Pretreatment of bagasse by UCT-Solvent for the enzymatic hydrolysis

A thermochemical pretreatment of bagasse for the enzymatic hydrolysis has been carried out, in which pretreatment bagasse was autoclaved with binary solvent, composed of Water and organic solvent having upper critical temperature (UCT) on the mutual solubility curve. The pretreatment was named “UCT-solvent pretreatment.” The hydrophobic decomposition products from lignin and hemicellulose, that dissolved in organic phase at room temperature, could be easily separated from the solid and sugars in the aqueous phase. By using UCT-solvent instead of only water, the sugar recoveries from bagasse through the pretreatment and the enzymatic hydrolysis were much improved. There exists an optimal mixing ratio between organic solvent and water to maximize the effect of the pretreatment for enzymatic hydrolysis. The optimal ratio can be explained by the competitive effect between the ability of water as a reagent for the hydrolysis and the ability of solvent for the extraction of the decomposition product, and furthermore by the competitive effect between affinities of the solvent to hydrophilic hemicellulose and hydrophobic lignin. Decomposition of hemicellulose at lower temperature than 190°C was decreased, and hence the degradation of xylose during the pretreatment decreased. These favorable effects of UCT-solvent pretreatment are significantly attributed to the formation of the homogeneous single phase of organic solvent and water at high temperature and the phase separation at room temperature.     

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

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.     

Soaking Pretreatment of Corn Stover for Bioethanol Production Followed by Anaerobic Digestion Process

The production of ethanol and methane from corn stover (CS) was investigated in a biorefinery process. Initially, a novel soaking pretreatment (NaOH and aqueous-ammonia) for CS was developed to remove lignin, swell the biomass, and improve enzymatic digestibility. Based on the sugar yield during enzymatic hydrolysis, the optimal pretreatment conditions were 1?% NaOH?+?8?% NH₄OH, 50°C, 48?h, with a solid-to-liquid ratio 1:10. The results demonstrated that soaking pretreatment removed 63.6?% lignin while reserving most of the carbohydrates. After enzymatic hydrolysis, the yields of glucose and xylose were 78.5?% and 69.3?%, respectively. The simultaneous saccharification and fermentation of pretreated CS using Pichia stipitis resulted in an ethanol concentration of 36.1?g/L, corresponding only to 63.3?% of the theoretical maximum. In order to simplify the process and reduce the capital cost, the liquid fraction of the pretreatment was used to re-soak new CS. For methane production, the re-soaked CS and the residues of SSF were anaerobically digested for 120?days. Fifteen grams CS were converted to 1.9?g of ethanol and 1337.3?mL of methane in the entire process.     

Evaluation of different biomass materials as feedstock for fermentable sugar production

Saline crops and autoclaved municipal organic solid wastes were evaluated for their potential to be used as feedstock for fermentable sugar production through dilute acid pretreatment and enzymatic hydrolysis. The saline crops included two woods, athel (Tamarix aphylla L) and eucalyptus (Eucalyptus camaldulensis), and two grasses, Jose tall wheatgrass (Agropyron elongatum), and creeping wild rye (Leymus triticoides). Each of the biomass materials was first treated with dilute sulfuric acid under selected conditions (acid concentration =1.4% (w/w), temperature =165 degrees C, and time =8 min) and then treated with the enzymes (cellulases and beta-glucosidase). The chemical composition (cellulose, hemicellulose, and lignin contents) of each biomass material and the yield of total and different types of sugars after the acid and enzyme treatment were determined. The results showed that among the saline crops evaluated, the two grasses (creeping wild rye and Jose tall wheatgrass) had the highest glucose yield (87% of total cellulose hydrolyzed) and fastest reaction rate during the enzyme treatment. The autoclaved municipal organic solid wastes showed reasonable glucose yield (64%). Of the two wood species evaluated, Athel has higher glucose yield (60% conversion of cellulose) than eucalyptus (38% conversion of cellulose).     

