Ozone pretreatment and fermentative hydrolysis of wheat straw

Principles of the ozone pretreatment of wheat straw for subsequent fermentation into sugars are investigated. The optimum moisture contents of straw in the ozonation process are obtained from data on the kinetics of ozone absorbed by samples with different contents of water. The dependence of the yield of reducing sugars in the fermentative reaction on the quantity of absorbed ozone is established. The maximum conversion of polysaccharides is obtained at ozone doses of around 3 mmol/g of biomass, and it exceeds the value for nonozonated samples by an order of magnitude. The yield of sugar falls upon increasing the dose of ozone. The process of removing lignin from the cell walls of straw during ozonation is visualized by means of scanning electron microscopy.     

Physicochemical patterns of ozone absorption by wood

Results from studying aspen and pine wood ozonation are presented. The effect the concentration of ozone, the reagent residence time, and the content of water in a sample of wood has on ozone consumption rate and ozone demand are analyzed. The residence time is shown to determine the degree of ozone conversion degree and the depth of substrate destruction. The main patterns of ozone absorption by wood with different moisture content are found. Ways of optimizing the ozonation of plant biomass are outlined.     

Acid Hydrolysis of Hemicellulose in Green Liquor Pre-Pulping Extract of Mixed Northern Hardwoods

Forest biomass is a promising resource for future biofuels and bioproducts. Pre-pulping extraction of hemicellulose by alkaline (Green Liquor) pretreatment produces a neutral-pH extract containing hemicellulose-derived oligomers. A near-term option for use of this extract is to hydrolyze the oligomers to fermentable monomer sugars. Chips of mixed northern hardwoods were cooked in a rocking digester at 160 °C for 110 min in Green Liquor at a concentration of 3% Na₂O equivalent salts on dry wood. The mass of wood extracted into the Green Liquor extract was approximately 11.4% of the debarked wood mass, which resulted in a dilute solution of oligomeric hemicelluloses sugars. The concentration of the extract was increased through partial evaporation prior to hydrolysis. Dilute sulfuric acid hydrolysis was applied at conditions ranging from 100 to 160 °C, 2% to 6% (w/v) H₂SO₄, and 2- to 258-min residence time. The maximum fermentable sugar concentration achieved from evaporated extract was 10.7 g/L, representing 90.7% of the maximum possible yield. Application of the biomass pretreatment severity function to the hydrolysis results proved to offer a relatively poor prediction of temperature and reaction time interaction. The combined severity function, which incorporates reaction time, temperature, and acid concentration, did prove to provide a useful means of trading off the combined effects of these three variables on total sugar yields.     

The oxidative destruction of lignin in the ozonation of wood

The oxidative destruction of lignin in the ozonation of aspen wood was studied. The kinetic curves of ozone consumption for samples with different contents of water were obtained. The consumption of ozone increased as the content of water grew. The second derivatives of the UV absorption spectra of lignin were obtained to show that the principal direction of lignin transformations under the action of ozone was the destruction of its aromatic constituents with the formation of carboxyl- and carbonyl-containing compounds. Measurements of the UV diffuse reflectance and EPR spectra of wood showed that the ozonation of wood caused the destruction of lignin quinoid structures. Part of lignin remained unchanged under the action of ozone. A key role in the destruction of wood lignin was played by ozone dissolved in water. Varying the content of water in wood samples allows various lignin transformation products to be obtained through ozonation.     

Effects of cell-wall acetate,xylan backbone,and lignin on enzymatic hydrolysis of aspen wood

Aspen wood substrates with varying degrees of deacetylation, xylan, and lignin removal have been prepared and submitted to enzymatic hydrolysis with a cellulase/hemicellulase preparation for an extended constant period of hydrolysis. Controlled deacetylation has been achieved by treating wood with various alkali metal hydroxide solutions, at various alkali/wood ratios. It has been found that samples with the same extent of deacetylation produce the same sugar yields upon enzymatic hydrolysis. Increased degree of deacetylation increases the yield of sugars obtained from enzymatic hydrolysis, all other compositional parameters held constant. The acetyl group removal is proportional to the stoichiometric relation between added base and wood acetyl content, i.e., the same number of milliequivalents of base/weight of wood remove the same extent of acetyl groups, regardless of the concentration of the base solution. No cation effects are found among Li, Na, and K alkali hydroxide solutions, suggesting that swelling is not as important a parameter as is the removal of the acetyl groups from the xylan backbone in determining the extent of hydrolyzability of the resulting sample.     

