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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.106, #1-3)
d-xylose transport by Candida succiphila and Kluyveromyces marxianus by Boris U. Stambuk; Mary Ann Franden; Arjun Singh; Min Zhang (pp. 255-263).
The kinetics and regulation of d-xylose uptake were investigated in the efficient pentose fermentor Candida succiphila, and in Kluyveromyces marxianus, which assimilate but do not ferment pentose sugars. Active high-affinity (K m ∼ 3.8 mM; V max ∼ 15 nmol/[mg·min]) and putative facilitated diffusion low-affinity (K m ∼ 140 mM; V max ∼ 130 nmol/[mg·min]) transport activities were found in C. succiphila grown, respectively, on xylose or glucose. K. marxianus showed facilitated diffusion low-affinity (K m ∼ 103 mM; V max ∼ 190 nmol/[mg·min]) transport activity when grown on xylose under microaerobic conditions, and both a low-affinity and an active high-affinity (K m ∼ 0.2 mM; V max ∼ 10 nmol/[mg·min]) transport activity when grown on xylose under fully aerobic conditions.
Keywords: d-Xylose; transport kinetics; fermentation; Candida succiphila ; Kluyveromyces marxianus
Molecular characterization of a gene for aldose reductase (CbXYL1) from Candida boidinii and its expression in Saccharomyces cerevisiae by Min Hyung Kang; Haiying Ni; Thomas W. Jeffries (pp. 265-276).
Candida boidinii produces significant amounts of xylitol from xylose, and assays of crude homogenates for aldose (xylose) reductase (XYL1p) have been reported to show relatively high activity with NADH as a cofactor even though XYL1p purified from this yeast does not have such activity. A gene coding for XYL1p from C. boidinii (CbXYL1) was isolated by amplifying the central region using primers to conserved domains and by genome walking. CbXYL1 has an open reading frame of 966 bp encoding 321 amino acids. The C. boidinii XYL1p is highly similar to other known yeast aldose reductases and is most closely related to the NAD(P)H-linked XYL1p of Kluyveromyces lactis. Cell homogenates from C. boidinii and recombinant Saccharomyces cerevisiae were tested for XYL1p activity to confirm the previously reported high ratio of NADH:NADPH linked activity. C. boidinii grown under fully aerobic conditions showed an NADH:NADPH activity ratio of 0.76, which was similar to that observed with the XYL1p from Pichia stipitis XYL1, but which is much lower than what was previously reported. Cells grown under low aeration showed an NADH:NADPH activity ratio of 2.13. Recombinant S. cerevisiae expressing CbXYL1 showed only NADH-linked activity in cell homogenates. Southern hybridization did not reveal additional bands. These results imply that a second, unrelated gene for XYL1p is present in C. boidinii.
Keywords: Candida boidinii ; Saccharomyces cerevisiae ; aldose reductase; CbXYL1 ; xylose reductase; NADH; NADPH; gene cloning; gene expression
Changing flux of xylose metabolites by altering expression of xylose reductase and xylitol dehydrogenase in recombinant Saccharomyces cerevisiae by Yong-Su Jin; Thomas W. Jeffries (pp. 277-285).
We changed the fluxes of xylose metabolites in recombinant Saccharomyces cerevisiae by manipulating expression of Pichia stipitis genes (XYL1 and XYL2) coding for xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively. XYL1 copy number was kept constant by integrating it into the chromosome. Copy numbers of XYL2 were varied either by integrating XYL2 into the chromosome or by transforming cells with XYL2 in a multicopy vector. Genes in all three constructs were under control of the strong constitutive glyceraldehyde-3-phosphate dehydrogenase promoter. Enzymatic activity of XR and XDH in the recombinant strains increased with the copy number of XYL1 and XYL2. XR activity was not detected in the parent but was present at a nearly constant level in all of the transformants. XDH activity increased 12-fold when XYL2 was on a multicopy vector compared with when it was present in an integrated single copy. Product formation during xylose fermentation was affected by XDH activity and by aeration in recombinant S. cerevisiae. Higher XDH activity and more aeration resulted in less xylitol and more xylulose accumulation during xylose fermentation. Secretion of xylulose by strains with multicopy XYL2 and elevated XDH supports the hypothesis that d-xylulokinase limits metabolic flux in recombinant S. cerevisiae.
