Check out our New Publishers' Select for Free Articles

Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.121, #1-3)

Feedstock supply, logistics, processing, and composition by Hans-Joachim G. Jung; David N. Thompson (pp. 3-4).

Biomechanics of wheat/barley straw and corn stover by Christopher T. Wright; Peter A. Pryfogle; Nathan A. Stevens; Eric D. Steffler; J. Richard Hess; Thomas H. Ulrich (pp. 5-19).

The lack of understanding the mechanical characteristics of cellulosic feedstocks is a limiting factor in economically collecting and processing crop residues, primarily wheat and barley stems and corn stover. Several testing methods—compression, tension, and bend—were investigated to increase the understanding of the biomechanical behavior of cellulosic feedstocks. Biomechanical data from these tests can provide required input to numerical models and help advance harvesting, handling, and processing techniques. In addition, integrating the models with the complete data set from this study can identify potential tools for manipulating the biomechanical properties of plant varieties in such a manner as to optimize their physical characteristics to produce higher-value biomass and more energy-efficient harvesting practices.

Keywords: Modulus of elasticity; biomechanics; wheat straw; corn stover; feedstock development

Effect of additions on ensiling and microbial community of senesced wheat straw by David N. Thompson; Joni M. Barnes; Tracy P. Houghton (pp. 21-46).

Crop residues collected during or after grain harvest are available once per year and must be stored for extended periods. The combination of air, high moisture, and high microbial loads leads to shrinkage during storage and risk of spontaneous ignition. Ensiling is a wet preservation method that could be used to store these residues stably. To economically adapt ensiling to biomass that is harvested after it has senesced, the need for nutrient, moisture, and microbial additions must be determined. We tested the ensiling of senesced wheat straw in sealed columns for 83 d. The straw was inoculated with Lactobacillus plantarum and amended with several levels of water and free sugars. The ability to stabilize the straw polysaccharides was strongly influenced by both moisture and free sugars. Without the addition of sugar, the pH increased from 5.2 to as much as 9.1, depending on moisture level, and losses of 22% of the cellulose and 21% of the hemicellulose were observed. By contrast, when sufficient sugars were added and interstitial water was maintained, a final pH of 4.0 was attainable, with correspondingly low (<5%) losses of cellulose and hemicellulose. The results show that ensiling should be considered a promising method for stable storage of wet biorefinery feedstocks.

Keywords: Wheat straw; lignocellulose; biorefinery feedstock; silage; ensiling; wet storage

Large-scale ethanol fermentation through pipeline delivery of biomass by Amit Kumar; Jay B. Cameron; Peter C. Flynn (pp. 47-58).

Issues of traffic congestion and community acceptance limit the size of biomass-processing plants based on truck delivery to about 2 million (M) dry t/yr or less. In this study, the cost of ethanol from an ethanol fermentation plant processing 2 M dry t/yr of corn stover supplied by truck is compared with that of larger plants in the range of 4–38 M dry t/yr supplied by a combination of trucks plus pipelines. For corn stover, a biomass source with a low yield per gross hectare, the cost of ethanol from larger plants is always higher. For wood chips from the boreal forest, a biomass source with a relatively high yield per gross hectare, a plant processing 14–38 M dry t/yr produces ethanol at a 13% reduction in cost compared with a plant producing 2 M dry t/yr supplied by truck. Processing of value-added products, such as chemicals from lignin, would be enabled by larger-scale plants.

Keywords: Ethanol fermentation; pipeline delivery; wood chips; corn stover; economies of scale

Perspectives on bioenergy and biotechnology in Brazil by Adalberto Pessoa-Jr; Inêes Conceição Roberto; Marcelo Menossi; Raphael Revert dos Santos; Sylvio Ortega Filho; Thereza Christina Vessoni Penna (pp. 59-70).

Brazil is one of the world’s largest producers of alcohol from biomass at low cost and is responsible for more than 1 million direct jobs. In 1973, the Brazilian Program of Alcohol (Proalcool) stimulated the creation of a bioethanol industry that has led to large economic, social, and scientific improvements. In the year 1984, 94.5% of Brazil’s cars used bioethanol as fuel. In 2003/2004, 350.3 million of sugarcane produced 24.2 million t of sugar and 14.4 billion L of ethanol for an average 4.3 million cars using ethanol. Since its inception, cumulative investment in Proalcool totals US$11 billion, and Brazil has saved US$27 billion in oil imports. The ethanol production industry from sugarcane generates 152 times more jobs than would have been the case if the same amount of fuel was produced from petroleum, and the use of ethanol as a fuel is advantageous for environmental reasons. In 2003, one of the biggest Brazilian ethanol industries started consuming 50% of the residual sugarcane bagasse to produce electrical energy (60 MW), a new alternative use of bioenergy for the Brazilian market. Other technologies for commercial uses of bagasse are in development, such as in the production of natural fibers, sweeteners (glucose and xylitol), single-cell proteins, lactic acid, microbial enzymes, and many other products based on fermentations (submerged and semisolid). Furthermore, studies aimed at the increase in the biosynthesis of sucrose and, consequently, ethanol productivity are being conducted to understand the genetics of sugarcane. Although, at present, there remain technical obstacles to the economic use of some ethanol industry residues, several research projects have been carried out and useful data generated. Efficient utilization of ethanol industry residues has created new opportunities for new value-added products, especially in Brazil, where they are produced in high quantities.

Keywords: Ethanol; bioenergy; sugarcane; agroindustry; sugar powder

Structural analysis of wheat stems by Kurt D. Hamman; Richard L. Williamson; Eric D. Steffler; Christopher T. Wright; J. Richard Hess; Peter A. Pryfogle (pp. 71-80).

Design and development of improved harvesting, preprocessing, and bulk handling systems for biomass requires knowledge of the biomechanical properties and structural characteristics of crop residue. Structural analysis of wheat stem cross-sections was performed using the theory of composites and finite element analysis techniques. Representative geometries of the stem’s structural components including the hypoderm, ground tissue, and vascular bundles were established using microscopy techniques. Material property data for the analysis was obtained from measured results. Results from the isotropic structural analysis model were compared with experimental data. Future work includes structural analysis and comparison with experimental results for additional wheat stem models and loading configurations.

