Showing papers in "Applied Biochemistry and Biotechnology in 2002"

Cellulase adsorption and an evaluation of enzyme recycle during hydrolysis of steam-exploded softwood residues.

Abstract: The sugar yield and enzyme adsorption profile obtained during the hydrolysis of SO2-catalyzed steam-exploded Douglas-fir and posttreated steam-exploded Douglas-fir substrates were determined. After hot alkali peroxide posttreatment, the rates and yield of hydrolysis attained from the posttreated Douglas-fir were significantly higher, even at lower enzyme loadings, than those obtained with the corresponding steam-exploded Douglas-fir. The enzymatic adsorption profiles observed during hydrolysis of the two substrates were significantly different. Ultrafiltration was employed to recover enzyme in solution (supernatant) and reused in subsequent hydrolysis reactions with added, fresh substrate. These recycle findings suggested that the enzyme remained relatively active for three rounds of recycle. It is likely that enzyme recovery and reuse during the hydrolysis of posttreated softwood substrates could lead to reductions in the need for the addition of fresh enzyme during softwood-based bioconversion processes.

A model explaining declining rate in hydrolysis of lignocellulose substrates with cellobiohydrolase I (cel7A) and endoglucanase I (cel7B) of Trichoderma reesei.

Abstract: It is commonly observed that the rate of enzymatic hydrolysis of solid cellulose substrates declines markedly with time. In this work the mechanism behind the rate reduction was investigated using two dominant cellulases of Trichoderma reesei: exoglucanase Cel7A (formerly known as CBHI) and endoglucanase Cel7B (formerly EGI). Hydrolysis of steam-pretreated spruce (SPS) was performed with Cel7A and Cel7B alone, and in reconstituted mixtures. Throughout the 48-h hydrolysis, soluble products, hydrolysis rates, and enzyme adsorption to the substrate were measured. The hydrolysis rate for both enzymes decreases rapidly with hydrolysis time. Both enzymes adsorbed rapidly to the substrate during hydrolysis. Cel7A and Cel7B cooperate synergistically, and synergism was approximately constant during the SPS hydrolysis. Thermal instability of the enzymes and product inhibition was not the main cause of reduced hydrolysis rates. Adding fresh substrate to substrate previously hydrolyzed for 24 h with Cel7A slightly increased the hydrolysis of SPS; however, the rate increased even more by adding fresh Cel7A. This suggests that enzymes become inactivated while adsorbed to the substrate and that unproductive binding is the main cause of hydrolysis rate reduction. The strongest increase in hydrolysis rate was achieved by adding Cel7B. An improved model is proposed that extends the standard endo-exo synergy model and explains the rapid decrease in hydrolysis rate. It appears that the processive action of Cel7A becomes hindered by obstacles in the lignocellulose substrate. Obstacles created by disordered cellulose chains can be removed by the endo activity of Cel7B, which explains some of the observed synergism between Cel7A and Cel7B. The improved model is supported by adsorption studies during hydrolysis.

Dilute-acid hydrolysis of sugarcane bagasse at varying conditions.

Abstract: Sugarcane bagasse, a byproduct of the cane sugar industry, is an abundant source of hemicellulose that could be hydrolyzed to yield a fermentation feedstock for the production of fuel ethanol and chemicals. The effects of sulfuric acid concentration, temperature, time, and dry matter concentration on hemicellulose hydrolysis were studied with a 20-L batch hydrolysis reactor using a statistical experimental design. Even at less severe conditions considerable amounts (>29%) of the hemicellulose fraction could be extracted. The percentage of soluble oligosaccharides becomes very low in experiments with high yields in monosaccharides, which indicates that the cellulose fraction is only slightly affected. For the sugar yields, acid concentration appears to be the most important parameter, while for the formation of sugar degradation products, temperature shows the highest impact. It could be demonstrated that the dry matter concentration in the reaction slurry has a negative effect on the xylose yield that can be compensated by higher concentrations of sulfuric acid owing to a positive interaction between acid concentration and dry matter contents.

Comparison of the fermentability of enzymatic hydrolyzates of sugarcane bagasse pretreated by steam explosion using different impregnating agents.

