Hydrogen fluoride saccharification of wood: lignin fluoride content, isolation of alpha-D-glucopyranosyl fluoride and posthydrolysis of reversion products

Wood chips from bigtooth aspen (Populus grandidentata Michx.) were saccharified by reaction with liquid hydrogen fluoride either anhydrous or containing up to 10% v/v water. The reaction products were separated into a solid lignin fraction and a water-soluble saccharide fraction. The fluoride content of the lignin (determined after alkaline fusion) was initially about 1 mg/g wood, but was lowered to 0.1 mg/g wood by grinding and washing. Thus little or no chemical binding of fluoride to lignin occurred during hydrogen fluoride (HF) solvolysis. Analysis of the water-soluble fraction by gel filtration on Biogel P2 columns showed a range of low-molecular-weight oligosaccharides and only 10-20% sugar monomers. Thus considerable reversion occurred during HF evacuation. Posthydrolysis conditions were optimized for these reversion products by varying temperature and acid concentration. Optimal conditions at 1 h were 140 degrees C with 100mN sulfuric acid or 225mN Hydrofluoric acid resulting in monomer yields of > 90% for 0.5% sugar solutions and > 80% for 10% sugar solutions. After reaction of pure cellulose (Filter paper) with hydrogen fluoride in the absence of water, and terminating the reaction with calcium carbonate, the reaction intermediate alpha-D-glucopyranosylfluoride was isolated with a maximal yield of 0.2 g/g paper. Upon purification via paper chromatography glucosylfluoride was identified by its specific rotation and also by gas chromatography-mass spectrometry of its tetra-O-trimethylsilyl derivative.     

Conversion of Japanese red pine wood (Pinus densiflora) into valuable chemicals under subcritical water conditions

A comparative study on the decomposition of Japanese red pine wood under subcritical water conditions in the presence and absence of phosphate buffer was investigated in a batch-type reaction vessel. Since cellulose makes up more than 40-45% of the components found in most wood species, a series of experiments were also carried out using pure cellulose as a model for woody biomass. Several parameters such as temperature and residence time, as well as pH effects, were investigated in detail. The best temperature for decomposition and hydrolysis of pure cellulose was found around 270 °C. The effects of the initial pH of the solution which ranged from 1.5 to 6.5 were studied. It was found that the pH has a considerable effect on the hydrolysis and decomposition of the cellulose. Several products in the aqueous phase were identified and quantified. The conditions obtained from the subcritical water treatment of pure cellulose were applied for the Japanese red pine wood chips. As a result, even in the absence of acid catalyst, a large amount of wood sample was hydrolyzed in water; however, by using phosphate buffer at pH 2, there was an increase in the hydrolysis and dissolution of the wood chips. In addition to the water-soluble phase, acetone-soluble and water-acetone-insoluble phases were also isolated after subcritical water treatment (which can be attributed mainly to the degraded lignin, tar, and unreacted wood chips, respectively). The initial wood:acid ratio in the case of reactions catalyzed by phosphate buffer was also investigated. The results showed that this weight ratio can be as high as 3:1 without changing the catalytic activity. The size of the wood chips as one of the most important experimental parameters was also investigated.     

Conversion of cellulose to glucose with cellulase of Trichoderma viride ITCC-1433

Trichoderma viride ITCC-1433 secretes a cellulase complex that is rich in β-glucosidase and therefore well suited for the saccharification of cellulosic materials. The cellulase was investigated with respect to optimum conditions of reaction and enzyme stability. Avicelase, CMCase, and β-glucosidase differed considerably in their physicochemical properties. At temperatures above 50°C, β-glucosidase is not very stable. Therefore, as a compromise the conditions of hydrolysis were chosen to be 50°C and pH 4.5. With the crude culture filtrate of T. viride ITCC-1433 a nearly pure glucose solution of 4% is reached from a 10% cellulose suspension. Wood pulp and newsprint are hydrolyzed to a much smaller extent. With an enzyme concentrate up to 8% glucose accumulated in the reaction fluid within 48 hr. At this time the glucose-cellobiose ratio was 75:1. Glucose was demonstrated to be the most potent inhibitor of total hydrolysis. The addition of glucose to the enzyme-substrate solution at zero time completely stopped its own formation and cellobiose and reducing groups (oligosaccharides) accumulated. By removing glucose through an ultrafilter device about 90% saccharification of cellulose to glucose was achieved in 48 hr without any accumulation of cellobiose.     