Keywords: Metabolic flux; metabolic engineering; xylose; xylose reductase; xylitol dehydrogenase
Effect of media on spore yield and thermal resistance of Bacillus stearothermophilus by Thereza Christina Vessoni Penna; Irene A. Machoshvili; Marina Ishii (pp. 287-294).
The interference of eight components in the yield of sporulation and thermal resistance to moist heat (121°C) of Bacillus stearothermophilus spores suspended in 0.02 M calcium acetate solution and inoculated on paper strips previously treated with calcium acetate/calcium hydroxide was studied. The spore yield of 1.0×108/mL was developed at 62°C in 17 media containing different concentrations of d-glucose, sodium chloride, l-glutamic acid, yeast extract, peptone, manganese sulfate, potassium phosphate, and ammonium phosphate. The combined effects of yeast extract, peptone, and glucose contributed positively to the spore yield and to the stability of the thermal resistance of both spores in suspension and on strips.
Keywords: Bacillus stearothermophilus ; thermoresistance; sporulation; bioindicator
Cassava flour wastewater as a substrate for biosurfactant production by Marcia Nitschke; Glaucia M. Pastore (pp. 295-301).
Five cassava flour wastewater (manipueira) preparations were tested as culture media for biosurfactant production by a wild-type Bacillus sp. isolate. No-solids (F), no-solids diluted (F/2), natural (I), natural diluted (I/2), and decanted (IPS) were the tested manipueira media. The microorganism was able to grow and to produce biosurfactant on all manipueira preparations. The media whose solids were removed (F and F/2) showed better results than preparations with the presence of solids (I, I/2, and IPS). No-solids medium (F) showed a surface tension of 26,59 mN/m and reciprocal of critical micelle concentration of over 100 and was selected as a potential substrate for biosurfactant production.
Keywords: Biosurfactant; Bacillus sp.; cassava flour wastewater
A new oxygen sensitivity and its potential application in photosynthetic H2 production by James W. Lee; Elias Greenbaum (pp. 303-313).
We have discovered a new competitive pathway for O2 sensitivity in algal H2 production that is distinct from the O2 sensitivity of hydrogenase per se. This O2 sensitivity is apparently linked to the photosynthetic H2 production pathway that is coupled to proton translocation across the thylakoid membrane. Addition of the proton uncoupler carbonyl cyanide-p-trifluoromethoxy-phenylhydrazone eliminates this mode of O2 inhibition on H2 photoevolution. This newly discovered inhibition is most likely owing to background O2 that apparently serves as a terminal electron acceptor in competition with the H2 production pathway for photosynthetically generated electrons from water splitting. This O2-sensitive H2 production electron transport pathway was inhibited by 3[3,4-dichlorophenyl]1,1-dimethylurea. Our experiments demonstrated that this new pathway is more sensitive to O2 than the traditionally known O2 sensitivity of hydrogenase. This discovery provides new insight into the mechanism of O2 inactivation of hydrogenase and may contribute to the development of a more-efficient and robust system for photosynthetic H2 production.
Keywords: Oxygen sensitivity; H2 production; photosynthetic H2 production; H2 production pathways; hydrogenase
Optimization of SO2-catalyzed steam pretreatment of corn fiber for ethanol production by Renata Bura; Rodney J. Bothast; Shawn D. Mansfield; John N. Saddler (pp. 319-335).
