Check out our New Publishers' Select for Free Articles

Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.91-93, #1-9)

Twenty years of trials, tribulations, and research progress in bioethanol technology by Charles E. Wyman (pp. 5-21).

The projected cost of ethanol production, from cellulosic biomass has been reduced by almost a factor of four over the last 20 yr. Thus, it is now competitive for blending with gasoline, and several companies are working to build the first plants. However, technology development faced challenges at all levels. Because the benefits of bioethanol were not well understood, it was imperative to clarify and differentiate its attributes. Process engineering was invaluable in focusing on promising opportunities for improvements, particularly in light of budget reductions, and in tracking progress toward a competitive goal. Now it is vital for one or more commercial projects to besuccessful, and improving our understanding of process fundamentals will reduce the time and costs for commercialization. Additionally, the cost of bioethanol, must be cut further to be competitive as a pure fuel in the open market, and aggressive technology advances are required to meet this target.

Keywords: biomass; biotechnology; ethanol; fuel; hydrolysis

Genetic improvement of poplar feedstock quality for ethanol production by Ronald J. Dinus (pp. 23-34).

Opportunities for matching chemical and physical properties of woody feedstocks to ethanol production process requirements via genetic improvement have long been recognized. Exploitation is now feasible owing to advances in trait measurement, breeding, and gene transfer technologies. Poplar genetic parameters are favorable largely for reducing lignin and increasing cellulose contents and specific gravity. Transgenic poplars with decreased lignin and increased cellulose contents, but otherwise normal growth and development, have been produced via genetic transformation. The long-standing debate on feasibility has thus become one of when, not if, designer varieties will become available.

Keywords: Biomass feedstock quality; lignin; cellulose; wood specific gravity; selection and breeding; genetic transformation

Detoxification of lignocellulose hydrolysates with ion-exchange resins by Nils-Olof Nilvebrant; Anders Reimann; Simona Larsson; Leif J. Jönsson (pp. 35-49).

Lignocellulose hydrolysates contain fermentation inhibitors causing decreased ethanol production. The inhibitors include phenolic compounds, furan aldehydes, and aliphatic acids. One of the most efficient methods for removing inhibiting compounds prior to fermentation is treatment of the hydrolysate with ion-exchange resins. The performance and detoxification mechanism of three different resins were examined: an anion exchanger, a cation exchanger, and a resin without charged groups (XAD8). A dilute acid hydrolysate of spruce was treated with the resins at pH 5.5 and 10.0 prior to ethanolic fermentation with Saccharomyces cerevisiae. In addition to the experiments with hydrolysate, the effect of the resins on selected model compounds, three phenolics (vanillin, guaiacol, and coniferyl aldehyde) and two furan aldehydes (furfural and hydroxymethyl furfural), was determined. The cation exchanger increased ethanol production, but to a lesser extent than XAD-8, which in turn was less effective than the an ion exchanger. Treatment at pH 10.0 was more effective than at pH 5.5. At pH 10.0, the anion exchanger efficiently removed both anionic and uncharged inhibitors, the latter by hydrophobic interactions. The importance of hydrophobic interactions was further indicated by a substantial decrease in the concentration of model compounds, such as guaiacol and furfural, after treatment with XAD-8.

Keywords: Detoxification; inhibition; ethanol production; Saccharomyces cerevisiae ; softwood; ion exchange

Fourier transform infrared quantitative analysis of sugars and lignin in pretreated softwood solid residues by Melvin P. Tucker; Quang A. Nguyen; Fannine P. Eddy; Kiran L. Kadam; Lynn M. Gedvilas; John D. Webb (pp. 51-61).

Hydrolysates were obtained from dilute sulfuric acid pretreatment of whole-tree softwood forest thinnings and softwood sawdust. Mid-infrared (IR) spectra were obtained on sample sets of wet washed hydrolysates, and 45°C vacuum-dried washed hydrolysates, using a Fourier transform infrared (FTIR) spectrophotometrer equipped with a diamond-composite attenuated total reflectance (ATR) cell. Partial least squares (PLS) analyiss of spectra from each sample set was performed. Regression analyses for sugar components and lignin were generated using results obtained from standard wet chemical and high-performance liquid chromatography methods. The correlation coefficients of the predicted and measured values were >0.9. The root mean square standard error of the estimate for each component in the residues was generally within 2 wt% of the measured value except where reported in the tables. The PLS regression analysis of the wet washed solids was similar to the PLS regression analysis on the 45°C vacuum-dried sample set. The FTIR-ATR technique allows mid-IR spectra to be obtained in a few minutes from wet washed or dried washed pretreated biomass solids. The prediction of the solids composition of an unknown washed pretreated solid is very rapid once the PLS method has been calibrated with known standard solid residues.

Keywords: Fourier transform infrared; biomass; softwood; dilute-acid pretreatment; acid hydrolysis

Bleachability and characterization by fourier transform infrared principal component analysis of acetosolv pulps obtained from sugarcane bagasse by Adilson R. Gonçalves; Denise S. Ruzene (pp. 63-70).

Sugarcane bagasse Acetosolv pulps were bleached by xylanase and the pulps classified by using Fourier transform infrared (FTIR) spectroscopy and principal component analysis (PCA). Pulp was treated with xylanase for 4–8 h with stirring at 30°C. Some samples were further extracted with NaOH for 1 h at 65°C. FTIR spectra were recorded directly from the dried pulp samples by using the diffuse reflectance technique. Reduction in kappa number of 69% was obtained after sequence xylanase (4 h)-alkaline extraction. During bleaching the viscosity decreased only 12%. FTIR-PCA showed that the first three principal components (PCs) explained more than 90% of the total variance of the pulp spectra. PC2×PC1 plot showed that the points related to pulps from sequence xylanase (4 h)-alkaline extraction are different from the other. This group isenlarged by plotting PC3×PC1 or PC3×PC2 containing all pulps submitted to alkaline extraction. PC2 and PC3 are the principal factor for differentiation of the pulps. These PCs suffer influence of the ester bands (1740 and 1244 cm⁻¹). On the other hand, the pulps bleached only with xylanase could not be differentiated from the nonbleached pulps.

Keywords: Xylanase bleaching; Fourier transform infrared spectra of pulps; sugarcane bagasse pulps; Acetosoly pulping

Production of oxychemicals from precipitated hardwood lignin by Qian Xiang; Y. Y. Lee (pp. 71-80).

Lignin is a major byproduct in the biomass-to-ethanol process. The lignin produced from acid treatment of biomass has characteristics suitable for further conversion to organic chemicals. It is free of contaminants and has a relatively low molecular weight. In this study, catalytic oxidative conversion of the acid-soluble lignin precipitated from acid hydrolysates of hardwood was investigated. The process is based on aqueous alkaline oxidation of lignin with dissolved O₂ in the presence of Fe³⁺ and Cu²⁺ catalysts at moderate reaction temperatures (160–180°C). Aromatic aldehydes, ketones, and organic acids are found to be the primary products identifiable on extraction with ether. The combined weight yield of the total ether extractable products is about 20–25% of the initial lignin. The yield of the aldehydes (vanillin + syringaldehyde) is in the vicinity of 15% with an additional 3 to 4% of aromatic ketones. The yields of aldehydes plus ketones observed in this work far exceeded those obtainable from the conventional alkaline air oxidation of spent sulfite liquors. This article also provides comprehensive batch reaction data on conversion and product distribution.

Keywords: Lignin; oxidation; degradation; oxygen

Effect of pretreatment reagent and hydrogen peroxide on enzymatic hydrolysis of oak in percolation process by Sung Bae Kim; Byung Hwan Um; Soon Chul Park (pp. 81-94).

The effect of pretreatment reagent and hydrogen peroxide on enzymatic digestibility of oak was investigated to compare pretreatment performance. Pretreatment reagents used were ammonia, sulfuric acid, and water. These solutions were used without or in combination with hydrogen peroxide in the percolation reactor. The reaction was carried out at 170°C for the predetermined reaction time. Ammonia treatment showed the highest delignification but the lowest digestibility and hemicellulose removal among the three treatments. Acid treatment proved to be a very effective method in terms of hemicellulose recovery and cellulose digestibility. Hemicellulose recovery was 65–90% and digestibilities were >90% in the range of 0.01–0.2% acid concentration. In both treatments, hydrogen peroxide had some effect on digestibility but decomposed soluble sugars produced during pretreatment. Unlike ammonia and acid treatments, hydrogen peroxide in water treatment has a certain effect on hemicellulose recovery as well as delignification. At 1.6% hydrogen peroxide concentration, both hemicellulose recovery and digestibility were about 90%, which were almost the same as those of 0.2% sulfuric acid treatment. Also, digestibility was investigated as a function of hemicellulose removal or delignification. It was found that digestibility was more directly related to hemicellulose removal rather than delignification.

Keywords: Pretreatment; ammonia; acid; water; hydrogen peroxide; enzymatic hydrolysis

Fingerprinting Trichoderma reesei hydrolases in a commercial cellulase preparation by T. B. Vinzant; W. S. Adney; S. R. Decker; J. O. Baker; M. T. Kinter; N. E. Sherman; J. W. Fox; M. E. Himmel (pp. 99-107).

Polysaccharide degrading enzymes from commercial T. reesei broth have been subjected to “fingerprint” analysis by high-resolution 2-D gelelectrophoresis. Forty-five spots from 11×25 cm Pharmacia gels have been analyzed by LC-MS/MS and the resulting peptide sequences were compared toexisting databases. Understanding the roles and relationships of component enzymes from the T. reesei cellulase system acting on complex substrates is key to the development of efficient artificial cellulase systems for the conversion of lignocellulosic biomass to sugars. These studies suggest follow-on work comparing induced and noninduced T. reesei cells at the proteome level, which may elucidate substrate-specific gene regulation and response.

