Sulphite addition during steam pretreatment enhanced both enzyme-mediated cellulose hydrolysis and ethanol production.

Sulphite addition during steam pretreatment of softwoods under acidic, neutral and alkaline conditions was assessed to try to minimize lignin condensation. Although pretreatment under neutral/alkaline conditions resulted in effective lignin sulphonation, non-uniform size reduction was observed. In contrast, acidic sulphite steam treatment at 210 °C for 10 min resulted in homogenous particle size reduction and water-insoluble component that was 62% carbohydrate and 33% lignin. This carbohydrate-rich substrate was readily hydrolyzed and fermented which indicated the lack of fermentation inhibitors in the steam-pretreated whole slurry. The use of high solid loading (25% w/v) resulted in a hydrolysis yield of 58% at an enzyme loading of 40 mg protein/g glucan and efficient fermentation (46.6 g/L of ethanol). This indicated that the addition of acidic sulphite at the steam pretreatment of softwoods improved both the enzymatic hydrolysis and fermentation of steam-pretreated whole slurries.

Interaction of carbohydrates with alcohol dehydrogenase: Effect on enzyme activity.

Alcohol dehydrogenase was covalently conjugated with three different oxidized carbohydrates i.e., glucose, starch and pectin. All the carbohydrates inhibited the enzyme. The inhibition was studied with respect to the inhibition rate constant, involvement of thiol groups in the binding, and structural changes in the enzyme. The enzyme activity decreased to half of its original activity at the concentration of 2 mg/mL of pectin, 4 mg/mL of glucose and 10 mg/mL of starch within 10 min at pH 7. This study showed oxidized pectin to be a potent inhibitor of alcohol dehydrogenase followed by glucose and starch. Along with the aldehyde-amino group interaction, thiol groups were also involved in the binding between alcohol dehydrogenase and carbohydrates. The structural changes occurring on binding of alcohol dehydrogenase with oxidized carbohydrates was also confirmed by fluorescence spectrophotometry. Oxidized carbohydrates could thus be used as potential inhibitors of alcohol dehydrogenase.

Thermal behaviour and tolerance to ionic liquid [emim]OAc in GH10 xylanase from Thermoascus aurantiacus SL16W.

GH10 xylanase from Thermoascus aurantiacus strain SL16W (TasXyn10A) showed high stability and activity up to 70-75 °C. The enzyme's half-lives were 101 h, 65 h, 63 min and 6 min at 60, 70, 75 and 80 °C, respectively. The melting point (T m), as measured by DSC, was 78.5 °C, which is in line with a strong activity decrease at 75-80 °C. The biomass-dissolving ionic liquid 1-ethyl-3-methylimidazolium acetate ([emim]OAc) in 30 % concentration had a small effect on the stability of TasXyn10A; T m decreased by only 5 °C. It was also observed that [emim]OAc inhibited much less GH10 xylanase (TasXyn10A) than the studied GH11 xylanases. The K m of TasXyn10A increased 3.5-fold in 15 % [emim]OAc with xylan as the substrate, whereas the approximate level of V max was not altered. The inhibition of enzyme activity by [emim]OAc was lesser at higher substrate concentrations. Therefore, high solid concentrations in industrial conditions may mitigate the inhibition of enzyme activity by ionic liquids. Molecular docking experiments indicated that the [emim] cation has major binding sites near the catalytic residues but in lower amounts in GH10 than in GH11 xylanases. Therefore, [emim] cation likely competes with the substrate when binding to the active site. The docking results indicated why the effect is lower in GH10.

Enhanced stability of alcohol dehydrogenase by non-covalent interaction with polysaccharides.

Non-covalent interaction of alcohol dehydrogenase with polysaccharides was studied using three neutral and three anionic polysaccharides. The process of interaction of alcohol dehydrogenase with gum Arabic was optimized with respect to the ratio of enzyme to gum Arabic, pH, and molarity of buffer. Alcohol dehydrogenase-gum Arabic complex formed under optimized conditions showed 93% retention of original activity with enhanced thermal and pH stability. Lower inactivation rate constant of alcohol dehydrogenase-gum Arabic complex within the temperature range of 45 to 60 °C implied its better stability. Half-life of alcohol dehydrogenase-gum Arabic complex was higher than that of free alcohol dehydrogenase. A slight increment was observed in kinetic constants (K(m) and V(max)) of gum Arabic-complexed alcohol dehydrogenase which may be due to interference by gum Arabic for the binding of substrate to the enzyme. Helix to turn conversion was observed in complexed alcohol dehydrogenase as compared to free alcohol dehydrogenase which may be responsible for observed stability enhancement.