A batch reactor was employed to steam explode corn fiber at various degrees of severity to evaluate the potential of using this feedstock as part of an enzymatically mediated cellulose-to-ethanol process. Severity was controlled by altering temperature (150–230°C), residence time (1–9 min), and SO2 concentration (0–6% [w/w] dry matter). The effects of varying the different parameters were assessed by response surface modeling. The results indicated that maximum sugar yields (hemicellulose-derived water soluble, and cellulose-derived following enzymatic hydrolysis) were recovered from corn fiber pretreated at 190°C for 5 minutes after exposure to 3% SO2. Sequential SO2-catalyzed steam explosion and enzymatic hydrolysis resulted in a conversion efficiency of 81% of the combined original hemicellulose and cellulose in the corn fiber to monomeric sugars. An additional posthydrolysis step performed on water soluble hemicellulose stream increased the concentration of sugars available for fermentation by 10%, resulting in the high conversion efficiency of 91%. Saccharomyces cerevisiae was able to ferment the resultant corn fiber hydrolysates, perhydrolysate, and liquid fraction from the posthydrolysis steps to 89, 94, and 85% of theoretical ethanol conversion, respectively. It was apparent that all of the parameters investigated during the steam explosion pretreatment had a significant effect on sugar recovery, inhibitory formation, enzymatic conversion efficiency, and fermentation capacity of the yeast.
Keywords: Corn fiber; steam pretreatment; enzymatic hydrolysis; posthydrolysis; fermentation; ethanol
A comprehensive kinetic model for dilute-acid hydrolysis of cellulose by Qian Xiang; Jun Seok Kim; Y. Y. Lee (pp. 337-352).
Acid-catalyzed hydrolysis is controlled not only by temperature and acid concentration but also by the physical state of the cellulose. Under low temperature and acid condition the cellulose structure stays in stable crystalline form. Therefore, the prevailing reaction mode is endwise hydrolysis. Glucose then becomes the main sugar product. However, when temperature and/or acid concentration is raised to a certain level, the cellulose structure becomes unstable by breakage of hydrogen bonding, the primary force that holds the cellulose chains. Once the crystalline structure of the cellulose is disrupted, acid molecules can penetrate into the inner layers of the cellulose chains. In support of this hypothesis, we have experimentally verified that a substantial amount of oligomers is formed as reaction intermediates under extremely low-acid and high-temperature conditions. We also found that the breakage of hydrogen bonds occurs rather abruptly in response to temperature. One such condition is 210°C, 0.07% H2SO4. Glucose, once it is formed in the hydrolysate, interacts with acid-soluble lignin, forming a lignin-carbohydrate complex. This occurs concurrently with other reactions involving glucose such as decomposition and reversion. On the basis of these findings, a comprehensive kinetic model is proposed. This model is in full compliance with our recent experimental data obtained under a broad range of reaction conditions.
Keywords: Acid hydrolysis; cellulose; kinetics; model; hydrogen bond; oligomer; lignin
Effect of agitation on removal of acetic acid from pretreated hydrolysate by activated carbon by Sarah A. Priddy; Thomas R. Hanley (pp. 353-364).
The effect of agitation on the adsorption of acetic acid by activated carbon was tested utilizing an external mass transfer-diffusion model. Simulated pretreated biomass was contacted with activated carbon under prescribed conditions of temperature and agitation. Adsorption isotherm studies are presented as well as batch kinetic rate studies. Use of these data enabled the determination of isotherm constants, an external mass transfer coefficient, and an effective diffusivity for each agitation rate studied. The external film coefficient results ranged from 33.62 µm/s to a complete absence of external mass transfer resistance, and the diffusivity results ranged from 0.8625 to 10.70 µm2/s. The optimum combination of no external film resistance, and highest diffusivity, 10.70 µm2/s, occurred at 250 rpm and 25°C. The results of these models and the experimental parameters suggested an efficacious method and conditions for the removal of this undesirable chemical.
Keywords: Adsorption; activated carbon; external mass transfer; effective diffusivity; detoxification
Enzymatic digestibility of used newspaper treated with aqueous ammonia-hydrogen peroxide solution by Sung Bae Kim; Nam Kyu Moon (pp. 365-373).
