Showing papers in "Applied Biochemistry and Biotechnology in 2001"

Thermozymes and their applications

Abstract: Enzymes from thermophilic microorganisms, thermozymes, have unique characteristics such as temperature, chemical, and pH stability. They can be used in several industrial processes, in which they replace mesophilic enzymes or chemicals. Thermozymes are often used when the enzymatic process is compatible with existing (high-temperature) process conditions. The main advantages of performing processes at higher temperatures are reduced risk of microbial contamination, lower viscosity, improved transfer rates, and improved solubility of substrates. However, cofactors, substrates, or products might be unstable or other side reactions may occur. Recent developments show that thermophiles are a good source of novel catalysts that are of great industrial interest. Thermostable polymer-degrading enzymes such as amylases, pullulanases, xylanases, proteases, and cellulases are expected to play an important role in food, chemical, pharmaceutical, paper, pulp, and waste-treatment industries. Considerable research efforts have been made to better understand the stability of thermozymes. There are no major conformational differences with mesophilic enzymes, and a small number of extra salt bridges, hydrophobic interactions, or hydrogen bounds seem to confer the extra degree of stabilization. Currently, overexpression of thermozymes in standard Escherichia coli allows the production of much larger quantities of enzymes, which are easy to purify by heat treatment. With wider availability and lower cost, thermophilic enzymes will see more application in industry.

Detoxification of lignocellulose hydrolysates with ion-exchange resins.

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

Oxidative lime pretreatment of high-lignin biomass: poplar wood and newspaper.

Abstract: Lime (Ca[OH]2) and oxygen (O2) were used to enhance the enzymatic digestibility of two kinds of high-lignin biomass: poplar wood and newspaper. The recommended pretreatment conditions for poplar wood are 150 degrees C, 6 h, 0.1 g of Ca(OH)2/g of dry biomass, 9 mL of water/g of dry biomass, 14.0 bar absolute oxygen, and a particle size of -10 mesh. Under these conditions, the 3-d reducing sugar yield of poplar wood using a cellulase loading of 5 filter paper units (FPU)/g of raw dry biomass increased from 62 to 565 mg of eq. glucose/g of raw dry biomass, and the 3-d total sugar (glucose + xylose) conversion increased from 6 to 77% of raw total sugars. At high cellulase loadings (e.g., 75 FPU/g of raw dry biomass), the 3-d total sugar conversion reached 97%. In a trial run with newspaper, using conditions of 140 degrees C, 3 h, 0.3 g of Ca(OH)2/g of dry biomass, 16 mL of water/g of dry biomass, and 7.1 bar absolute oxygen, the 3-d reducing sugar yield using a cellulase loading of 5 FPU/g of raw dry biomass increased from 240 to 565 mg of eq. glucose/g of raw dry biomass. A material balance study on poplar wood shows that oxidative lime pretreatment solubilized 38% of total biomass, including 78% of lignin and 49% of xylan; no glucan was removed. Ash increased because calcium was incorporated into biomass during the pretreatment. After oxidative lime pretreatment, about 21% of added lime could be recovered by CO2 carbonation.

Influence of culture conditions on lipopeptide production by Bacillus subtilis

Abstract: Bacillus subtilis produces various families of lipopeptides with different homologous compounds. To produce “new molecules” with improved activities and to select strains that produced a reduced number of homologs or isomers, we studied the effects of different media on the nature of the synthesis of fatty acid chains for each lipopeptide family. This study focused on two B. subtilis strains cultivated in flasks. Optimized medium for lipopeptide production and Landy medium modified by replacing glutamic acid with other α-amino acids were used. We found that the intensity of production of homologous compounds depends on the strain and the culture medium. Analysis of these lipopeptides by high-performance liquid chromatography showed that the strain B. subtilis NT02 yielded various homologous compounds when cultivated in Landy medium (L-Glu), but primarily one homologous product in high relative amounts when cultivated in the optimized medium. Mass spectrometric analysis and determination of the amino acid composition of this molecule enabled us to identify it as Bacillomycine L cl5.

Fingerprinting Trichoderma reesei hydrolases in a commercial cellulase preparation.

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

Lichenysin: a more efficient cation chelator than surfactin.