Keywords: Cellulase; Trichoderma reesei ; two-dimensional gel electrophoresis; liquid chromatography-mass spectrometry/mass spectrometry

Development of high-performance and rapid immunoassay for model food allergen lysozyme using antibody-conjugated bacterial magenetic particles and fully automated system by Reiko Sato; Haruko Takeyama; Tsuyoshi Tanaka; Tadashi Matsunaga (pp. 109-116).

A high-performance and rapid chemiluminescence immunoassay for model food allergen lysozyme, one of the major allergenic components in egg white, using antibody-conjugated bacterial magnetic particles and a fully automated system was developed. This system contians a reaction station, tip rack, and an eight-tip pipettor that is alble to attach and detach a strong magnet to the tip surface. The immunoreaction time was shortened to 5 min, and the assay was completed within 20 min. The lower detection limit for lysozyme was 10 ng/mL. This system can be used to perform 24 samples in 60 min within 10% coefficient of variation.

Keywords: Sandwich immunoassay; food allergen; lysozyme; bacterial magnetic particles; fully automated system

Fermentation performance assessment of a genomically integrated xylose-utilizing recombinant of Zymomonas mobilis 39676 by Hugh G. Lawford; Joyce D. Rousseau (pp. 117-131).

In pH-controlled batch fermentations with pure sugar synthetic hardwood hemicellulose (1% [w/v] glucose and 4% xylose) and corn stover hydrolysate (8% glucose and 3.5% xylose) lacking acetic acid, the xyloseutilizing, tetracycline (Tc)-sensitive, genomically integrated variant of Zymomonas mobilis ATCC 39676 (designated strain C25) exhibited growth and fermentation performance that was inferior to National Renewable Energy Laboratory's first-generation, Tc-resistant, plasmid-bearing Zymomonas recombinants. With C25, xylose fermentation following glucose exhaustion wasmarkellyslower, and the ethanol yield (based on sugars consumed) was lower, owing primarily to an increase in lactic acid formation. There was an apparent increased sensitivity to acetic acid inhibition with C25 compared with recombinants 39676:pZB4L, CP4:pZB5, and ZM4:pZB5. However, strain C25 performed well in continous ferm entation with nutrient-rich synthetic corn stover medium over the dilution range 0.03–0.06/h, with a maximum provess ethanol yield at D=0.03/h of 0.46 g/g and a maximum ethanol productivity of 3 g/(L·h). With 0.35% (w/v) acetic acid in the medium, the process yield at D=0.04/h dropped to 0.32 g/g, and the maximum productivity decreased by 50% to 1.5 g/(L·h). Under the same operating conditions, rec Zm Zm 4:pZB5 performed better; however, the medium contained 20 mg/L of Tc to constantly maintain selective pressure. The absence of any need for antibiotics and antiboitic resistance genes makes the chromosomal integrant C25 more com patible with current regulatory specifications for biocatalysts in large-scale commercial operations.

Keywords: Recombinant Zymomonas C25; genomic integrant; xylose; ethanol; biomass hydrolysate; acetate inhibition

Comparative ethanol productivities of different Zymomonas recombinants fermenting oat hull hydrolysate by Hugh G. Lawford; Joyce D. Rousseau; Jeffrey S. Tolan (pp. 133-146).

Iogen Corporation of Ottawa, Canada, has recently built a 50 t/d biomass-to-ethanol demonstration plant adjacent to its enzyme production facility. Iogen has partnered with the University of Toronto to test the C6/C5 cofermentation performance characteristics of National Renewable Energy Laboratory's metabolically engineered Zymomonas mobilis using its biomass hydrolysates. In this study, the biomass feedstock was an agricultural waste, namely oat hulls, which was hydrolyzed in a proprietary two-stage process involving pretreatment with dilute sulfuric acid at 200–250°C, followed by cellulase hydrolysis. The oat hull hydrolysate (OHH) contained glucose, xylose, and arabinose in a mass ratio of about 8:3:0.5. Fermentation media, prepared from diluted hydrolysate, were nutritionally amended with 2.5 mL/L of corn steep liquor (50% solids) and 1.2 g/L of diammonium phosphate. The estimated cost for large-scale ethanol production using this minimal level of nutrient supplementation was 4.4c/gal of ethanol. This work examined the growth and fermentation performance of xyloseutilizing, tetracycline-resistant, plasmid-bearing, patented, recombinant Z. mobilis cultures: CP4:pZB5, ZM4:pZB5, 39676:pZB4L, and a hardwood prehydrolysate-adapted variant of 39676:pZB4L (designated asthe “adapted” strain). In pH-stat batch fermentations with unconditioned OHH containing 6% (w/v) glucose, 3% xylose, and 0.75% acetic acid, rec Zm ZM4:pZB5 gave the best performance with a fermentation time of 30h, followed by CP4:pZB5 at 48h, with corresponding volumetric productivities of 1.4 and 0.89 g/(L·h), respectively. Based on the available glucose and xylose, the process ethanol yield for both strains was 0.47 g/g (92% conversion efficiency). At 48 h, the process yield for rec Zm 39676:pZB4L and the adapted strain was 0.32 and 0.34 g/g, respectively. None of the test strains was able to fermentarabinose. Acetic acid tolerance appeared to be a major determining factor in cofermentation performance.

Keywords: Recombinant Zymomonas ; oat hull hydrolysate; xylose; biomass hydrolysate; ethanol yield; acetic acid; productivity

Isolation of Magnetospirillum magneticum AMB-1 mutants defective in bacterial magnetic particle synthesis by transposon mutagenesis by Aris Tri Wahyudi; Haruko Takeyama; Tadashi Matsunaga (pp. 147-154).

Nonmagnetic mutants of Magnetospirillum magneticum AMB-1 were recovered following mini-Tn5 transposon mutagenesis. Transconjugants with kanamycin resistance were obtained at a frequency of 2.7 × 10⁻⁷ per recipient. Of 3327 transconjugants, 62 were defective for bacterial magnetic particle (BMP) synthesis. The frequency of independent transposition events for nonmagnetic mutants was about 1.4% in transconju gants. Further analysis of DNA sequences flanking transposon by inverted polymerase chain reaction allowed isolation of at least 10 genes or DNA sequences involved in BMP synthesis in M. magneticum AMB-1.

Keywords: Transposon mutagenesis; mini-Tn5; Magnetospirillum magneticum AMB-1; inverse polymerase chain reaction; bacterial magnetic particles; sequence analysis

Synthesis of bacterial magnetic particles during cell cycle of Magnetospirillum magneticum AMB-1 by Chen-Dong Yang; Haruko Takeyama; Tsuyoshi Tanaka; Aki Hasegawa; Tadashi Matsunaga (pp. 155-160).

We investigated the relationship between the synthesis of bacterial magnetic particles (BMPs) and the transcription of magA gene-encoding iron transport protein using synchronous culture of Magnetospirillum magneticum AMB-1. Synchronously cultured cells were subjected to transmission electron microscopic observation and fluorescence in situ hybridization. The average number of BMPs slowly increased in the cell with increasing cell size. A sharp increase in BMPs occurred just before cell division and resulted in maximum BMP production of 30 particles/cell. The transcription of magA was regulated immed iately before and after cell division

Keywords: Synchronous culture; Magnetospirillum magneticum AMB-1; bacterial magnetic particle; cell cycle; magA gene

Overexpression of glucose-6-phosphate dehydrogenase in genetically modified Saccharomyces cerevisiae by Fernando H. Lojudice; Daniel P. Silva; Nilson I. T. Zanchin; Carla C. Oliveira; Adalberto Pessoa Jr. (pp. 161-169).

Glucose-6-phosphate dehydrogenase (G6PD) (EC 1.1.1.49) is an abun dant enzyme in Saccharomyces cerevisiae. This enzyme is of great interest as an analytical reagent because it is used in a large number of quantitative assays. A strain of S. cerevisiae was genetically modified to improve G6PD production during aerobic culture. The modifications are based on cloning the G6PD sequence under the control of promoters that are upregulated by the carbon source used for yeast growth. The results showed that S. cerevisiae acquired from a commercial source and the same strain produced by aerobic cultivation under controlled conditions provided very similar G6PD. However, G6PD production by genetically modified S. cerevisiae produced very high enzyme activity and showed to be the most effective procedure to obtain glucose-6-phosphate dehydrogenase. As a consequence, the cost of producing G6PD can be significantly reduced by using strains that contain levels of G6PD up to 14-fold higher than the level of G6PD found in commercially available strains.

Keywords: Glucose-6-phosphate dehydrogenase; Saccharomyces cerevisiae ; aerobic culture; molecular biology

Biosorption of heavy metals by bacteria isolated from activated sludge by Wa C. Leung; Hong Chua; Waihung Lo (pp. 171-184).

Twelve aerobic bacteria from activated sludge were isolated and identified. These included both Gram-positive (e.g., Bacillus) and Gram-negative (e.g., Pseudomonas) bacteria. The biosorption capacity of these strains for three different heavy metals (copper, nickel, and lead) was determined at pH 5.0 and initial metal concentration of 100 mg/L. Among these 12 isolates, Pseudomonas pseudoalcaligenes was selected for further investigation owing to its high metal biosorption capacity. The lead and copper biosorption of this strain followed the Langmuir isotherm model quite well with maximum biosorption capacity (q _max) reaching 271.7mg of Pb²⁺/g of dry cell and 46.8 mg of Cu²⁺/g of dry cell at pH 5.0. Study of the effect of pH on lead and copper removal indicated that the metal biosorption increased with increasing pH from 2.0 to 7.0. A mutual inhibitory effect was observed in the lead-copper system because the presence of either ion affected the sorption capacity of the other. Unequal inhibitions were observed in all the nickel binary systems. The increasing order of affinity of the three metals toward P. pseudoalcaligenes was NiP. pseudoalcaligenes, may have possible applications in the removal and recovery of metals from industrial effluents.