Pretreatment of alfalfa stems by wood decay fungus <Emphasis Type="Italic">Perenniporia meridionalis</Emphasis> improves cellulose degradation and minimizes the use of chemicals

Enzymes of wood decay fungi can be exploited to degrade lignocellulosic wastes for sustainable production of bioethanol. Perenniporia meridionalis was tested for growing at different temperatures on stems of alfalfa. The process aims to produce fermentable sugars and can be divided into the following steps: (1) fungal treatment to degrade lignin, (2) microwave pretreatment in water or in phosphoric acid, and (3) enzymatic hydrolysis of cell wall carbohydrates. Thermogravimetric analysis assessed the biomass content of cellulose and lignin after the fungal treatment. Throughout all steps HPLC analysis of sugars, oligomers and by-products (furfural, hydroxymethylfurfural and acids) was performed. Scanning electron microscopy was used for visual inspection and characterization of the experimental material during the treatments. The P. meridionalis pretreatment enhanced the yield of fermentable sugars obtainable by enzymatic hydrolysis in samples subjected to microwave-assisted pretreatment in water, but not in those in acid medium. This is probably related to the very selective removal of lignin by P. meridionalis, exposing cellulose fibers without depleting them. Furthermore, microwave treatment in water produced less byproducts than in acid medium. By exploiting the P. meridionalis lignin degradation is therefore possible to avoid H₃PO₄ use during the alfalfa stem pre-treatment, reducing economic and environmental impacts.     

Kinetic Studies on the Product Inhibition of Enzymatic Lignocellulose Hydrolysis

In order to understand the product inhibition of enzymatic lignocellulose hydrolysis, the enzymatic hydrolysis of pretreated rice straw was carried out over an enzyme loading range of 2 to 30 FPU/g substrate, and the inhibition of enzymatic hydrolysis was analyzed kinetically based on the reducing sugars produced. It was shown that glucose, xylose, and arabinose were the main reducing sugar components contained in the hydrolysate. The mass ratio of glucose, xylose, and arabinose to the total reducing sugars was almost constant at 52.0?%, 29.7?% and 18.8?%, respectively, in the enzyme loading range. The reducing sugars exerted competitive inhibitory interferences to the enzymatic hydrolysis. Glucose, xylose, and arabinose had a dissociation constant of 1.24, 0.54 and 0.33?g/l, respectively. The inhibitory interferences by reducing sugars were superimposed on the enzymatic hydrolysis. The enzymatic hydrolysis could be improved by the removal of the produced reducing sugars from hydrolysate.     

Dilute Acid Pretreatment of Black Spruce Using Continuous Steam Explosion System

The pretreatment of lignocellulosic materials prior to the enzymatic hydrolysis is essential to the sugar yield and bioethanol production. Dilute acid hydrolysis of black spruce softwood chip was performed in a continuous high temperature reactor followed with steam explosion and mechanical refining. The acid-soaked wood chips were pretreated under different feeding rates (60 and 92 kg/h), cooking screw rotation speeds (7.2 and 14.4 rpm), and steam pressures (12 and 15 bar). The enzymatic hydrolysis was carried out on the acid-insoluble fraction of pretreated material. At lower feeding rate, the pretreatment at low steam pressure and short retention time favored the recovery of hemicellulose. The pretreatment at high steam pressure and longer retention time recovered less hemicellulose but improved the enzymatic accessibility. As a result, the overall sugar yields became similar no matter what levels of the retention time or steam pressure. Comparing with lower feeding rate, higher feeding rate resulted in consistently higher glucose yield in both liquid fraction after pretreatment and that released after enzymatic hydrolysis.     

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

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.     

Kinetic Study on the Pretreatment and Enzymatic Saccharification of Rice Hull for the Production of Fermentable Sugars

The production of fermentable sugars from rice hull was studied by dilute acid pretreatment and enzymatic saccharification. Rice hull (15%, w/v) was pretreated by 1% (v/v) sulfuric acid at high temperature (120∼160 °C) for 15, 30, 45, and 60 min, respectively. The maximum sugar concentration from rice hull in the prehydrolysate was obtained at 140 °C for 30 min, but the enzymatic saccharification yield from the corresponding pretreated rice hull is not high. To another aspect, the maximum enzymatic saccharification yield was achieved at 160 °C for 60 min, while the recovery of fermentable sugars was the poorest. To take account of fermentable sugars from pretreatment and enzymatic saccharification, the maximum yield of sugars was obtained only when rice hull was treated at 140 °C for 30 min. Under this condition, 72.5% (w/w) of all sugars generated from the raw material can be recovered. The kinetic study on the enzymatic saccharification of dilute acid pretreated rice hull was also performed in this work by a modified Michaelis–Menten model and a diffusion-limited model. After calculation by a linear and a non-linear regression analysis, both models showed good relation with the experimental results.     