Keywords: Wheat straw; biomechanics; structural analysis; modulus of elasticity; composite materials

Enzyme catalysis and engineering by Timothy C. Dodge; Gisella M. Zanin (pp. 83-83).

Activity and lifetime of urease immobilized using layer-by-layer nano self-assembly on silicon microchannels by Scott R. Forrest; Bill B. Elmore; James D. Palmer (pp. 85-91).

Urease has been immobilized and layered onto the walls of manufactured silicon microchannels. Enzyme immobilization was performed using layer-by-layer nano self-assembly. Alternating layers of oppositely charged polyelectrolytes, with enzyme layers “encased” between them, were deposited onto the walls of the silicon microchannels. The polycations used were polyethylenimine (PEI), polydiallyldimethylammonium (PDDA), and polyallylamine (PAH). The polyanions used were polystyrenesulfonate (PSS) and polyvinylsulfate (PVS). The activity of the immobilized enzyme was tested by pumping a 1 g/L urea solution through the microchannels at various flow rates. Effluent concentration was measured using an ultraviolet/visible spectrometer by monitoring the absorbance of a pH sensitive dye. The architecture of PEI/PSS/PEI/urease/PEI with single and multiple layers of enzyme demonstrated superior performance over the PDDA and PAH architectures. The precursor layer of PEI/PSS demonstrably improved the performance of the reactor. Conversion rates of 70% were achieved at a residence time of 26 s, on d 1 of operation, and >50% at 51 s, on d 15 with a six-layer PEI/urease architecture.

Keywords: Silicon microchannels; urease; architecture; polyelectrolytes; first-order constant

Production of cellulase/β-glucosidase by the mixed fungi culture of Trichoderma reesei and Aspergillus phoenicis on dairy manure by Zhiyou Wen; Wei Liao; Shulin Chen (pp. 93-104).

A cellulase production process was developed by growing the fungi Trichoderma reesei and Aspergillus phoenicis on dairy manure. T. reesei produced a high total cellulase titer (1.7 filter paper units [FPU]/mL, filter paper activity) in medium containing 10 g/L of manure (dry basis [w/w]), 2 g/L KH₂PO₄, 2 mL/L of Tween-80, and 2mg/L of CoCl₂. However, β-glucosidase activity in the T. reesei-enzyme system was very low. T. reesei was then cocultured with A. phoenicis to enhance the β-glucosidase level. The mixed culture resulted in a relatively high level of total cellulase (1.54 FPU/mL) and β-glucosidase (0.64 IU/mL). The ratio of β-glucosidase activity to filter paper activity was 0.41, suitable for hydrolyzing manure cellulose. The crude enzyme broth from the mixed culture was used for hydrolyzing the manure cellulose, and the produced glucose was significantly (p<0.01) higher than levels obtained by using the commercial enzyme or the enzyme broth of the pure culture T. reesei.

Keywords: Dairy manure; cellulase; β-glucosidase; Trichoderma reesei ; Aspergillus phoenicis

Lipase production by solid-state fermentation by Melissa L. E. Gutarra; Elisa D. C. Cavalcanti; Leda R. Castilho; Denise M. G. Freire; Geraldo L. Sant’Anna Jr. (pp. 105-116).

The production of lipase by Penicillium simplicissimum in solid-state fermentation was studied using babassu cake as the basal medium. Tray-type and packed-bed bioreactors were employed. In the former, the influence of temperature; content of the medium, and medium supplementation with olive oil, sugarcane molasses, corn steep liquor, and yeast hydrolysate was studied. For all combinations of supplements, a temperature of 30°C, a moisture content of 70%, and a concentration of carbon source of 6.25% (m/m, dry basis) provided optimum conditions for lipase production. When used as single supplements olive oil and molasses also were able to provide high lipase activities (20 U/g). Using packed-bed bioreactors and molasses-supplemented medium, optimum conditions for enzyme production were air superficial velocities above 55 cm/min and temperatures below 28°C. The lower temperature optimum found for these reactors is probably related to radial heat gradient formation inside the packed bed. Maximum lipase activities obtained in these bioreactors (26.4 U/g) were 30% higher than in tray-type reactors.

Keywords: Lipase; packed-bed bioreactor; tray-type bioreactor; Penicillium simplicissimum ; solid-state fermentation

Hydrolysis of new phthalimide-derived esters catalyzed by immobilized lipase by Juliana Vaz Bevilaqua; Lidia M. Lima; Aline Gomes Cunha; Eliezer J. Barreiro; Tito L. M. Alves; Lucia Moreira Campos Paiva; Denise M.Guimarães Freire (pp. 117-128).

The last step of the production of four phthalimide-derived acids, designed to act as antiasthma drugs, was performed by enzymatic hydrolysis of the respective methyl or ethyl esters. The esters 4-ethyl-[2-(1,3-dioxo-1,3-dihydro-2-isoindoylyl)]-phenoxyacetic methyl ester (PHT-MET), 4-ethyl-[2-(1,3-dioxo-1,3-dihydro-2-isoindoylyl)]-phenoxyacetic ethyl ester, 4-(1,3-dioxo-1,3-dihydro-2-isoindoylyl)-phenoxyacetic ethyl ester, and 2-(1,3-dioxo-1, 3-dihydro-2-isoindoylyl)-phenoxyacetic ethyl ester were hydrolyzed by immobilized lipase. The enzymatic reaction could be used only to produce the desired 4-substituted compounds. The best result that was found to hydrolysis of PHT-MET, and, therefore, that ester was selected for optimization experiments in a three-phase system. Reactions were performed with solid biocatalyst (Lipozyme® RM IM), organic solvent phase (ethyl acetate), and aqueous phase (saturated Na₂CO₃ solution). To optimize the reaction conditions, an experimental design optimization procedure was used. The variables studied were the amount of enzyme, the temperature, and the volume of the aqueous solution. Time course experiments were then performed for different initial enzyme concentrations (0.5, 0.9, and 1.4 U_H/mL of solvent). The optimized reaction conditions found were 20 mg of Lipozyme (0.9 U_H/mL_solvent) and 5.0 mL of Na₂CO_3(sat) at 40°C for 6 h.

