Semi-simultaneous Saccharification Research Articles

Bioethanol Production from Paper Sludge by Subcritical Water Pretreatment and Semi-simultaneous Saccharification and Fermentation

Bioethanol Production from Paper Sludge by Subcritical Water Pretreatment and Semi-simultaneous Saccharification and Fermentation

Dual assistance of surfactants in glycerol organosolv pretreatment and enzymatic hydrolysis of lignocellulosic biomass for bioethanol production

This study investigated an innovative strategy of incorporating surfactants into alkaline-catalyzed glycerol pretreatment and enzymatic hydrolysis to improve lignocellulosic biomass (LCB) conversion efficiency. Results revealed that adding 40 mg/g PEG 4000 to the pretreatment at 195 °C obtained the highest glucose yield (84.6%). This yield was comparable to that achieved without surfactants at a higher temperature (240 °C), indicating a reduction of 18.8% in the required heat input. Subsequently, Triton X-100 addition during enzymatic hydrolysis of PEG 4000-assisted pretreated substrate increased glucose yields to 92.1% at 6 FPU/g enzyme loading. High-solid fed-batch semi-simultaneous saccharification and co-fermentation using this dual surfactant strategy gave 56.4 g/L ethanol and a positive net energy gain of 1.4 MJ/kg. Significantly, dual assistance with surfactants rendered 56.3% enzyme cost savings compared to controls without surfactants. Therefore, the proposed surfactant dual-assisted promising approach opens the gateway to economically viable enzyme-mediated LCB biorefinery.

Application of Biorefinery Concept to the Production of Bromelain, Ethanol, and Xylitol from Pineapple Plant Waste

The massive potential of pineapple fruit production can produce a sizable amount of waste, around 75% (w/w) of pineapple weight, contributing to global environmental problems. For this reason, biorefinery techniques are urgently needed to convert pineapple plantation waste into high-value-added bioproducts including bromelain, various sugars, xylooligoscharide, xylitol, and ethanol. The purpose of this study was to examine the effectiveness of converting pineapple plantation waste into bromelain, xylitol, and ethanol. In this study, the activity of the bromelain enzyme was tested in each part of the pineapple plant waste. The configuration of the hydrolysis and fermentation processes used to make ethanol and xylitol from the rest of the pineapple plant waste from bromelain extraction was also investigated. Bromelain is a proteolytic enzyme found in pineapple plants and can be isolated from every part of pineapple plant waste. Enzyme activity under several conditions, such as crude extract, pure extract, and dried extract, has been studied to determine the best conditions for the downstream process of this enzyme’s production in the future. The purification of bromelain involved the utilization of the precipitation method followed by dialysis, whereas the drying process of bromelain employed the freeze-drying method. The bromelain enzyme specific activity is shown to be highest in the pineapple stem, as observed in crude-extract (1.45 ± 0.06 CDU/mg), purified-extract (10.38 ± 0.06 CDU/mg), and dried-extract (12.05 ± 0.43 CDU/mg) conditions. Using the pineapple stem to extract bromelain can produce lignocellulosic waste, which is made up of 39.47% starch, 19.96% hemicellulose, 36.44% cellulose, and 6.05% lignin. The high content of starch, cellulose, and hemicellulose has the potential to be used as feedstock for ethanol and xylitol fermentation. In this study, ethanol and xylitol fermentation were carried out using two methods: separate hydrolysis and fermentation methods (SHF) and semi-simultaneous saccharification and fermentation methods (semi-SSF). As a result, fermentation using the semi-SSF method produced ethanol with a higher titer and yield (22.12 ± 0.05 g/L and 0.44 ± 0.00 g/g, respectively). However, the production of xylitol was found to be insignificant, regardless of whether it was obtained using SHF or semi-SSF. The purification of bromelain involved the utilization of the precipitation method followed by dialysis, whereas the drying process of bromelain employed the freeze-drying method.

