. Polymer properties were analyzed by gas chromatography, differential scanning calorimetry, thermo-gravimetry, and nuclear magnetic resonance analysis.

Keywords: Activated sludge; copolymer; poly(3-hydroxybutyrate-co-3-hydroxyvalerate); polymer properties; Sphaerotilus natans

Effect of bubble size on foam fractionation of ovalbumin by Liping Du; Aleš Prokop; Robert D. Tanner (pp. 1075-1091).

The bubble size distribution and void fraction (ɛ g) (at two bulk liquid pool positions below the bulk liquid-foam interface and one lower foam phase position) in a continuous foam fractionation column containing ovalbumin were obtained using a photoelectric capillary probe. The bubble size and ɛ g data were gathered for different operating conditions (including the changes in the superficial gas velocity and feed flow rate) at a feed solution of pH 6.5 and used to calculate the specific area, a, of the bubbles. Thus, local enrichment (ER l), values of ovalbumin could be estimated and compared with directly obtained experimental results. The ER l results were also correlated with the bubble size and ɛ g to understand better the concentration mechanisms of foam fractionation. The high ER l in the lower foam phase was largely attributable to the abrupt increase in ɛ g (from 0.25 to 0.75), or the a (from about 12 to 25 cm²/cm³) from the bulk liquid to the foam phase. These changes correspond with enhanced gravity drainage. With an increase in the superficial gas velocity, the bubble size increased and the a decreased in both the bulk liquid and lower foam phases, resulting in a decrease in the local experimentally determined enrichments at high superficial gas velocities. At intermediate feed flow rates, the bubble size reached the maximum. The ɛ g and a, on the other hand, were the largest for the largest feed flow rate. The ER l in the lower foam phase was maximized at the lowest feed flow rate. It follows, therefore, that a alone is not sufficient to determine the magnitude of the ER l in the foam phase.

Keywords: Bubble size; void fraction; foam fractionation; ovalbumin

Continuous production of succinic acid by a fumarate-reducing bacterium immobilized in a hollow-fiber bioreactor by Young-Jung Wee; Jong-Sun Yun; Kui-Hyun Kang; Hwa-Won Ryu (pp. 1093-1104).

Enterococcus faecalis RKY1, a fumarate-reducing bacterium, was immobilized in an asymmetric hollow-fiber bioreactor (HFBR) for the continuous production of succinic acid. The cells were inoculated into the shell side of the HFBR, which was operated in transverse mode. Since the pH values in the HFBR declined during continuous operation to about 5.7, it was necessary to change the feed pH from 7.0 to 8.0 after 24 h of operation in order to enhance production of succinic acid. During continuous operation with a medium containing fumarate and glycerol, the productivity of succinate was 3.0–10.9 g/(L·h) with an initial concentration of 30 g/L of fumarate, 4.9–14.9 g/(L·h) with 50 g/L of fumarate, and 7.2–17.1 g/(L·h) with 80 g/L of fumarate for dilution rates between 0.1 and 0.4 h⁻¹. The maximum productivity of succinate obtained by the HFBR (17.1 g of succinate /[L·h]) was 1.7 times higher than that of the batch bioconversions (9.9 g of succinate /[L·h]) with 80 g/L of fumarate. Furthermore, the long-term stability of the HFBR was demonstrated with a continuously efficient production of succinate for more than 15 d (360 h).

Keywords: Succinic acid; fumaric acid; Enterococcus faecalis RKY1; hollow-fiber bioreactor; immobilization

Mixed submerged fermentation with two filamentous fungi for cellulolytic and xylanolytic enzyme production by O. Garcia-Kirchner; M. Muñoz-Aguilar; R. Pérez-Villalva; C. Huitrón-Vargas (pp. 1105-1114).