Keywords: Activated sludge; biosorption; copper adsorption; lead removal; bioremediation

Utilization of cyanobacteria in photobioreactors for orthophosphate removal from water by Alexea M. Gaffney; Sergei A. Markov; M. Gunasekaran (pp. 185-193).

The effectiveness of photosynthetic free-living and polyurethane foam (PU) immobilized Anabaena variabilis cells for, removal of orthophosphate (P) from water in batch cultures and in a photobioreactor was studied. Immobilization in PU foams was found to have a positive effect on P uptake by cyanobacteria in batch cultures. The efficiency of P uptake by immobilized cells was higher than by free-living cells. A laboratory scale photobioreactor was constructed for removal of P from water by the immobilized cyanobacteria. The photobioreactor was designed so that the growth medium (water) from a reservoir was pumped through a photobioreactor column with immobilized cyanobacteria and back to the reservoir. This created a closed system in which it was possible to measure P uptake. No leakage of cells into the photobioreactor medium reservoir was observed during the operation. The immobilized cells incorporated into a photobioreactor column removed P continuously for about 15 d. No measurable uptake was demonstrated after this period. Orthophosphate uptake efficiency of 88–92% was achieved by the photobioreactor.

Keywords: Orthophosphate; water clean-up; immobilization; cyanobacteria; photobioreactor

Enhancement of the conversion of toluene by Pseudomonas putida F-1 using organic cosolvents by Miguel Rodriguez; K. Thomas Klasson; Brian H. Davison (pp. 195-204).

Pseudomonas putida F-1 (ATCC700007) was used as a model organism in stirred tank reactors, to study conversion enhancement of poorly soluble substrates by organic cosolvents. After a literature study, silicone oil was used as a solvent system to enhance the mass transfer rate. To study the benefits of the organic solvent addition, batch experiments were conducted in two side-by-side fermentation vessels (experimental and control) at three different levels of silicone oil (10, 30, and 50%). Results showed that the presence of silicone oil resulted in a 100% increase in the toluene mass transfer compared to the control. Experiments in continuous stirred-tank reactors showed that improved conversion could beobtained, at higher agitation rates.

Keywords: Pseudomonas putida F-1; toluene; organic solvents; silicone oil

Effect of temperature on biofiltration of nitric oxide by K. Thomas Klasson; Brian H. Davison (pp. 205-211).

Biodegradation of formaldehyde by a formaldehyde-resistant bacterium isolated from seawater by Tomohioko Yamazaki; Wakako Tsugawa; Koji Sode (pp. 213-217).

A formaldehyde-tolerantbacterium designated as a DM-2 strain was used to biodegrade formaldehyde. The cells, precultivated in the presence of 400 ppm of formaldehyde, were able to degrade formaldehyde in a minimal medium supplemented with up to 400 ppm of formaldehyde in the presence of 3% NaCl. The rate of formaldehyde degradation achieved, in this study was 45 ppm/h when the DM-2 culture's optical density at 660 nm was 1.2.

Keywords: Biodegradation; formaldehyde; marine bacterium; screening

Dissemination of catabolic plasmids among desiccation-tolerant bacteria in soil microcosms by Frederic Weekers; Christian Rodriguez; Philippe Jacques; Maximilien Mergeay; Philippe Thonart (pp. 219-232).

The dissemination of catabolic plasmids was compared to bioaugmentation by strain inoculation in microcosm experiments. When Rhodococcus erythropolis strain T902, bearing a plasmid with trich loroethene and isopropylbenzene degradation pathways, was used as the inoculum, no transconjugant was isolated but the strain remained in the soil. This plasmid had a narrow host range. Pseudomonas putida strain C8S3 was used as the inoculum in a second approach. It bore a broad host range conjugative plasmid harboring a natural transposon, RP4∶Tn4371, responsible for biphenyl and 4-chlorobiphenyl degradation pathways. The inoculating population slowly decreased from its original level (10⁶ colony-forming units [CFU]/g of dry soil) to approx 3×10² CFU/g of dry soil after 3 wk. Transconjugant populations degrading biphenyl appeared in constant humidity soil (up to 2×10³ CFU/g) and desiccating soil (up to 10⁴ CFU/g). The feasibility of plasmid dissemination as a bioaugmentation technique was demonstrated in desiccating soils. The ecologic significance of desiccation in bioaugmentation was demonstrated; it upset the microbial ecology and the development of transconjugants.

Keywords: Bioaugmentation; drought tolerance; conjugation; plasmid dissemination; microcosm; isopropylbenzene

Ethanol production from lignocellulosic byproducts of olive oil extraction by Ignacio Ballesteros; Jose Miguel Oliva; Felicia Saez; Mercedes Ballesteros (pp. 237-252).

The recent implementation of a new two-step centrifugation process for extracting olive oil in Spain has substantially reduced water consumption, thereby eliminating oil mill wastewater. However, a new high sugar content residue is still generated. In this work the two fractions present in the residue (olive pulp and fragm ented stones) were assayed as substrate for ethanol production by the simultaneous saccharification and fermentation (SSF) process. Pretreatment of fragmented olive stones by sulfuric acid-catalyzed steam explosion was the most effective treatment for increasing enzymatic digestibility; however, a pretreatment step was not necessary to bioconvert the olive pulp into ethanol. Theolive pulp and fragmented olive stones were tested by the SSF process using a fed-batch procedure. By adding the pulp three times at 24-h intervals, 76% of the theoretical SSF yield was obtained. Experiments with fed-batch pretreated olive stones provided SSF yields significantly lower than those obtained at standard SSF procedure. The preferred SSF conditions to obtain ethanol from olives stones (61% of theoretical yield) were 10% substrate and addition of cellulases at 15 filter paper units/g of substrate.

Keywords: Ethanol; olive oil extraction byproducts; pretreatment; enzymatic saccharification; fermentation

Continuous countercurrent extraction of hemicellulose from pretreated wood residues by Kyoung Heon Kim; Melvin P. Tucker; Fred A. Keller; Andy Aden; Quang A. Nguyen (pp. 253-267).

Two-stage dilute acid pretreatment followed by enzymatic cellulose hydrolysisis an effectivemethod for obtaining high sugar yields from wood residuessuchassoftwood forest thinnings. In the first-stage hydrolysis step, most of the hemicellulose is solubilized using relatively mild conditions. The soluble hemicellu losic sugars are recovered from the hydrolysateslurry by washing with water. The washed solids are then subjected tomoresevere hydrolysis conditions to hydrolyze approx 50% of the cellulose to glucose. The remaining cellulose can further be hydrolyzed with cellulase enzyme. Our process simulation indicates that the amount of water used in the hemicellulose recovery step has a significan tim pact on the cost of ethanol production. It is important to keep water usage as low as possible while mainta ining relatively high recovery of solublesugars. To achieve this objective, a prototype pilot-scale continuous countercurrent screw extractor was evaluated for the recovery of hemicellulose from pretreated forest thinnings. Using the 274-cm (9-ft) long extractor, solubles recoveries of 98, 91, and 77% were obtained with liquid-to-insoluble solids (L/1S) ratios of 5.6, 3.4, and 2.1, respectively. An empirical equation was developed to predict the performance of the screwextractor. This equation predicts that soluble sugar recovery above 95% can be obtained with an L/IS ratio as low as 3.0.

Keywords: Extraction; hemicellulose; softwood; pretreatment; acid hydrolysis

Enzymatic hydrolysis of ammonia-treated sugar beet pulp by Brian L. Foster; Bruce E. Dale; Joy B. Doran-Peterson (pp. 269-282).

Sugar beet pulp is a carbohydrate-rich coproduct generated by the table sugar industry. Beet pulp has shown promise as a feedstock for ethanol production using enzymesto hydroly zepolymeric carbohydrates and engineered bacteria to ferment sugars to ethanol. In this study, sugar beet pulp underwent an ammonia pressurization depressurization (APD) pretreatment in which the pulp was exploded by the sudden evaporation of ammonia in a reactor vessel. APD was found to substantially increase hydrolysis efficiency of the cellulose component, but when hemicellulose- and pectindegrading enzymes wereadded, treated pulp hydrolysis was no better than the untreated control.

Keywords: Biomass; sugar beet pulp; enzyme hydrolysis; ammonia pretreatment; fuel alcohol

Ethanol production in a membrane bioreactor by Jose M. Escobar; Kishore D. Rane; Munir Cheryan (pp. 283-296).

Pilot plant trials were conducted in a corn wet mill with a 7000-L membrane recycle bioreactor (MRB) that integrated ceramic microfiltration membranes in a semi-closed loop configuration with a stirred-tank reactor. Residence times of 7.5–10 h with ethanol outputs of 10–11.5% (v/v) were obtained when the cell concentration was 60–100 g/L drywt of yeast, equivalent to about 10⁹−10¹⁰ cells/mL. The performance of the membrane was dependent on the startup mode and pressure management techniques. A steady flux of 70 L/(m²·h) could be maintained for several days before cleaning was necessary. The benefits of the MRB include better productivity; a clear productstream containing no particulates or yeast cells, which should improve subsequent stripping and distillation operations; and substantially reduced stillage handling. The capital cost of the MRB is $21–$34/(m³·yr) ($0.08–$0.13/[gal·yr]) of ethanol capacity. Operating cost, including depreciation, energy, membrane replacement, maintenance, labor, and cleaning, is $4.5–9/m³ ($0.017–$0.034/gal) of ethanol.

Keywords: Ethanol; membrane; corn wet mill; yeast

Cellulase recovery via membrane filtration by Wendy D. Mores; Jeffrey S. Knutsen; Robert H. Davis (pp. 297-309).