Integrating surfactants with low enzyme loading to increase the glucan conversion and ethanol concentration of reed after combined pretreatment

In this study, in order to obtain high concentration bioethanol, reed pretreated with liquid hot water and ammonia oxygen (LHW-NH3·H2O/O2) was used as the substrate for fermentation. Enzymatic hydrolysis becomes difficult by the ineffective lignin adsorption and the decline in cellulase activity. It is worth noting that even under low enzyme loading (9 FPU/g pretreated solid), this study was able to maintain a higher glucan conversion rate (97.60%). In addition, under high solid loading conditions, the combined effect of PEG 6000 and rhamnolipids increased ethanol yield (78.40%) and ethanol concentration (71.5 g/L). The combined LHW-NH3·H2O/O2 pretreatment with fed-batch semi-simultaneous saccharification and fermentation technique may be utilized to industrialize the production of bioethanol from reed since we concentrated on the maximum exploitation of biomass resources and reduction in energy consumption.

Valorization of Corn Cobs for Xylitol and Bioethanol Production through Column Reactor Process

Corncobs are a plentiful lignocellulosic material that can be utilized for energy production as well as the generation of other high-value products. Within the modern concept of biorefineries, we present processes conducted in a column reactor for the valorization of corncobs as a substrate for ethanol and xylitol production. In the first step, corncobs were subjected to acid hydrolysis, resulting in a hemicellulosic hydrolysate rich in xylose sugars intended for xylitol production by Candida tropicalis UFMGBX12-a. The YP/S (yield coefficient of product to substrate) and QP (productivity) values were approximately 0.2 g/g and 0.15 g/L·h, respectively, for the assays conducted in the column reactor. Next, the remaining solid portion of cellulignin was used for ethanol production through semi-simultaneous saccharification and fermentation process by Scheffersomyces parashehatae UFMG-HM 52.2. This approach involved an intensified successive process consisting of alkaline pretreatment of cellulignin, followed by enzymatic hydrolysis and fermentative processes conducted in the same reactor without biomass transfer. After obtaining the enzymatic hydrolysate, a QP value of 0.4 g/L·h for ethanol production was observed in the fermentation process conducted in the column reactor. The results demonstrate the potential of corncobs as a carbon source for biomolecules production, utilizing a process conducive to scale-up.

Semi-Simultaneous Saccharification and Fermentation Improved by Lignin and Extractives Removal from Sugarcane Bagasse

Lignocellulosic biomass and agro-industrial residues are a source of fermentable sugars; however, pretreatments are needed to overcome biomass recalcitrance. This study evaluated the effect of sugarcane bagasse hydrolysis and fermentation in response to dilute acid pretreatment. In natura bagasse, extractive-free bagasse, partially delignified bagasse, and bagasse with added butylated hydroxytoluene antioxidant were pretreated with diluted acid and investigated in semi-simultaneous saccharification and fermentation (S-SSF). The effect of butylated hydroxytoluene (BHT) resulted in lower yields of inhibitors in the liquid fraction of the acid pretreatment (0.01 g L−1 of furfural, 0.01 g L−1 of 5-hydroxymethylfurfural, and 0.68 g L−1 of acetic acid). Partially delignified material and material with BHT resulted in biomass with low hemicellulose and lignin contents, indicating that BHT influenced lignin removal. Extractives removal showed benefits for the acid pretreatment, decreasing the dioxane-soluble material, and a higher yield of glucose and ethanol via S-SSF for the partially delignified material. Enzymatic saccharification of partially delignified material showed 87% of cellulose conversion (24 h with 15 FPU/g), and after 48 h of S-SSF (25 FPU/g), residual 7.06 g L−1 of glucose and production of 15.17 g L−1 of ethanol were observed. The low content of extractives, lignin, and dioxane soluble material resulted in better cellulose accessibility and ethanol yield. Chemical compounds can help remove lignin from biomass favoring ethanol production by S-SSF.