Abstract: Sugarcane bagasse is a potential lignocellulosic feedstock for ethanol production, since it is cheap, readily available, and has a high carbohydrate content. In this work, bagasse was subjected to steam explosion pretreatment with different impregnation conditions. Three parallel pretreatments were carried out, one without any impregnation, a second with sulfur dioxide, and a third with sulfuric acid as the impregnating agent. The pretreatments were performed at 205 degrees C for 10 min. The pretreated material was then hydrolyzed using cellulolytic enzymes. The chemical composition of the hydrolyzates was analyzed. The highest yields of xylose (16.2 g/100 g dry bagasse), arabinose (1.5 g/100 g), and total sugar (52.9 g/100 g) were obtained in the hydrolysis of the SO2 -impregnated bagasse. The H2SO4 -impregnated bagasse gave the highest glucose yield (35.9 g/100 g) but the lowest total sugar yield (42.3 g/100 g) among the three methods. The low total sugar yield from the H2SO4-impregnated bagasse was largely due to by-product formation, as the dehydration of xylose to furfural. Sulfuric acid impregnation led to a three-fold increase in the concentration of the fermentation inhibitors furfural and 5-hydroxymethylfurfural (HMF) and a two-fold increase in the concentration of inhibitory aliphatic acids (formic, acetic, and levulinic acids) compared to the other two pretreatment methods. The total content of phenolic compounds was not strongly affected by the different pretreatment methods, but the quantities of separate phenolic compounds were widely different in the hydrolyzate from the H2SO4-impregnated bagasse compared with the other two hydrolyzates. No major differences in the content of inhibitors were observed in the hydrolyzates obtained from SO2-impregnated and non-impregnated bagasse. The fermentability of all three hydrolyzates was tested with a xylose-utilizing Saccharomyces cerevisiae strain with and without nutrient supplementation. The hydrolyzates of SO2-impregnated and nonimpregnated bagasse showed similar fermentability, whereas the hydrolyzate of H2SO4-impregnated bagasse fermented considerably poorer.

Cofermentation of glucose, xylose, and arabinose by genomic DNA-integrated xylose/arabinose fermenting strain of Zymomonas mobilis AX101.

Abstract: Cofermentation of glucose, xylose, and arabinose is critical for complete bioconversion of lignocellulosic biomass, such as agricultural residues and herbaceous energy crops, to ethanol. We have previously developed a plas-mid-bearing strain of Zymomonas mobilis (206C[pZB301]) capable of cofer-menting glucose, xylose, and arabinose to ethanol. To enhance its genetic stability, several genomic DNA—integrated strains of Z. mobilis have been developed through the insertion of all seven genes necessay for xylose and arabinose fermentation into the Zymomonas genome. From all the integrants developed, four were selected for further evaluation. The integrants were tested for stability by repeated transfer in a nonselective medium (containing only glucose). Based on the stability test, one of the integrants (AX101) was selected for further evaluation. A series of batch and continuous fermentations was designed to evaluate the cofermentation of glucose, xylose, and L-arabinose with the strain AX101. The pH range of study was 4.5, 5.0, and 5.5 at 30°C The cofermentation process yield was about 84%, which is about the same as that of plasmid-bearing strain 206C(pZB301). Although cofermentation of all three sugars was achieved, there was a preferential order of sugar utilization: glucose first, then xylose, and arabinose last.

Two-step steam pretreatment of softwood with SO2 impregnation for ethanol production.

Abstract: Two-step steam pretreatment of softwood was investigated with the aim of improving the enzymatic digestibility for ethanol production. In the first step, softwood was impregnated with SO2 and steam pretreated at different severities. The first step was performed at low severity to hydrolyze the hemicellulose and release the sugars into the solution. The combination of time and temperature that yielded the highest amount of hemicellulosic sugars in the solution was determined. In the second step, the washed solid material from the optimized first step was impregnated once more with SO2 and steam pretreated under more severe conditions to enhance the enzymatic digestibility. The investigated temperature range was between 180 and 220°C, and the residence times were 2, 5 and 10 min. The effectiveness of pretreatment was assessed by both enzymatic hydrolysis of the solids and simultaneous saccharification and fermentation (SSF) of the whole slurry after the second pretreatment step, in the presence of antibiotics. For each pretreatment combination, the liquid fraction was fermented to determine any inhibiting effects. At low severity in the second pretreatment step, a high conversion of cellulose was obtained in the enzymatic hydrolysis step, and at a high severity a high conversion of cellulose was obtained in the second pretreatment step. This resulted in an overall yield of sugars that was nearly constant over a wide range of severity. Compared with the one-step steam pretreatment, the two-step steam pretreatment resulted in a higher yield of sugar and in a slightly higher yield of ethanol. The overall sugar yield, when assessed by enzymatic hydrolysis, reached 80%. In the SSF configuration, an overall ethanol yield of 69% was attained.

Degradation of textile dyes mediated by plant peroxidases.