Analysis of carbohydrates in wood and pulps employing enzymatic hydrolysis and subsequent capillary zone electrophoresis

An efficient method for determining the carbohydrate composition of extractive-free delignified wood and pulp is described here. The polysaccharides in the sample are first hydrolyzed using a mixture of commercially available preparations of cellulase and hemicellulase. The reducing saccharides in the hydrolysate thus obtained are subsequently derivatized with 4-aminobenzoic acid ethyl ester and thereafter quantitated by capillary zone electrophoresis (CZE) in an alkaline borate buffer with monitoring of the absorption at 306 nm. All reducing sugars (i.e., neutral monosaccharides and uronic acids) which occur as structural elements in the polysaccharides of wood and pulp can be quantitated in a single such analytical run, which can also determine the contents of 4-deoxy-beta-L-threo-hex-4-enopyranosyluronic acid (HexA) residues present in pulps obtained from alkaline processes. CZE analyses were performed using linear regression of standard curves over a concentration range spanning approximately three orders of magnitude. Carbohydrate constituents constituting approximately 0.1% of the dry mass of the sample could be quantitated. The overall precision of this analytical procedure--involving enzymatic hydrolysis, derivatization and CZE--was good (RSD=2.2-7.5%), especially considering the heterogeneity of the wood and pulp samples. The total yield of carbohydrates (93-97%) obtained employing the procedure developed here was consistently higher than that obtained upon applying the traditional procedure for carbohydrate analysis (85-93%) (involving acid hydrolysis and gas chromatographic analysis) to the same pulps. The trisaccharide HexA-xylobiose was the only HexA-containing saccharide detected using the conditions for enzymatic hydrolysis developed here (i.e., 30 h incubation at pH 4 and 40 degrees C); whereas mixtures of HexA-xylobiose and HexA-xylotriose were obtained when the incubation was performed at pH 5 or 6.     

Optimization of Brewery's spent grain dilute-acid hydrolysis for the production of pentose-rich culture media

Dilute-acid hydrolysis of brewery's spent grain to obtain a pentose-rich fermentable hydrolysate was investigated. The influence of operational conditions on polysaccharide hydrolysis was assessed by the combined severity parameter (CS) in the range of 1.39–3.06. When the CS increased, the pentose sugars concentration increased to a maximum at a CS of 1.94, whereas the maximum glucose concentration was obtained for a CS of 2.65. The concentrations of furfural, hydroxymethylfurfural (HMF), as well as formic and levulinic acids and total phenolic compounds increased with severity. Optimum hydrolysis conditions were found at a CS of 1.94 with >95% of feedstock pentose sugars recovered in the monomeric form, together with a low content of furfural, HMF, acetic and formic acids, and total phenolic compounds. This hydrolysate containing glucose, xylose, and arabinose (ratio 10∶67∶32) was further supplemented with inorganic salts and vitamins and readily fermented by the yeast Debaryomyces hansenii CCMI 941 without any previous detoxification stage. The yeast was able to consume all sugars furfural, HMF, and acetic acid with high biomass yield, 0.68C-mol/C-mol, and productivity, 0.92 g/(L·h). Detoxification with activated charcoal resulted in a similar biomass yield and a slight increase in the volumetric productivity (11%).     

Increased Saccharification Yields from Aspen Biomass Upon Treatment with Enzymatically Generated Peracetic Acid

The recalcitrance of lignocellulosic biomass to enzymatic release of sugars (saccharification) currently limits its use as feedstock for biofuels. Enzymatic hydrolysis of untreated aspen wood releases only 21.8% of the available sugars due primarily to the lignin barrier. Nature uses oxidative enzymes to selectively degrade lignin in lignocellulosic biomass, but thus far, natural enzymes have been too slow for industrial use. In this study, oxidative pretreatment with commercial peracetic acid (470 mM) removed 40% of the lignin (from 19.9 to 12.0 wt.% lignin) from aspen and enhanced the sugar yields in subsequent enzymatic hydrolysis to about 90%. Increasing the amount of lignin removed correlated with increasing yields of sugar release. Unfortunately, peracetic acid is expensive, and concentrated forms can be hazardous. To reduce costs and hazards associated with using commercial peracetic acid, we used a hydrolase to catalyze the perhydrolysis of ethyl acetate generating 60–70 mM peracetic acid in situ as a pretreatment to remove lignin from aspen wood. A single pretreatment was insufficient, but multiple cycles (up to eight) removed up to 61.7% of the lignin enabling release of >90% of the sugars during saccharification. This value corresponds to a predicted 581 g of fermentable sugars from 1 kg of aspen wood. Improvements in the enzyme stability are needed before the enzymatically generated peracetic acid is a commercially viable alternative.     