Keywords: Immobilized lipase; asthma drug; phthalimide compounds; organic media; biocatalysis

Catalytically enhanced endocellulase cel5a from Acidothermus cellulolyticus by John O. Baker; James R. McCarley; Rebecca Lovett; Ching-Hsing Yu; William S. Adney; Tauna R. Rignall; Todd B. Vinzant; Stephen R. Decker; Joshua Sakon; Michael E. Himmel (pp. 129-148).

When Tyr245 in endocellulase Cel5A from Acidothermus cellulolyticus was changed to Gly (Y245G) by designed mutation, the value of K i for inhibition of the enzyme by the product cellobiose was increased more than 1480%. This reduction in product inhibition enabled the mutant enzyme (used in conjunction with Trichoderma reesei cellobiohydrolase-I) to release soluble sugars from biomass cellulose at a rate as much as 40% greater than that achieved by the wild-type (WT) enzyme. The mutant was designed on the basis of the previously published crystal structure of the WT enzyme/substrate complex (at a resolution of 2.4 Å), which provided insights into the enzyme mechanism at the atomic level and identified Tyr245 as a key residue interacting with a leaving group. To determine the origin of the change in activity, the crystal structure of Y245G was solved at 2.4-Å resolution to an R-factor of 0.19 (R-free=0.25). To obtain additional information on the enzyme-product interactions, density functional calculations were performed on representative fragments of the WT Cel5A and Y245G. The combined results indicate that the loss of the platform (Y245G) and of a hydrogen bond (from a conformational change in Gln247) reduces the binding energy between product and enzyme by several kilo calories per mole. Both kinetic and structural analyses thus relate the increased enzymatic activity to reduced product inhibition.

Keywords: Bioethanol; biomass conversion; protein crystallography; endoglucanase; Acidothermus cellulolyticus ; Trichoderma reesei

Oxidation of glucose to gluconic acid by glucose oxidase in a membrane bioreactor by Ester Junko Tomotani; Luiz Carlos Martins das Neves; Michele Vitolo (pp. 149-162).

Glucose oxidase (GO) (EC 1.1.3.4) was used as catalyst for oxidizing glucose into gluconic acid utilizing a 10-mL Bioengineering Enzyme Membrane Reactor® or a 400-mL Millipore Stirred Ultrafiltration Cell (MSUC) coupled with a Millipore UF membrane (cutoff of 100 kDa) and operated for 12 h under an agitation of 100 rpm, pH 5.5, and 30°C. The effect of feeding rate (0.10, 0.15, or 0.20 min⁻¹), glucose (2.5 or 5.0 mM), and GO (1.0 or 2.0 mg/mL) concentrations on the catalysis were studied. A yield of about 75% was attained when the MSUC filled with 1.0 mg/mL of GO was fed with 2.5 mM glucose solution at a rate of 0.15 min⁻¹.

Keywords: Glucose oxidase; membrane bioreactor; gluconic acid; stability; feeding rate

Weak lignin-binding enzymes by Alex Berlin; Neil Gilkes; Arwa Kurabi; Renata Bura; Maobing Tu; Douglas Kilburn; John Saddler (pp. 163-170).

Economic barriers preventing commercialization of lignocellulose-to-ethanol bioconversion processes include the high cost of hydrolytic enzymes. One strategy for cost reduction is to improve the specific activities of cellulases by genetic engineering. However, screening for improved activity typically uses “ideal” cellulosic substrates, and results are not necessarily applicable to more realistic substrates such as pretreated hardwoods and softwoods. For lignocellulosic substrates, nonproductive binding and inactivation of enzymes by the lignin component appear to be important factors limiting catalytic efficiency. A better understanding of these factors could allow engineering of cellulases with improved activity based on reduced enzyme-lignin interaction (“weak lignin-binding cellulases”). To prove this concept, we have shown that naturally occurring cellulases with similar catalytic activity on a model cellulosic substrate can differ significantly in their affinities for lignin. Moreover, although cellulose-binding domains (CBDs) are hydrophobic and probably participate in lignin binding, we show that cellulases lacking CBDs also have a high affinity for lignin, indicating the presence of lignin-binding sites on the catalytic domain.

Keywords: Cellulase; lignin; unproductive binding; softwood; hydrolysis

Sugarcane bagasse pulps by Regina Y. Moriya; Adilson R. Gonçalves; Marta C. T. Duarte (pp. 171-181).

Organosolv (ethanol/water and acetosolv) pulps were treated with Bacillus pumilus xylanase for 4, 8, and 12 h and compared with commercial Cartazyme HS xylanase-treated pulps. Treatment of ethanol/water pulps with B. pumilus xylanase increased viscosity by 40% in 8 h of treatment compared with pulps treated without enzyme. However, acetosolv pulps treated with B. pumilus xylanase lost viscosity. Ethanol/water pulps treated with Cartazyme had a viscosity of 18.5 cP in 4 h of treatment. In the acetosolv pulps treated with commercial enzyme, the loss of viscosity was 20% compared with pulps treated without enzyme. Ethanol/water pulps treated with B. pumilus and Cartazyme had similar effects: a 44% reduction in kappa number for pulps treated with enzyme followed by alkaline extraction compared with pulps treated with alkaline extraction. In acetosolv pulps treated with B. pumilus, the kappa number was from 12 to 18, compared with pulps treated without enzyme, which had a 40% reduction in 4 and 12 h and a 60% reduction in 8 h. Cartazyme-treated acetosolv pulps had a kappa number of 14 in 4 and 8 h of treatment. For 12 h of treatment, the kappa number was 8. Fourier transform infrared spectra of the pulps showed that enzyme-treated pulps had changes in the 1000 cm⁻¹ absorption owing to a C-O bond present in esters. Using principal component analysis, it is possible to differentiate the unbleached pulps and enzyme-treated pulps.

Keywords: Bacillus pumilus ; sugarcane bagasse pulps; cartazyme; viscosity; xylanase

Partial purification and characterization of protease enzyme from Bacillus subtilis and Bacillus cereus by Elif Orhan; Didem Omay; Yküsel Gvüenilir (pp. 183-194).