Impact of varying lignocellulosic sugars on continuous solvent production in an immobilized column reactor.

The effect of varying glucose, mannose and xylose concentrations on continuous solvent production at various dilution rates was studied by multiple linear regression (MLR) modeling using an immobilized column reactor. The factors affecting the solvent production were dilution rate and concentrations of glucose and mannose. MLR-models also showed a preference of glucose as well as its inhibitory effect on xylose consumption. The fermentation process was studied at bigger scale with a volume factor of 17 with an added recirculation loop in the system. The up-scaled reactor produced 12.5 g/l of acetone-butanol-ethanol (ABE) solvents at a dilution rate of 0.23 h(-1), as compared to 13.4 g/l with a smaller column reactor. The xylose utilization was significantly higher in the modified reactor (73%) as compared to the small scale (43%).

Oil palm empty fruit bunch to biofuels and chemicals via SO2-ethanol-water fractionation and ABE fermentation.

A process has been developed for conversion of spent liquor produced by SO2-ethanol-water (SEW) fractionation of oil palm empty fruit bunch (OPEFB) fibers to biofuels by ABE fermentation. The fermentation process utilizes Clostridia bacteria that produce butanol, ethanol and acetone solvents at a total yield of 0.26 g/g sugars. A conditioning scheme is developed, which demonstrates that it is possible to utilize the hemicellulose sugars from this agricultural waste stream by traditional ABE fermentation. Fractionation as well as sugar hydrolysis in the spent liquor is hindered by the high cation content of OPEFB, which can be partly removed by acidic leaching suggesting that a better deashing method is necessary. Furthermore, it is inferred that better and more selective lignin removal is needed during conditioning to improve liquor fermentability.

The two stage immobilized column reactor with an integrated solvent recovery module for enhanced ABE production.

The production of acetone, butanol, and ethanol (ABE) by fermentation is a process that had been used by industries for decades. Two stage immobilized column reactor system integrated with liquid-liquid extraction was used with immobilized Clostridium acetobutylicum DSM 792, to enhance the ABE productivity and yield. The sugar mixture (glucose, mannose, galactose, arabinose, and xylose) representative to the lignocellulose hydrolysates was used as a substrate for continuous ABE production. Maximum total ABE solvent concentration of 20.30 g L(-1) was achieved at a dilution rate (D) of 0.2h(-1), with the sugar mixture as a substrate. The maximum solvent productivity (10.85 g L(-1)h(-1)) and the solvent yield (0.38 g g(-1)) were obtained at a dilution rate of 1.0 h(-1). The maximum sugar mixture utilization rate was achieved with the present set up which is difficult to reach in a single stage chemostat. The system was operated for 48 days without any technical problems.

Wheat flour based propionic acid fermentation: an economic approach.

A process for the fermentative production of propionic acid from whole wheat flour using starch and gluten as nutrients is presented. Hydrolysis of wheat flour starch using amylases was optimized. A batch fermentation of hydrolysate supplemented with various nitrogen sources using Propionibacterium acidipropionici NRRL B 3569 was performed. The maximum production of 48.61, 9.40, and 11.06 g of propionic acid, acetic acid and succinic acid, respectively, was found with wheat flour hydrolysate equivalent to 90 g/l glucose and supplemented with 15 g/l yeast extract. Further, replacement of yeast extract with wheat gluten hydrolysate showed utilization of gluten hydrolysate without compromising the yields and also improving the economics of the process. The process so developed could be useful for production of animal feed from whole wheat with in situ production of preservatives, and also suggest utilization of sprouted or germinated wheat for the production of organic acids.

Wood pulp as an immobilization matrix for the continuous production of isopropanol and butanol.

