Showing papers in "Applied Biochemistry and Biotechnology in 1997"

Lime pretreatment of switchgrass

Abstract: Lime (calcium hydroxide) was used as a pretreatment agent to enhance the enzymatic digestibility of switchgrass. After studying many conditions, the recommended pretreatment conditions are: time = 2 h, temperature = 100°C and 120°C, lime loading = 0.1 g Ca(OH)2/g dry biomass, water loading = 9 mL/g dry biomass. Studies on the effect of particle size indicate that there was little benefit of grinding below 20 mesh; even coarse particles (4–10 mesh) digested well. Using the recommended pretreatment conditions, the 3-d reducing sugar yield was five times that of untreated switchgrass, the 3-d total sugar (glucose + xylose) yield was seven times, the 3-d glucose yield was five times, and the 3-d xylose yield was 21 times. A material balance study showed that little glucan (approx 10%) was solubilized as a result of the lime pretreatment, whereas about 26% of xylan and 29% of lignin became solubilized.

Molecular chaperones, folding catalysts, and the recovery of active recombinant proteins from E. coli. To fold or to refold.

Abstract: The high-level expression of recombinant gene products in the gram-negative bacterium Escherichia coli often results in the misfolding of the protein of interest and its subsequent degradation by cellular proteases or its deposition into biologically inactive aggregates known as inclusion bodies. It has recently become clear that in vivo protein folding is an energy-dependent process mediated by two classes of folding modulators. Molecular chaperones, such as the DnaK-DnaJ-GrpE and GroEL-GroES systems, suppress off-pathway aggregation reactions and facilitate proper folding through ATP-coordinated cycles of binding and release of folding intermediates. On the other hand, folding catalysts (foldases) accelerate rate-limiting steps along the protein folding pathway such as the cis/trans isomerization of peptidyl-prolyl bonds and the formation and reshuffling of disulfide bridges. Manipulating the cytoplasmic folding environment by increasing the intracellular concentration of all or specific folding modulators, or by inactivating genes encoding these proteins, holds great promise in facilitating the production and purification of heterologous proteins. Purified folding modulators and artificial systems that mimic their mode of action have also proven useful in improving the in vitro refolding yields of chemically denatured polypeptides. This review examines the usefulness and limitations of molecular chaperones and folding catalysts in both in vivo and in vitro folding processes.

Alginate properties and heavy metal biosorption by marine algae

Abstract: The physical properties of the alginate component in four different brown seaweeds (Sargassumfluitans, Ascophyllum nodosum, Fucus vesiculo-sus, andLaminaria japonica) were characterized using potentiometric titration,13C-nuclear magnetic resonance (NMR), chemical analysis, and viscosity measurements. The heavy metal binding capacities of the corresponding seaweeds were directly proportional to their respective total carboxyl group content, and related to the electronegativity of the elements investigated (Ca, Zn, Cd, Cu, and Pb). The uronic acid composition or sequence of the alginate component did not affect the metal uptake properties of the biosorbents studied here. However, the alginate leaching owing to its solubilization by Na ions was observed to decrease with increasing intrinsic viscosity of the extracted alginate, related to its molecular weight, and with increasing apparent acidic dissociation constant, related to the alginate density inside the biomass.

Oxygen Sensitivity of Algal H2-Production

Abstract: Photoproduction of H2 by green algae utilizes electrons originating from the photosynthetic oxidation of water and does not require metabolic intermediates. However, algal hydrogenases are extremely sensitive to O2, which limits their usefulness in future commercial H2-production systems. We designed an experimental technique for the selection of O2-tolerant, H2-producing variants of Chlamydomonas reinhardtii based on the ability of wild-type cells to survive a short (20 min) exposure to metronidazole in the presence of controlled concentrations of O2. The number of survivors depends on the metronidazole concentration, light intensity, preinduction of the hydrogenase, and the presence or absence of O2. Finally, we demonstrate that some of the selected survivors in fact exhibit H2-production capacity that is less sensitive to O2 than the original wild-type population.