Effects of SPORL and Dilute Acid Pretreatment on Substrate Morphology, Cell Physical and Chemical Wall Structures, and Subsequent Enzymatic Hydrolysis of Lodgepole Pine

The effects of pretreatment by dilute acid and sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL) on substrate morphology, cell wall physical and chemical structures, along with the subsequent enzymatic hydrolysis of lodgepole pine substrate were investigated. FE-SEM and TEM images of substrate structural morphological changes showed that SPORL pretreatment resulted in fiber separation, where SPORL high pH (4.2) pretreatment exhibited better fiber separation than SPORL low pH (1.9) pretreatment. Dilute acid pretreatment produced very poor fiber separation, consisting mostly of fiber bundles. The removal of almost all hemicelluloses in the dilute acid pretreated substrate did not overcome recalcitrance to achieve a high cellulose conversion when lignin removal was limited. SPORL high pH pretreatment removed more lignin but less hemicellulose, while SPORL low pH pretreatment removed about the same amount of lignin and hemicelluloses in lodgepole pine substrates when compared with dilute acid pretreatment. Substrates pretreated with either SPORL process had a much higher cellulose conversion than those produced with dilute acid pretreatment. Lignin removal in addition to removal of hemicellulose in SPORL pretreatment plays an important role in improving the cellulose hydrolysis of the substrate.     

Evaluation of wet oxidation pretreatment for enzymatic hydrolysis of softwood

The wet oxidation pretreatment (water, oxygen, elevated temperature, and pressure) of softwood (Picea abies) was investigated for enhancing enzymatic hydrolysis. The pretreatment was preliminarily optimized. Six different combinations of reaction time, temperature, and pH were applied, and the compositions of solid and liquid fractions were analyzed. The solid fraction after wet oxidation contained 58–64% cellulose, 2–16% hemicellulose, and 24–30% lignin. The pretreatment series gave information about the roles of lignin and hemicellulose in the enzymatic hydrolysis. The temperature of the pretreatment, the residual hemicellulose content of the substrate, and the type of the commercial cellulase preparation used were the most important factors affecting the enzymatic hydrolysis. The highest sugar yield in a 72-h hydrolysis, 79% of theoretical, was obtained using a pretreatment of 200°C for 10 min at neutral pH.     

Removal of Water-Soluble Extractives Improves the Enzymatic Digestibility of Steam-Pretreated Softwood Barks

Softwood bark contains a large amounts of extractives—i.e., soluble lipophilic (such as resin acids) and hydrophilic components (phenolic compounds, stilbenes). The effects of the partial removal of water-soluble extractives before acid-catalyzed steam pretreatment on enzymatic digestibility were assessed for two softwood barks—Norway spruce and Scots pine. A simple hot water extraction step removed more than half of the water-soluble extractives from the barks, which improved the enzymatic digestibility of both steam-pretreated materials. This effect was more pronounced for the spruce than the pine bark, as evidenced by the 30 and 11% glucose yield improvement, respectively, in the enzymatic digestibility. Furthermore, analysis of the chemical composition showed that the acid-insoluble lignin content of the pretreated materials decreased when water-soluble extractives were removed prior to steam pretreatment. This can be explained by a decreased formation of water-insoluble “pseudo-lignin” from water-soluble bark phenolics during the acid-catalyzed pretreatment, which otherwise results in distorted lignin analysis and may also contribute to the impaired enzymatic digestibility of the barks. Thus, this study advocates the removal of extractives as the first step in the processing of bark or bark-rich materials in a sugar platform biorefinery.     

Pretreatment of softwood by acid-catalyzed steam explosion followed by alkali extraction

A process for converting lignocellulosic biomass to ethanol hydrolyzes the hemicellulosic fraction to soluble sugars (i.e., pretreatment), followed by acid- or enzyme-catalyzed hydrolysis of the cellulosic fraction. Enzymatic hydrolysis may be improved by using an alkali to extract a fraction of the lignin from the pretreated material. The removal of the lignin may increase the accessibility of the cellulose to enzymatic attack, and thus improve overall economics of the process, if the alkali-treated material can still be effectively converted to ethanol. Pretreated Douglas fir produced by a sulfuric-acid-catalyzed steam explosion was treated with NaOH, NH₄OH, and lime to extract some of the lignin. The treated material, along with an untreated control sample, was tested by an enzymatic-digestion procedure, and converted to ethanol by simultaneous saccharification and fermentation using a glucose-fermenting yeast. NaOH was most effective at removing lignin (removed 29%), followed by NH4OH and lime. However, the susceptibility of the treated material to enzymatic digestion was lower than the control and decreased with increasing lignin removal. Ethanol production was similar for the control and NaOH-treated material, and lower for NH₄OH- and lime-treated material.     