The aim of this experimental study was to isolate and partially purify protease enzyme from Bacillus cereus and Bacillus subtilis. Protease enzyme is obtained by inducing spore genesis of bacteria from Bacillus species in suitable nutrient plates. The partial purification was realized by applying, respectively, ammonium sulfate precipitation, dialysis, and DEAE-cellulose ion-exchange chromatography to the supernatant that was produced later. Optimum pH, optimum temperature, pH stability, and temperature stability were determined, as well as the effects of pH, temperature, substrate concentration, reaction time, and inhibitors and activators on enzyme activity. In addition, the molecular mass of the obtained enzyme was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The specific activity of partially purified enzyme from B. subtilis was determined to be 84 U/mg. The final enzyme preparation was eight-fold more pure than the crude homogenate. The molecular mass of the partially purified enzyme was found to be 45 kDa by using SDS-PAGE. The protease enzyme that was partially purified from B. cereus was purified 1.2-fold after ammonium sulfate precipitation. The molecular mass of the partially purified enzyme was determined to be 37 kDa by using SDS-PAGE.

Keywords: Enzyme; protease; Bacillus subtilis ; Bacillus cereus ; purification; isolation

Enzymatic bleaching of organosolv sugarcane bagasse pulps with recombinant xylanase of the fungus Humicola grisea and with commercial cartazyme HS xylanase by Regina Y. Moriya; Adilson R. Gonçalves; Fabrícia P. Faria (pp. 195-203).

Organosolv (ethanol/water and acetosolv) pulps were treated with Humicola grisea var. thermoidea and compared with Cartazyme HS xylanase-treated pulp. The ethanol/water pulps treated with H. grisea had the same viscosity as unbleached pulps (8 cP). Ethanol/water pulps treated with Cartazyme had higher viscosity than H. grisea-treated pulps (12 cP). Acetosolv pulps treated with H. grisea and Cartazyme presented a reduction in viscosity; however, the pulps treated with H. grisea had a lower reduction in viscosity than Cartazyme-treated pulps. Ethanol/water pulps treated with H. grisea had a 23% reduction in kappa number in 4 and 8 h of treatment, compared with the unbleached pulps. Cartazyme-treated pulps had a kappa number similar to that of the control pulps for 4 h of treatment. Extending the treatment time to 12 h resulted in a reduction of 33%. The acetosolv pulp treated with H. grisea had a kappa number reduced to 23% in 4 h. Cartazyme treatment resulted in a reduction of 55 and 44% in kappa number for 4 and 8 h of treatment, respectively, when compared with control pulp. Extending the treatment time to 12 h decreased the kappa number 72%. Fourier transform infrared spectra and principal component analysis showed differences among unbleached, H. grisea-treated, and Cartazyme-treated pulps.

Keywords: Humicola grisea ; sugarcane bagasse pulps; cartazyme; viscosity; xylanase

Intracellular fate of hydrocarbons by Hortencia Silva-Jiménez; Roberto Zazueta-Sandoval (pp. 205-217).

In previous work, purification procedures and zymogram analysis conducted with supernatants of crude extracts from aerobic mycelium of the YR-1 strain of Mucor circinelloides isolated from petroleum-contaminated soils indicated the existence of only one soluble alcohol oxidase (sAO) activity. In the present work enzymatic activity of alcohol oxidase (AO) was also detected in the mixed membrane fraction (MMF) of a high-speed centrifugation procedure after drastic ballistic cellular homogenization to break the mycelium from strain YR-1. When mycelial cells were gently broken by freezing the mycelium with liquid nitrogen, smashing in a mortar, and submitting the samples to an isopycnic sucrose gradients (10–60% sucrose), AO activity was detected in particular and discrete fractions of the gradient, showing specific density values quite different from the density of peroxisomes. The results suggest that there could be a different intracellular pattern of distribution of the microsomal fraction in aerobically grown mycelium depending on the carbon source used in the culture media, including alcohols and hydrocarbons, but not in glucose. In working with particulate fractions, we found two AO activities: a new membrane alcohol oxidase (mAO) activity and the sAO. Both activities appear to be located in the inner of the cells in specific compartments different from the peroxisomes, so mAO could be in the membrane of these compartments and sAO in the lumen of the vesicles. We also assayed other enzymatic activities involved in hydrocarbon biodegradation to establish its intracellular location and other enzymatic activities such as peroxidase to use them as intracellular markers of different organelles. In the case of monooxygenase, the first enzymatic step in the hydrocarbon biodegradation pathway, its location was in the same fractions where AOs were located, suggesting the existance of a specific organelle that contains the enzymatic activities involved in hydrocarbon biodegradation.

Keywords: Filamentous fungi; hydrocarbon biodegradation; petroleum contamination; microsomes; peroxisomes; alcohol oxidase

Enzymatic hydrolysis of steam-exploded and ethanol organosolv-pretreated douglas-fir by novel and commercial fungal cellulases by Arwa Kurabi; Alex Berlin; Neil Gilkes; Douglas Kilburn; Renata Bura; Jamie Robinson; Aleksandr Markov; Anton Skomarovsky; Aleksandr Gusakov; Oleg Okunev; Arkady Sinitsyn; David Gregg; Dan Xie; John Saddler (pp. 219-230).

Softwood residues are the most abundant feedstock available for bioconversion in many northern countries. However, the high costs for delignification and enzymatic hydrolysis currently deter commercialization of softwood bioconversion processes. This study evaluates the abilities of two novel fungal preparations (MSUBC1 and MSUBC2) and two commercial cellulase preparations (TR1 and TR2) to hydrolyze cellulose in Douglas-fir pretreated by steam explosion or ethanol organosolv process. MSUBC1 showed significantly better performance than the other preparations on both lignocellulosic substrates. In particular, MSUBC1 achieved >76% cellulose conversion for hydrolysis of steam-exploded Douglas-fir (∼44% lignin) after 72 h at low enzyme loading (10 filter paper units/g of cellulose) and without β-glucosidase supplementation.

