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.     

Variations in Physicochemical Properties and Bioconversion Efficiency of <Emphasis Type="Italic">Ulva lactuca</Emphasis> Polysaccharides After Different Biomass Pretreatment Techniques

Green macroalgae are an abundant and undervalued biomass with a specific cell wall structure. In this context, different pretreatments, namely ethanol organosolv (Org), alkaline, liquid hot water (LHW), and ionic liquid (IL) pretreatments, were applied to the green macroalgae Ulva lactuca biomass and then evaluated. Their effects on chemical composition, biomass crystallinity, enzymatic digestibility, and theoretical ethanol potential were studied. The chemical composition analysis showed that the Org and LHW pretreatments allowed the highest glucan recovery (80.8 ± 3.6 and 62.9 ± 4.4 g/100 g DM, respectively) with ulvan (80.0 and 99.1%) and hemicellulose (55.0 and 42.3%) removal. These findings were in agreement with both thermogravimetric analysis and scanning electron microscopy results that confirm significant structural changes of the pretreated biomasses. It was found that the employed pretreatments did not significantly affect the cellulose crystallinity; however, they both increased the whole crystallinity and the enzymatic digestibility. This later reached 97.5% in the case of LHW pretreatment. Our results showed high efficiency saccharification of Ulva lactuca biomass that will constitute the key step of the implementation of a biorefinery process.     

Lime pretreatment of crop residues bagasse and wheat straw

Lime (calcium hydroxide) was used as a pretreatment agent to enhance the enzymatic digestibility of two common crop residues: bagasse and wheat straw. A systematic study of pretreatment conditions suggested that for short pretreatment times (1–3 h), high temperatures (85-135°C) were required to achieve high sugar yields, whereas for long pretreatment times (e.g., 24 h), low temperatures (50–65°C) were effective. The recommended lime loading is 0.1 g Ca(OH)₂/g dry biomass. Water loading had little effect on the digestibility. Under the recommended conditions, the 3-d reducing sugar yield of the pretreated bagasse increased from 153 to 659 mg Eq glucose/g dry biomass, and that of the pretreated wheat straw increased from 65 to 650 mg Eq glucose/g dry biomass. A material balance study on bagasse showed that the biomass yield after lime pretreatment is 93.6%. No glucan or xylan was removed from bagasse by the pretreatment, whereas 14% of lignin became solubilized. A lime recovery study showed that 86% of added calcium was removed from the pretreated bagasse by ten washings and could be recovered by carbonating the wash water with CO₂ at pH 9.5.     

Effect of pretreatment severity on the enzymatic hydrolysis of bamboo in hydrothermal deconstruction

Bamboo was subjected to hydrothermal deconstruction to release xylans for the enhancement of enzymatic hydrolysis. The de-waxed and de-starched bamboo culm was non-isothermally pretreated in a batch reactor at a solid to liquid ratio of 1:10 g/mL at 120–240 °C. With the increase of the maximum heating temperature from 120 to 240 °C, the pH value of the liquor decreased from 5.98 to 2.71. A maximum yield of the non-volatile components in the liquid was achieved at a pretreatment severity of 4.20. With the increase of the pretreatment severity from 1.18 to 4.82, the yield of the solid residue decreased from 99.52 to 59.91 %, accompanying a decrease of xylan content from 28.86 to 0 %, an increase of glucan content from 42.80 to 59.14 % and an increase of lignin content from 28.10 to 40.57 %. The solid residues after the hydrothermal pretreatment were comprehensively characterized by FT IR, XRD, and element analysis. Enzymatic hydrolysis of the solid residues was assayed by commercial cellulase. Under enzymatic hydrolysis for 96 h, the enzymatic hydrolysis of the pretreated bamboo at the pretreatment severity of 4.82 was 81.16 %, which equaled to 4.7 times of that of the untreated bamboo. This study provided an environmentally friendly process to pretreat biomass for the production of energy.     