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.     

Enhancement of the Enzymatic Digestibility and Ethanol Production from Sugarcane Bagasse by Moderate Temperature-Dilute Ammonia Treatment

In this study, sugarcane bagasse was pretreated with ammonium hydroxide, and the effectiveness of the pretreatment on enzyme hydrolysis and ethanol production was examined. Bagasse was soaked in ammonium hydroxide and water at a ratio of 1:0.5:8 for 0–4 days at 70 °C. Approximately, 14–45 % lignin, 2–6 % cellulose, and 13–22 % hemicellulose were removed during a 0.5- to 4-day ammonia soaking period. The highest glucan conversion of sugarcane bagasse soaked in dilute ammonia at moderate temperature by cellulase was accomplished at 78 % with 75 % of the theoretical ethanol yield. Under the same conditions, untreated bagasse resulted in a cellulose digestibility of 29 and 27 % of the theoretical ethanol yield. The increased enzymatic digestibility and ethanol yields after dilute ammonia pretreatment was related to a combined effect of the removal of lignin and increase in the surface area of fibers.     

Enhancement of Enzymatic Hydrolysis and Klason Lignin Removal of Corn Stover Using Photocatalyst-Assisted Ammonia Pretreatment

Photocatalyst-assisted ammonia pretreatment was explored to improve lignin removal of the lignocellulosic biomass for effective sugar conversion. Corn stover was treated with 5.0–12.5 wt.% ammonium hydroxide, two different photocatalysts (TiO₂ and ZnO) in the presence of molecular oxygen in a batch reactor at 60 °C. Various solid-to-liquid ratios (1:20–1:50) were also tested. Ammonia pretreatment assisted by TiO₂-catalyzed photo-degradation removed 70 % of Klason lignin under the optimum condition (12.5 % ammonium hydroxide, 60 °C, 24 h, solid/liquid?=?1:20, photocatalyst/biomass?=?1:10 with oxygen atmosphere). The enzymatic digestibilities of pretreated corn stover were 85 % for glucan and 75 % for xylan with NH₃-TiO₂-treated solid and 82 % for glucan and 77 % for xylan with NH₃-ZnO-treated solid with 15 filter paper units/g-glucan of cellulase and 30 cellobiase units/g-glucan of β-glucosidase, a 2–13 % improvement over ammonia pretreatment alone.     

Effects of Liquid Hot Water Pretreatment on Enzymatic Hydrolysis and Physicochemical Changes of Corncobs

Liquid hot water (LHW) pretreatment is an efficient chemical-free strategy for enhancing enzymatic digestibility of lignocellulosic biomass for conversion to fuels and chemicals in biorefinery. In this study, effects of LHW on removals of hemicelluloses and lignin from corncobs were studied under varying reaction conditions. LHW pretreatment at 160 °C for 10 min promoted the highest levels of hemicellulose solubilization into the liquid phase, resulting into the maximized pentose yield of 58.8% in the liquid and more than 60% removal of lignin from the solid, with 73.1% glucose recovery from enzymatic hydrolysis of the pretreated biomass using 10 FPU/g Celluclast?. This led to the maximal glucose and pentose recoveries of 81.9 and 71.2%, respectively, when combining sugars from the liquid phase from LHW and hydrolysis of the solid. Scanning electron microscopy revealed disruption of the intact biomass structure allowing increasing enzyme’s accessibility to the cellulose microfibers which showed higher crystallinity index compared to the native biomass as shown by x-ray diffraction with a marked increase in surface area as revealed by BET measurement. The work provides an insight into effects of LHW on modification of physicochemical properties of corncobs and an efficient approach for its processing in biorefinery industry.     