Ethanol production from Agave salmiana leaves by semi and simultaneous saccharification and fermentation at high temperature using Kluyveromycesmarxianus

Second-generation ethanol production by separate hydrolysis and fermentation (SHF) currently remains economically unviable, and thus more profitable alternatives are being sought. In the present study, the lignocellulosic biomass (LB) of Agave salmiana leaves was evaluated in the production of bioethanol through the processes of SHF, semi-simultaneous saccharification and fermentation (SSSF), and simultaneous saccharification and fermentation (SSF). The leaves of A. salmiana were subjected to acid pretreatment (1% H2SO4) and then to saccharification at temperatures of 40 and 50 °C. The processes of SHF, SSSF, and SSF were evaluated at 40 °C using the yeast Kluyveromyces marxianus OFF1. The results show that saccharification at 50 °C released up to 103.12 ± 1.07 g/L of reducing sugars, while at a temperature of 40 °C, the value was 78.41 ± 0.80 g/L, and thus the enzyme retained 76% of its activity at the latter temperature. In the fermentation processes, sugar consumption was up to 89% and ethanol yield up to 94%. The maximum ethanol production was recorded with the process of SSF, with concentrations of 51.5 g/L, which were significant and at least 13% higher compared to the other processes. The results show that the thermotolerance of K. marxianus OFF1 allows fermentation at 40 °C and a more efficient SSF process for transforming the sugars from this agro-industrial waste into ethanol.

Pretreatment Strategies to Enhance Enzymatic Hydrolysis and Cellulosic Ethanol Production for Biorefinery of Corn Stover.

There is a rising interest in bioethanol production from lignocellulose such as corn stover to decrease the need for fossil fuels, but most research mainly focuses on how to improve ethanol yield and pays less attention to the biorefinery of corn stover. To realize the utilization of different components of corn stover in this study, different pretreatment strategies were used to fractionate corn stover while enhancing enzymatic digestibility and cellulosic ethanol production. It was found that the pretreatment process combining dilute acid (DA) and alkaline sodium sulfite (ASS) could effectively fractionate the three main components of corn stover, i.e., cellulose, hemicellulose, and lignin, that xylose recovery reached 93.0%, and that removal rate of lignin was 85.0%. After the joint pretreatment of DA and ASS, the conversion of cellulose at 72 h of enzymatic hydrolysis reached 85.4%, and ethanol concentration reached 48.5 g/L through fed-batch semi-simultaneous saccharification and fermentation (S-SSF) process when the final concentration of substrate was 18% (w/v). Pretreatment with ammonium sulfite resulted in 83.8% of lignin removal, and the conversion of cellulose and ethanol concentration reached 86.6% and 50 g/L after enzymatic hydrolysis of 72 h and fed-batch S-SSF, respectively. The results provided a reference for effectively separating hemicellulose and lignin from corn stover and producing cellulosic ethanol for the biorefinery of corn stover.

An Integrated Process for the Xylitol and Ethanol Production from Oil Palm Empty Fruit Bunch (OPEFB) Using Debaryomyces hansenii and Saccharomyces cerevisiae.

Oil palm empty fruit bunch (OPEFB) is the largest biomass waste from the palm oil industry. The OPEFB has a lignocellulose content of 34.77% cellulose, 22.55% hemicellulose, and 10.58% lignin. Therefore, this material’s hemicellulose and cellulose content have a high potential for xylitol and ethanol production, respectively. This study investigated the integrated microaerobic xylitol production by Debaryomyces hansenii and anaerobic ethanol semi simultaneous saccharification and fermentation (semi-SSF) by Saccharomyces cerevisiae using the same OPEFB material. A maximum xylitol concentration of 2.86 g/L was obtained with a yield of 0.297 g/gxylose. After 96 h of anaerobic fermentation, the maximum ethanol concentration was 6.48 g/L, corresponding to 71.38% of the theoretical ethanol yield. Significant morphological changes occurred in the OPEFB after hydrolysis and xylitol and ethanol fermentation were shown from SEM analysis.