Wastepaper constitutes approximately half of municipal solid waste, making it a potential source of bioenergy. Newspaper was pretreated with an ammonia-hydrogen peroxide (H2O2) mixture in a shaking bath from room temperature to 80°C, and then its enzymatic digestibility was measured. A significant amount of ink was removed from the newspaper slurry by the reciprocating movement of the shaking bath. In addition, the ammonia-H2O2 significantly swelled the substrate, thereby greatly increasing its susceptibility to enzymatic digestion. After pretreating the newspaper with conditions of 40°C, 3 h, 130 strokes/min, and 4 wt% ammonia-2 wt% H2O2, the enzymatic digestibility was almost 90% of theoretical, or about 25% higher than that of untreated substrate. Digestibility was also investigated as a function of ammonia concentration, H2O2 concentration, shaking speed, pretreatment temperature, and time.
Keywords: Pretreatment; newspaper; ammonia; hydrogen peroxide; enzymatic digestibility
Continuous production of butanol from starch-based packing peanuts by Thaddeus C. Ezeji; Marisa Groberg; Nasib Qureshi; Hans P. Blaschek (pp. 375-382).
Acetone, butanol, ethanol (ABE, or solvents) were produced from starch-based packing peanuts in batch and continuous reactors. In a batch reactor, 18.9 g/L of total ABE was produced from 80 g/L packing peanuts in 110 h of fermentation. The initial and final starch concentrations were 69.6 and 11.1 g/L, respectively. In this fermentation, ABE yield and productivity of 0.32 and 0.17 g/(L·h) were obtained, respectively. Compared to the batch fermentation, continuous fermentation of 40 g/L of starch-based packing peanuts in P2 medium resulted in a maximum solvent production of 8.4 g/L at a dilution rate of 0.033 h−1. This resulted in a productivity of 0.27 g/(L·h). However, the reactor was not stable and fermentation deteriorated with time. Continuous fermentation of 35 g/L of starch solution resulted in a similar performance. These studies were performed in a vertical column reactor using Clostridium beijerinckii BA101 and P2 medium. It is anticipated that prolonged exposure of culture to acrylamide, which is formed during boiling/autoclaving of starch, affects the fermentation negatively.
Keywords: Starch-based packing peanuts; continuous fermentation; butanol; Clostridium beijerinckii BA101; reactor; dilution rate
Measurement of rheological properties of corn stover suspensions by Natalia V. Pimenova; Thomas R. Hanley (pp. 383-392).
Corn stover is currently being evaluated as a feedstock for ethanol production. The corn stover suspensions fed to reactors typically range between 10 and 40% solids. To simulate and design bioreactors for processing highly loaded corn stover suspensions, the rheologic properties of the suspension must be measured. In systems with suspended solids, rheologic measurements are difficult to perform owing to settling in the measurement devices. In this study, viscosities of corn stover suspensions were measured using a helical ribbon impeller viscometer. A calibration procedure is required for the impeller method in order to obtain the shear rate constant, k, which is dependent on the geometry of the measurement system. The corn stover suspensions are described using a power law flow model.
Keywords: Corn stover; rheological properties; helical impeller; cone-and-plate impeller; power law parameters
Effect of Bacillus circulans D1 thermostable xylanase on biobleaching of eucalyptus kraft pulp by Daniela A. Bocchini; Valquiria B. Damiano; Eleni Gomes; Roberto Da Silva (pp. 393-401).
The alkalophilic Bacillus circulans D1 was isolated from decayed wood. It produced high levels of extracellular cellulase-free xylanase. The enzyme was thermally stable up to 60°C, with an optimal hydrolysis temperature of 70°C. It was stable over a wide pH range (5.5—10.5), with an optimum pH at 5.5 and 80% of its activity at pH 9.0. This cellulase-free xylanase preparation was used to biobleach kraft pulp. Enzymatic treatment of kraft pulp decreased chlorine dioxide use by 23 and 37% to obtain the same kappa number (κ number) and brightness, respectively. Separation on Sephadex G-50 isolated three fractions with xylanase activity with distinct molecular weights.