Sulfolane pretreatment of shrub willow to improve enzymatic saccharification

Pretreatment has been regarded as the most efficient strategy for conversion of lignocellulosic biomass to fermentable sugars. In this work, sulfolane pretreatment was performed to break the intricate structure of shrub willow for inhabitation of the enzymatic accessibility to holocellulose. The effects of varying pretreatment parameters on enzymatic hydrolysis of shrub willow were investigated. It was found that sulfolane was more compatible with lignin instead of carbohydrate, and the loss of carbohydrate could be attributed to water and acid generated from sulfolane. The optimum conditions leading to maximal sugar recovery from enzymatic saccharification were confirmed. After pretreatment of shrub willow powder in sulfolane at 170 °C for 1.5 h with mass ratio of sulfolane to substrate of 5, the sugar release could reach 555 mg/g raw materials (352 mg glucose, 203 mg xylose) when combining 20 FPU cellulase, 20 CBU β-glucosidase, and 1.5 FXU xylanase, representing 78.2 % of glucose and 56.6 % of xylose in shrub willow. This enhanced enzymatic saccharification was due to delignification and removal of a proportion of hemicelluloses, as confirmed by X-ray diffraction analysis, scanning electron microscopy, Fourier-transform infrared spectroscopy, thermogravimetric analysis, gas chromatography, and ionic chromatography. Thus, these studies prove sulfolane pretreatment to be an effective and promising approach for biomass to biofuel processing.     

Organosolv-pulping III

Formic acid pretreatment onPinus radiata D. Don was studied in order to improve the cellulose hydrolysis by commercial cellulase. The formic acid treatment effectively removed the lignin. A low substitution (formylation) and a crystallinity decrease of the cellulose in the pulp were observed. As consequence of these parameter changes, owing to the formic acid pretreatment on sawdust, a higher saccharification value was observed. The degree of saccharification increased when the degree of substitution (measured by titration) decreased and the portion of amorphous cellulose (measured via an X-ray technique) increased.Trichoderma reesei cellulase hydrolyzed the untreated and pretreated Pinus sawdust with formic acid in 25% and 56% of saccharification, respectively.     

The influence of lignin on the self-assembly behaviour of xylan rich fractions from birch (Betula pendula)

A crude xylan isolate obtained by prehydrolysis and mild alkaline extraction from birch wood chips (Betula pendula), and a carefully delignified xylan fraction from the same source, were examined by dynamic light scattering (DLS) and cryogenic transmission electron microscopy (cryo-TEM) with regard to their propensity to self-assemble in water into insoluble aggregates. The delignification involved the extraction with chloroform of a crude xylan solution in a pyridine/acetic acid/water mixture. It resulted in a purified xylan fraction in a yield of 23% in which 75 and 90% of the lignin had been removed as indicated by Klason and UV-determination, respectively. It was found that both xylan fractions formed agglomerates by self-assembly in water. However, DLS and cryo-TEM indicated that the aggregates were larger in size (90 vs. 40 nm) and greater in mass when more lignin was present. The addition of an alkaline solution of isolated lignin (obtained by steam explosion) to increasing concentrations of a delignified xylan revealed increasing turbidity. Our conclusion is that lignin induces agglomeration of xylan in aqueous solutions, but xylan concentration plays an active role in the aggregation phenomena. An agglomeration mechanism for lignin rich xylan fractions is proposed.     

Selective multistage extraction process of biomolecules from vine shoots by a combination of biological,chemical, and physical treatments

A multistep extraction process was proposed to recover polyphenols, reducing sugars, and soluble lignin from vine shoots. A physical pretreatment by high voltage electrical discharges (HVED) was followed by an enzymatic hydrolysis and a final delignification step by alkaline hydrolysis. HVED before enzymatic hydrolysis enhanced the extraction of polyphenols (+72%), reducing sugars (+43%), and soluble lignin (+104%) as compared to control experiments (enzymatic hydrolysis). HVED also reinforced the subsequent delignification process by reducing 10% lignin content in exhausted residues. Identification and quantification of ferulic acid, resveratrol, p-coumaric acid, and hydroxybenzoic acid were carried out using high-performance liquid chromatography.     