Abstract: The peroxidase enzyme from the plants Ipomea palmata (1.003 IU/g of leaf) and Saccharum spontaneum (3.6 IU/g of leaf) can be used as an alternative to the commercial source of horseradish and soybean peroxidase enzyme for the decolorization of textile dyes, mainly azo dyes. Eight textiles dyes currently used by the industry and seven other dyes were selected for decolorization studies at 25-200 mg/L levels using these plant enzymes. The enzymes were purified prior to use by ammonium sulfate precipitation, and ion exchange and gel permeation chromatographic techniques. Peroxidase of S. spontaneum leaf (specific activity of 0.23 IU/mg) could completely degrade Supranol Green and Procion Green HE-4BD (100%) dyes within 1 h, whereas Direct Blue, Procion Brilliant Blue H-7G and Chrysoidine were degraded >70% in 1 h. Peroxidase of Ipomea (I. palmata leaf; specific activity of 0.827 U/mg) degraded 50 mg/L of the dyes Methyl Orange (26%), Crystal Violet (36%), and Supranol Green (68%) in 2-4 h and Brilliant Green (54%), Direct Blue (15%), and Chrysoidine (44%) at the 25 mg/L level in 1 to 2 h of treatment. The Saccharum peroxidase was immobilized on a hydrophobic matrix. Four textile dyes, Procion Navy Blue HER, Procion Brilliant Blue H-7G, Procion Green HE-4BD, and Supranol Green, at an initial concentration of 50 mg/L were completely degraded within 8 h by the enzyme immobilized on the modified polyethylene matrix. The immobilized enzyme was used in a batch reactor for the degradation of Procion Green HE-4BD and the reusability was studied for 15 cycles, and the half-life was found to be 60 h.

Predicting digestibility of ammonia fiber explosion (AFEX)-treated rice straw.

Abstract: The enzymatic digestibility of ammonia fiber explosion (AFEX)-treated rice straw was modeled by statistically correlating the variability of samples to differences in treatment using several different analytical techniques. Lignin content and crystallinity index of cellulose affect enzymatic hydrolysis the most. X-ray diffraction was used to measure the crystallinity index (CrI), while fluorescence and diffuse reflectance infrared (DRIFT) spectroscopy measured the lignin content of the samples. Multivariate analysis was applied to correlate the enzymatic hydrolysis results of the various samples with X-ray diffraction and spectroscopic data. Principal component analysis (PCA) and multilinear regression (MLR) techniques did not accurately predict the digestibility of the rice straw samples. The best correlation (R value of 0.775) was found between the treatment conditions of the AFEX process and the concentration of xylose at 24 h after enzymatic hydrolysis.

Hydrogen production by the thermophilic bacterium Thermotoga neapolitana.

Abstract: Virtually all members of the order Thermotogales have demonstrated the ability to produce hydrogen; however, some members of this order produce considerably greater quantities than others. With one representative of this order, Thermotoga neapolitana, we have consistently obtained accumulation of 25–30% hydrogen with 12–15% carbon dioxide as the only other prominent product in the batch reaction. In contradistinction to information widely disseminated in the literature, we have also found that most members of this order tolerate and appear to utilize the moderate amounts of oxygen present in the gaseous phase of batch reactors (6–12%), with no apparent decrease in hydrogen production. Hydrogen accumulation has been widely reported to inhibit growth of Thermotogales. While this may be true at very high hydrogen tensions, we have observed log phase bacterial morphology (rods) even in the presence of 25–35% hydrogen concentrations. To maximize hydrogen production and minimize production of hydrogen sulfide, inorganic sulfur donors are avoided and the cysteine concentration in the medium is increased. We and others have demonstrated that different members of the order Thermotogales utilize a wide variety of feedstocks, including complex carbohydrates and proteins. Thus, it appears that organisms within this order have the potential to utilize a variety of organic wastes and to cost-effectively generate hydrogen.

Chemical pretreatments of corn stover for enhancing enzymatic digestibility.

Abstract: Corn stover, the most abundant agricultural residue in Hungary, is a potential raw material for the production of fuel ethanol as a result of its high content of carbohydrates, but a pretreatment is required for its efficient hydrolysis. In this article, we describe the results using various chemicals such as dilute H2SO4, HCl, and NaOH separately as well as consecutively under relative mild conditions (120°C, 1h). Pretreatment with 5% H2SO4 or 5% HCl solubilized 85% of the hemicellulose fraction, but the enzymatic conversion of pretreated materials increased only two times compared to the untreated corn stover. Applying acidic pretreatment following a 1-d soaking in base achieved enzymatic conversion that was nearly the theoretical maximum (95.7%). Pretreatment with 10% NaOH decreased the lignin fraction >95%, increased the enzymatic conversion more than four times, and gave a 79.4% enzymatic conversion. However, by increasing the reaction time, the enzymatic degradability could also be increased significantly, using a less concentrated base. When the time of pretreatment was increased three times (0.5% NaOH at 120°C), the amount of total released sugars was 47.9 g from 100 g (dry matter) of untreated corn stover.

Production of 1,3-propanediol by Klebsiella pneumoniae.