Keywords: Bacillus circulans ; biobleaching; kraft pulp; thermophilic; xylanase
Fungi allergens produced by solid-state fermentation process by Salah D. M. Hasan; Walderez Gambale; Ricardo L. Zollner; Maria H. A. Santana (pp. 403-412).
Allergenic extracts were produced from Drechslera (Helminthosporium) monoceras biomass cultured by solid-state fermentation using wheat bran as the substrate. The main fermentation variables were selected by statistical design, and the optimized biomass yield (1.43 mg/[g of dry substrate · d]) was obtained at pH 9.5 and 45.8% moisture. The allergenic extracts were produced from crude extract by protein precipitation and polyphenol removal. Proteins in the range of 16–160 kDa were identified in the extracts. Their reactions in patients were characterized by in vivo cutaneous tests (positive in 40% of the atopic patients) and by dot-blotting assays.
Keywords: Allergenic extract; Drechslera (Helminthosporium) monoceras ; solid-state fermentation; statistical experimental design; wheat bran; proteins; biomass
Hybrid model for an enzymatic reactor by Ruy Sousa Jr.; Mariam M. Resende; Raquel L. C. Giordano; Roberto C. Giordano (pp. 413-422).
Cheese whey proteolysis, carried out by immobilized enzymes, can either change or evidence functional properties of the produced peptides, increasing the potential applications of this byproduct of the dairy industry. Optimization and scale-up of the enzymatic reactor relies on its mathematical model—a set of mass balance equations, with reaction rates usually given by Michaelis-Menten-like kinetics; no information about the distribution of peptides’ molecular sizes is supplied. In this article, a hybrid model of a batch enzymatic reactor is presented, consisting of differential mass balances coupled to a “neural-kinetic model,” which provides the molecular weight distributions of the resulting peptides.
Keywords: Cheese whey proteolysis; enzymatic reactor; hybrid model; mass balance equations; artificial neural networks
Preliminary investigation of fungal bioprocessing of wheat straw for production of straw-thermoplastic composites by David N. Thompson; Tracy P. Houghton; Jeffrey A. Lacey; Peter G. Shaw; J. Richard Hess (pp. 423-436).
Straw utilization for composites is limited by poor resin and polymer penetration, and excessive resin consumption owing to the straw cuticle, fines, and lignin-hemicellulose matrix. White-rot fungi degrade these components of straw and could, therefore, potentially be used to improve resin penetration and resin binding without the use of physical or chemical pretreatments. Although long treatment times and large footprints the limit use of fungal treatments on a large scale, distributed fungal pretreatments could alleviate land requirements. In this article, we present progress toward the development of a passive fungal straw upgrading system utilizing whiterot fungi.
Keywords: Fungal upgrading; white-rot fungi; wheat straw; Pleurotus ostreatus ; straw composite
Simulation of aerated lagoon using artificial neural networks and multivariate regression techniques by Karla Patricia Oliveira-Esquerre; Aline C. da Costa; Roy Edward Bruns; Milton Mori (pp. 437-449).
The aim of this study was to develop an empirical model that provides accurate predictions of the biochemical oxygen demand of the output stream from the aerated lagoon at International Paper of Brazil, one of the major pulp and paper plants in Brazil. Predictive models were calculated from functional link neural networks (FLNNs), multiple linear regression, principal components regression, and partial least-squares regression (PLSR). Improvement in FLNN modeling capability was observed when the data were preprocessed using the PLSR technique. PLSR also proved to be a powerful linear regression technique for this problem, which presents operational data limitations.
Keywords: Biochemical oxygen demand; functional link neural networks; partial least squares; principal components regression; multiple linear regression
Simplistic modeling approach to heterogeneous dilute-acid hydrolysis of cellulose microcrystallites by Pär O. Pettersson; Robert W. Torget; Robert Eklund; Qian Xiang; Y. Y. Lee; Guido Zacchi (pp. 451-455).