Keywords: Softwood; bioconversion; pretreatment; steam explosion; organosolv; lignin; cellulose; hydrolysis

Kinetics of enzyme-catalyzed alcoholysis of soybean oil in n-hexane by Débora de Oliveira; Irajá do Nascimento Filho; Marco di Luccio; Carina Faccio; Clarissa Dalla Rosa; João Paulo Bender; Nádia Lipke; Cristiana Amroginski; Cláudio Dariva; José Vladimir de Oliveira (pp. 231-241).

This work investigated the production of fatty acid ethyl esters (FAEEs) from soybean oil using n-hexane as solvent and two commercial lipases as catalysts, Novozym 435 and Lipozyme IM. A Taguchi experimental design was adopted considering the variables temperature (35–65°C), addition of water (0–10 wt/wt%), enzyme (5–20 wt/wt%) concentration, and oil-to-ethanol molar ratio (1:3–1:10). It is shown that complete conversion in FAEE is achieved for some experimental conditions. The effects of process variables on reaction conversion and kinetics of the enzymatic reactions are presented for all experimental conditions investigated in the factorial design.

Keywords: Alcoholysis; soybean oil; immobilized lipases; reaction kinetics; biodiesel

β-Glucosidase production by Trichoderma reesei by Tamás Juhász; Anita Egyházi; Kati Réczey (pp. 243-254).

The hydrolysis of cellulose to the water-soluble products cellobiose and glucose is achieved via synergistic action of cellulolytic proteins. The three types of enzymes involved in this process are endoglucanases, cellobiohydrolases, and β-glucosidases. One of the best fungal cellulase producers is Trichoderma reesei RUT C30. However, the amount of β-glucosidases secreted by this fungus is insufficient for effective cellulose conversion. We investigated the production of cellulases and β-glucosidases in shake-flask cultures by applying three pH-controlling strategies: (1) the pH of the production medium was adjusted to 5.8 after the addition of seed culture with no additional pH adjustment performed, (2) the pH was adjusted to 6.0 daily, and (3) the pH was maintained at 6.0 by the addition of Tris-maleate buffer to the growth medium. Different carbon sources—Solka Floc 200, glucose, lactose, and sorbitol—were added to standard Mandels nutrients. The lowest β-glucosidase activities were obtained when no pH adjustment was done regardless of the carbon source employed. Somewhat higher levels of β-glucosidase were measured in the culture filtrates when daily pH adjustment was carried out. The effect of buffering the culture medium on β-glucosidase liberation was most prominent when a carbon source inducing the production of other cellulases was applied.

Keywords: β-Glucosidase; cellulase; Trichoderma reesei RUT C30; Tris-maleate buffer; endoglucanase activities

Wood cellulignin as an alternative matrix for enzyme immobilization by Fabricio M. Gomes; Grazielle S. Silva; Daltro G. Pinatti; Rosa A. Conte; Heizir F. de Castro (pp. 255-268).

The objective of this work was to select an efficient methodology for preparing active samples of Candida rugosa lipase immobilized in wood cellulignin, to be applied in hydrolysis and ester reactions. For this purpose, lipase was immobilized in the matrix by physical adsorption (pure cellulignin) and covalent binding (activated cellulignin with glutaraldeyde or carbonyldiimidazole [CDI]) in the presence or absence of polyethylene glycol (PEG) (Molecular mass of 1500 Daltons) as stabilizing agent. The activating agent and the presence of PEG-1500 in the immobilization procedure showed a strong influence on enzyme retention in the support. The values for enzyme retention ranged from 20 to 68%, and the highest yield was obtained when the enzyme was immobilized in cellulignin activated with CDI in the presence of PEG-1500. This immobilized derivative presented high hydrolytic (193.27 µM/[mg·min]) and synthetic (522.92 µM/[g·min]) activities when compared with those obtained by other techniques. The superiority of this immobilized system was confirmed by additional analyses, such as infrared spectroscopy and elemental analysis, which demonstrated an appropriate enzyme fixation and the highest level of protein incorporation in the support. Further information on the immobilized derivative was obtained by assessing the recycle potential in both aqueous and nonaqueous media.

Keywords: Cellulignin; immobilizing support; lipase; Candida rugosa ; polyethylene glycol; carbondiimidazole

Utilization of methyloleate in production of microbial lipase by Jacqueline Destain; Patrick Fickers; Frédéric Weekers; Benoît Moreau; Philippe Thonart (pp. 269-277).

In this article, we report the development and optimization of an industrial culture medium for the production of extracellular lipase in the yeast Yarrowia lipolytica. Until now olive oil in combination with glucose was used as the carbon source and inducer for the production of lipase. Our results demonstrate that methyloleate, a cheap hydrophobic compound, could efficiently substitute olive oil as the inducer and carbon source for lipase production. A new process of lipase production was developed yielding a twofold increase in the level of production compared with the levels in previous reports.

Keywords: Yarrowia lipolytica ; lipase; bioreactor; methyloleate; olive oil; oleic acid

Detection of NAD⁺-dependent alcohol dehydrogenase activities in YR-1 strain of Mucor circinelloides, a potential bioremediator of petroleum contaminated soils by Arelí Durón-Castellanos; Vanesa Zazueta-Novoa; Hortencia Silva-Jiménez; Yolanda Alvarado-Caudillo; Eduardo Peña Cabrera; Roberto Zazueta-Sandoval (pp. 279-288).

Different soluble NAD⁺-dependent alcohol dehydrogenase (ADH) isozymes were detected in cell-free homogenates from aerobically grown mycelia of YR-1 strain of Mucor circinelloides isolated from petroleumcontaminated soil samples. Depending on the carbon source present in the growth media, multiple NAD⁺-dependent ADHs were detected when hexadecane or decane was used as the sole carbon source in the culture media. ADH activities from aerobically or anaerobically grown mycelium or yeast cells, respectively, were detected when growth medium with glucose added was the sole carbon source; the enzyme activity exhibited optimum pH for the oxidation of different alcohols (methanol, ethanol, and hexadecanol) similar to that of the corresponding aldehyde (≈7.0). Zymogram analysis conducted with partially purified fractions of extracts from aerobic mycelium or anaerobic yeast cells of the YR-1 strain grown in glucose as the sole carbon source indicated the presence of a single NAD⁺-dependent ADH enzyme in each case, and the activity level was higher in the yeast cells. ADH enzyme from mycelium grown in different carbon sources showed high activity using ethanol as substrate, although higher activity was displayed when the cells were grown in hexadecane as the sole carbon source. Zymogram analysis with these extracts showed that this particular strain of M. circinelloides has four different isozymes with ADH activity and, interestingly, one of them, ADH4, was identified also as phenanthrene-diol-dehydrogenase, an enzyme that possibly participates in the aromatic hydrocarbon biodegradation pathway.