Fermentability of the hemicellulose‐derived sugars from steam‐exploded softwood (douglas fir)

Steam explosion ofDouglas fir wood chips under low‐severity conditions (log Ro = 3.08 corresponding to 175°C, 7.5 min, and 4.5% SO₂) resulted in the recovery of around 87% of the original hemicellulose component in the water‐soluble stream. More than 80% of the recovered hemicellulose was in a monomeric form. As the pretreatment severity increased from 3.08 to 3.76, hemicellulose recovery dropped to 43% of the original hemicellulose found in Douglas fir chips while the concentration of glucose originating from cellulose hydrolysis increased along with the concentration of sugar degradation products such as furfural and hydroxymethylfurfural. Despite containing a higher concentration of hexose monomers (mainly glucose originating from cellulose degradation), the water‐soluble fraction prepared under high‐severity conditions (log Ro = 3.73 corresponding to 215°C, 2.38 min, and 2.38% SO₂) was not readily fermented. Only the two hydrolyzates obtained at low and medium (195°C, 4.5 min, and 4.5% SO₂) severities were fermented to ethanol using a spent sulfur liquor adapted strain of Saccharomyces cerevisiae. High ethanol yields were obtained for these two hydrolyzates with 0.44 g of ethanol produced per gram of hexose utilized (86% of theoretical). However, the best results of hemicellulose recovery and fermentability were obtained for the low‐severity water‐soluble fraction which was fermented significantly faster than the fraction obtained after medium‐severity treatment probably because it contained higher amounts of fermentation inhibitors. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 284–289, 1999.     

Auto-hydrolysis of lignocellulosics under extremely low sulphuric acid and high temperature conditions in batch reactor

Batch reactors were employed to investigate the kinetics of cellulose hydrolysis under extremely low acid (ELA) and high temperature condition. The sawdust was pretreated by Autohydrolysis prior to the batch reaction. The maximum yield of glucose obtained from the batch reactor experiment was about 70% for the pretreated sawdust, this occurred at 210 and 220°C. The maximum glucose yield from the untreated sawdust was much lower at these temperatures, about 55%. The maximum yields of glucose from the lignocellulosics were obtained between 15th and 20th minutes after which gradual decrease was observed.     

Ethanol production in simultaneous saccharification and fermentation of cellulose with temperature profiling

The effects of temperature on enzymatic saccharification of cellulose and simulataneous saccharification and fermentation (SSF) were investigated with 100 g·l⁻¹ Solka Floc, 5g·l⁻¹Trichoderma reesei cellulase, and Zymomonas mobilis ATCC 29191. The following results were obtained: 1) Ethanol fermentation under glucose dificient conditions can proceed for more than 100 h at 30°C but gradually ceases after 50 h of operation at 40°C. 2) Equivalent glucose yield based on cellulose for SSF operated at its optimum temperature (37°C) is higher than that for enzymatic saccharification of cellulose at the same temperature by 32%. However, the same equivalent glucose yields were obtained for both processes if they were operated at their respective optimum temperature. 3) SSF with temperature cycling increased the ethanol productivity but gave similar ethanol yield to SSF at 37°C. 4) SSF with temperature profiling gave an ethanol yield of 0.32 g·g⁻¹ and cellulose use of 0.86 g·g⁻¹ which were increased by 39% and 34% over SSF with temperature cycling and at 37°C.     

A microapparatus for liquid hydrogen fluoride solvolysis: Sugar and amino sugar composition of Erysiphe graminis and Triticum aestivum cell walls

The assembly and use of a simple and safe apparatus for HF solvolysis of microgram amounts of cell walls, polysaccharides, or glycoproteins are described. Using this apparatus the cell wall composition of Erysiphe graminis was compared with that of its wheat host. The HF solvolysis combined with TFA posthydrolysis considerably increased sugar yields compared with TFA hydrolysis alone, due mainly to increased yields of glucose from wheat, and glucosamine from Erysiphe, corresponding to cellulose and chitin, respectively. A potentially useful method for determining amounts of fungal hyphae in plant tissue is also provided.     

The production of xylitol by enzymatic hydrolysis of agricultural wastes

Agricultural waste products, beech wood and walnut shells, were hydrolyzed at 40°C using mixed crude enzymes produced byPenicillium sp. AHT-1 andRhizomucor pusillus HHT-1.d-xylose, 4.1 g and 15.1 g was produced from the hydrolysis of 100 g of beech wood and walnut shells, respectively. For xylitol production,Candida tropicalis IFO0618 and the waste product hydrolyzed solutions were used. The effects on xylitol production, of adding glucose as a NADPH source,d-xylose and yeast extract, were examined. Finally, a 50% yield of xylitol was obtained by using the beech wood hydrolyzed solution with the addition of 1% yeast extract and 1% glucose at an initial concentration.     