Membrane-mediated extractive fermentation for lactic acid production from cellulosic biomass.

Abstract: Lactic acid production from cellulosic biomass by cellulase and Lactobacillus delbrueckii was studied in a fermenter-extractor employing a microporous hollow fiber membrane (MHF). This bioreactor system was operated under a fed-batch mode with continuous removal of lactic acid by an in situ extraction. A tertiary amine (Alamine 336) was used as an extractant for lactic acid. The extraction capacity of Alamine 336 is greatly enhanced by addition of alcohol. Long-chain alcohols serve well for this purpose since they are less toxic to micro-organism. Addition of kerosene, a diluent, was necessary to reduce the solvent viscosity. A solvent mixture of 20% Alamine 336, 40% oleyl alcohol, and 40% kerosene was found to be most effective in the extraction of lactic acid. Progressive change of pH from an initial value of 5.0 down to 4.3 has significantly improved the overall performance of the simultaneous saccharification and extractive fermentation over that of constant pH operation. The change of pH was applied to promote cell growth in the early phase, and extraction in the latter phase.

Pretreatment of yellow poplar sawdust by pressure cooking in water

Abstract: The pretreatment of yellow poplar wood sawdust using liquid water at temperatures above 220°C enhances enzyme hydrolysis. This paper reviews our prior research and describes the laboratory reactor system currently in use for cooking wood sawdust at temperatures ranging from 220 to 260°C. The wood sawdust at a 6–6.6% solid/liquid slurry was treated in a 2 L, 304 SS, Parr reactor with three turbine propeller agitators and a proportional integral derivative (PID) controller, which controlled temperature within ±1°C. Heat-up times to the final temperatures of 220, 240, or 260°C were achieved in 60–70 min. Hold time at the final temperature was less than 1 min. A serpentine cooling coil, through which tap water was circulated at the completion of the run, cooled the reactor’s contents within 3 min after the maximum temperature was attained. A bottoms port, as well as ports in the reactor’s head plate, facilitated sampling of the slurry and measuring the pH, which changes from an initial value of 5 before cooking to a value of approx 3 after cooking. Enzyme hydrolysis gave 80–90% conversion of cellulose in the pretreated wood to glucose. Simultaneous saccharification and fermentation of washed, pretreated lignocellulose gave an ethanol yield that was 55% of theoretical. Untreated wood sawdust gave less than 5% hydrolysis under the same conditions.

Identification of inhibitory components toxic toward zymomonas mobilis CP4(pZB5) xylose fermentation

Abstract: Zymomonas mobilis CP4(pZB5) is a recombinant bacterium that can produce ethanol from both xylose and glucose. The ethanol-producing efficiency of this organism is substantially impeded by toxic substances present in pretreated hydrolyzates or solid biomass substrates. Acetic acid and furfural (a pentose degradation product) are highly toxic to this organism at levels envisioned for a pretreated-hardwood liquid hydrolyzate. In addition, lignin degradation products and 5-hydroxymethylfurfural (a hexose degradation product) have a moderately toxic effect on the organism. Of the compounds studied, organic acids and aldehydes were found to be more inhibitory than lignin acids or the one alkaloid studied. Acetone:water and methanol extracts of solid biomass samples from red oak, white oak, and yellow poplar are toxic toZymomonas cell growth and ethanol production, with the extracts from white oak being the most toxic.

Lipase production by Penicillium restrictum in a bench-scale fermenter : effect of carbon and nitrogen nutrition, agitation, and aeration.