Abstract: The lipopeptide lichenysin (cyclo-[L-Gln1-->D-Leu2-->L-Leu3-->L-Val4--> L-Asp5-->D-Leu6-->L-Ile7-beta-OH fatty acid]) produced by Bacillus licheniformis structurally resembles surfactin from Bacillus subtilis. The main difference is the presence of a glutaminyl residue in position 1 of the peptide sequence in place of glutamic acid in surfactin. This local variation causes significant changes in the properties of the molecule compared to surfactin. Lichenysin has a higher surfactant power, the critical micellar concentration (c.m.c.) being strongly reduced from 220 to 22 microM and a much higher hemolytic activity because 100% hemolysis was observed with only 15 microM instead of 200 microM. Lichenysin is also a better chelating agent because its association constants with Ca2+ and Mg2+ are increased by a factor of 4 and 16, respectively. This effect is assigned to an increase in the accessibility of the carboxyl group to cations owing to a change in the side chain topology induced by the Glu/Gln exchange. Additionally, the propensity of the lipopeptide for extensive hydrophobic interactions, as illustrated by its low c.m.c., contributes to further stabilization of the cation and an increase in the partitioning of lichenysin into the erythrocyte membrane. Our data support the formation of a lichensyin-Ca2+ complex in a molar ratio of 2:1 instead of 1:1 with surfactin, suggesting an intermolecular salt bridge between two lichenysin molecules. Therefore, when Ca2+ ions are present in the solution, micellization occurs via a dimer assembly, with a possible long-range effect on the spatial arrangement of the micelles or other supramolecular structures. Finally, among all the surfactin peptidic variants so far known, lichenysin is the one for which the three tested activities are the most substantially improved.

Pretreatment with ammonia water for enzymatic hydrolysis of corn husk, bagasse, and switchgrass

Abstract: Bagasse, corn husk, and switchgrass were pretreated with ammonia water to enhance enzymatic hydrolysis. The sample (2 g) was mixed with 1–6 mL ammonia water (25–28% ammonia) and autoclaved at 120°C for 20 min. After treatment, the product was vacuum-dried to remove ammonia gas. The dried solid could be used immediately in the enzymatic hydrolysis without washing. The enzymatic hydrolysis was effectively improved with more than 0.5 and 1 mL ammonia water/g for corn husk and bagasse, respectively. In bagasse, glucose, xylose, and xylobiose were the main products. The adsorption of CMCase and xylanase was related to the initial rate of enzymatic hydrolysis. In corn husks, arabinoxylan extracted by pretreatment was substantially unhydrolyzed because of the high ratio of arabinose to xylose (0.6). The carbohydrate yields from cellulose and hemicellulose were 72.9% and 82.4% in bagasse, and 86.2% and 91.9% in corn husk, respectively. The ammonia/water pretreatment also benefited from switchgrass (Miscanthus sinensis and Solidago altissima L.) hydrolysis.

Cellulose Hydrolysis Under Extremely Low

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

Twenty years of trials, tribulations, and research progress in bioethanol technology: selected key events along the way.

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

Cellulose Hydrolysis Under Extremely Low Sulfuric Acid and High-Temperature Conditions

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

Kinetic studies of lipase from Candida rugosa: a comparative study between free and immobilized enzyme onto porous chitosan beads.

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

Immobilization and stabilization of biomaterials for biosensor applications.

Abstract: Biosensors are finding applications in a variety of analytical fields. A biosensor basically consists of a transducer in conjunction with a biologically active molecule that converts a biochemical signal into a quantifiable electric response. The specificity of the biosensor depends on the selection of the biomaterial. Enzymes, antibodies, DNA, receptors, organelles, microorganisms as well as animal and plant cells or tissues have been used as biologic sensing materials. Advances in biochemistry, molecular biology, and immunochemistry are expected to lead to a rapid expansion in the range of biologic recognition elements to be used in the field of biosensors. Biomaterials that are stable and function even in highly acidic, alkaline, hydrophobic, or oxidizing environments as well as stable to high temperature and immune to toxic substrates in the processing stream will play an important role. Techniques for immobilization of the biomaterials have played a significant role in the biosensor field. Immobilization not only brings about the intimate contact of the biologic catalysts with the transducer, but also helps in the stabilization of the biologic system, thus enhancing its operational and storage stability. A number of techniques have been developed in our laboratory for the immobilization of enzymes, multienzyme systems, cells, and enzyme-cell conjugates. Some of these aspects that are of significance in biosensor applications have been highlighted.

Coimmobilization of urease and glutamate dehydrogenase in electrochemically prepared polypyrrole-polyvinyl sulfonate films.