A combined sedimentation and membrane filtration process was investigated for recycling cellulase enzymes in the biomass-to-ethanol process. In the first stage, lignocellulose particles longer than approx 50 μm were removed by means of sedimentation in an inclined settler. Microfiltration was then utilized to remove the remaining suspended solids. Finally, the soluble cellulase enzymes were recovered by ultrafiltration. The perm eate fluxes obtained in microfiltration and ultrafiltration were approx 400 and 80 L/(m²·h), respectively. A preliminary economic analysis shows that the cost benefit of enzyme recycling may be as much as 18 cents/gal of ethanol produced, provided that 75% of the enzyme is recycled in active form.

Keywords: Sedimentation; microfiltration; ultrafiltration; cellulase enzyme

Effect of yeast extract on growth kinetics of Monascus purpureus by Daniela Gerevini Pereira; Beatriz Vahan Kilikian (pp. 311-316).

Growth kinetics and red pigment production of Monascus purpureus CCT 3802 was studied. A reproducible inoculum with extremely dispersed hyphae for bioreactor runs was obtained through a two-step cultivation in a shaker. First, the spores were cultivated in a complex medium rendering a suspension of vegetative cells. In the second step these cells were grown in a semisynthetic medium. Two types of media were employed in the bioreactor runs: a semisynthetic (glucose, salts, and yeast extract), and a synthetic, without yeast extract. The inclusion of yeast extract, caused an increase in cell yield on glucose (Y_s/s) as high as 40%. Also, yeast extract probably yielded a higher proportion of red pigment associated with the cell, relative to the synthetic medium. On the other hand, cells grown on the synthetic medium were slightly higher producers of red soluble pigments.

Keywords: Monascus purpureus ; yeast extract; red pigment; kinetics

Severity function describing the hydrolysis of xylan using carbonic acid by G. Peter van Walsu (pp. 317-329).

Beech wood derived xylan to hydrolyzed to predominantly xylose monomer units after exposure to hot, compressed liquid water saturated with carbon dioxide. Similar treatment without CO₂ saturation resulted in only minor hydrolysis and a smaller fraction of monomers among the hydrolysis products. Severity of the hydrolysis reaction was correlated to reaction time, temperature, and carbon dioxide partial pressure and followed a function similar to those used to characterize mineral acid systems. Results from parallel hydrolysis experiments with an aqueous system and a very dilute sulfuric acid system allowed an approximation of the dissociation constant of carbonic acid in the temperature range of 170–230°C. Results suggest that carbonic acid may be a viable reagent for promoting hydrolysis without mineral acids, especially in the case of a bioprocessing plant that produces carbon dioxide.

Keywords: Xylan; carbonic acid; severity; hydrolysis; biomass conversion; pretreatment; carbon dioxide

Cellulose hydrolysis under extremely low sulfuric acid and high-temperature conditions by Jun Seok Kim; Y. Y. Lee; Robert W. Torget (pp. 331-340).

The kinetics of cellulose hydrolysis under extremely low acid (ELA) conditions (0.07 wt%) and at temperatures >200°C was investigated using batch reactors and bed-shrinking flow-through (BSFT) reactors. The maximum yield of glucose obtained from batch reactor experiments was about 60% for α-cellulose, which occurred at 205 and 220°C. The maximum glucose yields from yellow poplar feedstockswere substantially lower, falling in the range of 26–50%. With yellow poplar feedstocks, a large amount of glucose was unaccounted for at the latter phase of the batch reactions. It appears that a substantial amount of released glucose condenses with nonglucosidic substances. in liquid. The rate of glucan hydrolysis under ELA was relatively insensitive to temperature in batch experiments for all three substrates. This contradicts the traditional concept of cellulose hydrolysis and implies that additional factors influence the hydrolysis of glucan under ELA. Inexperiments using BSFT reactors, the glucose yields of 87.5, 90,3, and 90.8% were obtained for yellow poplar feedstocks at 205, 220, and 235°C, respectively. The hydrolysis rate for glucan was about three times higher with the BSFT than with the batch reactors. The difference of observed kinetics and performance data between the BSFT and the batch reactors was far above that predicted by the reactor theory.

Keywords: Yellow poplar; cellulose hydrolysis; bed-shrinking flow-through reactor; kinetics

A hybrid neuroal network algorithm for on-line state inference that accounts for differences in inoculum of Cephalosporium acremonium in fed-batch fermentors by Rosineide G. Silva; Antonio J. G. Cruz; Carlos O. Hokka; Raquel L. C. Giordano; Roberto C. Giordano (pp. 341-352).

One serious difficulty in modeling a fermentative process is the forecasting of the duration of the lag phase. The usual approach to model biochemical reactors relies on first-principles, unstructured mathematical models. These models are not able to take into account changes in the process response caused by different incubation times or by repeated fed batches. Toover come this problem, we have proposed a hybrid neural network algorithm. Feedforward neural networks were used to estimate rates of cell growth, substrate consumption, and product formation from on-line measurements during cephalosporin C production. These rates were included in the mass balance equations to estimate key process variables: concentrations of cells, substrate, and product. Data from fed-batch fermentation runs in a stirred aerated bioreactor employing the microorganism Cephalosporium acremonium ATCC 48272 were used. On-line measurements strongly related to the mass and activity of the cells used. They include carbon dioxide and oxygen concentrations in the exhausted gas. Good results were obtained using this approach.

Keywords: Neural networks; hybrid model; cephalosporin C production; state inference

Kinetics of ethanol fermentation with high biomass concentration considering the effect of temperature by Daniel I. P. Atala; Aline C. Costa; Rubens Maciel; Francisco Maugeri (pp. 353-365).

A model of ethanol fermentation considering the effect of temperature was developed and validated. Experiments were performed in a temperature range from 28 to 40°C in continuous mode with total cell recycling using a tangential microfiltration system. The developed model considered substrate, product and biomass inhibition, as well as an active cell phase (viable) and an inactive (dead) phase. The kinetic parameters were described as functions of temperature.

Keywords: Ethanol fermentation; high biomass concentration; temperature

Fermentation of xylose into acetic acid by Clostridium thermoaceticum by Niru Balasubramanian; Jun Seok Kim; Y. Y. Lee (pp. 367-376).

For optimum fermentation, fermenting xylose into acetic acid by Clostridium thermoaceticum (ATCC 49707) requires adaptation of the strain to xylose medium. Exposed to a mixture of glucose and xylose, it preferentially consumesxylose over glucose. The initial concentration of xylose in the medium affects the final concentration and the yield of acetic acid. Batch fermentation of 20 g/L of xylose with 5g/L of yeast extract as the nitrogen source results in a maximum acetate concentration of 15.2 g/L and yield of 0.76 g of acid/g of xylose. Corn steep liquor (CLS) is a good substitute for yeast extract and results in similar fermentation profiles. The organism consumes fructose, xylose, and glucose from a mixture of sugars in batch fermentation. Arabinose, mannose, and galactose are consumed only slightly. This organism loses viability on fed-batch operation, even with supplementation of all the required nutrients. In fed-batch fermentation with CSL supplementation, d-xylulose (an intermediate in the xylose metabolic pathway) accumulates in large quantities.

Keywords: Xylose; fermentation; Clostridium thermoaceticum ; a cetic acid

Heat transfer considerations in design of a batch tube reactor for biomass hydrolysis by Sigrid E. Jacobsen; Charles E. Wyman (pp. 377-386).

Biologic conversion of inexpensive and abundant sources of cellulosic biomass offers a low-cost route to production of fuels and commodity chemicals that can provide unparalleled environmental, economic, and strategic benefits. However, low-cost, high-yiel technologies are needed to recover sugars from the hemicellulose fraction of biomass and to prepare the remaining cellulose fraction for subsequent hydrolysis. Uncatalyzed hemicellulose hydrolysis in flow-through systems offers a number of important advantages for removal of hemicellulose sugars, and it is believed that oligomers could play an important role in explaining why the performance of flow-through systems differs from uncatalyzed steam explosion approaches. Thus, an effort is under way to study oligomer formation kinetics, and a small batch reactor is being applied to capture these important intermediates in a closed system that facilitates material balance closure for varying reaction conditions. In this article, heat transfer for batch tubes is analyzed to derive temperature profiles for different tube diameters and assess the impact on xylan conversion. It was found that the tube diameter must be <0.5 in, for xylan hydrolysis to follow the kinetics expected for a uniform temperature system at typical operating conditions.

Keywords: Reactors; heat transfer; hydrolysis; kinetics; pretreatment

Measurement of bubble size distribution in protein foam fractionation column using capillary probe with photoelectric sensors by Liping Du; Yuqing Ding; Aleš Prokop; Robert D. Tanner (pp. 387-404).

Bubble size is a key variable for predicting the ability to separate and concentrate proteins in a foam fraction ation process. It is used to characterize not only the bubble-specific interfacial a rea but also coalescence of bubbles in the foam phase. This article describes the development of a photoelectric method for measuring the bubble size distribution in both bubble and foam columns for concentrating proteins. The method uses a vacuum to withdraw a stream of gas-liquid dispersion from the bubble or foam column through a capillary tube with a funnel-shaped inlet. The resulting sample bubble cylinders are detected, and their lengths are calculated by using two pairs of infrared photoelectric sensors that are connected with a high-speed data acquisition system controlled by a microcomputer. The bubble size distributions in the bubble column 12 and 1 cm below the interface and in the foam phase 1 cm above the interface are obtained in a continuous foam fractionation process for concentrating ovalbumin. The effects of certain operating conditions such as the feed protein concentration, superficial gas velocity, liquid flow rate, and solution pH are investigated. The results may prove to be helpful in understanding the mechanisms controlling the foam fractionation of proteins.

Keywords: Bubble size; capillary tube; photoelectric method; foam fractionation; ovalbumin

Effect of a natural contaminant on foam fractionation of bromelain by Samuel Ko; Justin Cherry; Aleŝ Prokop; Robert D. Tanner (pp. 405-411).