Abstract: A preliminary screening work selected Penicillium restrictum as a promising micro-organism for lipase production. The physiological response of the fungus towards cell growth and enzyme production upon variable carbon and nitrogen nutrition, specific air flow rate (Qa) and agitation (N) was evaluated in a 5-L bench-scale fermenter. In optimized conditions for lipase production meat peptone at 2% (w/v) and olive oil at 1% (w/v) were used in a growth medium with a C/N ratio of 9.9. Higher C/N ratios favored cell growth in detriment of enzyme production. Low extracellular lipase activities were observed using glucose as carbon source suggesting glucose regulation. Final lipase accumulation of 13,000 U/L was obtained, using optimized specific air flow rate (Qa) of 0.5 vvm and an impeller speed (N) of 200 rpm. Agitation showed to be an important parameter to ensure nutrient availability in a growth medium having olive oil as carbon source.

Production of Succinic Acid by Anaerobiospirillum succiniciproducens

Abstract: The effect of an external supply of carbon dioxide and pH on the production of succinic acid by Anaerobiospirillum succiniciproducens was studied. In a rich medium containing yeast extract and peptone, when the external carbon dioxide supply was provided by a 1.5M Na2CO3 solution that also was used to maintain the pH at 6.0, no additional carbon dioxide supply was needed. In fact, sparging CO2 gas into the fermenter at 0.025 L/L-min or higher rates resulted in significant decreases in both production rate and yield of succinate. Under the same conditions, the production of the main by-product acetate was not affected by sparging CO2 gas into the fermenter. The optimum pH (pH 6.0) for the production of succinic acid was found to be in agreement with results previously reported in the literature. Succinic acid production also was studied in an industrial-type inexpensive medium in which light steep water was the only source of organic nutrients. At pH 6.0 and with a CO2 gas sparge rate of 0.08 L/L-min, succinate concentration reached a maximum of 32 g/L in 27 h with a yield of 0.99 g succinate/g glucose consumed.

Isotherms for adsorption of cellobiohydrolase I and II from Trichoderma reesei on microcrystalline cellulose

Abstract: Adsorption to microcrystalline cellulose (Avicel) of pure cellobiohydrolase I and II (CBH I and CBH II) from Trichoderma reesei has been studied. Adsorption isotherms of the enzymes were measured at 4 degrees C using CBH I and CBH II alone and in reconstituted equimolar mixtures. Several models (Langmuir, Freundlich, Temkin, Jovanovic) were tested to describe the experimental adsorption isotherms. The isotherms did not follow the basic (one site) Langmuir equation that has often been used to describe adsorption isotherms of cellulases; correlation coefficients (R-2) were only 0.926 and 0.947, for CBH I and II, respectively. The experimental isotherms were best described by a model of Langmuir type with two adsorption sites and by a combined Langmuir-Freundlich model (analogous to the Hill equation); using these models the correlation coefficients were in most cases higher than 0.995. Apparent binding parameters derived from the two sites Langmuir model indicated stronger binding of CBH II compared to CBH I; the distribution coefficients were 20.7 and 3.7 L/g for the two enzymes, respectively. The binding capacity, on the other hand, was higher for CBH I, 1.0 mu mol (67 mg) per gram Avicel, compared to 0.57 mu mol/g (30 mg/g) for CBH II. The isotherms when analyzed with the combined Langmuir-Freundlich model indicated presence of unequal binding sites on cellulose and/or negative cooperativity in the binding of the enzyme molecules.

Biogas production from Jatropha curcas press-cake.

Abstract: Seeds of the tropical plant Jatropha curcas (purge nut, physic nut) are used for the production of oil. Several methods for oil extraction have been developed. In all processes, about 50% of the weight of the seeds remain as a press cake containing mainly protein and carbohydrates. Investigations have shown that this residue contains toxic compounds and cannot be used as animal feed without further processing. Preliminary experiments have shown that the residue is a good substrate for biogas production. Biogas formation was studied using a semicontinous upflow anaerobic sludge blanket (UASB) reactor; a contact-process and an anaerobic filter each reactor having a total volume of 110 L. A maximum production rate of 3.5 m3 m-3 d-1 was obtained in the anaerobic filter with a loading rate of 13 kg COD m-3 d-1. However, the UASB reactor and the contact-process were not suitable for using this substrate. When using an anaerobic filter with Jatropha curcas seed cake as a substrate, 76% of the COD was degraded and 1 kg degraded COD yielded 355 L of biogas containing 70% methane.