Reusing the acetone-butanol-ethanol separated broth as the lignocellulose pretreatment liquor for fresh corn stalk biorefinery

In this study, fresh corn stalk was used as the feedstock for the production of bio-butanol via acetone-butanol-ethanol (ABE) fermentation, in which process the wastewater from the first stage sugar containing juice fermentation was recycled by ex situ pervaporation and reused as the pulping liquor of the residual corn stalk bagasse (CSB), affording to effective ABE production in the subsequential second stage fermentation process. Results indicated that the juice fermentation wastewater could effectively depolymerize the CSB. Under optimized conditions of the pulping process (180 °C and 10% (w/v) of biomass loading), 32.45 ± 0.85 g/L of total monomer sugars was obtained in CSB enzymatic hydrolysate, which can be directly used as the substrate for the second-stage cellulosic ABE production. Semi-simultaneous saccharification and fermentation (S-SSF) was carried out using the CSB pulps, so that improving the cellulosic ABE production. 10.94 ± 0.65 g/L of ABE concentration with 0.18 ± 0.01 g/L h of productivity were realized. After hybrid the first-stage juice fermentation and the second-stage CSB biotransformation, total 54.07 ± 1.25 g (ABE) can be produced from 1 kg of fresh corn stalk. The alternative process showed great significance in reduction of the wastewater discharge and ensuring high pulping performances.

A Detoxification-Free Process for Enhanced Ethanol Production From Corn Fiber Under Semi-Simultaneous Saccharification and Fermentation

Corn fiber, a by-product from the corn-processing industry, is an attractive feedstock for cellulosic ethanol because of its rich carbohydrate content (mainly residual starch, cellulose, and hemicellulose), abundant reserves, easy collection, and almost no transportation cost. However, the complex structure and components of corn fiber, especially hemicellulose, make it difficult to be effectively hydrolyzed into fermentable sugars through enzymatic hydrolysis. This study developed a simple and easy industrialized process without detoxification treatment for high-yield ethanol produced from corn fiber. Corn fiber was pretreated by dilute acid under the conditions optimized by Box-Behnken design (0.5% H2SO4 at 105°C for 43 min), and 81.8% of total sugars, including glucose, xylose, and arabinose, could be recovered, then the mixture (solid and hydrolysates) was directly used for semi-simultaneous saccharification and fermentation without detoxification, and ethanol yield reached about 81% of the theoretical yield.

Column reactors in fluidized bed configuration as intensification system for xylitol and ethanol production from napier grass (Pennisetum Purpureum)

Currently, the modern concept biorefinery industries require a consolidated, robust and cost-effective bioprocess applied to multi-products manufacturing. In alignment, this work presents biomass pretreatment of Pennisetum purpureum grass and integrated developments for carbohydrates portions utilization as carbon source for xylitol and ethanol production in intensified process conducted in column reactor with fluidized bed configuration, homogenized simply by air insertion. Primarily, by using hemicellulosic portion from acid hydrolysis, xylitol production was evaluated in column fluidized bed reactor by using the novel-wild Brazilian yeast Candida Tropicalis UFMGBX12-a verifying YP/S and Qp of 0.26 g/g and 0.06 g/L.h, respectively. Forwardly, the remaining celulignin from the acid hydrolysis was subjected to alkaline pretreatment, followed by Semi-Simultaneous saccharification and Fermentation process, in an intensification approach performed in column fluidized bed reactor, applied to ethanol production by Scheffersomyces Shehatae UFMG-HM 52.2. Enzymatic hydrolysis of pretreated biomass comprised 90% of glucose yield in 72h of saccharification, while ethanol productivity of 0.21 g/L.h, was found in 24 h of fermentation process. These new developments conferred potential use of Pennisetum purpureum grass as carbon source for xylitol and ethanol production in column reactor towards sustainable intensification process implementation.