Foam fractionation is a simple, inexpensive method for separating and purifying proteins. Typically, a dilute bromelain solution with a pH ranging from 2.0 to 7.0 foams very well when bubbles are introduced into a foam fractionation column. It was observed, however, that the dilute enzyme solution only foamed between approximately pH 2.0 and 3.0 when the inner wall of the fractionation column was coated with a natural contaminant (okra residue). We studied the separation ratio and the protein mass recovery to explore the effect of a natural antifoaming agent on the foam fractionation of a dilute bromelain solution. The control variables used in this process were the initial bulk solution pH, which ranged from 2.0 to 7.0, and the superficial air velocity, which varied between 1.7 and 6.2 cm/s.

Keywords: Bromelain; foam fractionation; protein; protein separation; protease recovery; antifoaming agent; natural antifoaming agent

Application of factorial design to study of heavy metals biosorption by waste biomass from beverage distillery by Maristella A. Dias; Carlos A. Rosa; Valter R. Linardi; Rosa A. Conte; Heizir F. De Castro (pp. 413-422).

A full factorial design leading to 20 sets of sorption runs was conducted to study the influence of four variables (bleaching earth and biomass concentrations, pH, and sorption time) on the iron, nickel, and chromium removal from stainless steel effluent using waste biomass from a beverage industry. Similar factor effects and interactions were found for each metalinvolved in this biosorption study, and the main factors were pH (positive effect) and biomass concentration (negative effect). Response surface methodology was adopted and an empirical linear polynomial model constructed on the basis of the specific uptake (mg of metal/g of biomass as dry weight) for each metal species. Under optimized process conditions (pH 4.0, biomass concentration of 2.0 g/L, absence of Celite), uptake values of 155 mg of Fe/g, 38 mg of Cr/g, and 0.4 mg of Ni/g were achieved after 3 h. This corresponded to a reduction in heavy metals concentration of approx 94% for Cr, 57% for Fe, and 25% for Ni.

Keywords: Biosorption; waste biomass; heavy metals; experimental design; effluent detoxification

Model compound studies by Thomas Walthers; Patcharee Hensirisak; Foster A. Agblevor (pp. 423-435).

The influence of other hemicellulosic sugars (arabinose, galactose, mannose, and glucose), oxygen limitation, and initial xylose concentration on the fermentation of xylose to xylitol was in vestigated using experimental design methodology. Oxygen limitation and initial xylose concentration had strong influences on xylitol production by Candida tropicalis ATCC 96745. Under semiaerobic conditions, xylitol yield was highest (0.62 g/g), whereas under aerobic conditions volumetric productivity was highest (0.90g/[L·h]). In the presence of glucose, xylose utilization was strongly repressed and sequential sugar utilization was observed. Ethanol produced from the glucose caused a 50% reduction in xylitol yield when the ethanol con centration exceeded 30 g/L. When complex synthetic hemicellulosic sugars were fermented, glucose was initially consumed followed by a simultaneous uptake of the other sugars. The highest xylitol yield (0.84 g/g) and volumetric productivity (0.49 g/[L·h]) were obtained for substrates containing high arabinose and low glucose and mannose contents.

Keywords: Xylitol; fermentation; aeration; hemicellulose

Nitrification and denitrification processes for biologic treatment of industrial effluents by Célia Regina Granhen Taveres; Renata Ribeiro de Araújo Rocha; Terezinha Aparecida Guedes (pp. 437-446).

Nitrification process performance was evaluated using a three-phase fluidized-bed bioreactor. Asynthetic effluent was used for this experiment containing 180–230 mg/L of chemical oxygen demand (COD), 25–30 mg/L of N−NH ₄ ⁺ , 12 to 13 mg/L of total phosphorous, and micronutrients. The bioreactor used for denitrification behaved as completely mixed. The results indicate that the nitrification process was efficient, reaching efficiencies of about 98%. The best results related to the efficiency of the denitrification process were obtained when the processes were supplemented with the carbon source. The results indicated an efficiency of 86–98% COD removal.

Keywords: Nitrification; denitrification; three-phase fluidized-bed bioreactor; Nitrobacter ; Nitrosomonas

Removal and recovery of copper (II) ions by bacterial biosorption by Mui F. Wong; Hong Chua; Waihung Lo; Chu K. Leung; Peter H. F. Yu (pp. 447-457).

Studies were conducted toinvestigate the removal and recovery of copper (II) ions from aqueous solutions by Micrococcus sp., which was isolated from a local activated sludge process. The equilibrium of copper biosorption followed the Langmuir isotherm model very well with a maximum biosorption capacity (q_max) of 36.5 mg of Cu²⁺/gofdry cell at pH 5.0 and 52.1 mg of Cu²⁺/g of dry cell at pH 6.0. Cells harvested at exponential growth phase and stationary phase showed similar biosorption characteristics for copper, Copper uptake by cells was negligible at pH 2.0 and then increased rapidly with increasing pH un til 6.0. In multim etal systems, Micrococcus sp. exhibited a preferential biosorption order: Cu−Pb>Ni−Zn. There is virtually no interference with copper uptake by Micrococcus sp. from solutions bearing high concentrations of Cl⁻, SO ₄ ²⁻ , and NO3/− (0–500 mg/L). Sulfuric acid (0.05 M) was the most efficient desorption medium, recovering >90% of the initial copper sorbed. The copper capacity of Micrococcus sp. remained unchanged after five successive sorption and desorption cycles. Immobilization of Micrococcus sp. in 2% calcium alginate and 10% polyacrylamide gel beads increased copper uptake by 61%. Biomass of Micrococcus sp. may be applicable to the development of potentially cost-effective biosorbent for removing and recovering copper from effluents.

Keywords: Biosorption; copper removal; biorem ediation; immobilization; metal adsorption

Production of biosurfactant from a new and promising strain of Pseudomonas aeruginosa PA1 by L. M. Santa Anna; G. V. Sebastian; N. Pereira Jr.; T. L. M. Alves; E. P. Menezes; D. M. G. Freire (pp. 459-467).

The Pseudomonas aeruginosa PA1 strain, isolated from the water of oil production in Sergipe, Northeast Brazil, wasevaluated as a potential rhamnolipid type of biosurfactant producer. The production of biosurfactants was investigated using different carbon sources (n-hexadecane, paraffin oil, glycerol, and babassu oil) and inoculum concentrations (0.0016–0.008 g/L) The best results were obtained with glycerol as the substrate and an initial cell concentration of 0.004 g/L. AC:N ratio of 22.8 led to the greatest production of rhamnolipids (1700 mg/L) and efficiency (1.18 g of rhamnolipid/g of dry wt).

Keywords: Production of biosurfactants; glycolipids; rhamnolipids; Pseudomonas aeruginosa ; surface tension

Cassava starch maltodextrinization/monomerization through thermopressurized aqueous phosphoric acid hydrolysis by José D. Fontana; Mauricio Passos; Madalena Baron; Sabrina V. Mendes; Luiz P. Ramos (pp. 469-478).

Kinetic conditions were established for the depolymerization of cassava starch for the production of maltodextrins and glucose syrups. Thin-layer chromatography and high-performance liquid chromatography analyses corroborated that the proper H₃PO₄ strengthand thermopressurization range (e.g., 142–170°C; 2.8–6.8 atm) can be successfully explored for such hydrolytic purposes of native starch granules. Because phosphoric acid can be advantageously maintained in the hydrolysate and generates, after controlled neutralization with ammonia, the strategic nutrient triplet for industrial fermentations (C, P, N), this pretreatment strategy can be easily recognized as a recommended technology for hydrolysis and upgrading of starch and other plant polysaccharides. Compared to the classic catalysts, the mandatory desalting step (chloride removal by expensive anion-exchange resin or sulfate precipitation as the calcium-insoluble salt) can be avoided. Furthermore, properly diluted phosphoric acid is well known as an allowable additive in several popular soft drinks such as colas since its acidic feeling in the mouth is compatible and synergistic with both natural and artificial sweeteners. Glycosyrups from phosphorolyzed cassava starch have also been upgraded to high-value single-cell protein such as the pigmented yeast biomass of Xanthophyllomyces dendrorhous (Phaffia rhodozyma), whose astaxanthin (diketo-dihydroxy-β-carotene) content may reach 0.5–1.0 mg/g of dry yeast cell. This can be used as an ideal complement for animal feeding as well as a natural staining for both fish farming (meat) and poultry (eggs).

Keywords: Starch; hydrolysis; phosphoric acid; maltodextrinization; astaxanthin; byproducts

The effect of pretreatments on surfactin production from potato process effluent by Bacillus subtilis by David N. Thompson; Sandra L. Fox; Gregory A. Bala (pp. 487-501).

Pretreatments of low-solids potato process effluent were tested for their potential to increase surfactin yield. Pretreatments included heat, removal of starch particulates, and acid hydrolysis. Elimination of contaminating vegetative cells was necessary for surfactin production. After autoclaving, 0.40 g/L of surfactin was produced from the effluent in 72 h, vs 0.24 g/L in the purified potato starch control. However, surfactin yields per carbon consumed were 76% lower from process effluent. Removal of starch particulates had little effect on the culture. Acid hydrolysis decreased growth and surfactant production, except 0.5 wt% acid, which increased the yield by 25% over untreated effluent.

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

Production of bacterial cellulose from alternate feedstocks by David N. Thompson; Melinda A. Hamilton (pp. 503-513).

Production of bacterial cellulose by Acetobacter xylinum ATCC 10821 and 23770 in static cultures was tested from unamended food process effluents. Effluents in cluded low-solids (LS) and high-solids (HS) potato effluents, cheese whey permeate (CW), or sugar beet raffinate (CSB). Strain 23770 produced 10% less cellulose from glucose than did strain 10821 and diverted more glucose to gluconate. Unamended HS, CW, and CSB were unsuitable for cellulose production by either strain, and LS was unsuitable for production by strain 10821. However, strain 23770 produced 17% more cellulose from LS than from glucose, indicating that unamended LS could serve as a feedstock for bacterial cellulose.