Abstract: Immobilization of urease and glutamate dehydrogenase enzymes in electrochemically prepared polypyrrole-polyvinyl sulfonate films (PPY-PVS) was carried out using physical adsorption and electrochemical entrapment techniques. Detailed studies on optimum pH, Fourier transform infrared spectroscopy, cyclic voltammetry, and scanning electron microscopy of the enzymes in the immobilized state were conducted. The value of the apparent Michaelis-Menten constant was experimentally determined to be 2.5 and 2.7 for physically adsorbed and electrochemically entrapped urease in PPY-PVS films, respectively.

Manufacture of xylose-based fermentation media from corncobs by posthydrolysis of autohydrolysis liquors.

Abstract: Milled corncob samples were mixed with water and heated to obtain a liquid phase containing oligosaccharides, sugars, and acetic acid as main reaction products (autohydrolysis reaction). To hydrolyze the sugar oligomers to the correspondent monomers, sulfuric acid was added to the autohydrolysis liquors to reach 0.5-2 wt% of solution, and the reaction media were heated at 101.5-135 degrees C. With this operational procedure, sugar solutions suitable as fermentation media (containing xylose as the major component) were obtained. The kinetics of the posthydrolysis step was characterized on the basis of experimental data concerning the time courses of the concentrations of xylooligosaccharides, xylose, furfural, and acetic acid. The concentrations of other reaction byproducts (glucose or arabinose) were also measured.

Enzymatic hydrolysis of ammonia-treated sugar beet pulp.

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

Kinetics of Ethanol Fermentation with High Biomass Concentration Considering the Effect of Temperature

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

Effect of two broad-spectrum antibiotics on activity and stability of continuous nitrifying system

Abstract: The effects of two broad-spectrum antibiotics, chloramphenicol and oxytetracycline hydrochloride, on the microbial activity and biofilm stability of a mixed nitrifying culture were studied. These antibiotics are present in some wastewaters generated in cattle farms or pharmaceutical industries. A 1-L fermentor, in which nitrifiers grew both in suspension and in a biofilm, was used during the experiments. Chloramphenicol (10–250 mg/L) barely had any effect on biofilm stability and nitrification. Ammonia was fully oxidized to nitrate. However, oxytetracycline caused biofilm sloughing at concentrations of 10 mg/L, but nitrification was not inhibited at antibiotic concentrations up to 100 mg/L. When the concentration of oxytetracycline chlorohydrate was increased stepwise from 100 to 250 mg/L, nitrification was inhibited by 50%. The dissolved organic carbon measurements in both the influent and effluent showed that the antibiotics were neither mineralized by the mixed nitrifying culture nor accumulated in the system. Furthermore, the microbial tests did not reveal the presence of active antibiotics in the effluent. This fact indicates that both cloramphenicol and oxytetracycline were degraded by the nitrifying sludge but not mineralized.

Biosorption of heavy metals by bacteria isolated from activated sludge.

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

Production of oxychemicals from precipitated hardwood lignin

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

Optimization of inulinase production by Kluyveromyces marxianus using factorial design.

Abstract: Factorial design and response surface techniques were used to optimize the culture medium for the production of inulinase by Kluyveromyces marxianus. Sucrose was used as the carbon source instead of inulin. Initially, a fractional factorial design (25–1) was used in order to determine the most relevant variables for enzyme production. Five parameters were studied (sucrose, peptone, yeast extract, pH, and K2HPO4), and all were shown to be significant. Sucrose concentration and pH had negative effects on inulinase production, whereas peptone, yeast extract, and K2HPO4 had positive ones. The pH was shown to be the most significant variable and should be preferentially maintained at 3.5. According to the results from the first factorial design, sucrose, peptone, and yeast extract concentrations were selected to be utilized in a full factorial design. The optimum conditions for a higher enzymatic activity were then determined: 14 g/L of sucrose, 10 g/L of yeast extract, 20 g/L of peptone, 1 g/L of K2HPO4. The enzymatic activity in the culture conditions was 127 U/mL, about six times higher than before the optimization.

Bioemulsifier production in batch culture using glucose as carbon source by Candida lipolytica.

Abstract: The yeast Candida lipolytica IA 1055 produced an inducible extracellular emulsification activity while utilizing glucose at different concentrations as carbon source during batch fermentation at 27°C. In all glucose concentrations studied, maximum production of emulsification activity was detected in the stationary phase of growth, after pH reached minimal values. The bioemulsifier isolated was a complex biopolymer constituting proteins, carbohydrates, and lipids. The results obtained in this work show that the biosynthesis of a bioemulsifier is not simply a prerequisite for the degradation of extracellular hydrocarbon.

Severity Function Describing the Hydrolysis of Xylan Using Carbonic Acid

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

Twenty Years of Trials, Tribulations, and Research Progress in Bioethanol Technology

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

Cellulase recovery via membrane filtration.