The classic kinetic model for cellulose hydrolysis is often referred to as pseudo-homogeneous, a term revealing the insight that the process is actually heterogeneous. During the past 10–15 yr, the shortcomings of this model have been demonstrated in various studies and the interest in the heterogeneous aspects has increased. The present work presents a simplistic model in which the intrinsic, heterogeneous hydrolysis and transport rates are coupled by the assumption of a constant glucosidic surface concentration. The mechanisms affecting these two rates are largely unknown, but the model serves as a guideline for further exploration of the process.
Keywords: Dilute-acid hydrolysis; kinetic model; heterogeneous model
Cellulosic fuel ethanol by Hugh G. Lawford; Joyce D. Rousseau (pp. 457-469).
Iogen (Canada) is a major manufacturer of industrial cellulase and hemicellulase enzymes for the textile, pulp and paper, and poultry feed industries. Iogen has recently constructed a 40 t/d biomass-to-ethanol demonstration plant adjacent to its enzyme production facility. The integration of enzyme and ethanol plants results in significant reduction in production costs and offers an alternative use for the sugars generated during biomass conversion. Iogen has partnered with the University of Toronto to test the fermentation performance characteristics of metabolically engineered Zymomonas mobilis created at the National Renewable Energy Laboratory. This study focused on strain AX101, a xylose- and arabinose-fermenting stable genomic integrant that lacks the selection marker gene for antibiotic resistance. The “Iogen Process” for biomass depolymerization consists of a dilute-sulpfuric acid-catalyzed steam explosion, followed by enzymatic hydrolysis. This work examined two process design options for fermentation, first, continuous cofermentation of C5 and C6 sugars by Zm AX101, and second, separate continuous fermentations of prehydrolysate by Zm AX101 and cellulose hydrolysate by either wildtype Z. mobilis ZM4 or an industrial yeast commonly used in the production of fuel ethanol from corn. Iogen uses a proprietary process for conditioning the prehydrolysate to reduce the level of inhibitory acetic acid to at least 2.5 g/L. The pH was controlled at 5.5 and 5.0 for Zymomonas and yeast fermentations, respectively. Neither 2.5 g/L of acetic acid nor the presence of pentose sugars (C6:C5 = 2:1) appreciably affected the high-performance glucose fermentation of wild-type Z. mobilis ZM4. By contrast, 2.5 g/L of acetic acid significantly reduced the rate of pentose fermentation by strain AX101. For single-stage continuous fermentation of pure sugar synthetic cellulose hydrolysate (60 g/L of glucose), wild-type Zymomonas exhibited a four-fold higher volumetric productivity compared with industrial yeast. Low levels of acetic acid stimulated yeast ethanol productivity. The glucose-to-ethanol conversion efficiency for Zm and yeast was 96 and 84%, respectively.
Keywords: Genomic integration; recombinant Zymomonas AX101; Zymomonas mobilis ; arabinose; xylose; ethanol; prehydrolysate; biomass hydrolysate; acetic acid; yeast
Effects of pressure pulsation on oxygen transfer rate measured by sulfite method by Wei-Cho Huang; Cheng S. Gong; George T. Tsao (pp. 471-480).
Pressure pulsation (PP) was investigated for its effects on oxygen transfer rate (OTR) measured by sulfite oxidation. By manipulating airflow rate, 0.41–1.2 vvm, and a control valve in a 4-L bioreactor, the frequency of PP was varied at different gas pressures3–15 psig. A mathematical model of OTR was built and compared to experimental data. OTR was also examined at constant gas pressure, 4.5–15.0 psig. The results indicate a good agreement between measurement and model prediction. OTR above 9 psig during PP showed significant enhancement at 25°C. This proves that PP not only affects the elevation of DO level, but also increases the interfacial area and mass transfer coefficient.
Keywords: Oxygen transfer; pressure pulsation; sulfite oxidation