Enhanced enzymatic cellulose degradation by cellobiohydrolases via product removal

Product inhibition by cellobiose decreases the rate of enzymatic cellulose degradation. The optimal reaction conditions for two Emericella (Aspergillus) nidulans-derived cellobiohydrolases I and II produced in Pichia pastoris were identified as CBHI: 52 °C, pH 4.5–6.5, and CBHII: 46 °C, pH 4.8. The optimum in a mixture of the two was 50 °C, pH 4.9. An almost fourfold increase in enzymatic hydrolysis yield was achieved with intermittent product removal of cellobiose with membrane filtration (2 kDa cut-off): The conversion of cotton cellulose after 72 h was ~19 % by weight, whereas the conversion in the parallel batch reaction was only ~5 % by weight. Also, a synergistic effect, achieving ~27 % substrate conversion, was obtained by addition of endo-1,4-β-d-glucanase. The synergistic effect was only obtained with product removal. By using pure, monoactive enzymes, the work illustrates the profound gains achievable by intermittent product removal during cellulose hydrolysis.     

Hot-water pretreatment of cattails for extraction of cellulose

To date in the US, production of renewable fuels, particularly ethanol, is primarily from food crops that are high in sugar and starch. The use of arable land for fuel rather than food production and the use of a food source for fuel rather than food have created issues in pricing and availability of traditional foods and feed. The use of cattails to produce biofuel will add value to land and also reduce emissions of greenhouse gases by replacing petroleum products. In order to investigate the feasibility of converting cattails into cellulosic ethanol, a hot-water pretreatment process was studied using a Dionex accelerated solvent extractor (ASE) varying treatment temperature and time. The pretreatment at 190°C for more than 10 min could effectively dissolve the xylan fraction of cattails as soluble oligomers. Both the glucose yield and xylose yield obtained from the pretreated cattails increased with the escalation of the final pretreatment temperature, treatment time or enzyme loading. When cattails were pretreated at 190°C for 15 min, the highest glucose yield of 77.6% from the cellulose was achieved in 48 h using a cellulase loading of 60 FPU/g glucan. The yeast Saccharomyces cerevisiae (ATCC 24858) was able to ferment glucose released by cattail cellulose, resulting in approximately 88.7 ± 2.8% of the theoretical ethanol yield. The higher enzyme loading of 60 FPU/g glucan will significantly increase costs. It is recommended that further studies be carried out using cattails as a feedstock for bio-fuels, especially to optimize the economics of biological conversion processes for cattails with regard to reducing enzyme usage, energy input, glucose yield and xylose yield.     

Transformation of wheat straw polysaccharides under dynamic conditions of subcritical autohydrolysis

Transformation of wheat straw polysaccharides under dynamic conditions of subcritical autohydrolysis has been examined at a pressure of 30 MPa in a temperature range of 150—290°C. The dependence of the yield of gaseous, liquid, and solid products on the process temperature has been studied. The formation of liquid products occurs mainly due to the hydrolysis of pentosans and partially of cellulose in a range of 150–200°C. The main components of liquid products are oligo- and monosaccharides. Xylose and arabinose comprise more than 65% of monosaccharides. A temperature rise in a range of 200–290°C is accompanied by a decrease in medium pH, cellulose hydrolysis, and intensive gasification. Glucose predominates in monosaccharides of liquid products isolated at 270°C; a portion of pentoses does not exceed 25% of the total of monosaccharides. The maximal yield of sugars is observed at 200°C and then decreases from 29.6 to 5.3% per straw oven-dry weight at 270°C. A decrease in reactivity of straw cellulose treated at 200°C to enzymatic hydrolysis has been found.     

Study of cellulases from a newly isolated thermophilic and cellulolytic <Emphasis Type="Italic">Brevibacillus</Emphasis> sp. strain JXL

A potentially novel aerobic, thermophilic, and cellulolytic bacterium designated as Brevibacillus sp. strain JXL was isolated from swine waste. Strain JXL can utilize a broad range of carbohydrates including: cellulose, carboxymethylcellulose (CMC), xylan, cellobiose, glucose, and xylose. In two different media supplemented with crystalline cellulose and CMC at 57°C under aeration, strain JXL produced a basal level of cellulases as FPU of 0.02 IU/ml in the crude culture supernatant. When glucose or cellobiose was used besides cellulose, cellulase activities were enhanced ten times during the first 24 h, but with no significant difference between these two simple sugars. After that time, however, culture with glucose demonstrated higher cellulase activities compared with that from cellobiose. Similar trend and effect on cellulase activities were also obtained when glucose or cellobiose served as a single substrate. The optimal doses of cellobiose and glucose for cellulase induction were 0.5 and 1%. These inducing effects were further confirmed by scanning electron microscopy (SEM) images, which indicated the presence of extracellular protuberant structures. These cellulosome-resembling structures were most abundant in culture with glucose, followed by cellobiose and without sugar addition. With respect to cellulase activity assay, crude cellulases had an optimal temperature of 50°C and a broad optimal pH range of 6–8. These cellulases also had high thermotolerance as evidenced by retaining more than 50% activity at 100°C after 1 h. In summary, this is the first study to show that the genus Brevibacillus may have strains that can degrade cellulose.     