Abstract: 1,3-Propanediol (1,3-PD) has numerous applications from polymers to cosmetics, foods, lubricants, and medicines. Recently, there are strong industrial interests in a new kind of polyester, polytrimethylene terephthalate, with 1,3-PD as a monomer. This new polyester shows significant promise for use in carpeting and textiles. In this article we introduce a mild aerobic fermentation process using a strain screened from Klebsiella pneumoniae ATCC 25955, which is insensitive to oxygen, to produce 1,3-PD. We also describe a two-step fermentation process starting with glucose that was converted into glycerol with a glycerol-producing yeast, followed by K. pneumoniae that converts glycerol into 1,3-PD without intermediate isolation and purification of glycerol.

Enhancing the enzymatic hydrolysis of cellulosic materials using simultaneous ball milling

Abstract: One of the limiting factors restricting the effective and efficient bioconversion of softwood-derived lignocellulosic residues is the recalcitrance of the substrate following pretreatment. Consequently, the ensuing enzymatic process requires relatively high enzyme loadings to produce monomeric carbohydrates that are readily fermentable by ethanologenic microorganisms. In an attempt to circumvent the need for larger enzyme loadings, a simultaneous physical and enzymatic hydrolysis treatment was evaluated. A ball-mill reactor was used as the digestion vessel, and the extent and rate of hydrolysis were monitored. Concurrently, enzyme adsorption profiles and the rate of conversion during the course of hydrolysis were monitored. α-Cellulose, employed as a model substrate, and SO2-impregnated steam-exploded Douglas-fir wood chips were assessed as the cellulosic substrates. The softwood-derived substrate was further posttreated with water and hot alkaline hydrogen peroxide to remove >90% of the original lignin. Experiments at different reaction conditions were evaluated, including substrate concentration, enzyme loading, reaction volumes, and number of ball beads employed during mechanical milling. It was apparent that the best conditions for the enzymatic hydrolysis of α-cellulose were attained using a higher number of beads, while the presence of air-liquid interface did not seem to affect the rate of saccharification. Similarly, when employing the lignocellulosic substrate, up to 100% hydrolysis could be achieved with a minimum enzyme loading (10 filter paper units/g of cellulose), at lower substrate concentrations and with a greater number of reaction beads during milling. It was apparent that the combined strategy of simultaneous ball milling and enzymatic hydrolysis could improve the rate of saccharification and/or reduce the enzyme loading required to attain total hydrolysis of the carbohydrate moieties.

Performance Testing of Zymomonas mobilis Metabolically Engineered for Cofermentation of Glucose, Xylose, and Arabinose

Abstract: IOGEN Corporation of Ottawa, Canada, has recently built a 40t/d biom-ass-to-ethanol demonstration plant adjacent to its enzyme production facility. It has partnered with the University of Toronto to test the C6/C5 cofermenta-tion performance characteristics of the National Renewable Energy Labora-tory’s metabolically engineered Zymomonas mobilis using various biomass hydrolysates. IOGEN’s feedstocks are primarily agricultural wastes such as corn stover and wheat straw. Integrated recombinant Z. mobilis strain AX101 grows on D-xylose and/or L-arabinose as the sole carbon/energy sources and ferments these pentose sugars to ethanol in high yield. Strain AX101 lacks the tetracycline resistance gene that was a common feature of other recombinant Zm constructs. Genomic integration provides reliable cofermentation performance in the absence of antibiotics, another characteristic making strain AX101 attractive for industrial cellulosic ethanol production. In this work, IOGEN’s biomass hydrolysate was simulated by a pure sugar medium containing 6% (w/v) glucose, 3% xylose, and 0.35% arabinose. At a level of 3 g/L (dry solids), corn steep liquor with inorganic nitrogen (0.8 g/L of ammonium chloride or 1.2 g/L of diammonium phosphate) was a cost-effective nutritional supplement. In the absence of acetic acid, the maximum volumetric ethanol productivity of a continuous fermentation at pH 5.0 was 3.54 g/L·h. During prolonged continuous fermentation, the efficiency of sugar-to-ethanol conversion (based on total sugar load) was maintained at >85%. At a level of 0.25% (w/v) acetic acid, the productivity decreased to 1.17 g/L·h at pH 5.5. Unlike integrated, xylose-utilizing rec Zm strain C25, strain AX101 produces less lactic acid as byproduct, owing to the fact that the Escherichia coli arabinose genes are inserted into a region of the host chromosome tentatively assigned to the gene for D-lactic acid dehydrogenase. In pH-controlled batch fermentations with sugar mixtures, the order of sugar exhaustion from the medium was glucose followed by xylose and arabinose. Both the total sugar load and the sugar ratio were shown to be important determinants for efficient cofermentation. Ethanol at a level of 3% (w/v) was implicated as both inhibitory to pentose fermentation and as a potentiator of acetic acid inhibition of pentose fermentation at pH 5.5. The effect of ethanol may have been underestimated in other assessments of acetic acid sensitivity. This work underscores the importance of employing similar assay conditions in making comparative assessments of biocatalyst fermentation performance.