Promoted bioethanol production through fed-batch semisimultaneous saccharification and fermentation at a high biomass load of sodium carbonate-pretreated rice straw

A study on the fed-batch semi-simultaneous saccharification and fermentation (FB-S-SSF) of sodium carbonate-pretreated rice straw at biomass loads between 10.0 and 30.0% (m/v) was conducted. Three different feeding modes, i.e., enzyme feeding (E feeding), substrate feeding (SR feeding) and the combination of both E feeding and SR feeding as well as batch mode were conducted for comparison under identical biomass loads. High ethanol concentrations above 90 g/L were obtained in all modes at a biomass load ≥25.0%. The SR feeding modes achieved higher final ethanol concentrations than those of the E feeding mode and batch mode. At biomass loads of 25.0%, 116.8 ± 3.3 and 118.9 ± 3.6 g/L ethanol was obtained for the SR feeding mode and the combination of E feeding and SR feeding modes, respectively. For batch mode and E feeding mode, the ethanol concentrations were 112.3 ± 3.6 and 108.6 ± 3.0 g/L, respectively. The present study shows that the production cost of bioethanol from lignocellulose can be reduced by adopting in situ-produced crude enzymes produced by Aspergillus fumigatus combined with FB-S-SSF of sodium carbonate-pretreated rice straw at a high biomass load, which provides a new idea for effectively reducing the production cost of bioethanol from lignocellulose.

An improved enzymatic pre-hydrolysis strategy for efficient bioconversion of industrial pulp and paper sludge waste to bioethanol using a semi-simultaneous saccharification and fermentation process

The current study investigated an integrated fermentation process for efficient conversion of pulp and paper sludge (PPS) material to bio-ethanol. To achieve maximum yield of reducing sugar from the pretreated PPS, most influencing parameters such as enzyme dosages, surfactant dosages on PPS loadings were optimized in a batch saccharification process using response surface methodology. The experimental validation studies under optimal conditions of 6% (w/w) solid loading condition, 0.16% (w/w) of surfactant concentration and 158 FPU/gm of enzyme loading resulted in a maximum reducing sugar yield of 45 ± 3.75% (w/w). Separate batch saccharification studies were conducted with 4% to 7% (w/w) solid loading to formulate best operating conditions for fed-batch saccharification with 13% (w/w), 18% (w/w) and 22% (w/w) high solid loading. The PPS solid loading of 18% (w/w) in fed-batch saccharification resulted in maximal release of glucose and xylose of 79.56 g/L and 8.65 g/L respectively at 60 h. Further improvement in the conversion of PPS to bioethanol was established through adoption of fed-batch semi-simultaneous saccharification and co-fermentation (S-SSCF) process. Fermentation using co-cultivation of two yeast species namely P. stipitis NCIM 3499 and Baker’s yeast resulted in maximum ethanol concentration of 42.34 g/L with 0.53 g/g yield from 18% (w/w) solid loading. Thus, the current study demonstrated the potential application of PPS waste as a feedstock material for ethanol production, which can be adopted by industries as an environment-friendly alternative to common solid waste management.

Novel biorefining method for succinic acid processed from sugarcane bagasse

Sugarcane bagasse (SCB) was pretreated with hot water (HLW), ethanol (ETH), and sodium hydroxide (SH). The obtained residuals were hydrolyzed and applied as carbon sources for succinic acid (SA) fermentation, the residue digestibility and SA conversion rate of alkali-pretreated residual were superior to others. Considering the characteristics of alkali pretreatment, enzymatic hydrolysis and succinic acid fermentation, a novel in-situ semi-simultaneous saccharification and co-fermentation (SSSCF) procedure for SA production from SCB was developed. The yield, productivity, and conversion rates of SA from SCB raw material (DRM) processed by SSSCF were 41 g/L, 300 mg/L/h, and 320 mg/g dry, respectively. For every kilogram of SA production, the developed coupling method reduced the SH and water usages, energy consumption, and effluent emission by 0.14 kg, 233.5 L 14,000 kJ and 7 L, respectively, and enhanced the SA productivity by 1.7 times compared with the non-coupling procedure.