Keywords: Bacterial cellulose; Acetobacter xylinum ; potato effluent; beet raffinate; whey permeate

Production of polyhydroxybutyrate by Bacillus species isolated from municipal activated sludge by Kin-Ho Law; Yun-Chung Leung; Hugh Lawford; Hong Chua; Wai-Hung Lo; Peter Hoifu Yu (pp. 515-524).

Plastic wastes are considered to be severe environmental contaminants causing waste disposal problems. Widespread use of biodegradable plastics is one of the solutions, but it is limited by high production cost. Biologic wastewater treatment generates large quantities of biomass as activated sludge. Only a few reports focus on the potential of utilizing resident Bacillus species from activated sludge in polyhydroxbutyrate (PHB) production as well as the production of PHB from food wastes. They have attractive properties such as short generation time, absence of endotoxins, and secretion of both amylases and proteinases that can well utilize food wastes for nutrients, which can further reduce the cost of production of polyhydroxyalkanoates (PHAs). Two PHA-producing strains, HF-1 and HF-2, were isolated from activated sludge. HF-1 outperfomed HF-2 in terms of growth and PHB production in hydrolyzed soy and malt wastes. The isolated bacteria was characterized by DNA sequence alignment. Cell extracts of HF-1 were also compared to Bacillus megaterium cell extracts on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The biopolymers accumulated were analyzed by gas chromatography, nuclear magnetic resonance, and Fourier transform infrared methods.

Keywords: Polyhydroxybutyrates; malt waste; soy waste; Bacillus ; activated sludge; Fourier transform in frared; inclusion body; nuclear magnetic resonance

Characterization of bioconversion of fumarate to succinate by alginate immobilized Enterococcus faecalis RKY1 by Hwa-Won Ryu; Young-Jung Wee (pp. 525-535).

In this study, the immobilization characteristics of Enterococcus faecalis RKY1 for succinate production were examined. At first, three natural polymers—agar, κ-carrageenan, and sodium alginate—were tried as immobilizing matrices. Among these, sodium alginate was selected as the best gel for immobilization of E. faecalis RKY1. Efficient conditions for immobilization were established to be with a 2% (w/v) sodium alginate solution and 2-mm-diameter bead. The bioconversion characteristics of the immobilized cellsat various pH values and temperatures were examined and compared with those of free cells. The optimum pH and temperature of the immobilized cells were the same as for free cells, 7.0 and 38°C respectively, but the conversion ratio was higher by immobilization for all the other pH and temperature conditions tested. When the seed volume of the immobilized cells was adjusted to 10% (v/v), 30 g/L of fumarate was completely converted tosuccinate (0.973 g/g conversion ratio) after 12 h. In addition, the immobilized cells maintained a conversion ratio of >0.95 g/g during 4wk of storageat 4°C in a 2% (w/v) CaCl₂ solution. In repetitive bioconversion experiments, the activity of the immobilized cells decreased linearly according to the number of times of reuse.

Keywords: Enterococcus faecalis RKY1; succinate; fumarate; immobilized cells; bioconversion

Modeling and simulation of cephalosporin C production in a fed-batch tower-type bioreactor by Renata M. R. G. Almeida; Antonio J. G. Cruz; Maria Lucia G. C. Araujo; Roberto C. Giordano; Carlos O. Hokka (pp. 537-549).

Immobilized cell utilization in tower-type bioreactor is one of the main alternatives being studied to improve the industrial bioprocess. Other alternatives for the production of β-lactam antibiotics, such as a cephalosporin C fed-batch process in an aerated stirred-tank bioreactor with free cells of Cephalosporium acremonium, or a tower-type bioreactor with immobilized cells of this fungus, have proven to be more efficient than the batch process. In the fed-batch process, it is possible to minimize the catabolite repression exerted by the rapidly utilization of carbon sources (such as glucose) in the synthesis of antibiotics by utilizing a suitable flow rate of supplementary medium. In this study, several runs for cephalosporin C production, each lasting 200 h, were conducted in a fed-batch tower-type bioreactor using different hydrolyzed sucrose concentrations. For this study's model, modifications were introduced to take intoaccount the influence of supplementary medium flow rate. The balance equations considered the effect of oxygen limitation inside the bioparticles. In the Monod-type rate equations, cell concentrations, substrate concentrations, and dissolved oxygen were included as reactants affecting the bioreaction rate. The set of differential equations was solved by the numerical method, and the values of the parameters were estimated by the classic nonlinear regression method following Marquardt's procedure with a 95% confidence interval. The simulation results showed that the proposed model fit well with the experimental data, and based on the experimental data and the mathematical model, an optimal mass flow rate to maximize the bioprocess productivity could be proposed.

Keywords: Cephalosporin C; tower-type bioreactor; fed-batch; modeling; simulation

Influence of culture conditions on lipopeptide production by Bacillus subtilis by Eric Akpa; Philippe Jacques; Bernard Wathelet; Michel Paquot; Regine Fuchs; Herbert Budzikiewicz; Philippe Thonart (pp. 551-561).

Bacillus subtilis produces various families of lipopeptides with different homologous compounds. To produce “new molecules” with improved activities and to select strains that produced a reduced number of homologs or isomers, we studied the effects of different media on the nature of the synthesis of fatty acid chains for each lipopeptide family. This study focused on two B. subtilis strains cultivated in flasks. Optimized medium for lipopeptide production and Landymedium modified by replacing glutamic acid with other α-amino acids were used. We found that the intensity of production of homologous compounds depends on the strain and the culture medium. Analysis of these lipopeptides by high-performance liquid chromatography showed that the strain B. subtilis NT02 yielded various homologous compounds when cultivated in Landy medium (L-Glu), but primarily one homologous product in high relative amounts when cultivated in the optimized medium. Mass spectrometric analysis and determination of the amino acid composition of this molecule enabled us to identify it as Bacillomycine L c15.

Keywords: Bacillus subtilis ; lipopeptide; biosurfactant; bacillomycin; high-performance liquid chromatography; culture medium

Mathematical modeling of controlled-relase kinetics of herbicides in a dynamic-water-bath system by Félix M. Pereira; Adilson R. Gonçalves; André Ferraz; Flávio T. Silva; Samuel C. Oliveira (pp. 563-574).

Release of herbicides from lignin-based formulations follows a diffusion-controlled mechanism. For mathematical modeling of diffusive transport, the conventional approach is to assume sink conditions at both surfaces of polymeric matrix. This boundary condition proved to be inadequate to describeeexperimental data obtained in a water dynamic bath system. However, satisfactory descriptions for this system were obtained when astagnant unstirred layer of herbicide solution was used as the boundary condition. The adequacy of the model incorporating this new boundary condition was statistically tested using the Fisher test at a confidence level of 95% and plotting the residual distribution.

Keywords: Mathematical modeling; controlled release; herbicides; lignin; anetryn; diuron; diffusion

Do cellulose binding domains increase substrate accessibility? by Ali R. Esteghlalian; Vinit Srivastava; Neil R. Gilkes; Douglas G. Kilburn; R. Antony J. Warren; John N. Saddler (pp. 575-592).

This article provides an overview of various theories proposed during the past five decades to describe the enzymatic hydrolysis of cellulose highlighting the major shifts that these theories have undergone. It also describes the effect of the cellulose-binding domain (CBD) of an exoglucanase/xylanase from bacterium Cellulomonas fimi on the enzymatic hydrolysis of Avicel. Pretreatment of Avicel with CBD_Cex at 4 and 37°C as well as simultaneous addition of CBD_Cex to the hydrolytic enzyme (Celluclast, Novo, Nordisk) reduced the initial rate of hydrolysis owing to irreversible binding of CBD proteins to the substrate's binding sites. Nonetheless, near complete hydrolysis was achieved even in the presence of CBD_Cex. Protease treatment of both pure and CBD_Cex-treated Avicel reduced the substrates' hydrolyzability, perhapsowing to proteolysis of the hydrolyzing enzyme (Celluclast) by the residual Proteinase K remaining in the substrate. Better protocols for comptete removal of CBD proteins from the substrate need to be developed to investigate the effect of CBD adsorption on cellulose digestibility.

Keywords: Cellulose; enzymatic hydrolysis; cellulases; cellulose-binding domain; Cellulomonas fimi

Cobalt activation of Bacillus BR449 thermostable nitrile hydratase expressed in Escherichia coli by Sang-Hoon Kim; Rugmini Padmakumar; Patrick Oriel (pp. 597-603).

Expression of nitrile hydratase enzymes utilized in a new “green” process for acrylamide production has proven difficult in Escherichia coli owing to lack of a cobalt transport system to introduce the required cobaltion into this host. We describe the expression of a thermostable nitrile hydratase from a moderatethermophile Bacillus sp. BR449 in E. coli in which the cobaltrequired for enzyme activation is introduced by incubation, of the apoenzyme in the presence of Co⁺⁺ ion at 50°C, yielding active and thermostable, enzyme.

Keywords: Nitrile hydratase; cobalt; thermophile; acrylamide; enzyme activation

Effect of agitation and aeration on production of hexokinase by Saccharomyces cerevisiae by Daniel Pereira Silva; Adalberto Pessoa Jr.; Inês-Conceição Roberto; Michele Vitolo (pp. 605-613).

A batch culture of Saccharomyces cerevisiae for the production of hexokinase was carried out in a 5-L fermentor containing 3 L of culture medium, which was in oculated with cell suspension (about 0.7 g/L), and left ferm entingat 35°C and pH 4.0. The aeration and agitation were adjusted to attain k _La values of 15, 60, 135, and 230 h⁻¹. The highest hexokinase productivity (754.6 U/[L h]) and substrate-cell conversion yield (0.21 g/g) occurred for a k _La of 60 h⁻¹. Moreover, the formation of hexokinase and cell growth are coupled events, which is in accordance with the constitutive character of this enzyme. Hexokinase formation for k _La>60 h⁻¹ was not enhanced probably owing to saturation of the respiratory pathway by oxygen.