The study was focused on developing a continuous method to produce an alcohol mixture suitable to be used as a gasoline supplement. The immobilized column reactor with wood pulp fibers was successfully used for the continuous production of butanol and isopropanol using Clostridium beijerinckii DSM 6423. A sugar mixture (glucose, mannose, galactose, arabinose and xylose) representing lignocellulose hydrolysate was used as a substrate for the production of solvents. The effect of dilution rate on solvent production was studied during continuous operation. The maximum total solvent concentration of 11.99 g/l was obtained at a dilution rate of 0.16 h(-1). The maximum solvent productivity (5.58 g/l h) was obtained at a dilution rate of 1.5 h(-1). The maximum solvent yield of 0.45 g/g from sugar mixture was observed at 0.25 h(-1). The system will be further used for the solvent production using wood hydrolysate as a substrate.

Butanol production from lignocellulosics.

Clostridium spp. produce n-butanol in the acetone/butanol/ethanol process. For sustainable industrial scale butanol production, a number of obstacles need to be addressed including choice of feedstock, the low product yield, toxicity to production strain, multiple-end products and downstream processing of alcohol mixtures. This review describes the use of lignocellulosic feedstocks, bioprocess and metabolic engineering, downstream processing and catalytic refining of n-butanol.

Continuous two stage acetone-butanol-ethanol fermentation with integrated solvent removal using Clostridium acetobutylicum B 5313.

The objective of this study was to optimize continuous acetone-butanol-ethanol (ABE) fermentation using a two stage chemostat system integrated with liquid-liquid extraction of solvents produced in the first stage. This minimized end product inhibition by butanol and subsequently enhanced glucose utilization and solvent production in continuous cultures of Clostridium acetobutylicum B 5313. During continuous two-stage ABE fermentation, sugarcane bagasse was used as the cell holding material for the both stages and liquid-liquid extraction was performed using an oleyl alcohol and decanol mixture. An overall solvent production of 25.32g/L (acetone 5.93g/L, butanol 16.90g/L and ethanol 2.48g/L) was observed as compared to 15.98g/L in the single stage chemostat with highest solvent productivity and solvent yield of 2.5g/Lh and of 0.35g/g, respectively. Maximum glucose utilization (83.21%) at a dilution rate of 0.051/h was observed as compared to 54.38% in the single stage chemostat.

Continuous bio-catalytic conversion of sugar mixture to acetone-butanol-ethanol by immobilized Clostridium acetobutylicum DSM 792.

Continuous production of acetone, n-butanol, and ethanol (ABE) was carried out using immobilized cells of Clostridium acetobutylicum DSM 792 using glucose and sugar mixture as a substrate. Among various lignocellulosic materials screened as a support matrix, coconut fibers and wood pulp fibers were found to be promising in batch experiments. With a motive of promoting wood-based bio-refinery concept, wood pulp was used as a cell holding material. Glucose and sugar mixture (glucose, mannose, galactose, arabinose, and xylose) comparable to lignocellulose hydrolysate was used as a substrate for continuous production of ABE. We report the best solvent productivity among wild-type strains using column reactor. The maximum total solvent concentration of 14.32 g L(-1) was obtained at a dilution rate of 0.22 h(-1) with glucose as a substrate compared to 12.64 g L(-1) at 0.5 h(-1) dilution rate with sugar mixture. The maximum solvent productivity (13.66 g L(-1) h(-1)) was obtained at a dilution rate of 1.9 h(-1) with glucose as a substrate whereas solvent productivity (12.14 g L(-1) h(-1)) was obtained at a dilution rate of 1.5 h(-1) with sugar mixture. The immobilized column reactor with wood pulp can become an efficient technology to be integrated with existing pulp mills to convert them into wood-based bio-refineries.

Continuous acetone-butanol-ethanol fermentation using SO2-ethanol-water spent liquor from spruce.