Lime Pretreatment of Sugarcane Bagasse for Bioethanol Production

The pretreatment of sugarcane bagasse with lime (calcium hydroxide) is evaluated. The effect of lime pretreatment on digestibility was studied through analyses using central composite design (response surface), considering pretreatment time, temperature, and lime loading as factors. The responses evaluated were the yield of glucose from pretreated bagasse after enzymatic hydrolysis. Experiments were performed using the bagasse as it comes from an alcohol/sugar factory (non-screened bagasse) and bagasse in the size range from 0.248 to 1.397 mm (screened bagasse) (12-60 mesh). It was observed that the particle size presented influence in the release of fermentable sugars after enzymatic hydrolysis using low loading of cellulase and β-glucosidase (3.5 FPU/g dry pretreated biomass and 1.0 IU/g dry pretreated biomass, respectively).     

Hybrid SSF/SHF Processing of SO<Subscript>2</Subscript> Pretreated Wheat Straw—Tuning Co-fermentation by Yeast Inoculum Size and Hydrolysis Time

Wheat straw is one of the main agricultural residues of interest for bioethanol production. This work examines conversion of steam-pretreated wheat straw (using SO₂ as a catalyst) in a hybrid process consisting of a short enzymatic prehydrolysis step and a subsequent simultaneous saccharification and fermentation (SSF) step with a xylose-fermenting strain of Saccharomyces cerevisiae. A successful process requires a balanced design of reaction time and temperature in the prehydrolysis step and yeast inoculum size and temperature in the SSF step. The pretreated material obtained after steam pretreatment at 210 °C for 5 min using 2.5 % SO₂ (based on moisture content) showed a very good enzymatic digestibility at 45 °C but clearly lower at 30 °C. Furthermore, the pretreatment liquid was found to be rather inhibitory to the yeast, partly due to a furfural content of more than 3 g/L. The effect of varying the yeast inoculum size in this medium was assessed, and at a yeast inoculum size of 4 g/L, a complete conversion of glucose and a 90 % conversion of xylose were obtained within 50 h. An ethanol yield (based on the glucan and xylan in the pretreated material) of 0.39 g/g was achieved for a process with this yeast inoculum size in a hybrid process (10 % water-insoluble solid (WIS)) with 4 h prehydrolysis time and a total process time of 96 h. The obtained xylose conversion was 95 %. A longer prehydrolysis time or a lower yeast inoculum size resulted in incomplete xylose conversion.     

Influence of ionic-liquid incubation temperature on changes in cellulose structure,biomass composition,and enzymatic digestibility

Varying ionic liquid, 1-ethyl 3-methyl imidazolium acetate, pretreatment incubation temperature on lignocellulosic biomass substrates, corn stover, switchgrass and poplar, can have dramatic effects on the enzymatic digestibility of the resultant regenerated biomass. In order to delineate the chemical and physical changes resulting from the pretreatment process and correlate changes with enzymatic digestibility, X-ray powder and fiber diffraction, ¹³C cross polarization/magic angle spinning nuclear magnetic resonance spectroscopy, and compositional analysis was completed on poplar, corn stover and switchgrass samples. Optimal pretreatment incubation temperatures were most closely associated with the retention of amorphous substrates upon drying of regenerated biomass. Maximal glucan to glucose conversion for 24 h enzyme hydrolysis was observed for corn stover, switchgrass and poplar at ionic liquid incubation temperatures of 100, 110 and 120 °C, respectively. We hypothesize that effective pretreatment temperatures must attain lignin redistribution and retention of xylan for optimal enzyme digestibility.     