Production of ligninolytic enzymes for dye decolorization by cocultivation of white-rot fungi Pleurotus ostreatus and phanerochaete chrysosporium under solid-state fermentation.

Abstract: Lignocellulosic wastes such as neem hull, wheat bran, and sugarcane bagasse, available in abundance, are excellent substrates for the production of ligninolytic enzymes under solid-state fermentation by white-rot fungi. A ligninolytic enzyme system with high activity showing enhanced decomposition was obtained by cocultivation of Pleurotus ostreatus and Phanerochaete chrysosporium on combinations of lignocellulosic waste. Among the various substrate combinations examined, neem hull and wheat bran wastes gave the highest ligninolytic activity. A maximum production of laccase of 772 U/g and manganese peroxidase of 982 U/g was obtained on d 20 and lignin peroxidase of 656 U/g on d 25 at 28 +/- 1 degrees C under solid-state fermentation. All three enzymes thus obtained were partially purified by acetone fractionation and were exploited for decolorizing different types of acid and reactive dyes.

SO2-Catalyzed Steam Explosion of Corn Fiber for Ethanol Production

Abstract: Corn fiber, a by-product of the corn wet-milling industry, represents a renewable resource that is readily available in significant quantities and could potentially serve as a low-cost feedstock for the production of fuel-grade alcohol. In this study, we used a batch reactor to steam explode corn fiber at various degrees of severity to evaluate the potential of using this feedstock in the bioconversion process. The results indicated that maximum sugar yields (soluble and following enzymatic hydrolysis) were recovered from corn fiber that was pretreated at 190°C for 5 min with 6% SO2. Sequential SO2-catalyzed steam explosion and enzymatic hydrolysis resulted in very high conversion (81%) of all polysaccharides in the corn fiber to monomeric sugars. Subsequently, Saccharomyces cerevisiae was able to convert the resultant corn fiber hydrolysates to ethanol very efficiently, yielding 90–96% of theoretical conversion during the fermentation process.

Evaluation of Different Carbon and Nitrogen Sources in Production of Rhamnolipids by a Strain of Pseudomonas aeruginosa

Abstract: Culture conditions involving variations in carbon and nitrogen sources and different C:N ratios were examined with the aim of increasing productivity in the process of rhamnolipid synthesis by Pseudomonas aeruginosa. In addition to the differences in productivity, the use of different carbon sources resulted in several proportions related to the types of rhamnolipids synthesized (monorhamnolipids and dirhamnolipids). Furthermore, the variation in nutrients, mainly the nitrogen source, resulted in different amounts of virulence factors, as phenazines and extracellular proteins. The data point out a new concern in the choice of substrate to be used for rhamnolipid production by P. aeruginosa: toxic byproducts.

Potential use of cutinase in enzymatic scouring of cotton fiber cuticle

Abstract: The present study characterized the ability of a bacterial cutinase to improve the wettability of raw cotton fabrics by specific hydrolysis of the cutin structure of the cuticle. The effect of cutinase was studied alone and in coreaction with pectin lyase. The changes in both the fabric and the reaction fluid were measured and compared to enzymatic hydrolysis with polygalacturonase, and to chemical hydrolysis with boiling NaOH. Water absorbancy, specific staining, fabric weight loss, and evaporative light-scattering reversephase high-performance liquid chromatography analysis of chloroform extract of the reaction fluid were measured to assess the enzymatic hydrolysis of the cuticle waxy layer. The pattern and extent of hydrolysis of the major cuticle constituents depended on the enzyme type and titers employed and paralleled the degree of wettability obtained. The combination of cutinase and pectin lyase resulted in a synergistic effect. The use of detergents improved enzymatic scouring. The major products released to the reaction medium by the cutinase treatment were identified by gas chromatography/mass spectrometry analysis as C:16 and C:18 saturated fatty acid chains.

Solid-state fermentation for production of phytase by Rhizopus oligosporus.

Abstract: Solid-state fermentation of coconut oil cake has been carried out with Rhizopus oligosporus for the production of phytase. Phytase is used commercially in the animal feed industry to improve animal performance because there is a substantial and growing interest among swine and poultry producers in the application of phytase to improve the nutritional quality in animal feeds. Demonstrated benefits include improved feed yield ratios and reduction in the environmental costs associated with the disposal of animal wastes. We report the production of extracellular phytase by R. oligosporus under solid-state fermentation using coconut oil cake as substrate. Maximal enzyme production (14.29 U/g of dry substrate) occurred at pH 5.3, 30°C, and 54.5% moisture content after 96 h of incubation. The addition of extra nutrients to the substrate resulted in inhibition of product formation. The results indicate the scope for production of phytase using coconut oil cake as solid substrate without additional nutrients.