Enzymatic hydrolysis of high-moisture corn fiber pretreated by afex and recovery and recycling of the enzyme complex

Abstract: Corn fiber is a grain-processing residue containing significant amounts of cellulose, hemicellulose, and starch, which is collected in facilities where fuel ethanol is currently manufactured. Preliminary research has shown that corn fiber (30% moisture dry weight basis [dwb]) responds well to ammonia-fiber explosion (AFEX) pretreatment. However, an important AFEX pretreatment variable that has not been adequately explored for corn fiber is sample moisture. In the present investigation, we determined the best AFEX operating conditions for pretreatment of corn fiber at high moisture content (150% moisture dwb). The optimized AFEX treatment conditions are defined in terms of the moisture content, particle size, ammonia to biomass ratio, temperature, and residence time using the response of the pretreated biomass to enzymatic hydrolysis as an indicator. Approximate optimal-pretreatment conditions for unground corn fiber containing 150% (dwb) moisture were found to be: temperature, 90‡C; ammonia: dry corn fiber mass ratio, 1:1; and residence time 30 min (average reactor pressure under these conditions was 200 pounds per square inch [psig]). Enzymatic hydrolysis of the treated corn fiber was performed with three different enzyme combinations. More than 80% of the theoretical sugar yield was obtained during enzymatic hydrolysis using the best enzyme combination after pretreatment of corn fiber under the optimized conditions previously described. A simple process for enzyme recovery and reuse to hydrolyze multiple portions of AFEX-treated corn fiber by one portion of enzyme preparation is demonstrated. Using this process, five batches of fresh substrate (at a concentration of 5% w/v) were successfully hydrolyzed by repeated recovery and reuse of one portion of enzyme preparation, with the addition of a small portion of fresh enzyme in each subsequent recycling step.

Production of 2,3- butanediol from pretreated corn cob by Klebsiella oxytoca in the presence of fungal cellulase.

Abstract: A simple and effective method of treatment of lignocellulosic material was used for the preparation of corn cob for the production of 2,3-butanediol by Klebsiella oxytoca ATCC 8724 in a simultaneous saccharification and fermentation process. During the treatment, lignin, and alkaline extractives were solubilized and separated from cellulose and hemicellulose fractions by dilute ammonia (10%) steeping. Hemicellulose was then hydrolyzed by dilute hydrochloric acid (1%, wJv) hydrolysis at 100 degrees C at atmospheric pressure and separated from cellulose fraction. The remaining solid, with 90% of cellulose, was then used as the substrate. A butanediol concentration of 25 g/L and an ethanol concentration of 7 g/L were produced by K. oxytoca from 80 g/L of corn cob cellulose with a cellulase dosage of 8.5 IFPU/g corn cob cellulose after 72 h of SSF. With only dilute acid hydrolysis, a butanediol production rate of 0.21 g/L/h was obtained that is much lower than the case in which corn cob was treated with ammonia steeping prior to acid hydrolysis. The butanediol production rate for the latter was 0.36 g/L/h.

Corn Steep Liquor as a Cost-Effective Nutrition Adjunct in High-Performance Zymomonas Ethanol Fermentations

Abstract: The ethanologenic bacterium Zymomonas mobilis has been demonstrated to possess several fermentation performance characteristics that are superior to yeast. In a recent survey conducted by the National Renewable Energy Laboratory (NREL), Zymomonas was selected as the most promising host for improvement by genetic engineering directed to pentose metabolism for the production of ethanol from lignocellulosic biomass and wastes. Minimization of costs associated with nutritional supplements and seed production is essential for economic large-scale production of fuel ethanol. Corn steep liquor (CSL) is a byproduct of corn wet-milling and has been used as a fermentation nutrient supplement in several different fermentations. This study employed pH-controlled batch fermenters to compare the growth and fermentation performance of Z. mobilis in glucose media with whole and clarified corn steep liquor as sole nutrient source, and to determine minimal amounts of CSL required to sustain high-performance fermentation.