Tailoring Wet Explosion Process Parameters for the Pretreatment of Cocksfoot Grass for High Sugar Yields

The pretreatment of lignocellulosic biomass is crucial for efficient subsequent enzymatic hydrolysis and ethanol fermentation. In this study, wet explosion (WEx) pretreatment was applied to cocksfoot grass and pretreatment conditions were tailored for maximizing the sugar yields using response surface methodology. The WEx process parameters studied were temperature (160–210 °C), retention time (5–20 min), and dilute sulfuric acid concentration (0.2–0.5 %). The pretreatment parameter set E, applying 210 °C for 5 min and 0.5 % dilute sulfuric acid, was found most suitable for achieving a high glucose release with low formation of by-products. Under these conditions, the cellulose and hemicellulose sugar recovery was 94 % and 70 %, respectively. The efficiency of the enzymatic hydrolysis of cellulose under these conditions was 91 %. On the other hand, the release of pentose sugars was higher when applying less severe pretreatment conditions C (160 °C, 5 min, 0.2 % dilute sulfuric acid). Therefore, the choice of the most suitable pretreatment conditions is depending on the main target product, i.e., hexose or pentose sugars.     

Effects of Low Moisture Anhydrous Ammonia (LMAA) Pretreatment at Controlled Ammoniation Temperatures on Enzymatic Hydrolysis of Corn Stover

Corn stover was treated using low-moisture anhydrous ammonia (LMAA) at controlled ammoniation temperature. Moisturized corn stover (50 % moisture) was contacted with anhydrous ammonia (0.1 g NH₃/g-biomass) in a batch reactor at various temperatures (ambient to 150 °C). After ammoniation at elevated and controlled temperature, ammoniated corn stover was pretreated at various temperatures (60–150 °C) for 72–144 h. Change in composition was marginal at low pretreatment temperature but was relatively severe with pretreatment at high temperature (130–150 °C). The latter resulted in low enzymatic digestibility. It was also observed that extreme levels (either high or low) of residual ammonia affected enzymatic digestibility, while residual ammonia improved by 1.0–1.5 %. The LMAA method enhanced enzymatic digestibility compared to untreated corn stover (29.8 %). The highest glucan and xylan digestibility (84.1 and 73.6 %, respectively) was obtained under the optimal LMAA conditions (i.e., ammoniation at 70 °C for 20 min, followed by pretreatment at 90 °C for 48 h).     

Evaluation of Selected White-Rot Fungal Isolates for Improving the Sugar Yield from Wheat Straw

Biological pretreatment of lignocellulosic biomass by fungi can represent a low-cost and eco-friendly alternative to physicochemical methods to facilitate enzymatic hydrolysis. However, fungal metabolism can cause cellulose loss and it is therefore necessary to use the appropriate fungal strain-biomass type combination. In this work, the effects of biological pretreatments carried out by five different fungi on enzymatic hydrolysis of wheat straw were investigated. The best results were obtained with a Ceriporiopsis subvermispora strain, which minimized weight and cellulose losses and gave the highest net sugar yield (calculated with respect to the holocellulose content of the untreated straw), up to 44 % after a 10-week pretreatment, more than doubling the yields obtained with the other isolates. Moreover, prolonging the pretreatment from 4 up to 10 weeks produced a 2-fold increase, up to 60 %, in digestibility (sugar yield, calculated considering the holocellulose content of the pretreated material). The hemicellulose content of the pretreated material resulted inversely correlated with digestibility, and it could thus be utilized as an index of the pretreatment efficacy. Finally, a correlation was also found between digestibility and the difference between the absorbance values at 290 and 320 nm of pretreated wheat straw extracts.     

