Production Of Fermentable Sugars Research Articles

Expression and characterization of a novel β-1,4-endoglucanase from Bacillus subtilis strain isolated from a pulp and paper mill wastewater.

The production of fermentable sugars from lignocellulosic biomass is achieved by the synergistic action of a group of enzymes called cellulases. Cellulose is a long chain of chemically linked glucoses by β-1,4 bonds. The enzyme β-1,4-endoglucanase is the first cellulase involved in the degradation, breaking the bond of the amorphous regions. A β-1,4-endoglucanase enzyme with high activity was obtained from a Bacillus subtilis strain isolated from wastewater of a pulp and paper mill. Sequencing and bioinformatic analysis showed that the gene was amplified by PCR consisting of 1,407 nucleotides and coding for a β-1,4-endoglucanase enzyme of approximately 55 kDa. The open reading frame (ORF) encoding the mature endoglucanase (eglS) was successfully inserted in a modified cloning plasmid (pITD03) and into the pYD1 plasmid used for its expression in yeast. Carboxymethylcellulose (CMC) plate assay, SDS-PAGE, and zymogram confirmed the production and secretion by the transformed E. coli BL21-SI strain of a 39 kDa β-1,4-endoglucanase consistent with the catalytic domain without the cellulose-binding module (CBM). The results showed that the truncated β-1,4-endoglucanase had higher activity and stability.

Enhancing Xylanase Production from Aspergillus tamarii Kita and Its Application in the Bioconversion of Agro-Industrial Residues into Fermentable Sugars Using Factorial Design

The endo-1,4-β-xylanases (EC 3.2.1.8) are the largest group of hydrolytic enzymes that degrade xylan, the major component of hemicelluloses, by catalyzing the hydrolysis of glycosidic bonds β-1,4 in this polymer, releasing xylooligosaccharides of different sizes. Xylanases have considerable potential in producing bread, animal feed, food, beverages, xylitol, and bioethanol. The fungus Aspergillus tamarii Kita produced xylanases in Adams’ media supplemented with barley bagasse (brewer’s spent grains), a by-product from brewery industries. The culture extract exhibited two xylanase activities in the zymogram, identified by mass spectrometry as glycosyl hydrolase (GH) families 10 and 11 (GH 10 and GH 11). The central composite design (CCD) showed excellent predictive capacity for xylanase production (23.083 U mL−1). Additionally, other enzyme activities took place during the submerged fermentation. Moreover, enzymatic saccharification based on a mixture design (MD) of three different lignocellulosic residues was helpful in the production of fermentable sugars by the A. tamarii Kita crude extract.

Valorization of black carrot pomace and pea powder for co-production of polygalacturonase and pectin lyase

AbstractPectic enzyme groups, particularly polygalacturonase and pectin lyase, are vital components of a high-value microbial enzyme category widely employed in applications within the fruit juice and wine industries. The exploration of alternative carbon and nitrogen sources remains crucial for enhancing enzyme production while reducing costs. This study evaluates the impact of carbon (black carrot pomace) and nitrogen (pea protein) loading on fermentable sugar content, protein content, and enzyme activities during both batch and fed-batch cultivation. Additionally, three distinct valorization techniques—thermal (steam), thermochemical (steam assisted with dilute acid), and microwave-assisted with dilute acid pretreatments—were assessed for their effectiveness in hydrolyzing black carrot pomace as a carbon source. The findings indicate that microwave-assisted dilute acid, coupled with enzymatic saccharification, resulted in the highest fermentable sugar production (0.493 g/g), achieving an 87.3% conversion yield. Pea protein demonstrated more favorable outcomes with the highest polygalacturonase activity (20.50 ± 0.52 U/L) and pectin lyase activity (46.44 ± 3.45 U/L) compared to whey protein and yeast extract used as nitrogen sources. Meanwhile, the highest polygalacturonase and pectin lyase activity, along with the highest total protein content (52.25 ± 0.06 mg/L), was recorded under the same culture conditions, reaching 164.34 ± 2.26 and 188.22 ± 1.72 U/L, respectively, after 72 h, representing approximately 1.18- and 1.34-fold increases from the batch system. Consequently, these results prove that fed-batch cultivation, utilizing black carrot pomace hydrolyzate as a feeding substrate and pea protein as a nitrogen source, significantly increases polygalacturonase and pectin lyase activity compared to batch cultivation.