Autohydrolysis extraction process as a pretreatment of lignocelluloses for their enzymatic hydrolysis

Autohydrolysis was studied as a pretreatment to enhance sugar yields from enzymatic hydrolysis of wheat and rape straw, beech, birch and poplar sawdust. Reaction temperatures were 185°C to 212°C and the reaction time 20 min. The pretreated slurries were hydrolyzed with “Novo” cellulase and Fusarium sp. 27 cellulase at 45°C and pH 4.8 for 24 h with addition of Fusarium sp. 27 cellbound cellobiase. From 85% to 90% sugar content of substrates were converted to reducing sugars after 24 h enzymatic hydrolysis, with exception of poplar wood. 10.8 g biomass was obtained after cultivation of Fusarium sp. 27 with water solution hemicellulose fraction from 100 g beech sawdust autohydrolyzed at 200°C during 20 min.     

Two-stage acid saccharification of fractionated Gelidium amansii minimizing the sugar decomposition

Two-stage acid hydrolysis was conducted on easy reacting cellulose and resistant reacting cellulose of fractionated Gelidium amansii (f-GA). Acid hydrolysis of f-GA was performed at between 170 and 200 °C for a period of 0-5 min, and an acid concentration of 2-5% (w/v, H2SO4) to determine the optimal conditions for acid hydrolysis. In the first stage of the acid hydrolysis, an optimum glucose yield of 33.7% was obtained at a reaction temperature of 190 °C, an acid concentration of 3.0%, and a reaction time of 3 min. In the second stage, a glucose yield of 34.2%, on the basis the amount of residual cellulose from the f-GA, was obtained at a temperature of 190 °C, a sulfuric acid concentration of 4.0%, and a reaction time 3.7 min. Finally, 68.58% of the cellulose derived from f-GA was converted into glucose through two-stage acid saccharification under aforementioned conditions.     

The effect of sugar decomposed on the ethanol fermentation and decomposition reactions of sugars

A batch reactor was used to investigate the dilute acid hydrolysis reaction of alpha-cellulose and sugar decomposition reactions. Varying the sulfuric acid concentration from 0.07 to 5.0% for reaction temperatures between 180 and 220°C significantly affected glucose yields, which ranged from about 70% to below 10%. Increasing the reaction temperature enhanced this effect. Similar experimental results were obtained for the decomposition of xylose. For sugar decomposition reactions, less than 0.3 g/L of furfural and 5-hydroxymethylfurfural (5-HMF) were produced from glucose and xylose in the absence of sulfuric acid at 190°C and 15 min of reaction time, but adding a small amount of sulfuric acid (0.5%) dramatically increased the decomposition rate and led to the formation of four undesireable products: formic acid, 5-HMF, acetic acid, and furfural. In both hydrolysis and fermentation reactions formic acid, acetic acid, and 5-HMF severely inhibited ethanol fermentation, while furfural had less of an inhibition effect.     

On the dual role of starch,cellulose and their dialdehydes as fillers and accelerators in the tertiary amine catalysed curing of epoxy resin

Gel time studies of epoxy resins containing varying concentrations of water, starch, cellulose, or their dialdehydes were carried out at 120°C using the tertiary amines triethylamine (TEA) and hexamethylenetetramine (HEXA) as catalysts. In the 40 parts per 100 parts of resin (phr) polysaccharide-filled epoxy-HEXA system it was found that ～50% oxidised starch or cellulose produced unexpectedly high curing rate enhancements and gel times of 13 and 22 min, respectively, were obtained. With 100% oxidised starch the gel time increased to 37 min, while with 10% oxidised cellulose the gel time obtained was 91 min. The non-oxidised starch and cellulose gave even higher gel times. Thus, there seems to be some sort of a synergistic mechanism operating when the degree of oxidation of the polysaccharide is ～50% of the glucose monomer units. The difference in the effects of TEA and HEXA on the polysaccharide-filled epoxy curing reaction is explained on the basis of the decomposition of HEXA into its constituents (formaldehyde and ammonia/ammonia derivatives) under the reaction conditions employed, the formation of intermolecular and intramolecular hemiacetal and/or various hydrated aldehyde structures, and the difference in crystallinity and rigidity of the different polysaccharides affecting the availability of hydroxyl groups.     