Keywords: Hexokinase; Saccharomyces cerevisiae ; agitation and aeration; fermentation; volumetric coefficient of oxygen transfer

Comparison of catalytic properties of free and immobilized cellobiase Novozym 188 by Luiza P. V. Calsavara; Flávio F. De Moraes; Gisella M. Zanin (pp. 615-626).

The enzyme cellobiase from Novo was immobilized in controlled pore silica particles by covalent binding with the silane-glutaraldehyde method with protein and activity yields of 67 and 13.7%, respectively. The activity of the free enzyme (FE) and immobilized enzyme (IE) was determined with 2 g/L of cellobiose, from 40 to 75°C at pH 3.0–7.0 for FE and from 40 to 70°C at pH 2.2–7.0 for IE. At pH 4.8 the maximum specific activity for the FE and IE occurred at 65°C: 17.8 and 2.2 micromol of glucose/(min·mg of protein), respectively. For all temperatures the optimum pH observed for FE was 4.5 whereas for IE it was shifted to 3.5. The energy of activation was 11 kcal/mol for FE and 5 kcal/mol for IE at pH 4.5–5, showing apparent diffusional limitation for the latter. Thermal stability of the FE and IE was determined with 2 g/L of cellobiose (pH 4.8) at temperatures from 40 to 70°C for FE and 40 to 75°C for IE. Free cellobiase maintained its activity practically constant for 240 min at temperatures up to 55°C. The IE has shown higher stability, retaining its activity in the sametest up to 60°C. Half-life experimental results for FE were 14.1, 2.1, and 0.17 h at 60, 65, and 70°C, respectively, whereas IE at the same temperatures had half-lives of 245, 21.3, and 2.9 h. The energy of thermal deactivation was 80.6 k cal/mol for the free enzyme and 85.2 k cal/mol for the IE, suggesting stabilization by immobilization.

Keywords: Immobilized enzyme; cellobiase; cellobiose; thermalstability; energy of activation; energy of deactivation

Influence of operating conditions and vessel size on oxygen transfer during cellulase production by Daniel J. Schell; Jody Farmer; Jenny Hamilton; Bob Lyons; James D. McMillan; Juan C. Sáez; Arun Tholudur (pp. 627-642).

The production of low-cost cellulase enzyme is a key step in the development of an enzymatic-based process for conversion of lignocellulosic biomass to ethanol. Although abundant information is available on cellulase production, little of this work has examined oxygen transfer. We investigated oxygen transfer during the growth of Trichoderma reesei, a cellulase-producing microorganism, on soluble and insoluble substrates in vessel sizes from 7 to 9000 L. Oxygen uptake rates and volumetric mass transfer coefficients (k _La) were determined using mass spectroscopy to measure off gas composition. Experimentally measured k _La values were found to compare favorably with a k _La correlation available in the literature for a non-Newtonian fermentation broth during the period of heavy cell growth.

Keywords: Oxygen transfer; mass transfer coefficient; cellulase; cellulose; ethanol

Characterization of cyclodextrin glycosyltransferase from Bacillus firmus strain No. 37 by Graciette Matioli; Gisella M. Zanin; Flávio F. De Moraes (pp. 643-654).

The enzyme cyclod extringly cosyltransferase (CGTase), EC2.4.1.19, which produces cyclodextrins (CDs) from starch, was obtained from Bacillus firmus strain no. 37 isolated from Brazilian soil and characterized in the soluble form using as substrate 100 g/L of maltodextrin in 0.05 M Tris-HCl buffer, 5 mM CaCl₂, and appropriate buffers. Enzymatic activity and its activation energy were determined as a function of temperature and pH. The activation energy for the production of β- and γ-CD was 7.5 and 9.9 kcal/mol, respectively. The energy of deactivation was 39 kcal/mol. The enzyme showed little thermal deactivation in the temperature range of 35–60°C, and Arrhenius-type equations were obtained for calculating the activity, deactivation, and half-life as a function of temperature. The molecular weight of the enzyme was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, giving 77.6k Da. Results for CGTase activity as a function of temperature gave maximal activity for the production of β-CD at 65°C, pH 6.0, and 7 1.5 mmol of β-CD/(min·mg of protein), whereas for γ-CD it was 9.1 m mol of γ-CD/(min·mg of protein) at 70°C and pH 8.0. For long contact times, the bestuse of the enzymatic activity occurs at 60°C oratalower temperature, and the reaction pH may be selected to increase the vield of a desired CD.

Keywords: Cyclodextringlycosyltransferase; cyclodextrins; activation energy; deactivation energy

Physiological aspects involved in production of xylanolytic enzymes by deep-sea hyperthermophilic archaeon Pyrodictium abyssi by Carolina M. M. Carvalho Andrade; Wilson Bucker Aguiar; Garo Antranikian (pp. 655-669).

Xylanases (EC3.2.1.8) catalyze the hydrolysis of xylan, the major constituent of hemicellulose. The use of these enzymes could greatly improve the overall economics of processing lignocellulosic materials for the generation of liquid fuels and chemicals. The hyperthermophilic archaeon Pyrodictium abyssi, which was originally isolated from marine hot abyssal sites, grows optimally at 97°C and is a prospective source of highly thermostable xylanase. Its endoxylanase was shown to be highly thermostable (over 100 m in at 105°C) and active even at 110°C. The growth of the deep-sea archaeon P. abyssi was investigated using different culture techniques. Among the carbohydrates used, beech wood xylan, birch wood glucuronoxylan and the arabinoxylan from oats pelt appeared to be good inducers for endoxylanase and β-xylosidase production. The highest production of arabinofuranosidase, however, was detected in the cell extracts after growth on xylose and pyruvate, indicating that the intermediate of the tricarboxylic acid cycle acted as a nonrepressing carbon source for the production of thi enzyme. Electron microscopic studies did not show a significant difference in the cell surface (e.g., xylanosomes) when P. abyssi cells were grown on different carbohydrates. The main kinetic parameters of the organism have been determined. The cell yield was shown to be very low owing to incomplete substrate utilization, but a very high maximal specific growth rate was determined (μ_max=0.0195) at 90°C and pH 6.0. We also give information on the problems that arise during the fermentation of this hyperthermophilic archaeon at elevated temperatures.

Keywords: Xylanases; Pyrodictium abyssi ; hyperthermophilic; archaea

Preliminary kinetic characterization of xylose reductase and xylitol dehydrogenase extracted from Candida guilliermondii FTI 20037 cultivated in sugarcane bagasse hydrolysate for xylitol production by Luciane Sene; Maria G. A. Felipe; Silvio S. Silva; Michele Vitolo (pp. 671-680).

Candida guilliermondii FTI 20037 was cultured in sugarcane bagasse hydrolysate supplemented with 2.0 g/L of (NH₄)₂SO₄, 0.1 g/L of CaCl₂·2H₂O, and 20.0 g/L of rice bran at 35°C; pH 4.0; agitation of 300 rpm; and aeration of 0.4, 0.6, or 0.8 vvm. The high xylitol production (20.0 g/L) and xylose reductase (XR) activity (658.8 U/mg of protein) occurred at an aeration of 0.4 vvm. Under this condition, the xylitol dehydrogenase (XD) activity was low. The apparent K _M for XR and XD against substrates and cofactors were as follows: for XR, 6.4×10⁻² M (xylose) and 9.5×10⁻³ mM (NADPH); for XD, 1.6×10⁻¹ M (xylitol) and 9.9×10⁻² mM (NAD+). Because XR requires about 10-fold less xylose and cofactor than XD for the condition in which the reaction rate is half of the V _max, some interference on the overall xylitol production by the yeast could be expected.

Keywords: Xylitol; xylose reductase; xylitol dehydrogenase; Candida guilliermondii

Xylanase production by Aspergillus awamori in solid-state fermentation and influence of different nitrogen sources by Judith L. S. Lemos; Maria C. de A. Fontes; Nei Pereira Jr. (pp. 681-689).

The use of purified xylan as a substrate for bioconversion into xylanases increases the cost of enzyme production. Consequently, there have been attempts to develop a bioprocess to produce such enzymes using different lignocellulosic residues. Filamentous fungi have been widely used to produce hydrolytic enzymes for industrial applications, including xylanases, whose levels in fungi are generally much higher than those in yeast and bacteria. Considering the industrial importance of xylanases, the present study evaluated the use of milled sugarcane bagasse, without any pretreatment, as a carbon source. Also, the effect of different nitrogen sources and the C∶N ratio on xylanase production by Aspergillus awamori were investigated, in experiments carried out in solid-state fermentation. High extracellular xylanolytic activity was observed on cultivation of A. awamori on milled sugarcane bagasse and organic nitrogen sources (45 IU/mL for endoxylanase and 3.5 IU/mL for β-xylosidase). Endoxylanase and β-xylosidase activities were higher when sodium nitrate was used as the nitrogen source, when compared with peptone, urea, and ammonium sulfate at the optimized C∶N ratio of 10∶1. The use of yeast extract as a supplement to the these nitrogen sources resulted in considerable improvementin the production of xylanases, showing the importance of this organic nitrogen source on A. awamori metabolism.

Keywords: Aspergillus awamori ; xylanases; nitrogen nutrition; solid-state fermentation; sugarcane bagasse

Kinetic and mass transfer parameters of maltotriose hydrolysis catalyzed by glucoamylase immobilized on macroporous silica and wrapped in pectin gel by Luciana R. B. Gonçalves; Glória S. Suzuki; Roberto C. Giordano; Raquel L. C. Giordano (pp. 691-702).