Impacts of delignification and hot water pretreatment on the water induced cell wall swelling behavior of grasses and its relation to cellulolytic enzyme hydrolysis and binding

The relationships between biomass composition, water retention value (WRV), settling volume and enzymatic glucose yield and enzyme binding is investigated in this work by employing grasses pretreated with combinations of alkaline hydrogen peroxide (AHP) delignification and liquid hot water pretreatment that result in significant alterations of cell wall properties and subsequent enzymatic hydrolysis yields. Specifically, these cell wall treatments are performed on corn stover and switchgrass to generate material with a range of lignin (6–35 %) and xylan (2–28 %) contents as well as a range of other properties such as carboxylic acid content, water binding affinity and swellability. It was determined that WRV and settling volume are predictors of glucose yield (R² = 0.900 and 0.895 respectively) over the range of materials and treatment conditions used. It was also observed that mild AHP delignification can result in threefold increases in the WRV. Dynamic vapor sorption isotherms demonstrated that AHP-delignified corn stover exhibited an increased affinity for water sorption from the vapor phase relative to untreated corn stover. These results indicate that these water properties may be useful proxies for biomass susceptibility to enzymatic deconstruction.     

Comparison of Different Pretreatment Strategies for Enzymatic Hydrolysis of Wheat and Barley Straw

In biomass-to-ethanol processes a physico-chemical pretreatment of the lignocellulosic biomass is a critical requirement for enhancing the accessibility of the cellulose substrate to enzymatic attack. This report evaluates the efficacy on barley and wheat straw of three different pretreatment procedures: acid or water impregnation followed by steam explosion versus hot water extraction. The pretreatments were compared after enzyme treatment using a cellulase enzyme system, Celluclast 1.5 L from Trichoderma reesei, and a beta-glucosidase, Novozyme 188 from Aspergillus niger. Barley straw generally produced higher glucose concentrations after enzymatic hydrolysis than wheat straw. Acid or water impregnation followed by steam explosion of barley straw was the best pretreatment in terms of resulting glucose concentration in the liquid hydrolysate after enzymatic hydrolysis. When the glucose concentrations obtained after enzymatic hydrolyses were related to the potential glucose present in the pretreated residues, the highest yield, approximately 48% (g g-1), was obtained with hot water extraction pretreatment of barley straw; this pretreatment also produced highest yields for wheat straw, producing a glucose yield of approximately 39% (g g-1). Addition of extra enzyme (Celluclast 1.5 L+Novozyme 188) during enzymatic hydrolysis resulted in the highest total glucose concentrations from barley straw, 32-39 g L-1, but the relative increases in glucose yields were higher on wheat straw than on barley straw. Maldi-TOF MS analyses of supernatants of pretreated barley and wheat straw samples subjected to acid and water impregnation, respectively, and steam explosion, revealed that the water impregnated + steam-exploded samples gave a wider range of pentose oligomers than the corresponding acid-impregnated samples.     

The Effect of Ionic Liquid Pretreatment on the Bioconversion of Tomato Processing Waste to Fermentable Sugars and Biogas

Tomato pomace is an abundant lignocellulosic waste stream from industrial tomato processing and therefore a potential feedstock for production of renewable biofuels. However, little research has been conducted to determine if pretreatment can enhance release of fermentable sugars from tomato pomace. Ionic liquids (ILs) are an emerging pretreatment technology for lignocellulosic biomass to increase enzymatic digestibility and biofuel yield while utilizing recyclable chemicals with low toxicity. In this study, pretreatment of tomato pomace with the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) was investigated. Changes in pomace enzymatic digestibility were affected by pretreatment time and temperature. Certain pretreatment conditions significantly improved reducing sugar yield and hydrolysis time compared to untreated pomace. Compositional analyses suggested that pretreatment primarily removed water-soluble compounds and enriched for lignocellulose in pomace, with only subtle changes to the composition of the lignocellulose. While tomato pomace was effectively pretreated with [C2mim][OAc] to improve enzymatic digestibility, as of yet, unknown factors in the pomace caused ionic liquid pretreatment to negatively affect anaerobic digestion of pretreated material. This result, which is unique compared to similar studies on IL pretreatment of grasses and woody biomass, highlights the need for additional research to determine how the unique chemical composition of tomato pomace and other lignocellulosic fruit residues may interact with ionic liquids to generate inhibitors for downstream fermentation to biofuels.     