Keywords: Alcohol dehydrogenase; aromatic hydrocarbon biodegradation; contaminated soil; petroleum contamination; Mucor Circinelloides

Synthesis of polyhydroxyalkanoate (PHA) from excess activated sludge under various oxidation-reduction potentials (ORP) by using acetate and propionate as carbon sources by W. F. Hu; S. N. Sin; H. Chua; P. H. F. Yu (pp. 289-301).

Accumulation of poly hydroxyalkanoate (PHA) from excess activated sludge (EAS) was monitored and controlled via the oxidation-reduction potential (ORP) adjusting process. The ORP was adjusted and controlled by only regulating the gas-flow rate pumped into the cultural broth in which sodium acetate (C2) and propionate (C3) were used as carbon sources. Productivity of PHA and the PHA compositions at various C2 to C3 ratios were also investigated. When ORP was maintained at +30 mV, 35% (w/w) of PHA of cell dry weight obtained when C2 was used as sole carbon source. The PHA copolymer, poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), accumulated by EAS with different 3-hydroxyvalarate (3HV) molar fractions ranged from 8% to 78.0% when C2 and C3 was used as sole carbon source, By using ORP to monitor and control the fermentation process instead DO meter, the ORP system provided more precise control to the PHA accumulation process from EAS under low dissolved oxygen (DO) concentrations. Adjusting the C2 to C3 ratios in the media could control the composition such as the 3HV/3HB ratios of the PHBV. Furthermore, it might be an effective way to adjust the 3HV molar fractions in PHBV by controlling the DO concentration via the ORP monitoring system. The 3HV molar fractions in the PHBV declined with increasing ORP from −30 mV to +100 mV by adjusting the gas-flow rate (i.e. the DO concentration). It is concluded that the DO plays a very important role in the synthesis of 3HV subunits in PHBV co-polymer from the EAS. Therefore, a hypothetic metabolic model for PHA synthesis from EAS was proposed to try to explain the results in this study.

Keywords: Activated sludge; oxidation-reduction potential; ORP; polyhydroxyalkanoate; PHA

Enzyme pretreatment of grass lignocellulose for potential high-value co-products and an improved fermentable substrate by William F. Anderson; Joy Peterson; Danny E. Akin; W. Herbert Morrison III (pp. 303-310).

High-yield bacillus subtilis protease production by solid-state fermentation by Valeria F. Soares; Leda R. Castilho; Elba P. S. Bon; Denise M. G. Freire (pp. 311-319).

A Bacillus subtilis isolate was shown to be able to produce extracellular protease in solid-state fermentations (SSF) using soy cake as culture medium. A significant effect of inoculum concentration and physiological age on protease production was observed. Maximum activities were obtained for inocula consisting of exponentially growing cells at inoculum concentrations in the range of 0.7–2.0 mg g⁻¹. A comparative study on the influence of cultivation temperature and initial medium pH on protease production in SSF and in submerged fermentation (SF) revealed that in SSF a broader pH range (5–10), but the same optimum temperature (37°C), is obtained when compared to SF. A kinetic study showed that enzyme production is associated with bacterial growth and that enzyme inactivation begins before biomass reaches a maximum level for both SF and SSF. Maximum protease activity and productivity were 960 U g⁻¹ and 15.4 U g⁻¹ h⁻¹ for SSF, and 12 U mL⁻¹ and 1.3 U mL⁻¹ h⁻¹ for SF. When SSF protease activity was expressed by volume of enzyme extract, the enzyme level was 10-fold higher and the enzyme productivity 45% higher than in SF. These results indicate that this bacterial strain shows a high biotechnological potential for protease production in solid-state fermentation.

Keywords: Bacillus subtilis ; protease; solid-state fermentation; submerged fermentation; soy cake

Profile of enzyme production by trichoderma reesei grown on corn fiber fractions by Xin-Liang Li; Bruce S. Dien; Michael A. Cotta; Y. Victor Wu; Badal C. Saha (pp. 321-334).

Corn fiber is the fibrous by-product of wet-mill corn processing. It typically consists of about 20% starch, 14% cellulose, and 30% hemicellulose in the form of arabinoxylan. Crude corn fiber (CCF) was fractionated into de-starched corn fiber (DSCF), corn fiber with cellulose (CFC) enriched, and corn fiber arabinoxylan (CFAX), and these fractions were evaluated as substrates for enzyme production by Trichoderma reesei. T. reesei QM9414 and Rut C-30 grew on CCF, DSCF, CFC, or CFAX and secreted a number of hydrolytic enzymes. The enzymes displayed synergism with commercial cellulases for corn fiber hydrolysis.

Keywords: Trichoderma reesei ; corn fiber; cellulase; xylanase

Partial purification and characterization of protease enzyme from Bacillus subtilis megatherium by Ayse Gerze; Didem Omay; Yuksel Guvenilir (pp. 335-345).

Bacteria of genus Bacillus are active producers of extracellular proteases, and characteristics of enzyme production by Bacillus species have been well studied. The aim of this experimental study is isolation and partial purification of protease enzyme from the Bacillus subtilis megatherium bacteria species. Protease enzyme is obtained by inducing spore genesis of bacteria from Bacillus species on suitable media. The partial purification was reali-zed by applying successively ammonium sulfate precipitation, dialysis, DEAE-cellulose ion exchange chromatography to the supernatant. In this study, the effect of substrate concentration, reaction time, the effect of inhibitor and activator on the optimum pH, optimum temperature, pH stability, and temperature stability was determined. Molecular weight of the obtained enzyme was investigated by SDS-PAGE. In this study, the specific activity of the supernatant, which was partially purified from Bacillus subtilis megatherium bacteria, was 10.4 U/mg, specific activity of supernatant was 13.5 U/mg after 80% ammonium sulfate fractionation. The final enzyme preparation was 1.1-fold purer than the crude homogenate. Molecular weight of the protease was determined, and it was found that the weight of enzyme was 45 kDa by using SDS-PAGE.