Kinetic and mass transport parameters were estimated for maltotriose hydrolysis using glucoamylase immobilized on macroporous silica and wrapped in pectin gel at 30°C. Free enzyme assays were used to obtain the inrinsic kinetic parameters of a Michaelis-Menten equation, with product inhibition by glucose. The uptake method, based on transient experimental data, was employed in the estimation of mass transfer parameters. Effective diffusivities of maltotriose in pectin gel were estimated by fitting a classical diffusion model to experimental data of maltotriose diffusion into particles of pectin gel in the absence of silica. The effective diffusivities of maltotriose in silica were obtained after fitting a bidisperse model to experimental data of maltotriose hydrolysis using glucoamylase immobilized in silica and wrapped in pectin gel.

Keywords: Maltotriose hydrolysis; kinetic parameters; effective diffusivities; Macroporous silica; pectin gel

Selection of stabilizing additive for lipase immobilization on controlled pore silica by factorial design by Cleide M. F. Soares; Heizir F. De Castro; M. Hellena A. Santana; Gisella M. Zanin (pp. 703-718).

Candida rugosa lipase was covalently immobilized on silanized controlled poresilica (CPS) previously activated with glutaraldehyde in the presence of several additives to improve the performance of the immobilized from in long-term operation. Proteins (albumin and lecithin) and organic molecules (β-cyclodextrin and polyethylene glycol [PEG]-1500) were added during the immobilization procedure, and their effects are reported and compared to the behavior of the immobilized biocatalyst in the absence (lacking) of additive. The selection of the most efficient additive at different lipase loadings (150–450 U/g of dry support) was performed by experimental design. Two 2²full factorial designs with two repetitions at the center point were employed to evaluate the immobilization yield. A better, stabilizing effect was found when small amounts of albumin or PEG-1500, were added simul-taneou sly to the lipase on to the support. The catalytic activity had a maximum (193 U/mg) for lipase loading of 150 U/g of dry support using PEG-1500 as the stabilizing additive. This immobilized system was used to perform esterification reactions under repeated batch cycles (for the synthesis of butyl butyrate as a model). The half-life of the lipase immobilized on CPS in the presence of PEG-150 was found to increase fivefold compared with the control (immobilized lipase on CPS without additive).

Keywords: Controlled pore silica; immobilization; lipase; additive; factorial design

Screening of variables in β-xylosidase recovery using cetyl trimethyl ammonium bromide reversed micelles by Francislene-Andréia Hasmann; Adalberto Pessoa Jr.; Inês-Conceição Roberto (pp. 719-728).

β-Xylosidase recovery by micelles using cetyl trimethyl ammonium bromide (CTAB) cationic surfactant was verified under different experimental conditions. A 2⁵⁻¹ fractional factorial design with center points was employed to verify the influence of the following factors on enzyme extraction: pH (x ₁), CTAB concentration (x ₂), electrical conductivity (x ₃), hexanol concentration (x ₄), and butanol concentration (x ₅). Statistical analysis of the results shows that of the fivevariables studied only hexanol and electrical conductivity did not have significant effects on the recovery of β-xylosidase. The other factors had significant effects in increasing order: (x ₁)>(x ₂)>(x ₅). The model predicts a recovery value of about 45%, which is similar to that obtained experimentally (43.5%).

Keywords: Reversed micelles; β-xylosidase; liquid-liquid extraction; statistical design

Activity of xylose reductase from Candida mogii grown in media containing different concentrations of rice straw hydrolysate by Zea D. V. L. Mayerhoff; Inês C. Roberto; Telma T. Franco (pp. 729-737).

Xylose reductase (XR) activity was evaluated in extracts of Candida mogii grown in media containing different concentrations of rice straw hydrolysate. Results of X Ractivity were compared to xylitol production and a similar behavior was observed for these parameters. Highest values of specific production and productivity were found for xylose reductase (35 U/g of cell and 0.97 U/[g of cell·h], respectively) and for xylitol (5.63 g/g of cell and 0.13 g/[g of cell·h]) in fermentation conducted in medium containing 49.2 g of xylose/L. The maximum value of XR:XD ratio (1.82) was also calculated under this initial xylose concentration with 60 h of fermentation.

Keywords: Candida mogii ; hydrolysate concentration; rice straw; xylitol; xylose reductase

Kinetic studies of lipase from Candida rugosa by Ernandes B. Pereira; Heizir F. De Castro; Flávio F. De Moraes; Gisella M. Zanin (pp. 739-752).

The search for an in expensive support has motivated our group to undertake this work dealing with the use of chitosan as matrix for immobilizing lipase. In addition to its low cost, chitosan has several advantages for use as a support, including its lack of toxicity and chemical reactivity, allowing easy fixation of enzymes. In this article, we describe the immobilization of Canada rugosa lipase onto porous chitosan beads for the enzymatic hydrolysis of oliveoil. The binding of the lipase onto the support was performed by physicalad sorption using hexane as the dispersion medium. A comparativestudy between free and immobilized lipase was conducted in terms of pH, temperature, and thermal stability. A slightly lower value for optimum pH (6.0) was found for the immobilized form in comparison with that attained for the soluble lipase (7.0). The optimum reaction temperature shifted from 37°C for the free lipase to 50°C for the chitosan lipase. The patterns of heat stability indicated that the immobilization process tends to stabilize the enzyme. The half-life of the soluble free lipase at 55°C was equal to 0.71 h (K _d=0.98 h⁻¹), whereas for the immobilized lipase it was 1.10 h (K _d=0.63 h⁻¹). Kinetics was tested at 37°C following the hydrolysis of olive oil and obeys the Michaelis-Menten type of rate equation. The K _m was 0.15 mM and the V _max was 51 μmol/(min·mg), which were lower than for free lipase, suggesting that the apparent affinity toward the substrate changes and that the activity of the immobilized lipase decreases during the course of immobilization.

Keywords: Lipase; immobilization; chitosan; physical adsorption; characterization; hydrolysis

Extraction by reversed micelles of the intrecellular enzyme xylose reductase by Ely V. Cortez; Maria das Graças de Almeida Felipe; Niês C. Roberto; Adalberto Pessoa Jr; Michele Vitolo (pp. 753-759).

Xylose reductase enzyme (EC 1.1.1.21) produced by Candidaguilliermondii in sugarcane bagasse was extracted by reversed micelles of N-benzyl-N-dodecyl-N-bis (2-hydroxyethyl) ammonium chloride cationic surfactant. An experimental design was employed to evaluate the influences of the following factors on the enzyme extraction: temperature, cosolvent, and surfactant concentration. A model was used to represent the enzyme recovery and fit of the experimental data. The extraction yielded a total recovery of 130%, and the purity increased 4.8-fold. This study demonstrates that liquid-liquid extraction by reversed micelles is a process able to recover and increase the enzymatic activity and purity of XR produced by C. guilliermondii.

Keywords: Resersed micelles; xylose reductase; liquid-liquid extraction

Controlled hydrolysis of cheese whey proteins using trypsin and α-chymotrypsin by Célia Maria A. Galvão; Astréa F. Souza Silva; Marcos Franqui Custódio; Rubens Monti; Raquel de Lima C. Giordano (pp. 761-776).

This study examined the production of protein hydrolysates with controlled composition from cheese whey proteins. Cheese whey was characterized and several hydrolysis experiments were made using whey proteins and purified β-lactoglobulin, assubstrates, and trypsin and α-chymotrypsin, as catalysts, at two tem peratures and several enzyme concentrations. Maximum degrees of hydrolysis obtained experimentally were compared to the theoretical values and peptide compositions were calculated. For trypsin, 100% of yield was achieved; for α-chymotrypsin, hydrolysis seemed to be dependent on the oligopeptide size. The results showed that the two proteases could hydrolyze β-lactoglobulin. Trypsin and α-chymotrypsin were stable at 40°C, but a sharp decrease in the protease activity was observed at 55°C.

Keywords: Cheese whey; β-lactoglobulin; protein hydrolysates; trypsin; α-chymotrypsin

Solid-State fermentation of phytase from cassava dregs by Kui Hong; Yan Ma; Meiqui Li (pp. 777-785).

Phytases produced by numerous microorganisms and plants degrade phytic acid that has chelated with metal ions in food and feed. It is important to study phytase for the role of metal ions in nutrition of animals and humans as wellas in the reduction of organic phosphate content of a queous environment. This article reports on solid-state fermentation of phytase from a new substrate of cassava dregs. Large quantities of cassava dregs are produced in tropical areas as a by product of cassava starch processing. Protein and inorganic salts were found to be low in cassava dregs. Cassava dregs could be employed for phytase synthesis after the addition of a nitrogen source and mineral salts. Ammonium nitrate was the best nitrogen source among the nitrogen sources investigated, including beef extract, yeast extract, urea, ammonium nitrate, sodium nitrate, and ammonium sulfate. Sodium dodecyl sulfate promoted phytase production from cassava dregs. A maximum phytase yield of 6.73 U/g of dry mass was obtained. The obtained phytase was stable at feed-processing temperature, since 70% of initial enzyme activity was maintained after 30 min of treatment at 75°C.

Keywords: Phytase; cassava dregs; nitrogén source; therm ostability

The effect of additional autopolyploidization in a slow growing cellulase hyperproducer of Trichoderma by Hideo Toyama; Nobuo Toyama (pp. 787-790).

M14-2 is a cellulase hyperproducer derived from Trichderma recesei QM 6a, but with a growth rate lower than that of the original strain. When M14-2 was autopolyploidized followed by haploidization and selection, the strain with both a higher cellulase productivity per mycelia and a higher growth rate could be obtained as M14-2B. This strain seemed to be constructed using gene sources amplified by additional autopolyploidization.

Keywords: Trichoderma ; cellulase; colchicine; cellulose; benomyl

Sections

Personal tools

Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.91-93, #1-9)

Sections

Personal tools

Local resources

Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.91-93, #1-9)