Anaerobic Digestion of Yard Waste with Hydrothermal Pretreatment

The digestibility of lignocellulosic biomass is limited by its high content of refractory components. The objective of this study is to investigate hydrothermal pretreatment and its effects on anaerobic digestion of sorted organic waste with submerged fermentation. Hydrothermal pretreatment (HT) was performed prior to anaerobic digestion, and three agents were examined for the HT: hot compressed water, alkaline solution, and acidic solution. The concentrations of glucose and xylose were the highest in the sample pretreated in acidic solution. Compared with that of the untreated sample, the biogas yields from digesting the samples pretreated in alkaline solution, acidic solution, and hot water increased by 364, 107, and 79 %, respectively. The decrease of chemical oxygen demand (COD) in liquid phase followed the same order as for the biogas yield. The initial ammonia content of the treated samples followed the order sample treated in acidic solution > sample treated in alkaline solution > sample treated in hot water. The concentrations of volatile fatty acids (VFAs) were low, indicating that the anaerobic digestion process was running at continuously stable conditions.     

Impact of Pretreatment with Dilute Sulfuric Acid Under Moderate Temperature on Hydrolysis of Corn Stover with Two Enzyme Systems

Pretreatment of corn stover with dilute sulfuric acid at moderate temperature was investigated, and glucan digestibility by Cellic CTec2 and Celluclast on the pretreated biomass was compared. Pretreatments were carried out from 60 to 180 min at the temperature from 105 to 135 °C, with acid concentrations ranging from 0.5 to 2 % (w/v). Significant portion of xylan was removed during pretreatment, and the glucan digestibility by CTec2 was significantly better than that by Celluclast in all cases. Analysis showed that glucan digestibility by both two enzymes correlated directly with the extent of xylan removal in pretreatment. Confidence interval was built to give a more precise range of glucan conversion and to test the significant difference among pretreatment conditions. Response surface model was built to obtain the optimal pretreatment condition to achieve high glucan conversion after enzymatic hydrolysis. Considering the cost and energy savings, the optimal pretreatment condition of 1.75 % acid for 160 min at 135 °C was determined, and glucan conversion can achieve the range from 72.86 to 76.69 % at 95 % confidence level after enzymatic hydrolysis, making total glucan recovery up to the range from 89.42 to 93.25 %.     

Microbial Lipid Production from Enzymatic Hydrolysate of Pecan Nutshell Pretreated by Combined Pretreatment

Biodiesel is a fuel composed of monoalkyl esters of long-chain fatty acids derived from renewable biomass sources. In this study, biomass waste pecan nutshell (PS) was attempted to be converted into microbial oil. For effective utilization of PS, sequential pretreatment with ethylene glycol–H₂SO₄–water (78:2:20, wt:wt:wt) at 130 °C for 30 min and aqueous ammonia (25 wt%) at 50 °C for 24 h was used to enhance its enzymatic saccharification. Significant linear correlation was obtained about delignification-saccharification (R ² = 0.9507). SEM and FTIR results indicated that combination pretreatment could effectively remove lignin and xylan in PS for promoting its enzymatic saccharification. After 72 h, the reducing sugars from the hydrolysis of 50 g/L pretreated PS by combination pretreatment could be obtained at 73.6% yield. Using the recovered PS hydrolysates containing 20 g/L glucose as carbon source, microbial lipids produced from the PS hydrolysates by Rhodococcus opacus ACCC41043. Four fatty acids including palmitic acid (C16:0; 23.1%), palmitoleic acid (C16:1; 22.4%), stearic acid (C18:0; 15.3%), and oleic acid (C18:1; 23.9%) were distributed in total fatty acids. In conclusion, this strategy has potential application in the future.     