Production of high concentration bioethanol from reed by combined liquid hot water and sodium carbonate-oxygen pretreatment

This study aimed to establish a method of producing high-concentration bioethanol from reed with reduced distillation-energy demand. Reed was subjected to a pretreatment combining liquid hot water (LHW) and sodium carbonate with oxygen (LHW–Na2CO3/O2) to increase substrate concentration and further achieve high bioethanol concentration through fed-batch semi-simultaneous saccharification and fermentation (fed-batch S-SSF). An oxygen pressure of 0.8 MPa, a Na2CO3 dosage of 16%, a reaction temperature of 160 °C, and a reaction time of 60 min were determined to be optimal, resulting in the highest bioethanol concentration (66.5 g L−1). Bioethanol concentration increased by 75.9% and resulted in a 76.6% decrease in distillation energy compared with those obtained by single-stage LHW pretreatment at 170 °C. After the LHW–Na2CO3/O2 pretreatment, the rate of lignin removal was 71.4%, and almost all hemicellulose was removed. Lignin and hemicellulose removal weakened the interactions among cellulose, hemicellulose, and lignin. We focused on the maximum utilization of biomass resources and decrease in energy expenditure, which highlighted the potential use of the combined LHW–Na2CO3/O2 pretreatment with fed-batch S-SSF method in the industrialization of bioethanol production from reed.

Optimization of enzymatic hydrolysis for bioethanol production by semi-simultaneous saccharification and fermentation using mature coconut fibre

Alternative substrates to produce useful chemicals such as biofuel have been attractive, in particular, for cellulosic ethanol production. In this context, the objective of this work was optimized the synergistic mixture of enzymes and bioethanol production. The enzymes of Trichoderma reesei and crude enzyme extract from Lichtheimia ramosa were used in the hydrolysis of mature coconut fibre pretreated by sequential process of alkaline hydrogen peroxide (Alk-H2O2)-sodium hydroxide (NaOH). Furthermore, these enzymes and pretreated vegetable biomass were applied in the bioethanol production by Saccharomyces cerevisiae in semi-simultaneous saccharification and fermentation strategy (SSSF). Resulting in the yields and conversions of delignified mature coconut fibre into reducing sugars between 12.7-82.14% and 0.09-0.64 g reducing sugars/g dry biomass, respectively, with an initial hydrolysis rate at 12 h between 0.10-0.89 g/(L.h). Yields and conversions of delignified mature coconut fibre into glucose between 10.16-83.78% and 0.06-0.43 g glucose/g dry biomass, in that order, with an initial hydrolysis rate at 12 h between 0.03-0.35 g/(L.h). Bioethanol production by S. cerevisiae using delignified mature coconut fibre, enzymes from T. reesei and crude enzyme extract from L. ramosa resulted in the production of 4.62 g/L, yield of 0.41 g ethanol/g glucose and volumetric productivity of ethanol of 0.13 g/(L.h), respectively. The results showed synergistic effects between enzymes from T. reesei and crude enzyme extract from L. ramosa, without promoting inhibition in the alcoholic fermentation. Therefore, allowing to formulate an optimized enzymatic preparation aiming cellulosic ethanol production.

Multi-scale study of the integrated use of the carbohydrate fractions of sugarcane bagasse for ethanol and xylitol production

A multi-scale study of sugarcane bagasse (SB) pretreatments and integrated developments for use of its carbohydrates portions for ethanol and xylitol production, are presented. In a first approach, after SB acid hydrolysis, hemicellulosic ethanol production was investigated, verifying ethanol yield of 0.31 g/g and volumetric productivity (QP) of 0.1 g/L.h. Xylitol production was also studied, showing xylitol yield and QP of 0.61 g/g and 0.38 g/L.h, respectively. Thus, remaining cellulignin from acid pretreatment was submitted to alkaline hydrolysis followed by Semi-Simultaneous Saccharification and Co-fermentation in sequential processes to ethanol production, by using mixture of SB hemicellulosic and enzymatic hydrolysate as carbon source, verifying QP of 0.6 g/L.h. Moreover, a biomass multi-scale study was conducted by analysis of MEV, X-Ray, FTIR and Ramman Spectroscopy, verifying clear changes in SB structure along each pretreatment. These novel approaches indicated the potential of these integrated use of SB carbohydrates portion for bioproducts obtaining from SB in sustainable processes.