SO2-ethanol-water (SEW) spent liquor from spruce chips was successfully used for batch and continuous production of acetone, butanol and ethanol (ABE). Initially, batch experiments were performed using spent liquor to check the suitability for production of ABE. Maximum concentration of total ABE was found to be 8.79 g/l using 4-fold diluted SEW liquor supplemented with 35 g/l of glucose. The effect of dilution rate on solvent production, productivity and yield was studied in column reactor consisting of immobilized Clostridium acetobutylicum DSM 792 on wood pulp. Total solvent concentration of 12 g/l was obtained at a dilution rate of 0.21 h(-1). The maximum solvent productivity (4.86 g/l h) with yield of 0.27 g/g was obtained at dilution rate of 0.64 h(-1). Further, to increase the solvent yield, the unutilized sugars were subjected to batch fermentation.

Continuous production of isopropanol and butanol using Clostridium beijerinckii DSM 6423.

Clostridium beijerinckii DSM 6423 was studied using different continuous production methods to give maximum and stable production of isopropanol and n-butanol. In a single-stage continuous culture, when wood pulp was added as a cell holding material, we could increase the solvent productivity from 0.47 to 5.52 g L⁻¹ h⁻¹ with the yield of 54% from glucose. The overall solvent concentration of 7.51 g L⁻¹ (39.4% isopropanol and 60.6% n-butanol) with the maximum solvent productivity of 0.84 g L⁻¹ h⁻¹ was obtained with two-stage continuous culture. We were able to run the process for more than 48 overall retention times without losing the ability to produce solvents.

Cloning, sequencing, overexpression and characterization of L-rhamnose isomerase from Bacillus pallidus Y25 for rare sugar production.

The L-rhamnose isomerase gene (L-rhi) encoding for L-rhamnose isomerase (L-RhI) from Bacillus pallidus Y25, a facultative thermophilic bacterium, was cloned and overexpressed in Escherichia coli with a cooperation of the 6xHis sequence at a C-terminal of the protein. The open reading frame of L-rhi consisted of 1,236 nucleotides encoding 412 amino acid residues with a calculated molecular mass of 47,636 Da, showing a good agreement with the native enzyme. Mass-produced L-RhI was achieved in a large quantity (470 mg/l broth) as a soluble protein. The recombinant enzyme was purified to homogeneity by a single step purification using a Ni-NTA affinity column chromatography. The purified recombinant L-RhI exhibited maximum activity at 65 degrees C (pH 7.0) under assay conditions, while 90% of the initial enzyme activity could be retained after incubation at 60 degrees C for 60 min. The apparent affinity (K(m)) and catalytic efficiency (k(cat)/K(m)) for L-rhamnose (at 65 degrees C) were 4.89 mM and 8.36 x 10(5) M(-1) min(-1), respectively. The enzyme demonstrated relatively low levels of amino acid sequence similarity (42 and 12%), higher thermostability, and different substrate specificity to those of E. coli and Pseudomonas stutzeri, respectively. The enzyme has a good catalyzing activity at 50 degrees C, for D: -allose, L-mannose, D-ribulose, and L-talose from D-psicose, L-fructose, D-ribose and L-tagatose with a conversion yield of 35, 25, 16 and 10%, respectively, without a contamination of by-products. These findings indicated that the recombinant L-RhI from B. pallidus is appropriate for use as a new source of rare sugar producing enzyme on a mass scale production.

A rare sugar xylitol. Part I: the biochemistry and biosynthesis of xylitol.

The rare sugar xylitol is a five-carbon polyol (pentitol) that has beneficial health effects. Xylitol has global markets and, therefore, it represents an alternative to current dominant sweeteners. The research on microbial reduction of D-xylose to xylitol has been focused on metabolically engineered Saccharomycess cerevisiae and Candida strains. The Candida strains have an advantage over the metabolically engineered S. cerevisiae in terms of D-xylose uptake and maintenance of the intracellular redox balance. Due to the current industrial scale production of xylitol, it has become an inexpensive starting material for the production of other rare sugar. The first part of this mini-review concentrates on the biochemistry of xylitol biosynthesis and the problems related to intracellular redox balance.

A rare sugar xylitol. Part II: biotechnological production and future applications of xylitol.

Xylitol is the first rare sugar that has global markets. It has beneficial health properties and represents an alternative to current conventional sweeteners. Industrially, xylitol is produced by chemical hydrogenation of D-xylose into xylitol. The biotechnological method of producing xylitol by metabolically engineered yeasts, Saccharomyces cerevisiae or Candida, has been studied as an alternative to the chemical method. Due to the industrial scale of production, xylitol serves as an inexpensive starting material for the production of other rare sugars. The second part of this mini-review on xylitol will look more closely at the biotechnological production and future applications of the rare sugar, xylitol.