A biotechnological process involving filamentous fungi to produce natural crystalline vanillin from maize bran.

Abstract: A new process involving the filamentous fungi Aspergillus niger and Pycnoporus cinnabarinus has been designed for the release of ferulic acid by enzymic degradation of a cheap and natural agricultural byproduct (autoclaved maize bran) and its biotransformation into vanillic acid and/or vanillin with a limited number of steps. On the one hand, the potentialities of A. niger I-1472 to produce high levels of polysaccharide-degrading enzymes including feruloyl esterases and to transform ferulic acid into vanillic acid were successfully combined for the release of free ferulic acid from autoclaved maize bran. Then vanillic acid was recovered and efficiently transformed into vanillin by P. cinnabarinus MUCL39533, since 767 mg/L of biotechnologic vanillin could be produced in the presence of cellobiose and XAD-2 resin. On the other hand, 3-d-old high-density cultures of P. cinnabarinus MUCL39533 could be fed with the autoclaved fraction of maize bran as a ferulic acid source and A. niger I-1472 culture filtrate as an extracellular enzyme source. Under these conditions, P. cinnabarinus MUCL39533 was shown to directly biotransform free ferulic acid released from the autoclaved maize bran by A. niger I-1472 enzymes into 584 mg/L of vanillin. These processes, involving physical enzymic, and fungal treatments, permitted us to produce crystallin vanillin from autoclaved maize bran without any purification step.

Reduction of furfural to furfuryl alcohol by ethanologenic strains of bacteria and its effect on ethanol production from xylose.

Abstract: The ethanologenic bacteria Escherichia coli strains KO11 and LYO1, and Klebsiella oxytoca strain P2, were investigated for their ability to metabolize furfural Using high performance liquid chromatography and 13C-nuclear magnetic resonance spectroscopy, furfural was found to be completely biotransformed into furfuryl alcohol by each of the three strains with tryptone and yeast extract as sole carbon sources This reduction appears to be constitutive with NAD(P)H acting as electron donor Glucose was shown to be an effective source of reducing power Succinate inhibited furfural reduction, indicating that flavins are unlikely participants in this process Furfural at concentrations >10 mM decreased the rate of ethanol formation but did not affect the final yield Insight into the biochemical nature of this furfural reduction process may help efforts to mitigate furfural toxicity during ethanol production by ethanologenic bacteria

Enhanced formation of extracellular laccase activity by the white-rot fungus Trametes multicolor.

Abstract: The white-rot fungus Trametes multicolor MB 49 has been identified as an excellent producer of the industrially important enzyme laccase. The forma- tion of extracellular laccase could be considerably stimulated by the addition of Cu(II) to a simple, glycerol-based culture medium. In this study, optimal concentrations of copper were found to be 0.5–1 mM, which were added during the growth phase of the fungus. Other medium components impor- tant for laccase production are the carbon and nitrogen sources employed. When using an optimized medium containing glycerol (40 g/L), peptone from meat (15 g/L), and MgSO4 7H2O and stimulating enzyme formation by the addition of 1.0 mM Cu, maximal laccase activities obtained in shake-flask cultures were approx 85 U/mL. These results, however, could not be scaled up to a laboratory f ermentor cultivation. Laccase production by T. multicolor decreased considerably when the fungus was grown in a stirred-tank reactor, presumably because of damage of the mycelia caused by shear stress and/or changes in the morphology of the fungus.

Archaeal tetraether lipids: unique structures and applications.

Abstract: The extremely stable biomolecules manufactured by organisms from extreme environments are of great scientific and engineering interest in the development of robust and stable industrial biocatalysts. Identification of molecules that impart stability under extremes will also have a profound impact on our understanding of cellular survival. This review discusses isolation and characterization of archaeal tetraethers as well as target technologies for tetraether lipid application. The isolation and characterization of archaeal tetraether lipids has led to some interesting applications improving on ester lipid technologies. Potential applications include novel lubricants, gene-delivery systems, monolayer lipid matrices for sensor devices, and protein stabilization. Following this review, patent abstracts and additional literature pertaining to the isolation, characterization, and application of archaeal membrane lipids are listed.

Overexpression in Catharanthus roseus hairy roots of a truncated hamster 3-hydroxy-3-methylglutaryl-CoA reductase gene.

