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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.84-86, #1-9)

Fundamental factors affecting biomass enzymatic reactivity by Vincent S. Chang; Mark T. Holtzapple (pp. 5-37).

Poplar wood was treated with peracetic acid, KOH, and ball milling to produce 147 modellignocelluloses with a broad spectrum of lignin contents, acetyl contents, and crystallinity indices (CrIs), respectively. An empirical model was identified that describes the roles of these three properties in enzymatic hydrolysis. Lignin content and CrI have the greatest impact on biomass digestibility, whereas acetyl content has a minor impact. The digestibility of several lime-treated biomass samples agreed with the empirical model. Lime treatment removesallacetyl groups and a moderate amount of lignin and increases CrIslightly; lignin removal is the dominant benefit from lime treatment.

Keywords: Lignocellulose; enzymatic digestibility; lignin; acetyl groups; crystallinity; correlation; pretreatment; lime

Fourier transform infrared quantification of sugars in pretreated biomass liquors by Melvin P. Tucker; Ragheed K. Mitri; Fannie P. Eddy; Q. A. Nguyen; Lynn M. Gedvilas; John D. Webb (pp. 39-50).

The process of converting renewable lignocellulosic biomass to ethanol requires a number of steps, and pretreatment is one of the most important. Pretreatment usually in volves a hydrolysis of the easily hydrolyzed hemicellulosic component of biomass using some form of thermal/chemical/mechanical action that results in a product that can be further hydrolyzed by cellulase enzymes (the cellulosic portion). The sugars produced can then befermented to ethanol by fermentative microorganisms. If the pretreatment step is not severe enough, the resultant residue is not as easily hydrolyzed by the cellulase enzyme. More severe pretreatment conditions result in the production of degradation products that are toxic to the fermentative microorgan ism. In this article, wereport the quantitative analysis of glucose, mannose, xylose, and acetic acid using Fourier transform infrared (FTIR) spectroscopy on liquors from dilute-acid-pretreated softwood and hard wood slurries. Comparison of FTIR and high-performance liquid chromatography quantitative analyses of these liquorsare reported. Recent developments in infrared probe technology has enabled the rapid quantification of these sugars by FTIR spectroscopy in the batch reactor during optimization of the pretreatment conditions, or interfaced to the computer controlling a continuous reactor for on-line monitoring and control.

Keywords: Fourier transform infrared; biomass; hardwood; softwood; pretreatment; acid hydrolysis

Floating cultivation of marine cyanobacteria using coal fly ash by Mitsufumi Matsumoto; Eri Yoshida; Haruko Takeyama; Tadashi Matsunaga (pp. 51-57).

The aim of this study was to develop improved methodologies for bulk culturing of biotechnologically useful marine cyanobacteria in the open ocean. We have investigated the viability of using coal fly ash (CFA) blocks as the support medium in a novel floating culture system for marine microalgae. The marine cyanobacterium Synechococcus sp. NKBG 040607 was found to adhere to floating CFA blocks in liquid culture medium. Maximum density of attached cells of 2.0×10⁸ cells/cm² was achieved using sea water. The marine cyanobacterium Synechococcus sp. NKBG 042902 weaklyadhered to floating CFA blocks in BG-11 medium. Increasing the concentration of calcium ion in the culture medium enhanced adherence to CFA blocks.

Keywords: Marine cyanobacteria; floating culture; coal fly ash; immobilization; power plant; calcium ion

A kinetic study of lipase-catalyzed alcoholysis of palm kernel oil by Débora de Oliveira; Tito Lívio Mointinho Alves (pp. 59-68).

The use of lipases as biocatalysts in interesterification reactions has been the object of growing interest, owing to the importance of esters as emulsifiers, intermediates to produce oleochemicals, and fuel alternatives. We consider in this article a kinetic study of lipase-catalyzed alcoholysis of palm kernel oil, using n-hexane as the solvent. In a first step the ester production was maximized by using a Taguchi design, and then an empirical model was built to determine the influence of the process variables. Taking into account the results obtained in the first step, we performed a kinetic study and developed a simple model for this system.

Keywords: Alcoholysis; lipases; experimental design; vegetable oils; kinetics

Simultaneous saccharification and fermentation of steam-pretreated spruce to ethanol by Mónika Bollók; Kati Réczey; Guido Zacchi (pp. 69-80).

Ethanol production was studied in simultaneous saccharification and fermentation (SSF) of steam-pretreated spruce at 42°C, using a thermotolerant yeast. Three yeast strains of Kluyveromyces marxianus were compared in test fermentations. SSF experiments were performed with the best of these on 5% (w/w) of substrate at a cellulase loading of 37 filter paper units/g of cellulose, and a β-glucosidase loading of 38 IU/gof cellulose. The detoxification of the substrate and the lack of pH control in the experiments increased the final ethanol concentration. The final ethanol yield was 15% lower compared to SSF with Saccharomyces cerevisiae at 37°C, owing to the cessation of ethanol fermentation after the first 10 h.

Keywords: Simultaneous saccharification and fermentation; Kluyveromyces marxianus ; ethanol fermentation; steam-pretreated spruce

Cellulose and hemicellulose hydrolysis models for application to current and novel pretreatment processes by Sigrid E. Jacobsen; Charles E. Wyman (pp. 81-96).

Acids catalyze the hydrolysis of cellulose and hemicellulose to produce sugars that organisms can ferment to ethanol and other products. However, advanced low- and no-acid technologies are critical if we are to reduce bioethanol costs to be competitive as a pure fuel. We believe carbohy drate oligomers play a key role in explaining the performance of such hydrolysis processes and that kinetic models would help us understand their role. Various investigations have developed reaction rate expressions based on an Arrhenius temperature dependence that is first order in substrate concentration and close to first order in acid concentration. In this article, we evaluate these existing hydrolysis models with the goal of providing a foundation for a unified model that can predict performance of both current and novel pretreatment process configurations.

Keywords: Biomass; hydrolysis; kinetics; bioethanol; pretreatment

Effect of chip size on steam explosion pretreatment of softwood by I. Ballesteros; J. M. Oliva; A. A. Navarro; A. González; J. Carrasco; M. Ballesteros (pp. 97-110).

Although considerable progress has been made in technology for converting lignocellulosic biomass into ethanol, substantial opportunities still exist toreduce production costs. In biomass pretreatment, reducing milling power is a technological improvement that will substantially lower production costs for ethanol. Improving sugar yield from hemicellulose hydrolysis would also reduce ethanol production costs. Thus, it would be desirable to test innovative pretreatment conditions to improve the economics by reducing electrical power of the milling stage and by optimizing pretreatment recovery of hemicellulose, as well as to enhance cellulose hydrolysis. The objective of this study was to evaluate the effect of chip size (2–5, 5–8, and 8–12 mm) on steam-explosion pretreatment (190 and 210°C, 4 and 8 min) of softwood (Pinus pinater).

Keywords: Pretreatment; steam explosion; softwood; chip size; enzymatic hydrolysis

Simultaneous saccharification and cofermentation of peracetic acid-pretreated biomass by Lincoln C. Teixeira; James C. Linden; Herbert A. Schroeder (pp. 111-127).

Previous work in our laboratories has demonstrated the effectiveness of peracetic acid for improving enzymatic digestibility of lignocellulosic materials. The use of dilute alkali solutions as a pre-pretreatment prior to peracetic acid lignin oxidation increased carbohydrate hydrolysis yields in a synergistic as opposed to additive manner. Deacetylation of xylan is easily achieved using dilute alkali solutions under mild conditions. In this article, we evaluate the effectiveness of peracetic acid combined with an alkaline pre-pretreatment through simulataneous saccharification and cofermentation (SSCF) of pretreated hybrid poplar wood and sugar can ebagasse. Respective ethanol yields of 92.8 and 91.9% of theoretical are achieved using 6% NaOH/15% peracetic acid-pretreated substrates and recombinant Zymomonas mobilis CP4/p ZB5. Reduction of acetyl groups of the lignocellulosic materials is demonstrated following alkaline pre-pretreatments. Such processing may be helpful in reducing peracetic acid requirements. The influence of deacetylation is more significant in combined pretreatments using lower peracetic acid loadings.

Keywords: Pretreatmen; biomass; peracetic acid; Zymomonas mobilis ; simul taneous saccharification and cofermentation; ethanol

Pretreatment of wastepaper and pulp mill sludge by aqueous ammonia and hydrogen peroxide by Jun Seok Kim; Y. Y. Lee; S. C. Park (pp. 129-139).

Pretreatment of two different softwood-based lignocellulosic wastes (newsprint and Kraft pulp mill sludge) was investigated. Pretreatment was done by aqueous ammonia and hydrogen peroxide (H₂O₂), two delignifying reagents that are environmentally benign. Three different treatment schemes were employed: aqueous ammonia alone (ammonia recycled percolation [ARP]), mixed stream of aqueous ammonia and H₂O₂ and successive treatment with H₂O₂ and aqueous ammonia. In all cases there was a substantial degree of delignification ranging from 30 to 50%. About half of the hemicellulose sugars were dissolved into the process effluent. Retention of cellulose after pretreatment varied from 85 to 100% for newspaper feedstock and from 77 to 85% for the pulp mill sludge. After treatment with aqueous ammonia alone (ARP), the digestibility of newspaper and the pulp mill sludge was improved only by 5% (from 40 to 45% for the former and from 68 to 73% for the latter), despite a substantial degree of delignification occurring after the ARP process. The lign in content thus did not correlate with the digestibility for these substrates. Simultaneous treatment with H₂O₂ and aqueous ammonia did not bring about any significant improvement in the digestibility over that of the ARP. A succcessive treatment by H₂O₂ and ARP showed the most promise because it improved the digestibility of the newspaper from 41 to 75%, a level comparable to that of α-cellulose.

Keywords: Ammonia; hydrogen peroxide; newspaper; pulp millslu dge; pretreatment

Fermentations of pectin-rich biomass with recombinant bacteria to produce fuel ethanol by Joy Bethune Doran; Jennifer Cripe; Misty Sutton; Brian Foster (pp. 141-152).

Pectin-rich residues from sugar beet processing contain significant carbohydrates and insignificant amounts of lignin. Beet pulp was evaluated for conversion toethanol using recombinant bacteria as biocatalysts. Hydrolysis of pectin-rich residues followed by ethanolic fermentations by yeasts has not been productive because galacturonic acid and arabinose are not ferm entable toethanol by these organisms. The three recombinant bacteria evaluated in this study, Escherichia coli strain KO11, Klebsiella oxytoca strain P2, and Erwinia chrysanthemi EC 16 pLOI 555, ferment carbohydrates in beet pulp with varying efficiencies. E. coli KO11 is able to convert pure galactu ronic acid to ethanol with minimal acetate production. Using an enzyme loading of 10.5 filter paper un its of cellulase, 120.4 polygalactu ronase units of pectinase, and 6.4 g of cellobiase (per gram of dry wt sugar beet pulp), with substrate addition after 24 h of fermentation, 40 g of ethanol/L was produced. Other recombinants exhibited lower ethanol yields with increases in acetate and succinate production.

Keywords: Recombinant bacteria; sugar beet pulp; pectin coversion; ethanol; galacturonic acid

Oxidative cracking of precipitated hardwood lignin by hydrogen peroxide by Qian Xiang; Y. Y. Lee (pp. 153-162).

Precipitated hardwood lignin (PHL) is a major byproduct in the biomassto-ethanol process. Oxidativecracking of PHL by hydrogen peroxide in aqueous medium was investigated as a means to produce potentially useful chemicals. The cracking reaction takes place at moderate temperatures (80–160°C), giving mono-and dicarboxylic acids as the main products. The yields of these products are in the range of 30–50% of initial lignin. The reaction mechanism and the product distribution are dependent upon the reaction conditions, especially the pH. The reaction under strong alkaline condition proceeds well even at low reaction temperatures (80–90°C). Under acidic conditions, higher temperatures (130–160°C) are required to attain the same degrees of cracking. The reaction patterns of the oxidative cracking reaction involve the cleavage of lignin ring, aryl ether bond, or other linkages within lignin. By using the findings of this investigation and those of previous work, we have illustrated the reaction pathways for degradation of PHL under alkaline and acidic conditions. Aldehydes and aromatic acids are interm ediate products in the oxidative degradation of lignin. However, they were produced only in trace amounts owing to rapid degradation induced by hydrogen peroxide.

Keywords: Lignin; oxidation; degradation; hydrogen peroxide

Optimizing ammonia pressurization/depressurization processing conditions to enhance enzymatic susceptibility of dwarf elephant grass by Alexis Ferrer; Floyd M. Byers; Betzabé Sulbarán-de-Ferrer; Bruce E. Dale; Cateryna Aiello (pp. 163-179).

An ammonia pressurization/depressurization process was investigated to evaluate the potential of producing reducing sugars from dwarf elephant grass, a warm-season forage. Moisture, temperature, and ammonia loading affected sugar yield (p<0.0001). At optimal conditions, ammonia processing solubilized 50.9% of the hemicellulose and raised the sugar yield (percentage of theoretical) from 18 to 83%. Glucose and xylose production were increased 3.2-and 8.2-fold, respectively. The mild processing conditions of the ammonia treatment (90–100°C, 5 min), the low enzyme loading (2 international filter paper units/g), and the short hydrolysis time (24 h), greatly enhance the potential of using for ages to produce sugars valuable for several applications.

Keywords: Ammonia; dwarfelephant grass; enzymatic hydrolysis; sugars

Development of new ethanologenic Escherichia coli strains for fermentation of lignocellulosic biomass by Bruce S. Dien; Nancy N. Nichols; Patricia J. O'Bryan; Rodney J. Bothast (pp. 181-196).