Expression of Ascaris suum Malic Enzyme in a Mutant Escherichia coli Allows Production of Succinic Acid from Glucose

Abstract: The malic enzyme gene of Ascaris suum was cloned into the vector pTRC99a in two forms encoding alternative amino-termini. The resulting plasmids, pMEA1 and pMEA2, were introduced into Escherichia coli NZN111, a strain that is unable to grow fermentatively because of inactivation of the genes encoding pyruvate dissimilation. Induction of pMEA1, which encodes the native animoterminus, gave better overexpression of malic enzyme, approx 12-fold compared to uninduced cells. Under the appropriate culture conditions, expression of malic enzyme allowed the fermentative dissimilation of glucose by NZN111. The major fermentation product formed in induced cultures was succinic acid.

Hydrolysis by commercial enzyme mixtures of AFEX-treated corn fiber and isolated xylans

Abstract: Corn fiber is a coproduct produced during the corn wet-milling process and is similar to other high hemicellulose/cellulose-containing biomass such as grasses, straws, or bagasse, all of which represent potential fermentation feedstock for conversion into biofuels or other products. Corn fiber was subjected to ammonia-explosion (AFEX) treatment to increase degradability and then enzymatically digested with a combined mixture of commercial amylase, xylanase, and cellulase enzyme preparations. Whereas the starch and cellulose components were converted solely to glucose, oligosaccharides represented 30–40% of the xylan degradation products. This enzyme mixture also produced substantial oligosaccharides with xylans purified from corn fiber, corn germ, beechwood, oatspelt, or wheat germ. Commercial xylan-degrading enzyme preparations containing xylanase, xylosidase, and arabinosidase activities were then used alone or in varying combinations to attempt to maximize degradation of these isolated xylans of differing chemical compositions. The results showed that oatspelt and beechwood xylans were degraded most extensively (40–60%) with substantial amounts of xylose, xylobiose, and xylotriose as products depending on the enzyme combination used. Corn fiber and wheat germ xylans, which contain large amounts of arabinose and uronic acid sidechains, were poorly degraded and only small amounts of arabinose and xylose and large amounts of pentamer or longer oligosaccharides were produced by enzymatic degradation. The data suggest that whereas enzymatic digestion of biomass hemicellulose does not produce toxic products, the process is not effective in producing a suitable fermentable substrate stream because of the low levels of monosaccharides and high levels of oligosaccharides produced.

Optimization of methanol biosynthesis by Methylosinus trichosporium OB3b: An approach to improve methanol accumulation

Abstract: Methylosinus trichosporium OB3b is a methanotrophic bacterium containing methane mono-oxygenase, catalyzing hydroxylation of methane to methanol. When methane is oxidized, the product is subsequently oxidized by methanol dehydrogenase contained in the same bacterium. To prevent further oxidation of methanol, the cell suspension was treated by cyclopropanol, an irreversible inhibitor for methanol dehydrogenase, leading to extracellular methanol accumulation. However, the reaction was terminated at approx 3 h with a final methanol concentration below 2.96 mmol/g dry cell. The methanol production efficiency (the ratio of the produced methanol per methane consumption) was 2.90%. By selecting the culture conditions and the reaction conditions, the reaction continued for 100 h, resulting in a methanol concentration of 152 mmol/g dry cell. This level was 51 times higher than that of the conventional reaction, and the methanol production efficiency was 61%.

A stable lipase from Candida lipolytica: cultivation conditions and crude enzyme characteristics.