Combining hydrothermal-alkaline/oxygen pretreatment of reed with PEG 6,000-assisted enzyme hydrolysis promote bioethanol fermentation and reduce enzyme loading

This study aimed to gain high bioethanol yield and concentration using reed as a raw material at a low enzyme loading to reduce its production cost. Reed was pretreated with liquid hot water–alkaline/oxygen (LHW–NaOH/O2), and then hydrolyzed at a cellulase loading of 3 fiter paper units/g–substrate with polyethylene glycol (PEG) 6000 to promote fermentation. Approximately 40.2 g/L of bioethanol concentration and 83.7 % (w/w) yield were achieved via separate hydrolysis and fermentation under optimal conditions. PEG 6000 was effective for promoting a high reed-to-bioethanol conversion at a low enzyme loading in the presence of a LHW–NaOH/O2 pretreated substrate. Enzyme dosage in the treatment with PEG 6000 decreased by 88 % compared with that in the treatment without PEG 6,000. The total cost decreased by 72.8 % compared with fed-batch semi-simultaneous saccharification and fermentation. Hence, separate hydrolysis and fermentation with PEG 6000 added method was recommended for LHW–NaOH/O2 pretreatment. This study provided an optimal reed-to-bioethanol conversion with a reduced production cost.

Chaetomella raphigera \u03b2-glucosidase D2-BGL has intriguing structural features and a high substrate affinity that renders it an efficient cellulase supplement for lignocellulosic biomass hydrolysis

BackgroundTo produce second-generation biofuels, enzymatic catalysis is required to convert cellulose from lignocellulosic biomass into fermentable sugars. β-Glucosidases finalize the process by hydrolyzing cellobiose into glucose, so the efficiency of cellulose hydrolysis largely depends on the quantity and quality of these enzymes used during saccharification. Accordingly, to reduce biofuel production costs, new microbial strains are needed that can produce highly efficient enzymes on a large scale.ResultsWe heterologously expressed the fungal β-glucosidase D2-BGL from a Taiwanese indigenous fungus Chaetomella raphigera in Pichia pastoris for constitutive production by fermentation. Recombinant D2-BGL presented significantly higher substrate affinity than the commercial β-glucosidase Novozyme 188 (N188; Km = 0.2 vs 2.14 mM for p-nitrophenyl β-d-glucopyranoside and 0.96 vs 2.38 mM for cellobiose). When combined with RUT-C30 cellulases, it hydrolyzed acid-pretreated lignocellulosic biomasses more efficiently than the commercial cellulase mixture CTec3. The extent of conversion from cellulose to glucose was 83% for sugarcane bagasse and 63% for rice straws. Compared to N188, use of D2-BGL halved the time necessary to produce maximal levels of ethanol by a semi-simultaneous saccharification and fermentation process. We upscaled production of recombinant D2-BGL to 33.6 U/mL within 15 days using a 1-ton bioreactor. Crystal structure analysis revealed that D2-BGL belongs to glycoside hydrolase (GH) family 3. Removing the N-glycosylation N68 or O-glycosylation T431 residues by site-directed mutagenesis negatively affected enzyme production in P. pastoris. The F256 substrate-binding residue in D2-BGL is located in a shorter loop surrounding the active site pocket relative to that of Aspergillus β-glucosidases, and this short loop is responsible for its high substrate affinity toward cellobiose.ConclusionsD2-BGL is an efficient supplement for lignocellulosic biomass saccharification, and we upscaled production of this enzyme using a 1-ton bioreactor. Enzyme production could be further improved using optimized fermentation, which could reduce biofuel production costs. Our structure analysis of D2-BGL offers new insights into GH3 β-glucosidases, which will be useful for strain improvements via a structure-based mutagenesis approach.