The effect of chemical and physical characteristics of spruce SEW pulps on enzymatic hydrolysis

Conifers, which are the most abundant biomass species in Nordic countries, USA, Canada and Russia, exhibit strong resistance towards depolymerization by cellulolytic enzymes. At present, it is still not possible to isolate a single structural feature which would govern the rate and degree of enzymatic hydrolysis. On the other hand, the forest residues alone represent an important potential for biochemical production of biofuels. In this study, the effect of substrate properties on the enzymatic hydrolysis of softwood was studied. Stem wood spruce chips were fractionated by SO₂–ethanol–water (SEW) treatment to produce pulps of varying composition by applying different operating conditions. The SEW technology efficiently fractionates different types of lignocellulosic biomass by rapidly dissolving hemicelluloses and lignin. Cellulose remains fully in the solid residue which is then treated by enzymes to release glucose. The differences in enzymatic digestibility of the spruce SEW pulp fibers were interpreted in terms of their chemical and physical characteristics. A strong correlation between the residual lignin content of SEW pulp and enzymatic digestibility was observed whereas cellulose degree of polymerization and hemicellulose content of pulp were not as important. For the pulps containing about 1.5 % (w/w) lignin, 90 % enzymatic digestibility was achieved at 10 FPU enzyme charge and 24 h of hydrolysis time.     

Sequential Extrusion-Ozone Pretreatment of Switchgrass and Big Bluestem

Pretreatment is one of the biggest challenges in utilizing lignocellulosic feedstocks to meet the mandatory requirements for biofuels around the world. Earlier researchers evaluated extrusion and ozone pretreatment separately and found that sugar recovery can be improved significantly from 15–20 to 40–75 % for different feedstocks. To further improve sugar recoveries, extrusion-ozone sequential pretreatment was explored. Accordingly, optimal extruded switchgrass (176?°C, 155 rpm, 20 % moisture, and 8 mm) and big bluestem (180?°C, 155 rpm, 20 % moisture, and 8 mm) at 25–75 % moisture content were exposed to an ozone flow rate of 37–365 mg/h for 2.5 to 10 min. Pretreated samples were then subjected to enzymatic hydrolysis to determine sugar recovery. Statistical analyses confirmed significant effects of the independent variables and their interactions on sugar recoveries for both feedstocks. Maximum glucose, xylose, and total sugar recovery of 66.4, 82.3, and 70.4 %, respectively, were obtained when a low-moisture (25 %) extruded switchgrass was ozonated for 2.5 min at a flow rate of 37 mg/h. Respectively, this represents increases of 3.42, 5.01, and 3.42 times that of the control. When big bluestem at 25 % moisture was extruded and then ozonated for 2.5 min at a flow rate of 365 mg/h, resulting glucose, xylose, and total sugar recoveries of 90.8, 92.2, and 87.5 %, respectively, were obtained. These represent increases of 4.5, 2.7, and 3.9 times than that of the control. It is also noteworthy that furfural and hydroxymethyl furfural were not detected in any of the pretreatments, and only low levels (0.14–0.18 g/l) of acetic acid were measured. The results show that sequential pretreatment using extrusion and ozone is an efficient way to improve sugar recovery from herbaceous biomass feedstocks.     

Hydrothermal pretreatment conditions to enhance ethanol production from poplar biomass

Pretreatment has been recognized as a key step in enzyme-based conversion processes of lignocellulose biomass to ethanol. The aim of this study is to evaluate two hydrothermal pretreatments (steam explosion and liquid hot water) to enhance ethanol production from poplar (Populus nigra) biomass by a simultaneous saccharification and fermentation (SSF) process. The composition of liquid and solid fractions obtained after pretreatment, enzymatic digestibility, and ethanol production of poplar biomass pretreated at different experimental conditions was analyzed. The best results were obtained in steam explosion pretreatment at 210°C and 4 min, taking into account cellulose recovery above 95%, enzymatic hydrolysis yield of about 60%, SSF yield of 60% of theoretical, and 41% xylose recovery in the liquid fraction. Large particles can be used for poplar biomass in both pretreatments, since no significant effect of particle size on enzymatic hydrolysis and SSF was obtained.     

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.