Two new ethanologenic strains (FBR4 and FBR5) of Escherichia coli were constructed and used to ferment corn fiber hydrolysate. The strains carry the plasmid pLO1297, which contains the genes from Zymomonas mobilis necessary for efficiently converting pyruvate into ethanol. Both strains selectively maintained the plasmid when grown anaerobically. Each culture was serially transferred 10 times in anaerobic culture with sugar-limited medium containing xylose, but noselective antibiotic. An average of 93 and 95% of the FBR4 and FBR5 cells, respectively, maintained pLO1297 in anaerobic culture. The fermentation performances of the repeatedly transferred cultures were compared with those of cultures freshly revived from stock in pH-controlled batch fermentations with 10% (w/v) xylose. Fermentation results were similar for all the cultures. Fermentations were completed within 60 h and ethanol yields were 86–92% of theoretical. Maximal ethanol concentrations were 3.9–4.2% (w/v). The strains were also tested for their ability to ferment corn fiber hydrolysate, which contained 8.5% (w/v) total sugars (2.0% arabinose, 2.8% glucose, and 3.7% xylose). E. coli FBR5 produced more ethanol than FBR4 from the corn fiber hydrolysate. E. coli FBR5 fermented all but 0.4% (w/v) of the available sugar, whereas strain FBR4 left 1.6% unconsumed. The fermentation with FBR5 was completed within 55 h and yielded 0.46 g of ethanol/g of available sugar, 90% of the maximum obtainable.

Keywords: Alcohol; biofuel; Escherichia coli ; fermentation; pentoses

Characterization and complementation of a Pichia stipitis mutant unable to grow on d-xylose or l-arabinose by Nian-Qing Shi; Kristine Prahl; Jim Hendrick; Jose Cruz; Ping Lu; Jae-Yong Cho; Sharon Jones; Thomas Jeffries (pp. 201-216).

Pichia stipitis CBS 6054 will grow on d-xylose, d-arabinose, and l-arabinose. d-Xylose and l-arabinose are abundant in seed hulls of maize, and their utilization is important in processing grain residues. To elucidate the degradation pathway for l-arabinose, we obtained a mutant, FPL-MY30, that was unable to grow on d-xylose and l-arabinose but that could grow on d-arabinitol. Activity assays of oxidoreductase and pentulokinase enzymes involved in d-xylose, d-arabinose, and l-arabinose pathways indicated that FPL-MY30 is deficient in d-xylitol dehydrogenase (D-XDH), d- and l-arabinitol dehydrogenases, and d-ribitol dehydrogenase. Transforming FPL-MY30 with a gene for xylitol dehydrogenase (PsXYL2), which was cloned from CBS 6054 (Gen Bank AF127801), restored the D-XDH activity and the capacity for FPL-MY30 to grow on l-arabinose. This suggested that FPL-MY30 is critically deficient in XYL2 and that the d-xylose and l-arabinose metabolic pathways have xylitolas a common intermediate. The capacity for FPL-MY30 to grow on d-arabinitol could proceed through d-ribulose.

Keywords: d-Xylose; l-arabinose; Pichia stipitis ; fermentation; metabolism; mutant; complementation

Investigation of the cell-wall loosening protein expansin as a possible additive in the enzymatic saccharification of lignocellulosic biomass by John O. Baker; Michele R. King; William S. Adney; Steve R. Decker; Todd B. Vinzant; Suzanne E. Lantz; Rafael E. Nieves; Steven R. Thomas; Lian-Chao Li; Daniel J. Cosgrove; Michael E. Himmel (pp. 217-223).

Butanol production using Clostridium beijerinckii BA101 hyper-butanol producing mutant strain and recovery by pervaporation by N. Qureshi; H. P. Blaschek (pp. 225-235).

Clostridium beijerinckii BA101 (mutant strain) and C. beijerinckii 8052 (wild type) were compared for substrate and butanol inhibition. The wild-type strain is more strongly inhibited by added butanol than is the mutant strain. Acetone and butanol were removed from a fed-batch reactor inoculated with C. beijcrinckii BA101 by pervaporation using a silicone membrane. In the batch reactor, C. beijerinckii BA101 produced 25.3 g/L of total solvents, whereas in the fermentation-recovery experiment it produced 165.1 g/L of total solvents. Solvent productivity increased from 0.35 (batch reactor) to 0.98 g/L·h (fed-batch reactor). The fed-batch reactor wasfed with 500 g/L of glucose-based P2 medium. Acetone selectivities ranged from 2 to 10 whereas butanol selectivities ranged from 7 to 19. Total flux varied from 26 to 31 g/m²·h.

Keywords: Clostridium beijcrinckii BA101; acetone; butanol; fed-batch reactor; pervaporation

Enzyme production of Trichoderma reesei rut C-30 on various lignocellulosic substrates by Chul Seung Shin; Joon Pyo Lee; Jin Suk Lee; Soon Chul Park (pp. 237-245).

Economical production of cellulase enzyme is key for feasible bioethanol production from ligh ocellulosics using an enzyme-based process. On-site cellulase production can be more feasible with the process of separate hydrolysis and fermentation (SHF) than with simultaneous saccharification and fermentation, since the cost of enzyme is more important and a variety of substrates are available for the SHF process. Cellulase production using various biomass substrates available for SHF, including paper sludge, pretreated wood (steam exploded), and their hydrolysis residues, was investigated in shake flasks and a fermenter for their productivities and titers. Among the newspaper sludge, office paper sludge, and steam-exploded woods treated in various ways, the steam-exploded wood showed the best properties for substrate in cellulase production. The besttiter of 4.29 IU/mL was obtained using exploded wood of 2% (w/v) slurry in the shake flask, and the titer with the same substrate was duplicated to about 4.30 IU/mL in a 3.7-L fermenter. Also, the yield of enzyme reached 215 1U/g of substrate or 363 IU/g of cellulose. Despite various pretreatment attempts, newspaper and office paper substrate was inferior to the exploded-wood substrate for cellulase production. However, hydrolysis residues of papers showed quite promising results. The hydrolysis residue of office paper produced 2.48 IU/mL of cellulase in 7 d. Hence, the utilization of hydrolysis residues for cellulase production will be further investigated in the future.

Keywords: Cellulase production; lignocellulosic substrates; waste papers; steam-exploded wood

Nitrogen regulation of Saccharomyces cerevisiae invertase by Maria Cristina F. Silveira; Edna M. M. Oliveira; Elvira Carvajal; Elba P. S. Bon (pp. 247-254).

The regulation of extracellular enzymes is of great biotechnological interest. We studied the regulatory role of the URE 2 gene on the periplasmic invertase of Saccharomyces cerevisiae, because its periplasmic asparaginase is regulated by the URE2/GLN3 system. Enzymatic activity was measured in the isogenic strains P40-1B, the ure2 mutant P40-3C, and the P40-3C strain transformed with the pIC-CS plasmid carrying the URE2 gene. The assays were performed using midlog and stationary phase cells and nitrogen-starved cells from these growth phases. During exponential growth, the level of invertase in both wild-type and ure 2 mutant cells was comparable. However, the invertase activity in ure2 mutant cells from stationary phase was sixfold lower than in the wild-type cells. When P40-3C cells were transformed with the pIC-CS plasmid, the wild-type phenotype was restored. On nitrogen starvation in the presence of sucrose, the invertase activity in wild-type cells from midlog phase decreased three times, whereas in stationary cells, the activity decreased eight times. However, invertase activity doubled in ure 2 mutant cells from both phases. When these cells were trans-formed with the aforementioned plasmid, the wild-type phenotype was restored, although a significant invertase decrease in stationary cells was not observed. These results suggested that the URE2 protein plays a role in invertase activity.

Keywords: Saccharomyces cerevisiae ; nitrogen regulation; URE2 ; invertase; asparaginase

N-demethylation of methylene blue by lignin peroxidase from Phanerochaete chrysosporium by Viridiana S. Ferreira; Denise B. Magalhães; Sérgio H. Kling; José G. Da Silva Jr.; Elba P. S. Bon (pp. 255-265).

Phanerochaete chrysosporium lignin peroxidase (LiP) can degrade synthetic dyes such as heterocyclic, azo, and triphenylmethane on its activation by H₂O₂. Analysis of the reaction products indicated that N-demethylation reactions are involved in the degradation of crystal violet and methylene blue (MB). We studied LiP oxidation of methylene blue and azure B (AB) in reaction mixtures containing different dye: H₂O₂ stoichiometric relations aiming at the selective formation of N-demethylated derivatives. High yields, about 70%, of the mono- and didemethylated derivatives, azure B and azure A, were obtained with the use of 1:1 and 1:2 MB:H₂O₂, respectively. Using azure B as substrate in reaction mixtures containing 1:1 AB:H₂O₂, a yield of 70% was also observed in azure A. Reaction mixtures containing 1:3 MB:H₂O₂ and 1:2 AB:H₂O₂, originated several oxidation products in similar proportions. These results indicated that the process of enzymatic degradation of methylene blue and azure B initiates via N(CH₃)₂ oxidation. According to the yields that were obtained for azure B and azure A, this enzymatic route can be used for the synthesis of these dyes since these data compare favorably to the chemical route that has a yield of 35%. The use of a dye: H₂O₂ relation of 1:10 resulted in a decoloration level of about 85%, showing the usefulness of this procedure for wastewater treatment. The reaction products were followed by spectrophotometric analysis within the wavelength of 500–700 nm. The product identifications were performed using a reverse-phase

Endocellulase and exocellulase activities of two Streptomyces strains isolated from a forest soil by Luzia T. A. S. Semêdo; Rosana C. Gomes; Elba P. S. Bon; Rosangela M. A. Soares; Luiz F. Linhares; Rosalie R. R. Coelho (pp. 267-276).

Two Streptomyces strains, M7a and M23, from a Brazilian forest soil were evaluated for the cellulase production of their superna tants after growth in a microcrystalline cellulose medium, using carboxy methylcellulose and filter paper as substrates at different temperatures and pH values. Endoglucanase and exoglucanase activities were compared to a commercial Trichoderma reesei cellulase using fluorogenic conjugated substrates Similar specific activities were observed for the enzyme preparations of strain M23 and T. reesei. For M7a the activities were about seven times higher than those obtained for T. reesei. Extracellular or cell-associated cellobiase activities were not detected in both strains.

Keywords: Brazilian forest soil; cellulotytic strains; endocellulases; exocellulases; Streptomyces

Comparative energetics of glucose and xylose metabolism in recombinant Zymomonas mobilis by Hugh G. Lawford; Joyce D. Rousseau (pp. 277-293).

Recombinant Zymomonas mobilis CP4:pZB5 was grown with pH control in batch and continuous modes with either glucose or xylose as the sole carbon and energy source. In batch cultures in which the ratio of the final cell mass concentration to the amount of sugar in the medium was constant (i.e., under conditions that promote “coupled growth”), maximum specific rates of glucose and xylose consumption were 8.5 and 2.1 g/(g of cell…h), respectively; maximum specific rates of ethanol production for glucose and xylose were 4.1 and 1.0 g/(g of cell…h), respectively; and average growth yields from glucose and xylose were 0.055 and 0.034 g of dry cell mass (DCM)/g of sugar respectively. The corresponding value of Y_ATP for glucose and xylose was 9.9 and 5.1 g of DCM/mol of ATP, respectively. Y_ATP for the wild-type culture CP4 with glucose was 10.4g of DCM/mol of ATP. For single substratechem ostat cultures in which the growth rate was varied as the dilution rate (D), the maximum or “true” growth yield (max Y_a/s) was calculated from Pirt plots as the inverse of the slope of the best-fit linear regression for the specific sugar utilization rate as a function of D, and the “maintenance coefficient” (m) was determined as the y-axis intercept. For xylose, values of max Y _s/s and m were 0.0417g of DCM/g of xylose (Y_ATP=6.25) and 0.04g of, xylose/(g of cell…h), respectively. However, with glucose there was an observed deviation from linearity, and the data in the Pirt plot was best fit with a second-order polynomial in D. At D>0.1/h, Y_ATP=8.71 and m=2.05g of glu/(g of cell…h) whereas at D<0.1/h, Y_ATP=4.9g of DCM/mol of ATP and m=0.04g of glu/(g of cell…h). This observation provides evidence to question the validity of the unstructured growth model and the assumption that Pirt's maintenance coefficient is a constant that is in dependent of the growth rate. Collectively, these observations with individual sugars and the values assign ed to various growth and fermentation parameters will be useful in the development of models to predict the behavior of rec Zm in mixed substrate fermentations of the type associated with biomass-to-ethanol processes.

Keywords: Recombinant zymomonas ; xylose; growth yield; maintenance coefficient; Y_ATP ; ethanol; chemostat cultures

Continuous fermentation studies with xylose-utilizing recombinant Zymomonas mobilis by Hugh G. Lawford; Joyce D. Rousseau; Ali Mohagheghi; James D. McMillan (pp. 295-310).