Enhancing the Enzymatic Hydrolysis of Corn Stover by an Integrated Wet-milling and Alkali Pretreatment

An integrated wet-milling and alkali pretreatment was applied to corn stover prior to enzymatic hydrolysis. The effects of NaOH concentration in the pretreatment on crystalline structure, chemical composition, and reducing-sugar yield of corn stover were investigated, and the mechanism of increasing reducing-sugar yield by the pretreatment was discussed. The experimental results showed that the crystalline structure of corn stover was disrupted, and lignin was removed, while cellulose and hemicellulose were retained in corn stover by the pretreatment with 1% NaOH in 1 h. The reducing-sugar yield from the pretreated corn stovers increased from 20.2% to 46.7% when the NaOH concentration increased from 0% to 1%. The 1% NaOH pretreated corn stover had a holocellulose conversion of 55.1%. The increase in reducing-sugar yield was related to the crystalline structure disruption and delignification of corn stover. It was clarified that the pretreatment significantly enhanced the conversion of cellulose and hemicellulose in the corn stover to sugars.     

Evidence of char formation during wood heat treatment by mild pyrolysis

The behaviour of wood polymers during heat treatment carried out under inert atmosphere at 240 °C has been reinvestigated to understand the important decrease of the O/C ratio observed in a previous study using X-ray photoelectron spectroscopy (XPS). Heat treatment was performed not only on beech sawdust but also on its lignin and holocellulose fractions obtained after acidic hydrolysis of polysaccharides or delignification with sodium chlorite. CP/MAS ¹³C NMR spectra indicate as previously reported an important degradation of hemicelluloses after thermal treatment. However, assignments of the signals appearing in the range of 125-135 ppm and 35 ppm attributed up to now to thermal crosslinking of lignin and formation of methylene bridges should be reconsidered. Indeed, heat treatment of the holocellulose fraction indicates quite similar signals showing that these latter are not due to lignin modification. According to the literature, these new signals have been attributed to the beginning of char formation. Determination of Klason lignin and HPLC analysis of the sugars contained in the hydrolysate support the hypothesis of formation of carbonaceous materials within the wood structure during heat treatment by mild pyrolysis.     

Investigation of the chemical modifications of beech wood lignin during heat treatment

Holocellulose, Klason lignin and milled wood lignin (MWL) of beech wood were extracted before and after heat treatment and analysed using CP MAS ¹³C NMR, ¹³C NMR, ³¹P NMR and size exclusion chromatography (SEC). Experimental results showed that the thermal treatment degrades hemicelluloses and affects lignin polymer through depolymerisation due mainly to cleavage of β-aryl-ether linkages and recondensation reactions. The spectroscopic analysis of MWL demonstrated that these recondensation reactions involved mainly guaiacyl units through formation of 5,5′-biphenolic and diarylmethane structures.Analysis of molecular weight distribution of MWL by SEC indicated that average molecular weights of heat treated milled wood lignin were lower than those of native milled wood lignin.     

Chemical evaluation of seeded fruit biomass of oil pumpkin (<Emphasis Type="Italic">Cucurbita pepo</Emphasis> L. var. <Emphasis Type="Italic">Styriaca</Emphasis>)

Oil pumpkin (Cucurbita pepo L. var. Styriaca) is an economically important horticultural plant cultivated for oil production. After harvesting seeds, the residual biomass has a limited application and is usually left in the field. An experimental study was performed to evaluate the chemical composition of the seeded fruit oil pumpkin biomass (OP) dried by solvent-exchange using ethanol. The sugar composition of polysaccharides obtained by sequential extraction with water and dilute alkali indicated the prevalence of pectic polysaccharides. Hemicelulloses were released in higher amounts in the alkaline step. The chemical composition of OP and its individual tissues (peel, flesh and hairy flesh) was investigated and compared to the corresponding preparations of standard pumpkin (SP, Cucurbita pepo L.). The content of components (on oven-dry basis), calculated from the analysis data of the individual tissues, was estimated for OP: 7.9 % ash, 7.6 % Klason lignin, 19.3 % pectin (as uronic acids), 34.1 % neutral carbohydrates, and 27.4 % α-cellulose and for SP: 6.4 % ash, 4.0 % Klason lignin, 20.9% pectin (as uronic acids), 38.1% neutral carbohydrates, and 29.2 % α-cellulose, respectively. The OP biomass showed a higher proportion of hemicelluloses.