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

Ethanol production from lignocellulosic byproducts of olive oil extraction.

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

Production of native and recombinant lipases by Candida rugosa: a review.

Abstract: The yeast Candida rugosa produces multiple lipase isoenzymes sharing high sequence homology but with some differences in their catalytic properties. The regulation of C. rugosa lipase (CRL) synthesis and secretion in C. rugosa obeys a complex pattern. Fermentation processes for both wild-type and mutant C. rugosa strains are available for lipase production. Native CRL preparations have been extensively used for biotransformations. However, their inherent mixture of isoforms with variable profiles complicates interpretation and brings into question the reproducibility achieved between preparations. Although heterologous CRLs gene expression had been hampered owing to a nonuniversal codon usage, recent advances have made heterologous CRLs available. This will expand and improve the industrial utility of CRLs even further. The purpose of this review is to provide a summary of the recent advances on the production of native and recombinant lipases by C. rugosa.

Metabolic flux analysis of Clostridium thermosuccinogenes: effects of pH and culture redox potential.

Abstract: Clostridium thermosuccinogenes are anaerobic thermophilic bacteria that ferment various carbohydrates to succinate and acetate as major products and formate, lactate, and ethanol as minor products. Metabolic carbon flux analysis was used to evaluate the effect of pH and redox potential on the batch fermentation of C. thermosuccinogenes. In a first study, the effects of four pH values (6.50, 6.75, 7.00, and 7.25) on intracellular carbon flux at a constant redox potential of -275 mV were compared. The flux of carbon toward succinate and formate increased whereas the flux to lactate decreased significantly with a pH increase from 6.50 to 7.25. Both specific growth rate and specific rate of glucose consumption were unaffected by changes in pH. The fraction of carbon flux at the phosphoenolpyruvate (PEP) node flowing to oxaloacetate increased with an increase in pH. At the pyruvate node, the fraction of flux to formate increased with increasing pH. At the acetyl CoA node, the fraction of flux to acetate increased significantly with an increase in pH. A second study elucidated the effect of four controlled culture redox potentials (-225, -250, -275, and -310 mV) on metabolic carbon flux at a constant pH of 7.25. Lower values of culture redox potential were correlated with increased succinate, acetate, and formate fluxes and decreased ethanol and hydrogen fluxes in C. thermosuccinogenes. Lactate formation was not significantly influenced by redox potential. At the PEP node, the fraction of carbon to oxaloacetate increased with a decrease in redox potential. At the pyruvate node, the fraction of carbon to formate increased, while at the acetyl CoA node, the fraction of carbon flux to acetate increased with reduced redox potential. The presence of hydrogen in the headspace or the addition of nicotinic acid to the growth media resulted in increased hydrogen and ethanol fluxes and decreased succinate, acetate, formate, and lactate fluxes.

Synthesis and characterization of a novel extracellular polysaccharide by Rhodotorula glutinis.

Abstract: The aim of this work was to characterize an exopolysaccharide by Rhodotorula glutinis KCTC 7989 and to investigate the effect of the culture conditions on the production of this polymer. The extracellular polysaccharide (EPS) produced from this strain was a novel acidic heteropolysaccharide composed of neutral sugars (85%) and uronic acid (15%). The neutral sugar composition was identified by gas chromatography as mannose, fucose, glucose, and galactose in a 6.7:0.2:0.1:0.1 ratio. The molecular weight of purified EPS was estimated to be 1.0−3.8×105 Dalton, and the distribution of the molecular weight was very homogeneous (polydispersity index =1.32). The EPS solution showed a characteristic of pseudoplastic non-Newtonian fluid at a concentration >2.0% in distilled water. The maximum EPS production was obtained when the strain was grown on glucose (30 g/L). Ammonium sulfate was the best suitable nitrogen source for EPS production. The highest yield of EPS was obtained at a carbon to nitrogen ratio of 15. The EPS synthesis was activated at the acidic range of pH 3.0–5.0 and increased when the pH of the culture broth decreased naturally to <2.0 during the fermentation. When the yeast was grown on glucose (30 g/L) and ammonium sulfate (2 g/L) at 22°C at an initial pH of 4.0, EPS production was maximized (4.0 g/L), and the glucose-based production yield coefficient and carbon-based production yield coefficient were 0.30 g of EPS/g of glucose and 0.34 g (carbon of EPS)/g (carbon of glucose), respectively.

Kinetic Studies of Lipase from Candida rugosa

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

Continuous countercurrent extraction of hemicellulose from pretreated wood residues.

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