This study examined the continuous cofermentation performance characteristics of a dilute-acid “prehydrolysate-adapted” recombinant Zymomonas 39676:pZB4L and builds on the pH-stat batch fermentations with this recombinant that we reported on last year. Substitution of yeast extract by 1% (w/v) corn steep liquor (CSL) (50% solids) and Mg (2 mM) did not alter the coferm entation performance. Using declared assumptions, the cost of using CSL and Mg was estimated to be 12.5c/gal of ethanol with a possibility of 50% cost reduction using fourfold less CSL with 0.1% diammonium phosphate. Because of competition for a common sugar transporter that exhibits a higher affinity for glucose, utilization of glucose was complete whereas xylose was always present in the chemostat effluent. The ethanol yield, based on sugar used, was 94% of theoretical maximum. Altering the sugar ratio of the synthetic dilute acid hardwood prehydrolysate did not appear to significantly change the pattern of xylose utilization. Using a criterion of 80% sugar utilization for determining the maximum dilution rate (D _max), changing the composition of the feed from 4% xylose to 3%, and simultaneously increasing the glucose from 0.8 to 1.8% shifted D _max from 0.07 to 0.08/h. With equal amounts of both sugars (2.5%), D _max was 0.07/h. By comparison to a similar investigation with rec Zm CP4:pZB5 with a 4% equal mixture of xylose and glucose, we observed that at pH 5.0, the D _max was 0.064/h and shifted to 0.084/h at pH 5.75. At a level of 0.4% (w/v) acetic acid in the CSL-based medium with 3% xylose and 1.8% glucose at pH 5.75, the D _max for the adapted recombinant shifted from 0.08 to 0.048/h, and the corresponding maximum volumetric ethanol productivity decreased 45%, from 1.52 to 0.84 g/(L·h). Under these conditions of continuous culture, linear regression of a Pirt plot of the specific rate of sugar utilization vs D showed that 4 g/L of acetic acid did not affect the maximum growth yield (0.030 g dry cell mass/g sugar), but did increase the maintenance coefficient twofold, from 0.46 to 1.0 g of sugar/(g of cell·h).

Keywords: Recombinant Zymomonas ; continuous cofermentation; xylose; biomass hydrolysate; ethanol yield; acetic acid; maintenance coefficient

Improvement of enantioselectivity of chiral organophosphate insecticide hydrolysis by bacterial phosphotriesterase by Wakako Tsugawa; Hiroyuki Nakamura; Koji Sode; Shokichi Ohuchi (pp. 311-317).

The bacterial phosphotriesterase (PTE) isolated from Flavobacterium sp. can catalyze the cleavage of the P-O bond in a variety of organophosphate triesters and has been shown to be an effective catalyst for the degradation of toxic organophosphate esters. Ethyl 4-nitrophenyl phenylphosphono-thioate (EPN) is a chiral organophosphate. Optical isomers of EPN show differences in their toxicity. R-EPN is known to be more toxic to hens and houseflies than S-EPN. We determined the K _i value of each enantiomer toward electriceel acetylcholinesterase. R-EPN (K _i=6 μM) inhibited acetylcholinesterase much more effectively than S-EPN (K _i=52 μM) did in vitro. Since PTE has been found to hydrolyze only the S-isomer of EPN, we attempted to alter the enantioselectivity of PTE in order to degrade toxic EPN enantiomer effectively. When PTE hydrolyzed EPN in the presence of dimethyl sulfoxide (DMSO), enzymatic activity toward S-EPN decreased linearly, but enzymatic activity toward R-EPN increased as a function of DMSO concentration. At 20% DMSO, the maximum activity was observed. The kinetic parameters of PTE to EPN isomers clearly indicated that in the presence of 20% DMSO, the enantioselectivity of PTE changed. The K _is value for R-EPN decreased from 0.24 to 0.03 mM, and the V _max value increased from 0.25 to 0.60 U/mg of protein. V _max/K _is values indicated that PTE preferred R-EPN over S-EPN in the presence of DMSO by a factor of 2.

Keywords: Enantioselectivity; organophosphate insecticide; detoxification; phosphotriesterase

Enhanced alkaline protease production in addition to α-amylase via constructing a Bacillus subtilis strain by Taha I. Zaghloul; Abir E. Abdel Wahab; Mostafa H. Mostafa (pp. 319-327).

Bacillus subtilis Bios 11 strain was previously isolated and identified. This strain naturally produces a high level of α-amylase. The multicopy (pS1) plasmid that carries the complete alkaline protease aprA gene was introduced to this host strain by transformation. The newly constructed strain was found to express the aprA gene and produces a high level of alkaline protease. The level of α-amylase production was not affected compared with the parent strain. The pS1 plasmid in the new host was proved to be segregationally and structurally stable, and the multicopy aprA gene was expressed at the stationary phase. This expression did not affect growth rate and sporulation frequency. Moreover, the level of α-amylase was maintained. Both alkaline protease and α-amylase enzymes were purified using a single-step affinity chromatography column. The use of the newly constructed strain would be valuable to the enzyme industry and would promote recycling of some food-processing wastes.

Keywords: α-Amylase; aprA gene; B. subtilis ; alkaline protease

Methods to enhance tolerances of Chlorella KR-1 to toxic compounds in flue gas by Ju-No Lee; Jin-Suk Lee; Chul-Seung Shin; Soon-Chul Park; Seung-Wook Kim (pp. 329-342).

Possible methods to minimize the toxic effects of SO x and NO x on the growth of a highly CO₂ tolerant and fast-growing microalga, Chlorella sp. KR-1, were investigated. Maintaining the pH of the culturing media at an adequate value was quite important to enhancing the tolerances of the microalgae to SO x and NO x. Controlling the pH by adding an alkaline solution, using a low flow rate of gas fed to the culture, and using a high concentration of inoculating cells were effective methods to maintaining the proper pH of the culture. Controlling the pH was the most effective method but could be applied only for some specific microalgae.

Keywords: SO x and NO x tolerances; pH control; Chlorella KR-1; microalgae; flue gas

Characterization of a high-productivity recombinant strain of Zymomonas mobilis for ethanol production from glucose/xylose mixtures by Eva L. Joachimsthal; Peter L. Rogers (pp. 343-356).

The fermentation characteristics of a recombinant strain of Zymomonas mobilis ZM4(pZB5) capable of converting both glucose and xylose to ethanol have been further investigated. Previous studies have shown that the strain ZM4(pZB5) was capable of converting a mixture o 65 g/L of glucose and 65 g/L of xylose to 62 g/L of ethanol in 48 h with an overall yield of 0.46 g/g. Higher sugar concentrations (e.g., 75/75 g/L) resulted in incomplete xylose utilization (80 h). In the present study, further kinetic evaluations at high sugar levels are reported. Acetate inhibition studies and evaluation of temperature and pH effects indicated increased maximum specific uptake rates of glucose and xylose under stressed conditions with increased metabolic uncoupling. A high-productivity system was developed that involved a membrane bioreactor with cell recycling. At sugar concentrations of approx 50/50 g/L of glucose/xylose, an ethanol concentration of 50 g/L, an ethanol productivity of approx 5 g/(L·h), and a yield (Y _p/s) of 0.50 g/g were achieved. Decreases in cell viability were found in this system after attainment of an initial steady state (40–60 h); a slow bleed of concentrated cells may be required to overcome this problem.

Keywords: Recombinant Zymomonas mobilis ; xylose fermentation; lighocellulosic hydrolysates; inhibition; ethanol

Nuclear magnetic resonance studies of acetic acid inhibition of rec Zymomonas mobilis ZM4(pZB5) by In S. Kim; Kevin D. Barrow; Peter L. Rogers (pp. 357-370).

The fermentation characteristics and effects of lignocelulosic toxic compounds on recombinant Zymomonas mobilis ZM4(pZB5), which is capable of converting both glucose and xylose to ethanol, and its parental strain, ZM4, were characterized using ¹³C and ³¹P nuclear magnetic resonance (NMR) in vivo. From the ³¹P NMR data, the levels of nucleoside triphosphates (NTP) of ZM(pZB5) using xylose were lower than those of glucose. This can be related to the intrinsically slower assimilation and/or metabolism of xylose compared to glucose and is evidence of a less energized state of ZM4(pZB5) cells during xylose fermentation. Acetic acid was shown to be strongly inhibitory to ZM4(pZB5) on xylose medium, with xylose utilization being completely inhibited at pH 5.0 or lower in the presence of 10.9 g/L of sodium acetate. From the ³¹P NMR results, the addition of sodium acetate caused decreased NTP and sugar phosphates, together with acidification of the cytoplasm. Intracellular deenergization and acidification appear to be the major mechanisms by which acetic acid exerts its toxic effects on this recombinant strain.

Keywords: Recombinant Zymomonas ; xylose fermentation; ¹³C nuclear magnetic resonance; ³¹P nuclear magnetic resonance; acetic acid inhibition

Effects of environmental conditions on xylose reductase and xylitol dehydrogenase production by Candida guilliermondii by L. Sene; M. Vitolo; M. G. A. Felipe; S. S. Silva (pp. 371-380).

The effects of environmental conditions, namely initial pH (2.5–7.0) and temperature (25 and 35°C), on xylose reductase and xylitol dehydrogenase levels, as well as on xylitol production, were evaluated. Although the fermentative parameter values increased with an increase in pH and temperature (the maximum Y_P/s and Q p were 0.75 g/g and 0.95 g/[L·h], respectively, both attained at pH 6.0, 35°C), the highest xylose reductase activities (nearly 900 1U/mg of protein) were observed at an initial pH varying from 4.0 to 6.0. Xylitol dehydrogenase was favored by an increase in both initial pH and temperature of the medium. The highest xylitol dehydrogenase specific activity was attained at pH 6.5 and 35°C (577 1U/mg of protein).

Keywords: Xylose reductase; Xylitol dehydrogenase; sugarcane bagasse hydrolysate; Candida guilliermondii

Construction of recombinant Escherichia coli strains for polyhydroxybutyrate production using soy waste as nutrient by Kui Hong; Yun Chung Leung; Sui Yi Kwok; Kin Ho Law; Wai Hung Lo; Hong Chua; Peter Hoi Fu Yu (pp. 381-390).

Construction and comparison of recombinant Escherichia coli strains harboring the polyhydroxybutyrate (PHB) operon from Ralstonia entropha using vectors possessing different promotors, as well as the production of PHB from soy waste by the recombinant strain, are reported. The lac promotor was the most efficient on expression of the phb operon among the three promotors studied: i.e., lac promotor, T7 promotor and the normal σ⁷⁰ promotor. The pKS/PHB was the most efficient plasmid for phboperon expression among the three plasmids used: i.e., pKS⁻, pAED4, and pJM9131. It was observed that isopropyl-β-d-thiogalactopyranoside was not required for the induction of the expression of phb operon. The cell dry wt and polyhydroxyalkan cote content by E. coli XL-1 Blue (pKS/PHB) were 3.025 g/L and 27.83%, respectively.

Keywords: Escherichia coli ; polyhydroxybutyrates; lac promotor; T7 promotor; Fourier-transform infrared; soy waste

Effect of temperature on growth of psychrophilic and psychrotrophic members of Rhodotorula aurantiaca by A. Sabri; P. Jacques; F. Weekers; G. Baré; S. Hiligsmann; M. Moussaïf; P. Thonart (pp. 391-399).

The thermodependence of growth kinetic parameters was investigated for the Antarctic psychrophilic strain Rhodotorula aurantiaca and a psychrotrophic strain of the same species isolated in Belgium (Ardennes area). Cell production, maximum growth rate (μ_max), and half-saturation constant for glucose uptake (Ks) of both yeasts were temperature dependent. For the two yeasts, a maximum cell production was observed at about 0°C, and cell production decreased when temperature increased. The μ_max values for both strains increased with temperature up to a maximum of 10°C for the psychrophilic strain and 17°C for the psychrotrophic strain. For both yeasts, Ks for glucose was relatively constant at low temperatures. It increased at temperatures above 10°C for the psychrophilic strain and 17°C for the psychrotrophic strain. Although its glucose affinity was lower, the psychrotrophic strain grew more rapidly than the psychrophilicone. The difference in growth rate and substrate affinity was related to the origin of the strain and the adaptation strategy of R. aurantiaca to environmental conditions.

Keywords: Rhodotorula aurantiaca ; growth kinetics; psychrophilic; psychrotrophic; cold adaptation

Self-assembling photosynthetic reaction centers on electrodes for current generation by Chikashi Nakamura; Miki Hasegawa; Yoshiaki Yasuda; Jun Miyake (pp. 401-408).

Photosynthetic reaction centers (RCs) made from photosynthetic organisms can be used in solar batteries because their molecules cause light-induced charge separation. We present a simple immobilization system of the intact RCs from Rhodobacter sphaeroides on an electrode that uses nickel ligand binding by the hexameric histidine tag on H subunit (HHisRC). The binding constant of HHisRC to the nickel-nitrilotriacetic acid (Ni−NTA) chip measured with a surface plasmon resonance instrument was 1.6×10⁸M⁻¹. HHisRCs were immobilized on an indium tin oxide electrode overlaid with an Ni−NTA gold substrate. The photoinduced displacement current of this electrode was measured to estimate the orientation of HHisRC on the electrode, and the detachability of HHisRC from the electrode was determined by using an imidazole solution wash. The direction of the flash-light-induced displacement current suggested that the H subunit side of the immobilized HHisRC faced the surface of the electrode. The photoinduced current disappeared after the electrode was washed in the imidazole solution. This simple immobilization and detachment of HHisRC to the electrode might be useful for making a reproducible photocurrent device.

Keywords: Photosynthetic reaction center; purple bacteria; genetic manipulation; protein assembly; displacement current; surface plasmon resonance

Fabrication of an electrode-viologen-hydrogenase heterogeneous system and the electrochemical hydrogen evolution by Dong-Jin Qian; Chikashi Nakamura; Kazuyuki Noda; Nickolai A. Zorin; Jun Miyake (pp. 409-418).

An indium tinoxide (ITO) electrode was chemically modified by one layer of viologen (VIO) deri vative, which possessed a persistent and reproducible electrochemical response. A monolayer of a thermal stable hydrogenase from Thiocapsa roseopersic ina was stabilized on a synthesized poly-l-lysine subphase surface and transferred on to the electrode for fabrication of an ITO-VIO-hydrogenase heterogeneous system. Electrochemical properties of both the ITO-VIO monolayer and the heterogeneous ITO-VIO-hydrogenase system have been investigated. Hydrogen evolution could be measured by potentiostating the VIO-hydrogenase-covered ITO electrode to “electroplate” [(VIO⁺)_n]_surf and a large increase in hydrogen evolution was observed when using an electrolytesolution containing sodium dithionite. We discuss the possible electron transfer process.