Keywords: Enzyme; protease; Bacillus subtilis megatherium ; purification; isolation

Effect of media composition and growth conditions on production of β-glucosidase by Aspergillus niger C-6 by O. García-Kirchner; M. Segura-Granados; P. Rodríguez-Pascual (pp. 347-359).

The hydrolytic activity of fungal originated β-glucosidase is exploited in several biotechnological processes to increase the rate and extent of saccharification of several cellulosic materials by hydrolyzing the cellobiose which inhibits cellulases. In a previous presentation, we reported the screening and liquid fermentation with Aspergillus niger, strain C-6 for β-glucosidase production at shake flask cultures in a basal culture medium with mineral salts, corn syrup liquor, and different waste lignocellulosic materials as the sole carbon source obtaining the maximum enzymatic activity after 5–6 d of 8.5 IU/mL using native sugar cane bagasse. In this work we describe the evaluation of fermentation conditions: growth temperature, medium composition, and pH, also the agitation and aeration effects for β-glucosidase production under submerged culture using a culture media with corn syrup liquor (CSL) and native sugar cane bagasse pith as the sole carbon source in a laboratory fermenter. The maximum enzyme titer of 7.2 IU/mL was obtained within 3 d of fermentation. This indicates that β-glucosidase productivity by Aspergillus niger C-6 is function of culture conditions, principally temperature, pH, culture medium conditions, and the oxygen supply given in the bioreactor. Results obtained suggest that this strain is a potential microorganism that can reach a major level of enzyme production and also for enzyme characterization.

Keywords: Sugar cane bagasse pith; Aspergillus niger ; β-glucosidase; corn syrup liquor; oxygen supply

A sequential enzymatic microreactor system for ethanol detection of gasohol mixtures by Eliana M. Alhadeff; Andréa M. Salgado; Nei Pereira Jr.; Belkis Valdman (pp. 361-371).

A sequential enzymatic double microreactor system with dilution line was developed for quantifying ethanol from gasohol mixtures, using a colorimetric detection method, as a new proposal to the single micro reactor system used in previous work. Alcohol oxidase (AOD) and horseradish peroxidase (HRP) immobilized on glass beads, one in each microreactor, were used with phenol and 4-aminophenazone and the red-colored product was detected with a spectrophotometer at 555 nm. Good results were obtained with the immobilization technique used for both AOD and HRP enzymes, with best retention efficiencies of 95.3 ± 2.3% and 63.2 ± 7.0%, respectively. The two microreactors were used to analyze extracted ethanol from gasohol blends in the range 1–30 % v/v (10.0–238.9 g ethanol/L), with and without an on-line dilution sampling line. A calibration curve was obtained in the range 0.0034–0.087 g ethanol/L working with the on-line dilution integrated to the biosensor—FIA system proposed. The diluted sample concentrations were also determined by gas chromatography (GC) and high-pressure liquid chromatography (HPLC) methods and the results compared with the proposed sequential system measurements. The effect of the number of analysis performed with the same system was also investigated.

Keywords: Biosensors; ethanol; flow injection analysis; alcohol oxidase; horseradish peroxidase

Microbial catalysis and metabolic engineering by Johannes P. van Dijken; Gregory M. Luli (pp. 375-377).

Bioabatement to remove inhibitors from biomass-derived sugar hydrolysates by Nancy N. Nichols; Bruce S. Dien; Gema M. Guisado; Maria J. López (pp. 379-390).

Bioabatement is a potential method to remove inhibitory compounds from lignocellulose hydrolysates that could be incorporated into a scheme for fermentation of ethanol from cellulose. Coniochaeta ligniaria NRRL30616, an Ascomycete that metabolizes furfural and 5-hydroxymethylfurfural, is a unique strain that may be useful for detoxifying biomass sugars. NRRL30616 and 23 related fungal strains were screened for the ability to metabolize furans and grow in dilute-acid hydrolysate of corn stover. NRRL30616 was the best strain for removal of inhibitors from hydrolysate, and abatement of hydrolysate by inoculation with the strain allowed subsequent yeast fermentation of cellulose to ethanol.

Keywords: Biomass; lignocellulose; inhibitor abatement; bioremediation; fermentation; detoxification; ethanol

Cloning, expression, purification, and analysis of mannitol dehydrogenase gene mtlK from Lactobacillus brevis by Siqing Liu; Badal Saha; Michael Cotta (pp. 391-401).

The commercial production of mannitol involves high-pressure hydrogenation of fructose using a nickel catalyst, a costly process. Mannitol can be produced through fermentation by microorganisms. Currently, a few Lactobacillus strains are used to develop an efficient process for mannitol bioproduction; most of the strains produce mannitol from fructose with other products. An approach toward improving this process would be to genetically engineer Lactobacillus strains to increase fructose-to-mannitol conversion with decreased production of other products. We cloned the gene mtlK encoding mannitol-2-dehydrogenase (EC 1.1.1.67) that catalyzes the conversion of fructose into mannitol from Lactobacillus brevis using genomic polymerase chain reaction. The mtlK clone contains 1328 bp of DNA sequence including a 1002-bp open reading frame that consisted of 333 amino acids with a predicted molecular mass of about 36 kDa. The functional mannitol-2-dehydrogenase was produced by overexpressing mtlK via pRSETa vector in Escherichia coli BL21pLysS on isopropyl-β-d-thiogalactopyranoside induction. The fusion protein is able to catalyze the reduction of fructose to mannitol at pH 5.35. Similar rates of catalytic reduction were observed using either the NADH or NADPH as cofactor under in vitro assay conditions. Genetically engineered Lactobacillus plantarum TF103 carrying the mtlK gene of L. brevis indicated increased mannitol production from glucose. The evaluation of mixed sugar fermentation and mannitol production by this strain is in progress.