Novel substrate specificity of D-arabinose isomerase from Klebsiella pneumoniae and its application to production of D-altrose from D-psicose.

d-Arabinose isomerase from Klebsiella pneumoniae 40bXX was purified 12-fold with a 62.5% yield indicated by its electrophoretic homogeneity. The purified enzyme showed the highest activities toward d-arabinose and l-fucose as substrates at optimum conditions (50 mM glycine-NaOH, pH 9.0, 40 degrees C). The enzyme had a broad range of substrate specificities toward various d/l-aldoses, i.e., d-arabinose, l-fucose, d/l-xylose, d-mannose, d/l-lyxose, l-glucose, d-altrose and d/l-galactose. The equilibrium ratios between d-arabinose and d-ribulose, l-fucose and l-fuculose, d-altrose and d-psicose, and l-galactose and l-tagatose were 90:10, 90:10, 13:87 and 25:75, respectively. Using a combination of the immobilized d-tagatose 3-epimerase and d-arabinose isomerase, we achieved the production of d-altrose from d-fructose in a batch reactor. We successfully produced approximately 12 g of d-altrose from 200 g of d-fructose in a reaction series with an overall yield of 6%. The product obtained was confirmed to be d-altrose by HPLC and (13)C-NMR. To the best of our knowledge, this is the first report on the production of d-altrose from a cheap sugar, d-fructose, using an enzymatic method.

Efficient biosynthesis of D-allose from D-psicose by cross-linked recombinant L-rhamnose isomerase: separation of product by ethanol crystallization.

Mass production of a rare aldohexose D-allose from D-psicose was achieved in a batch reaction by crude recombinant L-rhamnose isomerase (L-RhI) cross-linked with glutaraldehyde. The D-psicose substrate was, in turn, mass produced from a naturally abundant ketohexose D-fructose by immobilized recombinant D-tagatose 3-epimerase (D-TE). At an equilibrium state, 25% of D-psicose was isomerized to D-allose, that is, 25 g of D-allose was obtained from 100 g of D-psicose. The D-allose product was easily separated and crystallized from the reaction mixture that contains 25%D-allose, 8%D-altrose and 67%D-psicose using ethanol. Empirically, approximately 338 g, that is, 90% of a theoretical overall yield for the purification of pure D-allose crystals was produced from 1.5 kg of D-psicose within 30 d using a constructed bioreactor. The cross-linked enzyme had an operative half-life of two months after repeated usages.

Novel reactions of L-rhamnose isomerase from Pseudomonas stutzeri and its relation with D-xylose isomerase via substrate specificity.

Escherichia coli strain JM 109 harboring 6 x His-tag L-rhamnose isomerase (L-RhI) from Pseudomonas stutzeri allowed a 20-fold increase in the volumetric yield of soluble enzyme compared to the value for the intrinsic yield. Detailed studies on the substrate specificity of the purified His-tagged protein revealed that it catalyzed previously unknown common and rare aldo/ketotetrose, aldo/ketopentose, and aldo/ketohexose substrates in both D- and L-forms, for instance, erythrose, threose, xylose, lyxose, ribose, glucose, mannose, galactose, altrose, tagatose, sorbose, psicose, and fructose. Using a high enzyme-substrate ratio in extended reactions, the enzyme-catalyzed interconversion reactions from which two different products from one substrate were formed: L-lyxose, L-glucose, L-tagatose and D-allose were isomerized to L-xylulose and L-xylose, L-fructose and L-mannose, L-galactose and L-talose, and D-psicose and D-altrose, in that order. Kinetic studies, however, showed that L-rhamnose with Km and Vmax values of 11 mM and 240 U/mg, respectively, was the most preferred substrate, followed by L-mannose, L-lyxose, D-ribose, and D-allose. Based on the observed catalytic mode of action, these new findings reflected a hitherto undetected interrelation between L-RhI and D-xylose isomerase (D-XI).