Keywords: Indium tin oxide-viologen monolayer; electrochemistry; hydrogenase; Langmuir-Blodgett film; hydrogen evolution

Successive construction of cellulase hyperproducers of Trichoderma using hyperpolyploids by Hideo Toyama; Nobuo Toyama (pp. 419-429).

When the swollen conidia of Trichoderma reesei QM 6a are treated with 0.1% (w/v) colchicine solution, huge autopolyploid nuclei can be formed in those swollen conidia. When a mycelial mat derived from such a conidum is treated with a haploidizing reagent, benomyl, many fan-shaped sectors are produced from the colony, and cellulase hyperproducers are selected from conidia on the colony. When colchicine and benomyl treatments are repeated on cellulase hyperproducers, new hyperproducers can be constructed successively and systematically. Moreover, when conidia derived from autopolyploids are treated with ethylmethanesulfonate solution, another type of cellulase hyperproducers (polyploids) can be obtained.

Keywords: Trichoderma ; cellulase; colchicine; cellulose; saccharification

Effect of light/dark cycle on bacterial hydrogen production by Rhodobacter sphaeroides RV by Tatsuki Wakayama; Eiju Nakada; Yasuo Asada; Jun Miyake (pp. 431-440).

Hydrogen production by photosynthetic bacteria provides an efficient energy conversion method under low light intensity. However, under strong illumination, such as midday sunlight, the efficiency drops. This prevents the method from being applied industrially. To overcome this problem, we examined a method to thin out the excessive illumination. Light was given intermittently to reduce the total energy flux. The on/off ratio was set at 1/1 throughout the study, so that the time average of the light energy flux became half the continuous illumination. By keeping the time-average light flux constant (0.6 kW·m⁻²), the effects of the cycle period were examined in the range of hours to seconds. The hydrogen production rate was greatly affected by the cycle period, but cell growth and substrate consumption rates remained almost constant. The 30-min light/dark cycle (30 min on and 30 min off) provided the highest rate of hydrogen production (22 L·m⁻²·24 h⁻¹). At the shorter cycles, the rate decreased except that there was a suboptimum at about 40 s. Under excessive light intensity (1.2 kW·m⁻²), the light-to-hydrogen conversion efficiency was greatly enhanced. The hydrogen production rate during the 30-min cycle was twice as high as during a 12-h cycle under the same conditions.

Keywords: Photosynthetic bacteria; hydrogen production; Rhodobacter sphaeroides ; light/dark cycle; intermittent irradiation

Two-dimensional analysis of proteins specific to the bacterial magnetic particle membrane from Magnetospirillum sp. AMB-1 by Yoshiko Okamura; Haruko Takeyama; Tadashi Matsunaga (pp. 441-446).

We report the identification of five proteins expressed specifically on the bacterial magnetic particle (BMP) membrane of Magnetospirillum sp. AMB-1. These proteins are major components of the BMP membrane. The molecular weights were determined to be 12.0, 16.0, 24.8, 35.6, and 66.2 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Of these five, the 16.0-kDa protein was the most abundant in the BMP membrane. Furthermore, the 16.0-kDa protein consisted of two components each of differing pI. The 35.6-kDa protein was the second most abundant protein of the five detected.

Keywords: Magnetic bacteria; bacterial magnetic particle; bacterial magnetic particle-specific protein

Salinity-regulated replication of the endogenous plasmid pSY10 from the marine cyanobacterium Synechococcus sp. by Haruko Takeyama; Hideki Nakayama; Tadashi Matsunaga (pp. 447-453).

The endogenous plasmid pSY10 in the marine cyanobacterium Synechococcus sp. NKBG042902 is maintained at a high copy number when cells are grown in seawater and at a low copy number when cultured in freshwater. The mechanism of salinity-regulated replication of this plasmid was investigated. Transcription of repA was depressed under freshwater, which was accompanied by a low copy number of pSY10 and the appearance of a new protein that was expressed only in cells cultured in freshwater. This protein was observed to bind to putative repA promoters (Prep1 and Prep2) on pSY10. Moreover, this protein was observed only in Synechococcus sp. NK BG042902. The data suggest that this protein(s) regulates repA transcription in pSY10, stress responsive and encoded by the host chromosome.

Keywords: Plasmid pSY10; replication; marine cyanobacterium; Synechococcus sp.; salinity stress; repA gene; band shift assay; copy number

Production of lactic acid from pulp mill solid waste and xylose using Lactobacillus delbrueckii (NRRL B445) by Susanna Thomas (pp. 455-468).

Using the simultaneoussaccharification and fermentation (SSF) technique, pulp mill solid waste cellulose was converted into glucose using cellulase enzyme and glucose into lacticacid using NRRL B445. SSF experiments were conducted at various pH levels, temperatures, and nutrient concentrations, and the lactic acid yield ranged from 86 to 97%. The depletion of xylose in SSF was further investigated by inoculating NRRL B445 into a xylose-only medium. On prolonged incubation, depletion of xylose with lactic acid production was observed. An experimental procedure with a nonglucose medium was developed to eliminate the lag phase. From xylose fermentation, Lactobacillus delbrueckii yielded 88–92% lactic acid and 2–12% acetic acid.

Keywords: Xylose; pulp mill solid waste; lactic acid; Lactobacillus ; fermentation

Design and performance of a fibrous bed bioreactor for odor treatment by Hong Chua; Xiang Z. Li; Peter H. F. Yu; Chung Y. Tam; Yu L. Huang; Shang T. Yang (pp. 469-478).

Biological processes have become popular for odor treatment. In this study, a novel fibrous bed bioreactor was applied for treatment of odorous gas. The column reactor was packed with spirally wound fibrous sheet material on which a consortium of microorganisms selected from activated sludge was immobilized. The first stage of this work comprised a preliminary study that aimed at investigating the feasibility of the fibrous bed bioreactor for treatment of odorous volatile fatty acids (VFAs). In this stage, the performance of a fibrous bed bioreactor at increasing mass loadings ranging from 9.7 to 104.2 g/(m³·h) was studied. VFA removal efficiencies above 90% were achieved at mass loadings up to 50.3 g/(m³·h). At a mass loading of 104.2 g/(m³·h), removal efficiency was found to be 87.7%. In the second stage of the work, the process was scaled up with design and operational considerations, namely, packing medium, process condition, and configuration selections. A trickling biofilter with synthetic fibrous packing medium was selected. It was operated under countercurrent flow of gas and liquid streams. The effects of inlet concentration and empty bed retention time on bioreactor performance were studied. The bioreactor was effective in treating odorous VFAs at mass loadings up to 32g/(m³·h), at which VFAs started to accumulate in the recirculation liquid, indicating that the biofilm was unable to degradeall the VFAs introduced. Although VFAs accumulated in the liquid phase, the removal efficency remained above 99%, implying that the biochemical reaction rate, rather than gas-to-liquid mass transfer rate, was the limiting factor of this process. The bioreactor was stable for longterm operation; no clogging and degeneration of the packing medium was observed during the 4-mo operation.

Keywords: Fibrous bed bioreactor; odor treatment; volatile fatty acids; trickling biofilter

α-amylase production by free and immobilized Bacillus subtilis by Enrique Duran-Paramo; Oscar Garcia-Kirchner; Jean-Franç ois Hervagault; Daniel Thomas; Jean-Noël Barbotin (pp. 479-485).

The effect of glucose on the α-amylase production by Bacillus subtilis ATCC-21556 was studied. Initial glucose concentrations up to 20 g/L were found to be directly proportional to the specific α-amylase production in an immobilized-cell batch system, whereas a free-cell batch system presented an inversely proportional relationship with the initial glucose concentration. This might be owing to the α-amylase repression by the glucose present in the culture medium. Three hundred eighty-five percent of the specific α-amylase production with the free-cell system was produced by the immobilized-cell batch culture.

Keywords: α-Amylase production; Bacillus subtilis ; catabolite repression; cells immobilization

Effects of trace levels of copper, chromium, and zinc ions on the performance of activated sludge by S. N. Sin; H. Chua; W. Lo; P. H. F. Yu (pp. 487-500).

The effects of copper, chromium, and zinc ions, at trace levels, on the performance of a simulated activated sludge process were investigated. The results of batch adsorption experiments showed that the adsorption of copper, chromium, and zinc ions followed both the Langmuir and Freundlich isotherms. The presence of trace levels of these three metals not only reduced the adsorption rate of organic matters but also the chemical oxygen demand adsorption capacity (CAC) of the activated sludge. Metal ions competed with the organic substrate for adsorption binding sites on the surfaces of activated sludge bioflocs and reduced the CAC. Studies performed in a sequential batch reactor (SBR) showed that the presence of trace levels of heavy metal ions in wastewater affected the SBR performance to different extents depending on the hydraulic retention time (HRT). When the reactors were operated at short HRTs of 2.5 d or less, the presence of trace levels of heavy metal ions reduced substantially the CAC of activated sludge, which, in turn, affected significantly the performance of the SBR. However, under longer HRTs (e.g., 5d), the heavy metal ions in the wastewater reduced the CAC but had not significant effect on the chemical oxygen demand removal efficiency.

Keywords: Heavy metals; chemical oxygen demand removal; activated sludge; adsorption capacity; sequencing batch reactor

Repeated solid-phase fermentation and extraction for enzyme production by George T. Tsao; Cheng S. Gong; Ning J. Cao (pp. 505-524).

Solid-phase fermentation has been found to have a much higher productivity than the popular liquid submerged fermentation in producing cellulase enzymes. The highest reported productivity in the literature for cellulases by Trichoderma cultures in submerged fermentation is 158 filter paper units (FPU)/(h·L) of fermenting liquid. From preliminary experiments of solid-phase fermentation in 1000-m L flasks, a productivity of 234 FPU of cellulases/(h·L) of solid-bed volume was obtained. When two novel techniques—pressure pulsation and repeated extraction—were applied, a productivity of 806 FPU/(h·L) was achieved. The same techniques also greatly enhanced the productivity of other enzymes by fungal cultures in solid-phase fermentation.

Keywords: Solid phase fermentation; pressure pulsation; repeated extraction; fungi; enzyme

Ethanol production from glucose and xylose by immobilized Zymomonas mobilis CP4 (pZB5) by Mahesh S. Krishnan; Maria Blanco; Christopher K. Shattuck; Nhuan P. Nghiem; Brian H. Davison (pp. 525-541).

Fermentation of glucose-xylose mixtures to ethanol was investigated in batch and continuous experiments using immobilized recombinant Zymomonas mobilis CP4(pZB5). This microorganism was immobilized by entrapment in κ-carrageenan beads having a diameter of 1.5–2.5 mm. Batch experiments showed that the immobilized cells cofermented glucose and xylose to ethanol and that the presence of glucose improved the xylose utilization rate. Batch fermentation of rice straw hydrolysate containing 76 g/L of glucose and 33.8 g/L of xylose gave an ethanol concentration of 44.3 g/L after 24 h, corresponding to a yield of 0.46 g of ethanol/g of sugars. Comparable results were achieved with a synthetic sugar control. Continuous fermentation experiments were performed in a laboratory-scale fluidized-bed bioreactor (FBR). Glucose-xylose feed mixtures were pumped through the FBR at residence times of 2–4 h. Glucose conversion to ethanol was maintained above 98% in all experiments. Xylose conversion to ethanol was highest at 91.5% for a feed containing 50 g/L of glucose and 13 g/L of xylose at a dilution rate of 0.24/h. The xylose conversion to ethanol decreased with increasing feed xylose concentration, dilution rate, and age of the immobilized cells. Volumetric ethanol productivities in the range of 6.5–15.3 g/L·h were obtained. The improved productivities achieved in the FBR compared to other bioreactor systems can help in reducing the production costs of fuel ethanol from lignocellulosic sugars.

Coproduction of ethanol and glycerol by Cheng S. Gong; Jiau X. Du; Ning J. Cao; George T. Tsao (pp. 543-559).

Ethanol and glycerol are both metabolic products of yeasts. There are occasions when coproduction of both is considered desirable in industrial operations. In this article, we describe the potential of integrating the two processes. A LORRE Y8 yeast culture isolated from molasses is capable of efficient glycerol production from glucose, and a yeast Culture 1400 is an excellent producer of ethanol. By controlling the process conditions, the ratio of ethanol and glycerol production can be varied.

Keywords: Glycerol; ethanol; coproduction; yeast fermentation

Two-stage dilute-acid pretreatment of softwoods by Quang A. Nguyen; Melvin P. Tucker; Fred A. Keller; Fannie P. Eddy (pp. 561-576).

Whole treechips obtained from softwood forest thinnings were pretreated via single-and two-stage dilute-sulfuric acid pretreatment. Whole-tree chips were impregnated with dilute sulfuric acid and steam treated in a 4-L steam explosion reactor. In single-stage pretreatment, wood chips were treated using a wide range of severity. In two-stage pretreatment, the first stage was carried out at low severity tomaximize hemicellulose recovery. Solubilized sugars were recovered from the first-stage prehydrolysate by washing with water. In the second stage, water-insoluble solids from first-stage prehydrolysate were impregnated with dilute sulfuric acid, then steam treated at more severe conditions to hydrolyze a portion of the remaining cellulose to glucose and to improve the enzyme digestibility. The total sugar yields obtained after enzymatic hydrolysis of two-stage dilute acid-pretreated samples were compared with sugar yields from single-stage pretreatment. The overall sugar yield from two-stage dilute-acid pretreatment was approx 10% higher, and the net enzyme requirement was reduced by about 50%. Simultaneous saccharification and fermentation using an adapted Saccharomyces cerevisiae yeast strain further improved cellulose conversion yield and lowered the enzyme requirement.