Combined steam pretreatment and enzymatic hydrolysis of starch-free wheat fibers

Steam treatment of an industrial process stream, denoted starch-free wheat fiber, was investigated to improve the formation of monomeric sugars in subsequent enzymatic hydrolysis for further bioconversion into ethanol. The solid fraction in the process stream, derived from a combined starch and ethanol factory, was rich in arabinose (21.1%), xylose (30.1%), and glucose (18.6%), in the form of polysaccharides. Various conditions of steam pretreatment (170–220°C for 5–30 min) were evaluated, and their effect was assessed by enzymatic hydrolysis with 2 g of Celluclast + Ultraflo mixture/ 100 g of starch-free fiber (SFF) slurry at 5% dry matter (DM). The highest overall sugar yield for the combined steam pretreatment and enzymatic hydrolysis, 52g/100 g of DM of SFF, corresponding to 74% of the theoretical, was achieved with pretreatment at 190°C for 10 min followed by enzymatic hydrolysis.     

Two-stage dilute-acid pretreatment of softwoods

Whole treechips obtained from softwood forest thinnings were pretreated via single-and two-stage dilute-sulfuric acid pretreatment. Whole-tree chips were impregnated with dilute sulfuric acid and steam treated in a 4-L steam explosion reactor. In single-stage pretreatment, wood chips were treated using a wide range of severity. In two-stage pretreatment, the first stage was carried out at low severity tomaximize hemicellulose recovery. Solubilized sugars were recovered from the first-stage prehydrolysate by washing with water. In the second stage, water-insoluble solids from first-stage prehydrolysate were impregnated with dilute sulfuric acid, then steam treated at more severe conditions to hydrolyze a portion of the remaining cellulose to glucose and to improve the enzyme digestibility. The total sugar yields obtained after enzymatic hydrolysis of two-stage dilute acid-pretreated samples were compared with sugar yields from single-stage pretreatment. The overall sugar yield from two-stage dilute-acid pretreatment was approx 10% higher, and the net enzyme requirement was reduced by about 50%. Simultaneous saccharification and fermentation using an adapted Saccharomyces cerevisiae yeast strain further improved cellulose conversion yield and lowered the enzyme requirement.     

Inhibition Effects of Dilute-Acid Prehydrolysate of Corn Stover on Enzymatic Hydrolysis of Solka Floc

Dilute-acid pretreatment liquor (PL) produced at NREL through a continuous screw-driven reactor was analyzed for sugars and other potential inhibitory components. Their inhibitory effects on enzymatic hydrolysis of Solka Floc were investigated. When the PL was mixed into the enzymatic hydrolysis reactor at 1:1 volume ratio, the glucan and xylan digestibility decreased by 63% and 90%, respectively. The tolerance level of the enzyme for each inhibitor was determined. Of the identified degradation components, acetic acid was found to be the strongest inhibitor for cellulase activity, as it decreased the glucan yield by 10% at 1 g/L. Among the sugars, cellobiose and glucose were found to be strong inhibitors to glucan hydrolysis, whereas xylose is a strong inhibitor to xylan hydrolysis. Xylo-oligomers inhibit xylan digestibility more strongly than the glucan digestibility. Inhibition by the PL was higher than that of the simulated mixture of the identifiable components. This indicates that some of the unidentified degradation components, originated mostly from lignin, are potent inhibitors to the cellulase enzyme. When the PL was added to a simultaneous saccharification and co-fermentation using Escherichia coli KO11, the bioprocess was severely inhibited showing no ethanol formation or cell growth.     