The efficient saccharification of lignocellulosic materials requires the cooperative actions of different cellulase enzyme activities: exoglucanase, endoglucanase, β-glucosidase, and xylanase. Previous studies with the fungi strains Aureobasidium sp. CHTE-18, Penicillium sp. CH-TE-001, and Aspergillus terreus CH-TE-013, selected mainly because of their different cellulolytic and xylanolytic activities, have demonstrated the capacity of culture filtrates of cross-synergistic action in the saccharification of native sugarcane bagasse pith. In an attempt to improve the enzymatic hydrolysis of different cellulosic materials, we investigated a coculture fermentation with two of these strains to enhance the production of cellulases and xylanases. The 48-h batch experimental results showed that the mixed culture of Penicillium sp. CH-TE-001 and A. terreus CH-TE-013 produced culture filtrates with high protein content, cellulase (mainly β-glucosidase), and xylanase activities compared with the individual culture of each strain. The same culture conditions were used in a simple medium with mineral salts, corn syrup liquor, and sugarcane bagasse pith as the sole carbon source with moderate shaking at 29°C. Finally, we compared the effect of the cell-free culture filtrates obtained from the mixed and single fermentations on the saccharification of different kinds of cellulosic materials.

Keywords: Cellulolytic activity; mixture fermentation; bagasse pith; saccharification

Studies on mass production of transformed Panax ginseng hairy roots in bioreactor by Gwi-Taek Jeong; Don-Hee Park; Baik Hwang; Kyungmoon Park; Si-Wouk Kim; Je-Chang Woo (pp. 1115-1127).

The growth properties of Panax ginseng hairy roots transformed by Agrobacterium rhizogenes were compared between flask and aerated column or stirred bioreactor. In flask cultures, sucrose, initially 30 g/L, was nearly exhausted after 45 d of culture. The pH of the medium dropped from 5.5 to 4.96 after 10 d, but afterward it gradually increased to 6.4. After 45 d, hairy roots grew about 16-folds. The growth rate of hairy roots in air-bubble column or stirred bioreactor cultures was 1.13 (1.11) to 1.23 (1.20) g fresh wt (dry wt)/(g of cells·d), respectively. For both bioreactors, growth was about three times as high as in the flask cultivation.

Keywords: Panax ginseng ; transformed hairy roots; ginseng crude saponin; polysaccharide; Agrobacterium rhizogenes

Optimum conditions for transformed Panax ginseng hairy roots in flask culture by Gwi-Taek Jeong; Don-Hee Park; Hwa-Won Ryu; Woo-Tai Lee; Kyungmoon Park; Choon-Hyoung Kang; Baik Hwang; Je-Chang Woo (pp. 1129-1139).

Panax ginseng hairy roots were transformed by Agrobacterium rhizogenes KTCT 2744. They showed an active branching pattern and fast growth in hormone-free medium, and good growth at 23°C, pH 5.8, 1/2 MS medium, and 3% sucrose. Sucrose provided the highest growth among seven carbon sources tested. Six complex media were also tested. In the combined sugar study, hairy roots grew better on sucrose without glucose or fructose than with glucose or fructose. In the 1/2 MS basal medium, 30 mM in nitrogen and 0.62 mM phosphate salt concentration was the optimum. The growth ratio was maximal at an inoculum size of 0.4% (w/v). Crude saponin and polysaccharide levels were also measured.

Keywords: Panax ginseng ; transformed hairy roots; optimal condition; ginseng crude saponin; polysaccharide

Molecular distillation process for recovering biodiesel and carotenoids from palm oil by C. B. Batistella; E. B. Moraes; R. Maciel Filho; M. R. Wolf Maciel (pp. 1149-1159).

Carotenoids and biodiesel from palm oil were recovered through a process involving neutralization and transesterification of palm oil followed by molecular distillation of the esters. The concentrated obtained contains more than 30,000 ppm of carotenoids and the distillate contains above 95% of light-colored biodiesel. The experimental data were obtained from falling film and centrifugal molecular distillators. It can be seen that each one has its own characteristics, which are a function of the operating temperatures and of the tendency of the material thermal decomposition. These characteristics can determine the type of equipment to be used, since they have different operating conditions. The experimental results were compared to the ones from simulations using the mathematical modeling for the falling film and centrifugal distillators developed.

Keywords: Molecular distillation; carotenoids; biodiesel; palm oil

Combined sedimentation and filtration process for cellulase recovery during hydrolysis of lignocellulosic biomass by Jeffrey S. Knutsen; Robert H. Davis (pp. 1161-1172).