Keywords: Softwood; pretreatment; acid hydrolysis; enzymatic hydrolysis; ethanol

Simulated dynamics and control of an extractive alcoholic fermentation by Aline C. Costa; Eduardo C. Dechechi; Flávio L. H. Silva; Francisco Maugeri; Rubens Maciel (pp. 577-593).

In this study, we investigated the dynamics of a computer simulation of a continuous alcoholic fermentation process combined with a flash column under vacuum. The alcohol was partially extracted in order to maintain its concentration at about 40 kg/m³ in the fermentor. The mathematical model of the fermentation was developed for industrial conditions and considers the effect of the temperature on the kinetic parameters. The performance of the dynamic matrix control algorithm, single input single output and multiple input multiple output, for the control of the extractive process was studied. The concepts of factorial design were used in a simulation study to determine the best control structures for the process.

Keywords: Dynamic matrix control; extractive alcoholic fermentation

Mathematical modeling of controlled-release systems of herbicides using lignins as matrices by Samuel C. Oliveira; Félix M. Pereira; André Ferraz; Flávio T. Silva; Adilson R. Goncalves (pp. 595-615).

The herbicides applied in soils can be easily lost, owing to leaching, volatilization, and bio-and photodegradation. Controlled-release systemsusing polymeric matrices claim to solve these problems. The movement of the herbicides in the soilisalso an important phenomenon to be studied in order to evaluate the loss processes. The development of mathematical models is a relevantrequirement for simulation and optimization of such systems. This study reviews mathematical models as an initial step for modeling data obtained for controlled-release systems of herbicides (diuron, 2,4-dichlorophenoxyacetic acid, and ametryn) using sugarcane bagasse lignin as a polymeric matrix. The release kinetic studies were carried out using several acceptorsystems includinga water bath, soil, and soil-packed columns. Generally, these models take into account phenomena such as unsteady-state mass transfer by diffusion (Fick'slaw) and convection, consumption by several processes, and partitioning processes, resulting in partial differential equations with respect to time and space variables.

Keywords: Mathematical modeling; controlled-release formulations; lignins

Influence of lignocellulose-derived aromatic compounds on oxygen-limited growth and ethanolic fermentation by Saccharomyces cerevisiae by Simona Larsson; Alexis Quintana-Sáinz; Andres Reimann; Nils-Olof Nilvebrant; Leif J. Jönsson (pp. 617-632).

Phenolic compounds released and generated during hydrolysis inhibit fermentation of lignocellulose hydrolysates to ethanol by Saccharomyces cerevisiae. A wide variety of aromatic compounds form from lignin, which is partially degraded during acid hydrolysis of the lignocellulosic raw material. Aromatic compounds may also form as a result of sugar degradation and dare present in wood as extractives. The influence of hydroxy-methoxy-benzaldehydes, diphenols/quinones, and phenylpropane derivatives on S. cerevisiae cell growth and ethanol formation was assayed using a defined medium and oxygen-limited conditions. The inhibition effected by the hydroxy-methoxy-benzaldehydes was highly dependent on the positions of the substituents. A major difference in inhibition by the oxidized and reduced form of a diphenol/quinone was observed, the oxidized form being the more inhibitory. The phenylpropane derivatives were examined with respect to difference in toxicity depending on the oxidation-reduction state of the γ-carbon, the presence and position of unsaturated bonds in the aliphatic side chain, and the number and identity of hydroxyl and methoxyl substituents. Transformations of aromatic compounds occuring during the fermentation included aldehyde reduction, quinone reduction, and double bond saturation. Aromatic alcohols were detected as products of reductions of the corresponding aldehydes, namely hydroxy-methoxy-benzaldehydes and coniferyl aldehyde. High molecular mass compounds and the corresponding diphenol were detected as products of quinone reduction. Together with coniferyl alcohol, dihydroconiferyl alcohol was identified as a major transformation products of conifery aldehyde.

Keywords: Aromatic compounds; inhibitors; S. cerevisiae ; ethanolic fermentation; growth

Effect of the oxygen transfer coefficient on xylitol production from sugarcane bagasse hydrolysate by continuous stirred-tank reactor fermentation by Ernesto Acosta Martínez; Silvio S. Silva; Maria G. A. Felipe (pp. 633-641).

The effect of the oxygen transfer coefficient on the production of xylitol by biocon version of xylose present in sugarcane bagasse hemicellulosic hydrolysate using the yeast Candiada guilliermondii was investigated. Continuous cultivation was carried out in a 1.25-L fermentor at 30°C, pH 5.5, 300 rpm, and a dilution rate of 0.03/h, using oxygen transfer coefficients of 10,20, and 30/h. The results showed that the microbial xylitol production (11 g/L) increased by 108% with the decrease in the oxygen volumetric transfer coefficient from 30 to 20/h. The maximum values of xylitol productivity (0.7g/[L…h]) and yield (0.58 g/g) were obtained at k _L a 20/h.

Keywords: Hemicellulosic hydrolysate; sugarcane bagasse; xylose; xylitol; Candida guilliermondii ; continuous fermentation

Study of biocatalyst to produce ethanol from starch by Raquel L. C. Giordano; Paulo C. Hirano; Luciana R. B. Gonçalves; Willibaldo Schmidell Netto (pp. 643-654).

This article presents a detailed study on the conditions for achieving a stable biocatalyst to be used in the production of ethanol from starch. Different pellets were used depending on which characteristic of the biocatalyst was being studied: (a) Saccharomyces cerevisiae entrapped in pectin or calcium alginate gel particles; (b) silica containing immobilized glucoamylase entrapped in pectin gel particles; or (c) pectin gel particles, with the silicaenzyme derivative and yeast coimmobilized. The influence of several variables on the mechanical resistance of the particle, on the viability of the microorganism, and on the rate of substrate hydrolysis was studied with biocatalyst. The best conditions found were 6% pectin gel, 2-mm particle diameter, and curein 0.2 M CaCl₂·2H₂O/60 mM acetate buffer, pH 4.2, for gel preparation; and 6.0 g/L of CaCl₂·2H₂O in the fermentation medium. Biocatalyst (c) was successfully tested for the production of ethanol from liquefield manioc flour syrup.

Keywords: Ethanol; biocatalyst; glucoamylase; yeast; coimmobilization; starch

Study of different media for production of penicillin G acylase from Bacillus megaterium ATCC 14945 by Laura M. Pinotti; Astrea F. S. Silva; Rosineide G. Silva; Raquel L. C. Giordano (pp. 655-663).

In this study, several fermentation media were tested for the production of penicillin G acylase (PGA) using Bacillus megaterium. The carbon sources studied were glucose and lactose. The nitrogen sources studied were enzymatic casein hydrolysates produced with proteases of different specificities. The replacement of glucose with cheese whey and the addition of free amino acids in the PGA production were also tested. The results showed a strong correlation between the nitrogen source and enzyme yield and the presence of glucose repression. The highest enzyme concentration achieved was 138 IU/L using casein hydrolyzed with 0.6 L of Alcalase^® and cheese whey.

Keywords: Bacillus megaterium ; penicillin G acylase; fermentation; medium optimization; enzymatic casein hydrolysates; cheese whey

High-yield fermentation of pentoses into lactic acid by Prashant V. Iyer; Susanna Thomas; Y. Y. Lee (pp. 665-677).

Lactobacillus species capable of fermenting glucose are generally incapable of utilizing xylose for growth or fermentation. In this study, a novel aspect of a well-known Lactobacillus strain, L. casei subsp. rhamnous (ATCC 10863), was uncovered: it can ferment xylose as efficiently as glucose. This strain is a registered organism, extremely stable on long-term operation. Fermentation by this strain is characterized by an initial lag phase lasting 24–72 h before xylose consumption takes place. The yield (grams/gram) of lactic acid from xylose is in excess of 80% with initial volumetric productivity of 0.38 g/(L-h). Acetic acid is the primary byproduct formed at the level of about 10% of the lactic acid. In addition to xylose, it can ferment all other minor sugars in hemicellulose except arabinose. Subjected to mixed sugar fermentation, this strain consumes glucose first, then mannose, followed by almost simultaneous utilization of xylose and galactose. It shows high tolerance for lactic acid as well as extraneous toxins. It can ferment the mixed sugars present in acid-treated hydrolysate of softwood, giving yields similar to that of pure sugar but at a slower rate.

Cellulase production of Trichoderma reesei rut C 30 using steam-pretreated spruce by Zsolt Szengyel; Guido Zacchi; Amaranta Varga; Kati Réczey (pp. 679-691).

Various techniques are available for the conversion of lignocellulosics to fuel ethanol. During the last decade processes based on enzymatic hydrolysis of cellulose have been investigated more extensively, showing good yield on both hardwood and softwood. The cellulase production of a filamentous fungi, Trichoderma reesei Rut C30, was examined on carbon sources obtained after steam pretreatment of spruce. These materials were washed fibrous steam-pretreated spruce (SPS), and hem icellulose hydrolysate. The hemicellulose hydrolysate contained, besides water-soluble carbohydrates, lignin and sugar degradation products, which were formed during the pretreatment and proved to be inhibitory to microorganisms. Experiments were performed in a 4-L laboratory fermentor. The hydrolytic capacity of the produced enzyme solutions was compared with two commercially available enzyme preparations, Celluclast and logen Cellulase, on SPS, washed SPS, and Solka Floc cellulose powder. There was no significant difference among the different enzymes produced by T. reesei Rut C30. However, the conversion of cellulose using these enzymes was higher than that obtained with logen or Celluclast cellulases using steam-pretreated spruce as substrate.

Keywords: Cellulase production; steam-pretreated spruce; enzymatic capacity of cellulases; filter paper activity measurement; Trichoderma

Steam pretreatment of douglas-fir wood chips by Abdel-Latif Boussaid; Ali R. Esteghlalian; David J. Gregg; Keun Ho Lee; John N. Saddler (pp. 693-705).

Douglas-fir sapwood and heartwood were impregnated with SO₂ and steam exploded at three severity levels, and the cellulose-rich, water-insoluble component was enzymatically hydrolyzed. The high-severity conditions resulted in near complete solubilization and some degradation of hemicelluloses and a significant improvement in the efficiency of enzymatic digestibility of the cell ulose component. At lower severity, some of the hemicellulose remained un hydrolyzed, and the cellulose present in the pretreated solids was not readily hydrolyzed. The medium-severity pretreatment conditions proved to be a good compromise because they improved the enzymatic hydrolyzability of the solids and resulted in the recovery of the majority of hemicellulose in a monomeric form within the water-soluble stream. Sapwood-derived wood chips exhibited a higher susceptibility to both pretreatment and hydrolysis and, on steam explosion, formed smaller particles as compared to heartwood-derived wood chips.

Keywords: Douglas fir; sapwood; heartwood; steam explosion; enzymatic hydrolysis; fiber coarseness; particle size distribution

Biotreatment of refinery spent-sulfidic caustic using an enrichment culture immobilized in a novel support matrix by Julie A. Conner; Robert R. Beitle; Kathleen Duncan; Ravi Kolhatkar; Kerry L. Sublette (pp. 707-719).

Sodium hydroxide solutions are used in petroleum refining to remove hydrogen sulfide (H₂S) and mercaptans from various hydrocarbon streams. The resulting sulfide-laden waste stream is called spent-sulfidic caustic. An aerobic enrichment culture was previously developed using a gas mixture of H₂S and methylmercaptan (MeSH) as the soleenergy source. This culture has now been immobilized in a novel support matrix, DuP ont BIO-SEP^TM beads, and is used to biotreat a refinery spent-sulfidic caustic containing both inorganic sulfide and mercaptans in a continuous flow, fluidized-bed column bioreactor. Complete oxidation of both inorganic and organic sulfur to sulfate was observed with no breakthrough of H₂S and <2 ppmv of MeSH produced in the bioreactor outlet gas. Excessive buildup of sulfate (>12 g/L) in the bioreactor medium resulted in an upset condition evidenced by excessive MeSH breakthrough. Therefore, bioreactor performance was limited by the steady-state sulfate concentration. Further improvement in volumetric productivity of a bioreactor system based on this enrichment culture will be dependent on maintenance of sulfate concentrations below inhibitory levels.

Keywords: Spent-sulfidic caustic; hydrogen sulfide; methylmercaptan; BIO-SEP^TM, immobilization

Lactobacillus plantarum amylase acting on crude starch granules by José A. Florêncio; Daura R. Eiras-Stofella; Carlos R. Soccol; Maurice Raimbault; Jean P. Guyot; José D. Fontana (pp. 721-730).

The microheterogeneous native amylolytic complex secreted by the isolate A6 of Lactobacillus plantarum revealed a selective enzyme specificity loss when submitted to a limited proteolysis under a suboptimum pH condition. A clear electrophoretic profile change toward just one shorter, more acidic, and equally active polypeptide fragment resulted from the pronase E pretreatment. Although the whole enzyme activity remained apparently unaffected for soluble starch, the native parallel activity on intact and nongelatinized starch granules either from cereals or tubers was dramatically reduced. This phenomenon was more clearly documented by scanning electron microscopy using the easiest accessible native substrate: wheat starch granules. The anion-exchange-purified native enzymes from L. plantarum displayed a different optimum pH curve when compared with the thermotolerant α-amylase from Bacillus licheniformis. The α-amylases from the lactic-acid-producing A6 isolate presented an electrophoretic profile easily distinguishable from those from B. liqueniformis and B. subtilis species.