Non-ionic Surfactants and Non-Catalytic Protein Treatment on Enzymatic Hydrolysis of Pretreated Creeping Wild Ryegrass

Our previous research has shown that saline Creeping Wild Ryegrass (CWR), Leymus triticoides, has a great potential to be used for bioethanol production because of its high fermentable sugar yield, up to 85% cellulose conversion of pretreated CWR. However, the high cost of enzyme is still one of the obstacles making large-scale lignocellulosic bioethanol production economically difficult. It is desirable to use reduced enzyme loading to produce fermentable sugars with high yield and low cost. To reduce the enzyme loading, the effect of addition of non-ionic surfactants and non-catalytic protein on the enzymatic hydrolysis of pretreated CWR was investigated in this study. Tween 20, Tween 80, and bovine serum albumin (BSA) were used as additives to improve the enzymatic hydrolysis of dilute sulfuric-acid-pretreated CWR. Under the loading of 0.1 g additives/g dry solid, Tween 20 was the most effective additive, followed by Tween 80 and BSA. With the addition of Tween 20 mixed with cellulase loading of 15 FPU/g cellulose, the cellulose conversion increased 14% (from 75 to 89%), which was similar to that with cellulase loading of 30 FPU/g cellulose and without additive addition. The results of cellulase and BSA adsorption on the Avicel PH101, pretreated CWR, and lignaceous residue of pretreated CWR support the theory that the primary mechanism behind the additives is prevention of non-productive adsorption of enzymes on lignaceous material of pretreated CWR. The addition of additives could be a promising technology to improve the enzymatic hydrolysis by reducing the enzyme activity loss caused by non-productive adsorption.     

Evaluation of Ethanol Production from Corncob Using Scheffersomyces (Pichia) stipitis CBS 6054 by Volumetric Scale-up

In scale-up, the potential of ethanol production by dilute sulfuric acid pretreatment using corncob was investigated. Pretreatments were performed at 170 °C with various acid concentrations ranging from 0% to 1.656% based on oven dry weight. Following pretreatment, pretreated biomass yield ranged from 59% to 67%. More than 90% of xylan was removed at 0.828% of sulfuric acid. At same pretreatment condition, the highest glucose yield obtained from pretreated biomass by enzymatic hydrolysis was about 76%, based on a glucan content of 37/100 g. In hydrolysate obtained by pretreatment, glucose concentration was low, while xylose concentration was significantly increased above 0.368% of sulfuric acid. At 1.656% of sulfuric acid, xylose and glucose concentration was highest. In subsequent, fermentation with hydrolysate, maximal ethanol yield was attained after 24 h with 0.368% of sulfuric acid. The fermentation efficiency of hydrolysate obtained by enzymatic hydrolysis reached a maximum of 75% at an acid charge of 0.368%.     

Influence of mixing regime on enzymatic saccharification of steam-exploded softwood chips

In an attempt to elucidate the effect of reduced mixing on the enzymatic hydrolysis of lignocellulosic feedstocks, a pretreated softwood substrate was hydrolyzed under various mixing regimes using a commercial cellulase mixture. The substrate was generated by SO₂-catalyzed steam explosion of Douglas fir wood chips followed by alkali-peroxide treatment to remove lignin. Three mixing regimes were tested; continuous mixing at low (25 rpm) and high (150 rpm) speeds, and mixing at low-speed interspersed with 5-min intervals of high-speed agitation at 150 rpm. At both substrate concentrations (7.5 and 10% [w/w]), the mixed-speed mixing was able to produce sufficiently high conversion rates and yields (93% after 96 h), close or slightly better than those obtained under vigorous mixing (150 rpm). The low-speed shaking produced appreciably lower conversion yields at both levels of substrate concentration. Therefore, the mixed-speed regime may be a viable process option, because it does not seem to have an adverse impact on the cellulose conversion yield and can be an effective means of reducing the mixing energy requirements of an enzymatic hydrolysis process.     

Imidazole Processing of Wheat Straw and Eucalyptus Residues—Comparison of Pre-Treatment Conditions and Their Influence on Enzymatic Hydrolysis

Biomass pre-treatment is a key step in achieving the economic competitiveness of biomass conversion. In the present work, an imidazole pre-treatment process was performed and evaluated using wheat straw and eucalyptus residues as model feedstocks for agriculture and forest-origin biomasses, respectively. Results showed that imidazole is an efficient pre-treatment agent; however, better results were obtained for wheat straw due to the recalcitrant behavior of eucalyptus residues. The temperature had a stronger effect than time on wheat straw pre-treatment but at 160 °C and 4 h, similar results were obtained for cellulose and hemicellulose content from both biomasses (ca. 54% and 24%, respectively). Lignin content in the pre-treated solid was higher for eucalyptus residues (16% vs. 4%), as expected. Enzymatic hydrolysis, applied to both biomasses after different pre-treatments, revealed that results improved with increasing temperature/time for wheat straw. However, these conditions had no influence on the results for eucalyptus residues, with very low glucan to glucose enzymatic hydrolysis yield (93% for wheat straw vs. 40% for eucalyptus residues). Imidazole can therefore be considered as a suitable solvent for herbaceous biomass pre-treatment.