Keywords: Lactic acid bacteria; mannitol-2-dehydrogenase; NAD(P)H; mtlK ; mannitol fermentation; Lactobacillus

Continuous hydrogen photoproduction by Chlamydomonas reinhardtii by Alexander S. Fedorov; Sergey Kosourov; Maria L. Ghirardi; Michael Seibert (pp. 403-412).

This study demonstrates, for the first time, that it is possible to couple sulfate-limited Chlamydomonas reinhardtii growth to continuous H₂ photoproduction for more than 4000 h. A two-stage chemostat system physically separates photosynthetic growth from H₂ production, and it incorporates two automated photobioreactors (PhBRs). In the first PhBR, the algal cultures are grown aerobically in chemostat mode under limited sulfate to obtain photosynthetically competent cells. Active cells are then continuously delivered to the second PhBR, where H₂ production occurs under anaerobic conditions. The dependence of the H₂ production rate on sulfate concentration in the medium, dilution rates in the PhBRs, and incident light intensity is reported.

Keywords: Green algae; sulfur deprivation; photobioreactor; chemostat; H₂ production; Chlamydomonas reinhardtii

Effects of aliphatic acids, furfural, and phenolic compounds on Debaryomyces hansenii CCMI 941 by Luís C. Duarte; Florbela Carvalheiro; Inês Neves; Francisco M. Gírio (pp. 413-425).

Debaryomyces hansenii is a polyol overproducing yeast that can have a potential use for upgrading lignocellulosic hydrolysates. Therefore, the establishment of its tolerance to metabolic inhibitors found in hydrolysates is of major interest. We studied the effects of selected aliphatic acids, phenolic compounds, and furfural. Acetic acid favored biomass production for concentrations <6.0 g/L. Formic acid was more toxic than acetic acid and induced xylitol accumulation (maximum yield of 0.21 g/g of xylose). All tested phenolics strongly decreased the specific growth rate. Increased toxicity was found for hydroquinone, syringaldehyde, and 4-methylcatechol and was correlated to the compound’s hydrophobicity. Increasing the amount of furfural led to longer lag phases and had a detrimental effect on specific growth rate and biomass productivity.

Keywords: Lignocellulosic byproducts; acetic acid; formic acid; hydroquinone; syringaldehyde; 4-methylcatechol; furfural; inhibition

Evaluation of inoculum of Candida guilliermondii grown in presence of glucose on xylose reductase and xylitol dehydrogenase activities and xylitol production during batch fermentation of sugarcane bagasse hydrolysate by Débora Danielle Virgínio da Silva; Maria das Graças de Almeida Felipe; Ismael Maciel de Mancilha; Sílvio Silvério da Silva (pp. 427-437).

The effect of glucose on xylose-xylitol metabolism in fermentation medium consisting of sugarcane bagasse hydrolysate was evaluated by employing an inoculum of Candida guilliermondii grown in synthetic media containing, as carbon sources, glucose (30 g/L), xylose (30 g/L), or a mixture of glucose (2 g/L) and xylose (30 g/L). The inoculum medium containing glucose promoted a 2.5-fold increase in xylose reductase activity (0.582 IU/mg_prot) and a 2-fold increase in xylitol dehydrogenase activity (0.203 IU/mg_prot) when compared with an inoculum-grown medium containing only xylose. The improvement in enzyme activities resulted in higher values of xylitol yield (0.56 g/g) and productivity (0.46 g/[L·h]) after 48 h of fermentation.

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

Effect of surface attachment on synthesis of bacterial cellulose by Barbara R. Evans; Hugh M. O’Neill (pp. 439-450).

Gluconacetobacter spp. synthesize a pure form of hydrophilic cellulose that has several industrial specialty applications. Literature reports have concentrated on intensive investigation of static and agitated culture in liquid media containing high nutrient concentrations optimized for maximal cellulose production rates. The behavior of these bacteria on semisolid and solid surfaces has not been specifically addressed. The species Gluconacetobacter hansenii was examined for cellulose synthesis and colony morphology on a range of solid supports, including cotton linters, and on media thickened with agar, methyl cellulose, or gellan. The concentration and chemical structure of the thickening agent were found to be directly related to the formation of contiguous cellulose pellicules. Viability of the bacteria following freezer storage was improved when the bacteria were frozen in their cellulose pellicules.

Keywords: Gluconacetobacter hansenii ; cellulose; pellicule; gellan; agar

Enhanced biotransformation of furfural and hydroxymethylfurfural by newly developed ethanologenic yeast strains by Z. Lewis Liu; Patricia J. Slininger; Steve W. Gorsich (pp. 451-460).

Furfural and hydroxymethylfurfural (HMF) are representative inhibitors among many inhibitive compounds derived from biomass degradation and saccharification for bioethanol fermentation. Most yeasts, including industrial strains, are susceptible to these inhibitory compounds, especially when multiple inhibitors are present. Additional detoxification steps add cost and complexity to the process and generate additional waste products. To promote efficient bioethanol production, we studied the mechanisms of stress tolerance, particularly to fermentation inhibitors such as furfural and HMF. We recently reported a metabolite of 2,5-bis-hydroxymethylfuran as a conversion product of HMF and characterized a dose-dependent response of ethanologenic yeasts to inhibitors. In this study, we present newly adapted strains that demonstrated higher levels of tolerance to furfural and HMF. Saccharomyces cerevisiae 307-12H60 and 307-12H120 and Pichia stipitis 307 10H60 showed enhanced biotransformation ability to reduce HMF to 2,5-bis-hydroxymethylfuran at 30 and 60 mM, and S. cerevisiae 307-12-F40 converted furfural into furfuryl alcohol at significantly higher rates compared to the parental strains. Strains of S. cerevisiae converted 100% of HMF at 60 mM and S. cerevisiae 307-12-F40 converted 100% of furfural into furfuryl alcohol at 30 mM. The results of this study suggest a possible in situ detoxification of the inhibitors by using more inhibitor-tolerant yeast strains for bioethanol fermentation. The development of such tolerant strains provided a basis and useful materials for further studies on the mechanisms of stress tolerance.

Keywords: Hydroxymethylfurfural; 2,5-bis-hydroxymethylfuran; biotransformation; furfural; furfuryl alcohol

Sections

Personal tools

Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.121, #1-3)

Sections

Personal tools

Local resources

Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.121, #1-3)