Keywords: Lactobacillus plantarum ; amylase; crude starch depolymerization; proteolysis; amylase zymogram

Development of a modified three-stage methane production process using food wastes by Si W. Kim; Jin Y. Park; Jung K. Kim; Jae H. Cho; Young N. Chun; In H. Lee; Jung S. Lee; Jin S. Park; Don-Hee Park (pp. 731-741).

Amodified three-stage system was developed for the rapid production of methane from food wastes. The primary stage wasa semianaerobic hydrolysis/aci dogenic system, in which approx 4100 mg/L of volatile fatty acids (VFAs) was produced at a hydraulic retention time (HRT) of 2 d. The operation temperature and pH were 30°C and 5.0–5.5, respectively. The nondegraded materials were removed through a holeat the bottom of the reactor. The secondary stage was an anaerobic aci dogenic system equipped with an upflow anaerobic sludge blanket (UASB) type of fermentor. VFA was accumulated up to 6100 mg/L by the addition of Clostridium butyricum to the reactor at an HRT of 2 d. The optimum temperature and pH range were 35°C and 5.0–5.5, respectively. The terliary methanogenic stage produced CH₄ and CO₂ from the VFA in the UASB reactor. Methane content was 72% of the total gas volume, and the yield was 0.45–0.50 m³/kg of volatile solids at an HRT of 12 d. The operation temperature and pH were 41°C and 7.6–7.9, respectively. The three-stage process exhibited an unusually high total chemical oxygen dem and reduction rate up to 95%. Total nitrogen decreased to 96% and <10 mg/L of total phosphorus remained in the final effluent.

Keywords: Anaerobic digestion; volatile fatty acid; modified threestage methane production process

Solid-state fermentation with Aspergillus niger for cellobiase production by George T. Tsao; Liming Xia; Ningjun Cao; Cheng S. Gong (pp. 743-749).

Aspergillus niger NRRL3 was cultivated in a moist wheat bran and ground corncob solid medium supplemented with inorganic minerals for the production of cellobiase (β-1,4-glucosidase, EC 3.2.1.21). With this method, A. niger NRRL3 was able to produce a high concentration of cellobiase (215 IU/gofsolid substrate) after 96 h of incubation. Temperature and moisture content affected final cellobiase titers. The best conditions for cell obiase production from solid substrate by A. niger NRRL3 were determined to be 70% moisture and 35°C.

Keywords: Aspergilus niger ; cellobiase; solid-state fermentation

Effects of initial pH on biological synthesis of xylitol using xylose-rich hydrolysate by Tihany A. Morita; Silvio S. Silva; Maria G. A. Felipe (pp. 751-759).

Sugarcane bagasse, an agricultural residue plentiful in Brazil, was utilized for xylitol production by a biotechnological process. Am edium fermentation prepared with this xylose-rich biomass at an oxygen transfer volmetric coefficient of 10/h¹ and different initial pH value was inoculated with cells of Candida guilliermondii FTI 20037.The maximum values of xylitol and cell volumetric productivities (Q _p=0.56 g/[L·h] and Q _p=0.11 g/[g·h]), xylitol yield factor (Y _p/s=0.79 g/g), and xylose uptake rate (qs=0.197 g/[g·h]) wereattained atp H 7.0 without further pH control. The results show that the yeast performance was influeced by the pH, an im portant bioengineering prameter in this fermentation process.

Keywords: Sugarcance bagasse hydrolysate; xylitol; detoxification method; pH; rermentative parameters

Effect of shear on human insulin in zinc suspension by Reid J. Grainger; Samuel Ko; Eugene Koslov; Ales Prokop; Robert D. Tanner; Veara Loha (pp. 761-768).

Human insulin in zinc suspension was used as a model protein to test the effect of shear on the settling rate of proteins, a possible inference for protein denaturation. The rate of settling was determined directly in a spectropho-tometer. Shear effects are important in retaining the activity of proteins and are present in bubble, foam, and droplet protein fractionation processes. A sim pletest, such as that conducted here, mayeven be useful for monitoring changes in protein structure caused by commercial shipping of the protein. The settling ratefor insulin was continously monitored in theoriginal bottle by spectrophotometric absorbance changes as a function of time. A settling curve was determined following each shear experiment, which included shaking the “worked” insulin solution in a vortex mixer for different lengths of time. It was determined, when comparing long shaking times with short ones, that the initial settling rate was less for the long-term shaking of the insulin samples and greater for the short-term shaking. The secondary effects of light and heat, along with shaking, a pparently did not produce differences from shaking alone.

Keywords: Insulin; human insulin; settling rate; shear; shear stress; shear stress on proteins

Screening of potential antibiotic action of cellulolytic fungi by Oscar Garcia-Kirchner; Marcela Segura-Granados; Ignacio Robledo-Bautista; Enrique Duran-Paramo (pp. 769-778).

Twenty different strains of filamentous fungi were initially selected for evaluation of cellulolytic activity using a single test in a simple mineral salts culture medium with filter paper as the only carbon source. Those fungi strains that were capable of completely breaking the filter paper strip within 4–8 d were assayed also for antimicrobial action, using Staphyloccocus a ureus ATCC 6538P according to the so-called agar piece method. We screened three different strains with both capacities: the production of cellulolytic activity and antibiotic action. The experimental results suggest that the fungi Pinicillium sp. FOPCO1, Aspergillus sp. F0Q001, and Cephalosporium sp. F03800 have both capabilities because they grew rapidly on cellulose as the only carbon source and were able to produce an area of growth inhibition in S. aureus of approx 2.04, 1.57, and 2.39 cm, respectively, on agar plates using the agar piece method. Subsequently, the antibiotic production obtained with those cellulolytic strains was evaluated by submerged fermentation at the flask level, in a simple culture medium containing lactose without biosynthesis precursor, obtaining 3670, 2830, and 4060 antibiotic units/mL, referred to as penicillin G, whereas for cellulolytic activity, the results were 1.34, 1.81 and 0.57 FPU/mL, respectively.

Keywords: Cellulolytic activity; antimicrobial action; filamentous fungi; bioassays

Mycelial pellet formation by Rhizopus oryzae ATCC 20344 by Ying Zhou; Jianxin Du; George T. Tsao (pp. 779-789).

Factors in a cultivation medium affecting fungal growth morphology and funmaric acid production by Rhizopus oryzae ATCC 20344 were investigated. These factors included the initial pH value and trace metals such as zinc, magnesium, iron, and manganese in the cultivation medium. It was found that a significant change in the growth morphology of R. oryzae ATCC 20344 occurs when the initial pH value is varied. A lower initial pH value in the cultivation medium was inhibitory to fungal growth, and fast growth in the cultivation medium at a higher initial pH value promoted, the formation of large pellets or filamentous forms. Trace metals in the cultivation media also had significant effects on pellet formation and fumaric acid fermentation.

Keywords: Pellet formation; fumaric acid; Rhizopus oryzae

l-DOPA production by immobilized tyrosinase by Gabriela M. J. Carvalho; Tito Lívio M. Alves; Denise M. G. Freire (pp. 791-800).

The production of l-DOPA using l-tyrosine as substrate, the enzyme tyrosinase (EC 1.14.18.1) as biocatalyst, and l-ascorbate as reducing agent for the o-quinones produced by the enzymatic oxidation of the substrates was studied. Tyrosinase immobilization was investigated on different supports and chemical agents: chitin flakes activated with hexamethylenediamine and glutaraldehyde as crosslinking agent, chitosan gel beads, chitosan gel beads in the presence of glutaraldehyde, chitosan gel beads in the presence of polyvinyl pyrrolidone, and chitosan flakes using glutaraldehyde as crosslinking agent. The last support was considered the best using as performance indexes the following set of immobilization parameters: efficiency (90.52%), yield (11.65%), retention (12.87%), and instability factor (0.00). The conditions of immobilization on chitosan flakes were optimized using a two-level full factorial experimental design. The independent variables were enzyme-support contact time (t), glutaraldehyde concentration (G), and the amount of enzyme units initially offered (U _C). The response variable was the total units of enzymatic activity shown by the immobilized enzyme (U _IMO). The optimal conditions were t=24 h, G=2% (v/v), and U _C=163.7 U. Under these conditions the total units of enzymatic activity shown by the immobilized enzyme (U _IMO) was 23.3 U and the rate of l-DOPA production rate was 53.97 mg/(L·h).

Keywords: Tyrosinase; enzymatic production; l-DOPA; immobilized enzyme

Inhibition of microbial xylitol production by acetic acid and its relation with fermentative parameters by Tihany A. Morita; Silvio S. Silva (pp. 801-808).

Precipitated sugarcane bagasse hemicellulosic hydrolysate containing acetic acid was fermented by Candida guilliermondii FTI 20037 under different operational conditions (pH 4.0 and 7.0, three aeration rates). At pH 7.0 and k _L a of 10 (0.75 vvm) and 22.5/h (3.0 vvm) the acetic acid had not been consumed until the end of the fermentations, whereas at the same pH and k _L a of 35/h (4.5 vvm) the acid was rapidly consumed and acetic acid inhibition was not important. On the other hand, fermentations at an initial pH of 4.0 and k _L a of 22.5 and 35/h required less time for the acid uptake than fermentations at k _L a of 10/h. The acetic acid assimilation by the yeast indicates the ability of this strain to ferment in partially detoxified medium, making possible the utilization of the sugarcane bagasse hydrolysate in this bioprocess. The effects on xylitol yield and production are reported.

Keywords: Xylitol; hydrolysate; acetic acid; k _L a ; pH

Anaerobic digestion from residue of industrial cassava industrialization with acidogenic and methanogenic physical separation phases by Marcos A. Paixão; Celia R. G. Tavares; Rosángela Bergamasco; André L. E. Bonifácio; Rodrigo T. Costa (pp. 809-819).

A trial was carried out in a continuous regimen, using a completely stirred tank reactor, at acidogenic phase, and a hybrid reactor (upflow anaerobic sludgeblanket+fixed bed) at methanogenic phase at room temperature. The residue to be treated came from a flour and cassava meal industry, and the reactors operated for 300 d with affluent chemical oxygen demand (COD) concentrations of 7500, 9000, 11,000, and 14,000 mg/L. The final results showed a biogas production with a content of 80% methane and an average reduction of COD and free cyanide of nearly 96 and 98%, respectively. The separation of phases selected bacterial groups. At acidogenic phase, a predominance of propionic, n-butyric, and n-valeric acids, as well as a biomass composed of 95% fermentative bacilli, which were responsible for a 90% reduction in free cyanide concentration, was observed. At methanogenic phase, a predominance of methanogenic bacteria that came only from the Methanothrix genus was observed. The bacteria were responsible for high levels of organic matter removal and methane production.

Keywords: Anaerobic digestion; cassava; methanogenic phase; acidogenic phase

Use of corncob for endoxylanase production by thermophilic fungus Thermomyces lanuginosus IOC-4145 by Mônica C. T. Damaso; Carolina M. M. C. Andrade; Nei Pereira Jr. (pp. 821-834).

The production of cellulase-free end oxylanase by the thermophilic fungus Thermomyces lanuginosus was investigated insemisolid fermentation and liquid fermentation. Different process variables were investigated in semisolid fermentation, employing corncobas the carbon source. The best results were with the following conditions: grain size=4.5 mm, solid:liquid ratio=1:2, and inoculum size=20% (v/v). Corncob, xylan, and xylose were the best inducers for endoxylanase production. Additionally, organic nitrogen sources were necessary for the production of high endoxylanase activities. The crude enzyme had optimum activity at pH 6.0 and 75°C, displaying a high thermostability. The apparent K ₂₅ and V _max were 1.77 mg of xylan/mL and 21.5 U/mg of protein, respectively.

Keywords: Xylanases; Thermomyces lanuginosus ; corncob; liquid fermentation; semisolid fermentation

Effect of protein denaturation on void fraction in foam separation column by Robert D. Tanner; Tobias Parker; Samuel Ko; Yuoing Ding; Veara Loha; Liping Du; Ales Prokop (pp. 835-842).

Foam fractionation is a cost-effective process that uses air to extract protein from a liquid (in this case “crude” dilute egg-albumin solution). This article deals with how the void fraction (fraction of air in the aerated solution) of foam is affected by heat denaturation of the protein. A 2-mm glass tube was used to sample the foam-liquid interface fluid in a 35-mm-diameter column in order to detect small changes in void fraction and foam production, which are not easily detected directly from the bulk foam. The main control variablein this study was the protein solution preheating time. As the preheating time increased, the initial void fraction in the column decreased. The initial void fraction of the undenatured solution ranged from about 0.73 to 0.80, and the void fraction for significant preheating times of 5 min ranged from approx 0.68 to 0.72. Furthermore, the period of foam production increased from 5 to 7 min for undenatured proteins in solution to as long 15 min for 5-min preheated solutions. Side-port sampling through a small capillary tube has the potential to be used as a rapid and inexpensive way to determine the level of protein denaturation by directly determining the void fraction and then estimating the effect of denaturation from a protein denaturation calibration curve of the void fraction.

Keywords: Egg albumin; protein denaturation; foam fractionation; hydrophobicity; void fraction

Microbial production of polyhydroxyalkanoates by bacteria isolated from oil wastes by Ai Ling Wong; Hong Chua; Peter Hoi Fu Yu (pp. 843-857).