Characterization of recombinantE. coli ATCC 11303 (pLOI 297) in the conversion of cellulose and xylose to ethanol

This work describes the characterization of recombinantEsherichia coli ATCC 11303 (pLOI 297) in the production of ethanol from cellulose and xylose. We have examined the fermentation of glucose and xylose, both individually and in mixtures, and the selectivity of ethanol production under various conditions of operation. Xylose metabolism was strongly inhibited by the presence of glucose. Ethanol was a strong inhibitor of both glucose and xylose fermentations; the maximum ethanol levels achieved at 37°C and 42°C were about 50 g/l and 25 g/l respectively. Simmultaneous sacharification and fermentation of cellulose with recombinantE. coli and exogenous cellulose showed a high ethanol yield (84% of theoretical) in the hydrolysis regime of pH 5.0 and 37°C. The selectivity of organic acid formation relative to that of ethanol increased at extreme levels of initial glucose concentration; production of succinic and acetic acids increased at low levels of glucose ( <1 g/l), and lactic acid production increased when initial glucose was higher than 100 g/l.     

Study of the Conditions for the Biosynthesis of Bacterial Cellulose by the Producer <Emphasis Type="Italic">Medusomyces gisevii</Emphasis> Sa-12

The effect of culture conditions on the bacterial cellulose (BC) yield synthesized by symbiotic culture Medusomyces gisevii Sa-12 grown in synthetic nutrient medium was studied for the first time. The conditions providing the maximum yield of bacterial cellulose were evaluated: the initial glucose concentration in the culture medium was 20–25 g/L, the temperature was 24–27°C, the ratio of the nutrient medium to the air volume was 1: 10, and the content of the black tea extracts comprised 1.6–4.8 g/L. A sample of chemically pure BC with the following characteristics was obtained under the selected culture conditions: moisture— 99.0%, degree of polymerization—4800, average width of microfibrillar ribbons—30.6 nm, degree of crystallinity— 86%, and the content of triclinic modification Iα—98%.     

Pretreatment of cellulosic wastes to increase enzyme reactivity

The enzymatic hydrolysis of cellulose to glucose is generally a slow reaction. Different pretreatments, such as ball milling to a ?200 mesh or swelling in 1–2% NaOH are reported to increase the reactivity considerably. In this work a fiber fraction from cattle manure was treated in an autoclave for 5–30 min at temperatures ranging from 130–200°C. The reactivity of the cellulose, measured by incubating samples with a commercial cellulase preparation for one hour at 50°C and pH 4.8, was increased by a factor of 4–6 compared to NaOH treatment and 10–12 compared to untreated fiber. The increased reaction rate is probably mostly due to an increase in cellulose availability to enzymatic attack, as structural hemicellulose is hydrolyzed and removed during the treatment. Sugars, produced by hemicellulosis, hydrolysis, will react further to give caramelization products. These side reactions were shown to be suppressed by short treatment times. The treated fiber could support growth of a mixed culture of Trichoderma viride and Candida utilis only after washing, indicating the formation of water soluble inhibitory products during treatment. The treatment with high-temperature steam can probably be used also with other cellulosic materials to increase reactivity. This may be an attractive way to prepare low-valued wastes such as manure fibers, straw, stalks, or corn cobs for fermentation processes to increase the protein content or for use directly as ruminant animal feed.     

Production of sugars from wood using high-pressure hydrogen chloride

Saturating wood particles with HCl gas under pressure was found to be an effective pretreatment prior to subjecting wood to dilute acid hydrolysis. Pretreament is necessary to release sugars from wood because of the tight lattice structure of cellulose. The HCl gas makes the cellulose more susceptible to subsequent acid hydrolysis and the glucose yield is doubled when dilute acid hydrolysis is preceded by HCl saturation at high pressure. The saturation was most effectively performed in a fluidized bed reactor, with pure HCl gas fluidizing an equal volume of ground wood plus inert particles. The fluidized bed effectively dissipated the large amount of heat released upon HCl absorption into the wood. Batch reaction times of 1 h at 315 psia gave glucose yields of 80 degrees and xylose yields of 95 degrees after dilute acid hydrolysis. A model was developed which proposed gas diffusing through the solid as limiting the reaction rate and this was found to effectively describe the HCl-wood reaction in the fluidized bed. The HCl was found to form a stable adduct with the lignin residue in the wood, in a ratio of 3.33 mole of lignin monomer. The adduct was broken upon the addition of water.