Abstract: Although lipases have been intensively studied, some aspects of enzyme production like substrate uptake, catabolite repression, and enzyme stability under long storage periods are seldom discussed in the literature. This work deals with the production of lipase by a new selected strain of Candida lipolytica. Concerning nutrition, it was observed that inorganic nitrogen sources were not as effective as peptone, and that oleic acid or triacylglycerides (TAG) were essential carbon sources. Repression by glucose and stimulation by oleic acid and long chain TAG (triolein and olive oil) were observed. Extracellular lipase activity was only observed at high levels at late stationary phase, whereas intracellular lipase levels were constant and almost undetectable during the cultivation period, suggesting that the produced enzyme was attached to the cell wall, mainly at the beginning of cultivation. The crude lipase produced by this yeast strain shows the following optima conditions: pH 8.0-10.0, temperature of 55 degrees C. Moreover, this preparation maintains its full activity for at least 370 d at 5 degrees C.

Utilization of glycosyltransferases to change oligosaccharide structures

Abstract: Carbohydrates on cell surfaces are important biomolecules in various biological recognition processes. Elucidation of the biological roles of complex oligosaccharides necessitates an efficient methodology to synthesize these compounds and their analogs. Enzymatic synthesis renders itself to be useful in the construction of an oligosaccharide structure owing to its mild reaction condition, high regio- and stereoselectivity. This review article focuses on the recent progress in oligosaccharide syntheses catalyzed by glycosyltransferases, namely sialyltransferase, galactosyltransferase, fucosyltransferase, and N-acetylglucosaminyltransferase. A survey of the latest patent and literature related to this field is also included.

Adsorption of heavy metal ions by immobilized phytic acid

Abstract: Phytic acid (myoinositol hexaphosphate) or its calcium salt, phytate, is an important plant constituent. It accounts for up to 85% of total phosphorus in cereals and legumes. Phytic acid has 12 replaceable protons in the phytic molecule, rendering it the ability to complex with multivalent cations and positively charged proteins. Poly 4-vinyl pyridine (PVP) and other strong-based resins have the ability to adsorb phytic acid. PVP has the highest adsorption capacity of 0.51 phytic acid/resins. The PVP resin was used as the support material for the immobilization of phytic acid. The immobilized phytic acid can adsorb heavy metal ions, such as cadmium, copper, lead, nickel, and zinc ions, from aqueous solutions. Adsorption isotherms of the selected ions by immobilized phytic acid were conducted in packed-bed column at room temperature. Results from the adsorption tests showed 6.6 mg of Cd2+, 7 mg of Cu2+, 7.2 mg of Ni2+, 7.4 mg of Pb2+, and 7.7 mg of Zn2+ can be adsorbed by each gram of PVP-phytic acid complex. The use of immobilized phytic acid has the potential for removing metal ions from industrial or mining waste water.

Production of glucose and bioactive aglycone by chemical and enzymatic hydrolysis of purified oleuropein fromOlea Europea

Abstract: Pure-grade oleuropein, a bitter, hypotensive, phenolic glucoside, was obtained from organic extracts of olive plant leaves by two Chromatographic steps. The purified compound was characterized by spectroscopic NMR and FAB-MS methods. The glucoside underwent chemical and enzymatic hydrolysis. Aglycone was characterized by spectroscopic methods (1H-NMR and FAB-MS). Glucose was measured by enzymatic methods. The enzymatic hydrolysis of oleuropein was carried out by a soluble β-glucosidase. The reaction was characterized in terms of kinetic parameters, optimal pH value, activation energy, inhibition constant by glucose, and thermal stability. Preliminary experiments were also performed in a continuousflow ultrafiltration membrane reactor. The cut-off of the membrane was lower than the molecular-weight of the enzyme, thus determining β-glucosidase confinement within the reactor. Under these conditions, β-glucosidase had a good long-term stability. This is an encouraging result in view of possible industrial applications.

Principles for efficient utilization of light for mass production of photoautotrophic microorganisms

Abstract: Outdoor production of microalgae could be set on a sound industrial basis if solar energy were utilized at a much higher efficiency than presently obtained. Many types of photobioreactors have been developed in the past in an attempt to answer this challenge, but their photosynthetic efficiency has been rather similar to the basically inefficient open raceway commonly used today. Efficient utilization of the oversaturating solar energy flux mandates that reactors should have a narrow lightpath to facilitate ultra-high cell densities, be maximally exposed to sunshine, and have an efficient mixing system to create strong turbulent streaming to affect dark-light cycles of the highest possible frequency.