A Gram-positive coccus-shaped bacterium capable of synthesizing higher relative molecular weight (M _r), polyhydroxybutyrate (PHB) was isolated from sesame oil and identified as Staphylococcus epidermidis (by Microbial ID, Inc., Newark, NJ). The experiment was conducted by shake flask fermentation culture using media containing fructose. Cell growth up to a dry mass of 2.5 g/L and PHB accumulation up to 15.02% of cell dry wt was observed. Apart from using single carbohydrate as a sole carbon source, various industrial food wastes including sesame oil, ice cream, malt, and soya wastes were investigated as nutrients for S. epidermidis to reduce the cost of the carbon source. As a result, we found that by using malt wastes as nutrient for cell growth, PHB accumulation of S. epidermidis was much better than using other wastes as nutrient source. The final dried cell mass and PHB production using malt wastes were 1.76 g/L and 6.93% polymer/cells (grams/gram), and 3.5 g/L and 3.31% polymer/cells (grams/gram) in shake flask culture and in fermentor culture, respectively. The bacterial polymer was characterized by ¹H-nuclear magnetic resonance (NMR), ¹³C-NMR, Fourier transform infrared, and differential scanning calorimetry. The results show that with different industrial food wastes as carbon and energy sources, the same biopolymer (PHB) was obtained. However, the use of sesame oil as the carbon source resulted in the accumulation of PHB with a higher melting point than that produced from other food wastes as carbon sources by this organism under similar experimental conditions.

Keywords: Staphylococcus epidermidis ; polyhydroxybutyrate; food wastes

Improvements in titer, productivity, and yield using solka-floc for cellulase production by Tammy Kay Hayward; Jenny Hamilton; Arun Tholudur; James D. McMillan (pp. 859-874).

Researchers studying cellulase enzymes for the economical production of fuel ethanol envision cellulose as the carbon source. However, submerged Trichoderma reesei cultures grown on cellulose exhibit high run-to-run variability. Thus, an investigation of 30 batch cellulase production experiments was instrumental in determining fermentation conditions that improved enzyme titers, yields, and productivities. Eighteen of the 30 batch experiments experienced minimal process upsets and were classified into eight groups based on agitation rate, gas sparge rate, and the use of oxygen supplementation. Comparing corn steep liquor with yeast extract/peptone also tested the effect of different sources of nitrogen in the media. Average 7-d enzyme titers were doubled from 4 to 8 FPU/mL primarily by increasing aeration.

Keywords: Cellulase; Trichoderma reesei L27; aeration; agitation; oxygen supplementation

Cloning and expression of the limonene hydroxylase of Bacillus stearothermophilus BR388 and utilization in two-phase limonene conversions by Tae K. Cheong; Patrick J. Oriel (pp. 903-915).

A 3.6-kb fragment of Bacillus stearo thermophilus Br388 chromosomal DNA that confers growth on limonene to Escherichia coli has been sequenced, revealing a single open reading frame encoding a single subunit limonene hydroxylase containing 444 amino acid residues. This enzyme proved capable of limonene hydroxylation to a mixture of carveol and perillyl alcohol as well as dehydrogenation of these products to carvone and perillyl aldehyde. Oxygen, FAD, and NADH were found to stimulate the hydroxylation reaction in cell extracts, and NAD⁺ stimulated the dehydrogenase reaction. In two-phase bioconversionsusing viable E. coli cells overexpressing the limonene hydroxylase, perillyl alcohol and carvone were the principal products observed.

Keywords: Limonene; carveol; perillyl alcohol; two-phase; Bacillus stearo thermophilus

Biosurfactants from potato process effluents by David N. Thompson; Sandra L. Fox; Gregory A. Bala (pp. 917-930).

High-solids (HS) and low-solids (LS) potato process effluents were tested as substrates for surfactin production. Tests used effluents diluted 1∶10, unamended and amended with trace minerals or corn steep liquor. Heat pretreatment was necessary for surfactin production from effluents due to indigenous bacteria, whose spores remained after autoclaving. Surfactin production from LS surpassed HS in all cases. Surfactin yields from LS were 66% lower than from a pure culture in an optimized potatostarch medium. LS could potentially be used without sterilization for surfactin production for low-value applications such as environmental remediation or oil recovery.

Keywords: Bacillus subtilis ; Biosurfactant; surfactin; alternatefeedstock; potato; enhanced oil recovery

A kinetic study of synthesis of amoxicillin using penicillin G acylase immobilized on agarose by Luciana R. B. GonÇalves; Roberto Fernandez-Lafuente; Jose M. Guisán; Raquel L. C. Giordano (pp. 931-945).

We presenta kinetic model for the synthesis of amoxicillin from p-hydroxyphenylglycine methyl ester and 6-aminopenicillanic acid, catalyzed by penicillin G acylase immobilized on agarose, at 25°C. Michaelis-Menten kinetic parameters (with and without inhibition) were obtained from initial velocity data (pH 7.5 and 6.5). Amoxicillin synthesis reactions were used to validate the kinetic model after checking mass transport effects. A reasonable representation of this system was achieved under some operational conditions, but the model failed under others. Nevertheless, it will be useful whenever a simplified model is required, e.g., in model-based control algorithms for the enzymatic reactor.

Keywords: Amoxicillin; kinetic model; penicillin G acylase; agarose; synthesis

Enzyme electrochemical preparation of a 3-keto derivative of 1,5-anhydro-d-glucitol using glucose-3-dehydrogenase by Koji Sode; Hiroto Sugiura; Wakako Tsugawa; Yoshihumi Watazu; Tetsuro Hamafuji (pp. 947-954).

A novel enzymatic organic synthesis was reported, utilizing glucose-3-dehydrogenase (G3DH) and its regeneration via electrochemical methods. We combined the water-soluble G3DH prepared from a marine bacterium, Halomonas sp. α-15, and electron mediator with the electrode system in order to regenerate the enzyme. Using this system, the conversion of 1,5-anhydro-d-glucitol (1,5AG), a diabetes marker in human blood, was investigated. The final yield of the product, 3-keto anhydroglucitol (3-ketoAG), which was identified by ¹³C nuclear magnetic resonance, was 82% based on the initial amount of 1,5AG. The electrochemical yield of the reaction proceeded almost stoichiometrically. The electrochemical conversion rate of 1,5AG was 1.24 mmol/(L·h), and the electrochemical yield of 1,5AG consumption was 80%, whereas that for 3-ketoAG was 60%.

Keywords: 1,5 Anhydro-d-glucitol; glucose-3-dehydrogenase; electrochemistry; 3-keto sugars

Enhancement of selectivity for producing γ-cyclodextrin by Graciette Matioli; Gisella M. Zanin; Flávio F. de Moraes (pp. 955-962).

The production of cyclodextrins (CDs) by cyclodextrin-glycosyl-transferase (CGTase) from Bacillus firmus was studied, with respect to the effect of the source of starch upon CD yield and on the selectivity for producing γ-CD. Cyclodextrin production tests were run for 24 h at 50°C, pH 8.0, and 1 mg/L of CGTase, and substrates were maltodextrin or the starches of rice, potato, cassava, and corn hydrolyzed up to D. E. 10. Cornstarch was the best substrate for producing γ-CD. Later, glycyrrhizin (2.5% [w/v]), which forms a stable complex with γ-CD, was added to the cornstarch reaction medium and increased the yield of γ-CD to about four times that produced with only maltodextrin, but the total yield of CDs remained practically unchanged. Therefore, the results showed that the studied CGTase is capable of giving relatively high yield of γ-CD in the presence of glycyrrhizin as complexant and cornstarch as substrate.

Keywords: Cyclodextrin-glycosyl-transferase; cyclodextrin; hydrolyzed starches; glycyrrhizin; cornstarch.

Simultaneous saccharification and fermentation of cellulosic biomass to acetic acid by Jacob R. Borden; Youn Y. Lee; Hyon-Hee Yoon (pp. 963-970).

Astrain of Clostridium thermoaceticum (ATCC 49707) was evaluated for its homoacetate potential. This thermophilic anaerobe best produces acetate from glucose at pH 6.0 and 59°C with a yield of 83% of theoretical. Enzyme hydrolysis of two substrates, a-cellulose and a pulp mill sludge, yielded 68% and 70% digestion, respectively. The optimum conditions for the simultaneous saccharification and fermentation (SSF) were substrate dependent: 55°C, pH 6.0 for α-cellulose, and 55°C, pH 5.5 for the pulp mill sludge. In the SSF with α-cellulose, the overall yield of acetate was strongly influenced by the enzyme loading. In a fed-batch operation of SSF with α-cellulose, an overall acetic acid yield of 60 wt% was obtained. Among the factors limiting the yields were incomplete digestion by the enzyme and the end-product inhibition. In the SSF of pulp mill sludge, inhibitors present in the sludge severely limited bacterial action. A large accumulation of glucose developed over the entire process, changing the intended SSF operation into a separate hydrolysis and fermentation operation. Despite a long lag phase of microbial growth, a terminal yield of 85% was obtained with this substrate.

Keywords: Biomass; SSF; acetic acid; Clostridium thermoaceticum

Effect of C∶N molar ratio on monomer composition of polyhdroxyalk anoates produced by Pseudomonas mendocina 0806 and Pseudomonas pseudoalkaligenus YS1 by Kui Hong; Guo Qiang Chen; Peter Hoi Fu Yu; Guang Zhang; Ye Liu; Hong Chua (pp. 971-980).

Polyhydroxyalkanoates (PHAs) are biodegradable polymers produced by bacteria. In this study, the effect of C∶N molar ratio on the monomer composition of PHAs was investigated, including medium chain length PHA produced by Pseudomonas mendocina 0806 and PHA blends consisting of monomers of 3-hydroxybutyrate and medium chain length hydroxyalkan⇘te produced by Pseudomonas pseudoalkaligenus YS1. It was observed that there were some fixed ranges of C∶N molar ratio that affect the monomer composition of PHA independently of the substrate. For strain 0806, the ranges were C∶N <20, 20200. The monomer composition was constant among these ranges when using glucose and octanoate as the sole substrate. For strain YS1, the ranges were C∶N<20, 2045. These results are useful for controlling monomer composition in PHA production.

Keywords: Polyhydroxyalkanoates; polyhydroxybutyrate; Pseudomonas mendocina ; Pseudomonas pscudoalkaligenus

Optimal production of polyhydroxyalkanoates in activated sludge biomass by C. K. Ma; H. Chua; P. H. F. Yu; K. Hong (pp. 981-989).

Polyhydroxyalkanoates (PHAs) have been recognized as good candidates for biodegradable plastics, but their high price compared with conventional plastics has limited their use. In this study, actiated sludge microorgan isms from a conventional wastewater treatment process were induced, bycontrol-lingthe carbon: nitrogen (C:N) ratioin the reacorliquor, toaccumulate PHAs. In addition, an intermittent nitrogen feeding program was established to optimize the volumetric PHA productivity in a wastewater treatment process. The optimal overall polymer production yield of 0.111 g of polymer/g of carbonaceous substrate consumed was achieved under a C:N ratio of 96:1 by feeding nitrogen in the reactor liquor onceevery four cycles. At the same time, the amount of excess sludge generated from the wastewater treatment process was reduced by22.9%.

Keywords: Activated sludge; polyhydroxyalkanoates; carbon: nitrogen ratio; intermittent nitrogen feeding; wastewater treatment

Polyhydroxybutyrate production from carbon dioxide by cyanobacteria by Masato Miyake; Kazuya Takase; Midori Narato; Emir Khatipov; Joerg Schnackenberg; Makoto Shirai; Ryuichiro Kurane; Yasuo Asada (pp. 991-1002).

Genetic characterization and enhancement of polyhydroxybutyrate (PHB) accumulation in cyanobacteria were investigated for efficient PHB production from CO₂. The genome DNAs in the PHB-accumulating strains Synechococcus sp. MA19 and Spirulina platensis NIES46 retained the highly homologous region to phaC of Synechocystis PCC6803, whereas low homology was detected in the nonaccumulating strains Synechococcus sp. PCC7942 and Anabaenacylindrica NIES19. Synechococcus sp. MA19, which accumulates PHB up to 30% of dry cell weight from CO₂ as the sole carbon source, was mutated by insertion of transposon Tn5 to enhance the PHB accumulation. Genetic and physiological analysis of the mutant indicated that decreased phosphotransacetylase activity could trigger an increase of acetyl coenzyme A leading to enhancement of PHB accumulation. PHB synthase in Synechococcus sp. MA19 was probably attached to thylakoid membrane since PHB granules were associated with pigments. A genetically engineered cyanobacteria retaining soluble PHB synthase from Ralstonia eutropha accumulated pigment-free PHB granules, which is an advantage for the purification of PHB.

Keywords: Polyhydroxyalkanoates; cyanobacteria; carbon dioxide

Production of cyclodextrins in a fluidized-bed reactor using cyclodextrin-glycosyl-transferase by Paulo W. Tardioli; Gisella M. Zanin; Flávio F. de Moraes (pp. 1003-1019).

Cyclodextrin-glycosyl-transferase (EC2.4.1.19), produced by Wacker (Munich, Germany), was purified by biospecific affinity chromatography with β-cyclodextrin (β-CD) as ligand, and immobilized into controlled pore silica particles (0.42 mm). This immobilized enzyme (IE) had 4.7 mg of protein/g of support and a specific activity of 8.6 μmol of β-CD/(min·g_IF) at 50°C, pH 8.0. It was used in a fluidized-bed reactor (FBR) at the same conditions for producing cyclodextrins (CDs) with 10% (w/v) maltodextrin solution as substrate. Bed expansion was modeled by the Richardson and Zaki equation, giving a good fit in two distin ctranges of bed porosities. The minimum fluidization velocity was 0.045 cm/s, the bed expansion coefficient was 3.98, and the particle terminal velocity was 2.4 cm/s. The FBR achieved high productivity, reaching in only 4 min of residence time the same amount of CDs normally achieved in a batch reactor with free enzyme after 24h of reaction, namely, 10.4 mM β-CD and 2.3 mM γ-CD.