Abstract: Catharanthus roseus (L.) G. Don hairy roots harboring hamster 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) (EC 1.1.1.88) cDNA without membrane-binding domain were evaluated by quantifying the levels of sterols and some indol-alkaloids. Clone 236, with the highest hybridization signal, had the lowest soluble and microsomal HMGR activity and produced more ajmalicine and catharanthine than the control but had reduced campesterol concentration. Clone 19, with low hybridization signal, had high soluble HMGR activity and produced high levels of campesterol and five to seven times more serpentine than the control but a low level of ajmalicine and no accumulation of catharanthine. These results suggest a possible role for HMGR in indole alkaloid biosynthesis and a possible cosuppression of both the endogenous and foreign HMGR genes in clone 236.

Scale-up of microbubble dispersion generator for aerobic fermentation

Abstract: A laboratory-scale microbubble dispersion (MBD) generator was shown to improve oxygen transfer to aerobic microorganisms when coupled to the conventional air-sparger. However, the process was not demonstrated on a large scale to prove its practical application. We investigated the scale-up of a spinning-disk MBD generator for the aerobic fermentation of Saccharomyces cerevisiae (baker's yeast). A 1-L spinning-disk MBD generator was used to supply air for 1- and 50-L working volume fermentation of baker's yeast. For the two levels investigated, the MBD generator maintained an adequate supply of surfactant-stabilized air microbubbles to the microorganisms at a relatively low agitation rate (150 rpm). There was a significant improvement in oxygen transfer to the microorganism relative to the conventional sparger. The volumetric mass transfer coefficient, kLa, for the MBD system at 150 rpm was 765 h(-1) compared to 937 h(-1) for the conventional sparger at 500 rpm. It is plausible to surmise that fermentation using larger working volumes may further improve the kLa values and the dissolved oxygen (DO) levels because of longer hold-up times and, consequently, improve cell growth. There was no statistically significant difference between the cell mass yield on substrate (0.43 g/g) under the MBD regime at an agitation rate of 150 rpm and that achieved for the conventional air-sparged system (0.53 g/g) at an agitation rate of 500 rpm. The total power consumption per unit volume of broth in the 50-L conventional air-sparged system was threefold that for the MBD unit for a similar product yield. Practical application of the MBD technology can be expected to reduce power consumption and therefore operating costs for aerobic fermentation.

Production of acetone butanol ethanol from degermed corn using Clostridium beijerinckii BA101

Abstract: In this article we report on acetone butanol ethanol (ABE) fermentation characteristics of degermed corn when using Clostridium beijerinckii BA101. Recent economic studies suggested that recovery of germ from corn and hence corn oil would help to make the ABE fermentation process more economical. C. beijerinckii BA101 ferments corn mash efficiently to produce ABE under appropriate nutritional and environmental conditions. Corn mash contains germ/corn oil that is, possibly, ancillary to the production of butanol during the ABE fermentation process. Since the presence of corn oil is not a critical factor in solvent fermentation, it can be removed and this will allow for byproduct credit. Batch fermentation of degermed corn resulted in 8.93 g/L of total ABE production as compared with 24.80 g/L of total ABE when supplemented with P2 medium nutrients. During the course of the germ separation process, corn steeping is required prior to grinding and removing the germ. It is likely that some nutrients from the corn are leached out during the steeping process. This may reduce the rate of fermentation and impact the final concentration of butanol/ABE that can be achieved. Fermentation of degermed corn with corn steep liquor resulted in the production of 19.28 g/L of ABE.

Heavy metal removal by biosorption using Phanerochaete chrysosporium

Abstract: Biosorption using microbial cells as adsorbents is being seen as a cost-effective method for the removal of heavy metals from wastewaters. Biosorption studies with Phanerochaete chrysosporium were performed for copper (II), lead (II), and cadmium (II) to evaluate the effectiveness and to optimize the operational parameters using response surface methodology. The operational parameters chosen were initial metal ion concentration, pH, and biosorbent dosage. Using this method, the metal removal could be correlated to the operational parameters, and their values were optimized. The results showed fairly high adsorptive capacities for all the metals within the settings of the operational parameters. The removal efficiencies followed the order Pb>Cu>Cd. As a general trend, metal removal efficiency decreased as the initial metal ion concentration increased, and the results fitted the Langmuir and Freundlich isotherms well.