Evaluation of PTMSP membranes in achieving enhanced ethanol removal from fermentations by pervaporation

Abstract: The use of membrane processes for the recovery of fermentation products has been gaining increased acceptance in recent years. Pervaporation has been studied in the past as a process for simultaneous fermentation and recovery of volatile products such as ethanol and butanol. However, membrane fouling and low permeate fluxes have imposed limitations on the effectiveness of the process. In this study, we characterize the performance of a substituted polyacetylene membrane, poly[(l-trimethylsilyl)-l-propyne] (PTMSP), in the recovery of ethanol from aqueous mixtures and fermentation broths. Pervaporation using PTMSP membranes shows a distinct advantage over conventional poly(dimethyl siloxane) (PDMS) membranes in ethanol removal. The flux with PTMSP is about threefold higher and the concentration factor is about twofold higher than the corresponding performance achieved with PDMS under similar conditions. The performance of PTMSP with fermentation broths shows a reduction in both flux and concentration factor relative to ethanol-water mixtures. However, the PTMSP membranes indicate initial promise of increased fouling resistance in operation with cell-containing fermentation broths.

Production of Xylitol from D-xylose by Debaryomyces hansenii

Abstract: Xylitol, a naturally occurring five-carbon sugar alcohol, can be produced from D-xylose through microbial hydrogenation. Xylitol has found increasing use in the food industries, especially in confectionary. It is the only so-called “second-generation polyol sweeteners” that is allowed to have the specific health claims in some world markets. In this study, the effect of cell density on the xylitol production by the yeast Debaryomyces hansenii NRRL Y-7426 from D-xylose under microaerobic conditions was examined. The rate of xylitol production increased with increasing yeast cell density to 3 g/L. Beyond this amount there was no increase in the xylitol production with increasing cell density. The optimal pH range for xylitol production was between 4.5 and 5.5. The optimal temperature was between 28 and 37°C, and the optimal shaking speed was 300 rpm. The rate of xylitol production increased linearly with increasing initial xylose concentration. A high concentration of xylose (279 g/L) was converted rapidly and efficiently to produce xylitol with a product concentration of 221 g/L was reached after 48 h of incubation under optimum conditions.

Production of fumaric acid by immobilized rhizopus using rotary biofilm contactor.

Abstract: Rotary biofilm contactor (RBC) is a reactor consisting of plastic discs that act as supports for micro-organisms. The discs are mounted on a horizontal shaft and placed in a medium-containing vessel. During nitrogen-rich growth phase, mycelia of Rhizopus oryzae ATCC 20344 grew on and around the discs and formed the “biofilm” of self-immobilized cells on the surface of the plastic discs. During the fermentation phase, the discs are slowly rotated, and the biofilms are exposed to the medium and the air space, alternately. With RBC, in the presence of CaCO3, Rhizopus biofilm consumes glucose and produces fumaric acid with a volumetric productivity of 3.78 g/L/h within 24 h. The volumetric productivity is about threefolds higher with RBC than with a stirred-tank fermenter with CaCO3. Furthermore, the duration of fermentation is one-third of the stirred-tank system. The immobilized biofilm is active for over a 2-wk period with repetitive use without loss of activity.

Lignin biodegradation by the ascomycete Chrysonilia sitophila.

Abstract: The lignin biodegradation process has an important role in the carbon cycle of the biosphere. The study of this natural process has developed mainly with the use of basidiomycetes in laboratory investigations. This has been a logical approach since most of the microorganisms involved in lignocellulosic degradation belong to this class of fungi. However, other microorganisms such as ascomycetes and also some bacteria, are involved in the lignin decaying process. This work focuses on lignin biodegradation by a microorganism belonging to the ascomycete class, Chrysonilia sitophila. Lignin peroxidase production and characterization, mechanisms of lignin degradation (lignin model compounds and lignin in wood matrix) and biosynthesis of veratryl alcohol are outstanding. Applications of C. sitophila for effluent treatment, wood biodegradation and single-cell protein production are also discussed.