Keywords: Cyclodextrin; cyclodextrin-glycosyl-transferase; fluidized-bed reactor; maltodextrin

Occlusion of transition metal ions by new adsorbents synthesized from plant polyphenols and animal fibrous proteins by Masatoshi Goto; Kyozo Suyama (pp. 1021-1038).

Removing and collecting heavy and rare metal ions from industrial effluents and waste aqueous solutions are improtant problems. Our previous studies showed that animal fibrous proteins (AFPs) such as hen eggshell membrane, chicken feather (CF), wool, and silk were stable and insoluble proteins and had an excellent ability to bind not only hard (Mn²⁺ and Fe³⁺) but alsosoft (Ag⁺, Au⁺, Pd²⁺, Pt²⁺, and Hg²⁺) acids from aqueous solutions. In this study, we syntheszed some adsorbents for transition (Cr⁶⁺, Mn²⁺, Co²⁺, Ni²⁺, and Cu²⁺) and heavy (Cd²⁺) metalions from AFPs (gelatin, CF, and wool) and plant polyphenols (lignin and tannin) by heating a mixture of AFPs and plant polyphenols under acidic conditions. In batch experiments, pH profile, time dependency, and isotherm analysis were performed to determine binding properties of adsorbents for transition and heavy metal ions. Columnexperiments were also performed to removecopper ion from aqueous solution. The results showed that the new adsorbents were effective for collecting and removing transition and heavy metal ions from aqueous solutions.

Keywords: Transition metal; animal fibrous protein; plant polyphenol

Phosphotransacetylase as a key factor in biological production of polyhydroxybutyrate by Masato Miyake; Joerg Schnackenberg; Ryuichiro Kurane; Yasuo Asada (pp. 1039-1044).

Phosphotransa cetylase (Pta) catalyzes the reversible conversion of, acetyl-coenzyme A (CoA) to acetyl phosphate. Polyhydroxybutyrate (PHB) synthase and accumulation were compared between a Pta-deficient mutant and the wild-type Escherichia coli, which were transformed with pAE100, coding for 3-ketothiolase, NADPH-dependenta cetoacetyl-CoA reductase, and PHB synthase from Ralstonia eutropha. During the growth period, PHB synthase activity in the Pta-deficient mutant was lower than that in the wild type. PHB accumulation in the Pta-deficient mutant, however, was higher than that in wild-type cells grown in Luria-Bertani (LB) medium containing 1% glucose (high C:N ratio). The Pta-deficient mutant showed PHB accumulation even in LB medium (low C:N ratio), whereas wild-type cells showed no PHB accumulation. These data suggest the activation of PHB synthase by acetyl phosphate that is synthesized by Pta. A decrease in Pta activity probably causes some increase in acetyl-CoA as substrate for the PHB synthesis pathway, resulting in increased PHB accumulation.

Keywords: Polyhydroxy butyrate; cyanobacteria; phosphotransacetylase; metabolic engineering; pta; ack

Recovery of volatile products from dilute high-fouling process streams by Philip W. Madson; David B. Lococo (pp. 1049-1061).

As biomass hydrolysis, and fermentation technologies approach commercial viability, advancements in product recovery technologies will be required. For cases in which fermentation products are more volatile than water, recovery by distillation is often the technology of choice. Distillation technologies that will allow the economic recovery of dilute volatile products from streams containing a variety of impurities have been developed and commercially demonstrated. Distillation tower and tray designs, along with specialized heat-exchanger designs, allowing for extended processing intervals on solutions containing lignocellulosic residues, organic acids, and inorganic salts concentrations >100 g/L are in commerical operation. In the case of ethanol, distillation energy consumption efficiencies for processing solutions containing <40 g/L of desired product can approach demonstrated energy consumption efficiencies for solutions containing concentrations >120 g/L. These proprietary technologies have been applied individually at commercial scale, and designs have been developed that incorporate the combined technologies with only a marginal increase in capital investment compared to traditional methods.

Keywords: Distillation; energy consumption; fouling; productrecovery; separation

Adsorption of inulinases in ion-exchange columns by F. -R. C. Silva; C. C. Santana (pp. 1063-1078).

Theuse of adsorption columns packed with ion-exchange resins for recovering, concentrating and purifying proteins is now widespread. The present work consists of a study on the dyamic behavior ofadsorption columns that uses two kinds of adsorbents: a cationic and an anionic resin. A frontal analysis of the columns was performed with experimental data obtained from Fructozyme, a mixture of inulinase en zymes. The parameters of a Langmuir type of isotherm and adsorption kinetics were obtained from experimental tests in a batch system. A numerical technique based on orthogonal colocation and a fourth-order Runge-Kutta method was coupled with a nonlinear optimization method to predict the coefficients of the rate equations, which are fundamental for scale-up purposes.

Keywords: Adsorption; enzyme adsorption; inulinases; ion-exchange; adsorption modeling

Sonic wave separation of invertase from a dilute solution to generated droplets by Robert D. Tanner; Samuel Ko; Veara Loha; Ales Prokop (pp. 1079-1086).

It has previously been shown that a droplet fractionation process, simulated by shaking a separatory funnel containing a dilute protein solution, can generate droplets richer in protein than present in the original dilute solution. In this article, we describe an alternative method that can increase the amount of protein transferred to the droplets. The new metho uses ultrasonic waves, enhanced by a bubble gas stream to create the droplets. The amount of protein in these droplets increases by about 50%. In this method, the top layer of the dilute protein solution (of the solution-air interface) becomes enriched in protein when air is bubbled into the solution. This concentrating procedure is called bubble fractionation. Once the protein has passed through the initial buildup, this enriched protein layer is transferred into droplets with the aid of a vacuum above the solution at the same time that ultrasonic waves are introduced. The droplets are then carried over to a condenser and coalesced. We found that this new method provides an easier way to remove the protein-enriched top layer of the dilute solution and generates more droplet within a shorter period than the separatory funnel droplet generation method. The added air creates the bubbles and carries the droplets, and the vacuum helps remove the effluent airstream from the condenser. The maximum partition coefficient, the ratio of the protein concentration in the droplets to that in the residual solution (approx 8.5), occurred at pH 5.0.

Keywords: Sonic waves; droplet fractionation; invertase; hydrophobicity; enzymatic activity

Modeling a protein foam fractionation process by Liping Du; Veara Loha; Robert D. Tanner (pp. 1087-1099).

A simple staged model for the protein foam fractionation process is proposed in this article. This simplified model does not detail the complex foam structure and gas-liquid hydrodynamics in the foam phase but, rather, is built on the conventional theoretical stage concept considering upward bubbles with entrained liquid and downward liquid (drainage) as counter-current flows. To simulate the protein concentration distribution in the liquid along the column by the model, the bubble size and liquid hold-up with respect to the position must be known, as well as the adsorption isotherm of the protein being considered. The model is evaluated for one stage by data from the semibatch foam fractionation of egg albumin and data from the continuous foam fractionation of bovine serum albumin. The effect of two significant variables (superficial gas velocity and feed protein concentration) on enrichment is well predicted by the model, especially for continuous operation and semibatch operation when initial concentration is high.

Keywords: Staged model; foam fractionation; egg albumin; Bovine serum albumin

β-Xylosidase recovery by reversed micelles by Francislene Andrea Hasmann; Adalberto Pessoa Jr.; Ines Conceicao Roberto (pp. 1101-1111).

β-Xylosidase, an enzyme produced by Penicillium janthinellum fungus, was prepurified by fractionated precipitation with ethanol and extracted by reversed micelles of N-benzyl-N-dodecyl-N-bis(2-hydroxyethyl) ammonium chloride (BDBAC) cationic surfactant. A 2^5-1 fractional factorial design was employed to evaluate the influence of the following factors on the enzyme extraction: pH (A), conductivity (B), surfactant concentration (C), cosolvent concentration (D) and temperature (E). A statistical analysis of the results revealed that, of the five variables studied, pH, surfactant concentration, and temperature had significant main effects (p<0.05) on the recovery of β-xylosidase (Y). However, the interactions between pH and temperature, conductivity and cosolvent concentration, conductivity and temperature, BDBAC concentration and temperature also had significant influences. A first-order model (R ²=0.98) expressed by the equation Y=7.73−5.55A−0.67B+3.49C−0.41D+5.26E+2.05AB−3.26AC+0.96AD−7.07AE−3.93BD−2.19BE+0.99CD+0.78CE was proposed to represent the enzyme recovery as a function of the effects that were really significant. This model predicts a recovery value of about 40%, which is similar to that obtained experimentally (39.5%).

Keywords: Reverse micelles; liquid-liquid extraction; β-xylosidase; cationic surfactant; downstream processing

A novel magnetite-immobilized cell process for heavy metal removal from industrial effluent by Wang Lei; H. Chua; W. H. Lo; P. H. F. Yu; Y. G. Zhao; P. K. Wong (pp. 1113-1126).

The sorption and desorption of copper (II) (Cu[II]) ions from the wastewater by magnetite-immobilized cells of Pseudomonas putida 5-x with acidic pretreatment were studied. Pretreating cells with 0.6 N HCl was found to enhance greatly the adsorption capacity of biomass up to 85.6 mg/g and had no significant effect on the loss of P. putida 5-x cells during biosorbent pretreatment. The biosorption capacity to Cu²⁺ of magnetite-immobilized cells of P. putida 5-x harvested during various growth phases was also investigated. The experimental results illustrated that the adsorption capacity to Cu²⁺ of P. putida 5-x cultured in sulfate-limiting medium reached maximum during the late stationary growth phase or early death phase, and reached minimum during the log growth phase. The mechanism of copper sequestering by this type of biomass was studied via transmission electron microscopy. A degradation of the peptidoglycan layer of the cell wall was observed in the acidic pretreatment, but no further degradation appeared after the adsorption-desorption cycle. Cu(II) accumulated mostly on the surface of the cell walls and was effectively desorbed by the acidic treatment during the desorption process.

Keywords: Magnetite-immobilized cells; copper; sorption and desorption mechanism; transmission electron microscopy analysis; grwoth phase; Pseudomonas putida 5-x

Effect of food: Microorganism ratio in activated sludge foam control by Hong Chua; Peter H. F. Yu; Shirley N. Sin; Kok N. Tan (pp. 1127-1135).

Foaming is a common operational problem in activated sludge processes that often adversely affects the quality of the treated effluent. Overgrowth of the filamentous Nocardia spp. in the microbial ecosystem was previously identified as the cause of foaming. In the present study, the specific growth rate of Nocardia amarae was found to be much higher than that of nonfilamentous bacteria under food:microorganism (F:M) ratios lower than 0.5 mg of biological oxygen demand (BOD)/(mg of mixed liquor suspended solids [MLor]·d). This indicated that filamentous overgrowth may occur in normal activated sludge processes that are continually operated under the usual F:M range of 0.2–0.6 mg of BOD/(mg of MLSS·d). A novel two-component feast-fast operation (FFO) that capitalized on the sensitivity of filamentous bacteria to F:M ratio was designed to prevent and control foaming problems. The F:M ratio in the “feasting” aeration unit was 0.8 mg of BOD/(mg of MLSS·d) whereas that in the “fasting” aeration unit was 0.2 mg of BOD/(mg of MLSS·d). The FFO resulted in an overall process F:M ratio that still remained within the normal range, while avoiding prolonged exposure of the activated sludge ecosystem to an F:M ratio below 0.5 mg of BOD/(mg of MLSS·d). The FFO suppressed the over growth of filamentous bacteria without adversely affecting the organic treatment efficiency of the modified process.

Keywords: Activated sludge; feast-fast operation; filamentous microorganisms; food:microorganism ratio; foam control

Lipase production by Penicillium restrictum using solid waste of industrial babassu oil production as substrate by Marcia B. Palma; Annette L. Pinto; Andreas K. Gombert; Karina H. Seitz; Silvia C. Kivatinitz; Leda R. Castilho; Denise M. G. Freire (pp. 1137-1145).

Lipase, protease, and amylase production by Penicillium restrictum in solid-state fermentation was investigated. The basal medium was an industrial waste of babassu oil (Orbignya oleifera) production. It was enriched with peptone, oliveoil, and Tween-80. The supplementation positively influenced both enzyme production and fungal growth. Media enriched with Tween-80 provided the highest protease activity (8.6 U/g), whereas those enriched with peptone and olive oil led to the highest lipase (27.8 U/g) and amylase (31.8 U/g) activities, respectively.

Keywords: Lipase; protease; amylase; solid-state fermentation; Penicillium restrictum

Brazilian bioethanol program by Gisella M. Zanin; Cesar C. Santana; Elba P. S. Bon; Raquel C. L. Giordano; Flávio F. de Moraes; Silvio R. Andrietta; Carlos Coelho De Carvalho Neto; Isaias C. Macedo; Djalma Lahr Fo.; Luiz P. Ramos; José D. Fontana (pp. 1147-1161).

Brazil is the largest producer of bioethanol, and sugarcane is the main raw material. Bioethanol, is produced by both batch and continuous processes, and in some cases, flocculating yeast is use. This article analyzes the Bracilian Ethanol Program. for the 1996–1997 havest, Brazil produced 14.16 billion L of ethanol and 13.8 million metrict of sugar, from 286 million metrict of sugarcane. These products were produced by 328 industries inactivity, with 101 autonomousethanol plants producing only ethanol, and 227 sugar mills producing sugar and ethanol. The sugar-ethanol market reaches about 7.5 billion US$/yr, accounting for direct and indirect revenues.

Keywords: Sugarcane; alcohol; bioethanol program; ethanol

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.84-86, #1-9)