Gibberellic acid production by solid-state fermentation in coffee husk

Abstract: Five strains of Gibberella fujikuroi and one of Fusarium moniliforme were screened for the production of gibberellic acid (GA3) in coffee husk, and based on the results, one strain, G. fujikuroi LPB-06, was selected. The comparative production of GA3 by solid-state fermentation and submerged fermentation indicated better productivity with the former technique, mainly with pretreated substrate. The GA3 accumulation was 6.1 times higher in the case of solid-state fermentation. Considering the C:N relation, higher yields of GA3 were achieved using a mixed substrate comprising coffee husk and cassava bagasse (7:3, dry wt), increasing the results twice. Supplementation of an optimized saline solution containing 0.03% FeSO4 and 0.01% (NH4)2SO4 enhanced the accumulation of GA3 1.7 times in the fermented substrate. Under the finally optimized condition, the culture gave a maximum of 492.5 mg of GA3/kg of dry substrate, with a pH of 5.3, moisture of 75%, and incubation temperature of 29 degrees C. GA3 yield was almost 13 times more than the initial results.

Synergism in binary mixtures of Thermobifida fusca cellulases Cel6B, Cel9A, and Cel5A on BMCC and Avicel.

Abstract: In an earlier binding study conducted in our laboratory using Thermobifida fusca cellulases Cel6B, Cel9A, and Cel5A (formally Thermomonospora fusca E3, E4, and E5), it was observed that binding capacities for these three cellulases were 18–30 times higher on BMCC than on Avicel. These results stimulated an interest in how the difference in accessibility between the two cellulosic substrates would affect synergism observed with cellulase mixtures. To explore the impact of substrate, accessibility on the extent of conversion and synergism, three binary T. fusca cellulase mixtures were tested over a range of cellulase ratios and total molar cellulase concentrations on Avicel and BMCC. Higher extents of conversion were observed for BMCC due to the higher enzyme to substrate ratio resulting from the higher binding The processive endoglucanase, Cel9A, had four times the extent of conversion of the end endocellulase Cel5A, while the exocellulase Cel6B had three times the extent of conversion of Cel5A. Approximately 500 nmol/g of the cel9A+Cel6B mixture was needed to obtain 80% conversion, while the Cel6B+Cel5A and Cel9A+Cel5A mixtures required 1500 and 1250 nmol/g, respectively, to obtain 80% conversion. Thus, it appears that the more accessible structure of BMCC, as reflected by its binding capacity, results in relative higher processive activity.

Production and characterization of pullulan from beet molasses using a nonpigmented strain of Aureobasidium pullulans in batch culture.

Abstract: The production of pullulan from beet molasses by a pigment-free strain of Aztreobasidium pullulans on shake-flask culture was investigated. Combined pretreatment of molasses with sulfuric acid and activated carbon to remove potential fermentation inhibitors present in molasses resulted in a maximum pullulan concentration of 24 g/L, a biomass dry wt of 14 g/L, a pullulan yield of 52.5%, and a sugar utilization of 92% with optimum fermentation conditions (initial sugar concentration of 50 g/L and initial pH of 7.0). The addition of other nutrients as carbon and nitrogen supplements (olive oil, ammonium sulfate, yeast extract) did not further improve the production of the exopolysaccharides. Structural characterization of the isolated polysaccharides from the fermentation broths by 13C-nuclear magnetic resonance spectroscopy and pullulanase digestion combined with size-exclusion chromatography confirmed the identity of pullulan and the homogeneity (>93% dry basis) of the elaborated polysaccharides by the microorganism. Using multiangle laser light scattering and refractive index detectors in conjunction with high-performance size-exclusion chromatography molecular size distributions and estimates of the molecular weight (Mw = 2.1-4.1 x 10(5)), root mean square of the radius of gyration (R = 30-38 nm), and polydispersity index (Mw/Mn = 1.4-2.4) were obtained. The fermentation products of molasses pretreated with sulfuric acid and/or activated carbon were more homogeneous and free of contaminating proteins. In the concentration range of 2.8-10.0 (w/v), the solution's rheologic behavior of the isolated pullulans was almost Newtonian (within 1 and 1200 s(-1) at 20 degrees C); a slight shear thinning was observed at 10.0 (w/v) for the high molecular weight samples. Overall, beet molasses pretreated with sulfuric acid and activated carbon appears as an attractive fermentation medium for the production of pullulan by A. pullulans.

Characterization of a keratinolytic metalloprotease from Bacillus sp. SCB-3.

Abstract: A keratinolytic protease-producing microorganism was isolated from soybean paste waste and was identified as a strain of Bacillus sp. The keratinase was purified by polyethylene glycol precipitation and two successive column chromatographies with DEAE-Toyopearl 650C and Sephacryl S-200 HR. The purified enzyme had overall 11 purification folds with an 18% yield. The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration on Sephacryl G-200 indicated that the purified enzyme was monomeric and had a molecular weight of 134 kDa. The optimum temperature and pH were 40°C and 7.0, respectively. This enzyme was completely inhibited by EDTA and EGTA, and it was restored by the addition of Ca+2 and Mg+2. These results suggested that it is a metalloprotease. The stimulated enzyme activity by reducing agents indicated that the reducing condition was important in the expression of the activity.