A combined sedimentation and ultrafiltration process was investigated for recovering cellulase enzymes during the hydrolysis of lignocellulosic biomass. Lignocellulosic particles larger than approx 50 µm in length were first removed via sedimentation using an inclined settler. Ultrafiltration was then used to retain the remaining lignocellulosic particles and the cellulose enzymes, while transmitting fermentable sugars and other small molecules. The permeate flux from the ultrafiltration step for a feed consisting of 0.22 w/v% cellulase is 64±5 L/m²-h, while that for a feed consisting of the settler overflow from a mixture 0.22 w/v% cellulase and 10 wt% lignocellulose fed to the settler is 130±20 L/m²-h. The higher permeate flux in the latter case is presumably due to binding of a portion of the cellulase enzymes to the lignocellulosic particles during hydrolysis and filtration, preventing the enzymes from fouling the membrane. A filter paper activity assay shows little loss in enzymatic activity throughout the combined sedimentation/ultrafiltration separation process.

Alkali hydrolysis of trinitrotoluene by Christian Karasch; Milan Popovic; Mohamed Qasim; Rakesh K. Bajpai (pp. 1173-1185).

Data for alkali hydrolysis of 2,4,6-trinitrotoluene (TNT) in aqueous solution at pH 12.0 under static (pH-controlled) as well as dynamic (pH-uncontrolled) conditions are reported. The experiments were conducted at two different molar ratios of TNT to hydroxyl ions at room temperature. The TNT disappeared rapidly from the solution as a first-order reaction. The complete disappearance of aromatic structure from the aqueous solution within 24 h was confirmed by the ultraviolet-visible (UV-VIS) spectra of the samples. Cuvet experiments in a UV-VIS spectrophotometer demonstrated the formation of Meisenheimer complex, which slowly disappeared via formation of aromatic compounds with fewer nitro groups. The known metabolites of TNT were found to accumulate only in very small quantities in the liquid phase.

Keywords: 2,4,6-Trinitrotoluene; alkali; hydrolysis; ultraviolet-visible spectra; metabolites

Molecular distillation by C. B. Batistella; E. B. Moraes; R. Maciel Filho; M. R. Wolf Maciel (pp. 1187-1206).

In this work, important results from simulations are presented, showing the potentiality of the molecular distillation process for recovering vitamin E from vegetal oils. Two types of molecular distillators are considered: falling film and centrifugal. The results emphasize the degree of recovery and factors that influence substantially the performance of the molecular distillators, such as feed flow rate, residence time, and process temperature. Moreover, they show that each type of molecular distillator enables one to operate under specific residence time and temperature. Therefore, a careful analysis must be made in order to determine the best equipment and operating conditions for obtaining products with high quality and concentration, and reduced problems of material thermal decomposition. Vitamin E (tocopherols) from vegetal oils, more specifically, from the deodorizer distillate of soya oil, was the studied case.

Keywords: Molecular distillation; vegetal oils; vitamin E; soya oil

Hydrogenolysis of lignins by Adilson R. Gonçalves; Ulf Schuchardt (pp. 1211-1219).

Hydrolytic eucalyptus lignin was converted to oils by hydrogenolysis. The lignin was obtained by acid hydrolysis of eucalyptus chips from two semi-industrial sources in Brazil: FTI and Coalbra. Hydrogenolysis was performed in an 1-L reactor using lignin/water ratio 1/4 (w/v), pH 9 (adjusted with NaOH), 0.1 g sodium formate/g lignin, 20 MPa (argon pressure) at 280°C for 15 min. After reaction, the products were filtered and the solids extracted with chloroform/ethanol 3/1 (v/v). The solvent was evaporated from the organic phase and a dark oil was obtained. The solid remaining after extraction was weighed to calculate the conversion. Without pretreatment conversions were 41.3 and 47.9% for Coalbra and FTI, respectively. The oil yields were 22.7–27.6% for Coalbra and FTI, respectively. Using microwave pretreatment (30 min of irradiation at 490 W) the conversions were 41.6–50.5% and the oil yields increased slightly to 25.2–31.4%. The polymeric chains in the lignin breakdown due to the action of water near the boiling point under microwave irraditation. On the other hand, by using ultrasound (30 min at 50°C in a ultrasound bath of 25 kHz and 0.8 W/cm²) the conversions were 35.2–46.9% and oil yields were 22.0–27.1%. Ultrasound favors the formation of radicals that probably caused the reticulation of lignins, decreasing the conversion and yield. Oils analyzed by infrared spectroscopy showed an increase in C=O bond intensities, compared with the original lignins.

Keywords: Hydrogenolysis of lignins; oils from biomass; microwave; ultrasound

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.98-100, #1-9)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.98-100, #1-9)