Maximizing the xylitol production from sugar cane bagasse hydrolysate by controlling the aeration rate.

Abstract: Batch fermentations of sugar cane bagasse hemicellulosic hydrolysate treated for removing the inhibitors of the fermentation were performed by Candida guilliermondii FTI 20037 for xylitol production. The fermentative parameters agitation and aeration rate were studied aiming the maximization of xylitol production from this agroindustrial residue. The maximal xylitol volumetric productivity (0.87 g/L·h) and yield (0.67 g/g) were attained at 400/min and 0.45 v.v.m. (KLa 27/h). According to the results, a suitable control of the oxygen input permitting the xylitol formation from sugar cane bagasse hydrolysate is required for the development of an efficient fermentation process for large-scale applications.

Simultaneous saccharification and fermentation of pretreated hardwoods - effect of native lignin content

Abstract: A series of correlations was made between the performance of 15 wood species in simultaneous saccharification and fermentation (SSF) and their respective chemical compositions. A compelling inverse trend (p < 0.001) was demonstrated between the percent conversion of glucan to ethanol during SSF and the Klason lignin content of the wood samples before dilute acid pretreatment. No significant relationships were found between the glucan, xylan, and ash compositions of the native wood samples and ethanol yield. This observation is unique and provides a convenient predictor of biomass conversion efficiency.

Asparaginase II of Saccharomyces cerevisiae. GLN3/URE2 regulation of a periplasmic enzyme.

Abstract: The production of some extracellular enzymes is known to be negatively affected by readily metabolized nitrogen sources such as NH4 + although there is no consensus regarding the involved mechanisms. Asparaginase II is a periplasmic enzyme of Saccharomyces cerevisiae encoded by the ASP3 gene. The enzyme activity is not found in cells grown in either ammonia, glutamine, or glutamate, but it is found in cells that have been subjected to nitrogen starvation or have been grown on a poor source of nitrogen such as proline. In this report it is shown that the formation of this enzyme is dependent upon the functional GLN3 gene and that the response to nitrogen availability is under the control of the URE2 gene product. In this respect the expression of ASP3 is similar to the system that regulates the GLN1,GDH2, GAP1, and PUT4 genes that codes for glutamine synthetase, NAD-linked glutamate dehydrogenase, general amino-acid permease, and high affinity proline permease, respectively.

Enhanced Cofermentation of Glucose and Xylose by Recombinant Saccharomyces Yeast Strains in Batch and Continuous Operating Modes

Abstract: Agricultural residues, such as grain by-products, are rich in the hydrolyzable carbohydrate polymers hemicellulose and cellulose; hence, they represent a readily available source of the fermentable sugars xylose and glucose. The biomass-to-ethanol technology is now a step closer to commercialization because a stable recombinant yeast strain has been developed that can efficiently ferment glucose and xylose simultaneously (coferment) to ethanol. This strain, LNH-ST, is a derivative of Saccharomyces yeast strain 1400 that carries the xylose-catabolism encoding genes of Pichia stipitis in its chromosome. Continuous pure sugar cofermentation studies with this organism resulted in promising steady-state ethanol yields (70.4% of theoretical based on available sugars) at a residence time of 48 h. Further studies with corn biomass pretreated at the pilot scale confirmed the performance characteristics of the organism in a simultaneous saccharification and cofermentation (SSCF) process: LNH-ST converted 78.4% of the available glucose and 56.1% of the available xylose within 4 d, despite the presence of high levels of metabolic inhibitors. These SSCF data were reproducible at the bench scale and verified in a 9000-L pilot scale bioreactor.