Enhancing fermentable sugar production from sugarcane bagasse through surfactant-assisted ethylene glycol pretreatment and enzymatic hydrolysis: Reduced temperature and enzyme loading

In this study, an efficient coupling surfactant to ethylene glycol pretreatment (EG) and enzymatic hydrolysis of sugarcane bagasse (SCB) was proposed to enhance fermentable sugar production with lower pretreatment energy consumption and reduced enzyme loading. Under optimized conditions, 5 % Tween 80-assisted EG pretreatment of SCB achieved 80.5 % delignification while retaining cellulose (91.6 %) and hemicellulose (81.6 %) content. This led to an enhanced glucose yield of 81.3 %, compared to 65.1 % without Tween 80. The addition of Tween 80 to pretreatment modified residual lignin through etherification, reduced phenolic hydroxyl groups and enhanced hydrophilicity by 23.7 % and 9.4 %, respectively, compared to the lignin sample without Tween 80. Consequently, this modification alleviated non-productive adsorption between lignin and enzymes, improving substrate hydrolyzability. Moreover, when 4.5 % Triton-X 100 was introduced during the hydrolysis of Tween 80-assisted EG-pretreated substrates, a maximum glucose yield of 91.8 % and xylose yield of 92.6 % were achieved. Energy and enzyme cost analyses revealed a reduction of 35 % in pretreatment energy consumption and a 58.8 % decrease in enzyme costs, thanks to the synergistic action of surfactants in EG pretreatment and enzymatic hydrolysis. Overall, integrating surfactants into pretreatment and enzymatic hydrolysis holds promise for highly efficient conversion of SCB into fermentable sugars in enzyme-mediated lignocellulosic biorefineries.

A novel seawater hydrothermal-deep eutectic solvent pretreatment enhances the production of fermentable sugars and tailored lignin nanospheres from Pinus massoniana

Lignocellulose biorefinery depended on effective pretreatment strategies is of great significance for solving the current global crisis of ecosystem and energy security. This study proposes a novel approach combining seawater hydrothermal pretreatment (SHP) and microwave-assisted deep eutectic solvent (MD) pretreatment to achieve an effective fractionation of Pinus massoniana into high value-added products. The results indicated that complex ions (Mg2+, Ca2+, and Cl−) in natural seawater served as Lewis acids and dramatically promoted the depolymerization of mannose and xylan into oligosaccharides with 40.17 % and 75.43 % yields, respectively. Subsequent MD treatment realized a rapid and effective lignin fractionation (~90 %) while retaining cellulose. As a result, the integrated pretreatment yielded ~85 % of enzymatic glucose, indicating an eightfold increase compared with untreated pine. Because of the increased hydrophobicity induced by the formation of acyl groups during MD treatment, uniform lignin nanospheres were successfully recovered from the DES. It exhibited low dispersibility (PDI = 2.23), small molecular weight (1889 g/mol), and excellent oxidation resistance (RSI = 5.94), demonstrating promising applications in functional materials. The mechanism of lignin depolymerization was comprehensively elucidated via FTIR, 2D-HSQC NMR, and GPC analyses. Overall, this study provides a novel and environmentally friendly strategy for lignocellulose biorefinery and lignin valorization.

Life cycle assessment of bioproducts from bamboo residues with biphasic phenoxyethanol-acid based biorefinery technology

Utilizing bamboo residues to produce decarbonization energy products or materials diverts wastes from low-value treatment and supports the circular bioeconomy at the same time. This study develops a cradle-to-grave life-cycle assessment (LCA) for a biorefinery converting bamboo residues into bioproducts (ethanol, polylactic acid (PLA), and bio-based wood adhesive) in China. To combat the low enzymatic hydrolysis performance of bamboo residues and valorize the lignin, this study adopts a biphasic pretreatment by coupling phenoxyethanol and sulfuric acid solution (1%) to achieve high fermentable sugar production. The LCA study is coupled with a process simulation for an industrial-scale biorefinery based on the experimental data. Our results show that the life-cycle Global Warming Potential (GWP) is 17.0–32.1 gCO2e/MJ for ethanol cases and 3.2–3.7 kgCO2e/kg for PLA cases, when biphasic pretreatment is adopted. The lower GWP values are achieved by using 1:1 phenoxyethanol: acid solution, though it has a lower product yield than 4:1 phenoxyethanol: acid solution cases. Without biphasic pretreatment, the GWP is 135.8 gCO2e/MJ ethanol and 4.4 kgCO2e/kg PLA. For the perspective of treating 1 dry t bamboo residues, with biphasic pretreatment, the GWP is −174 to −66 kgCO2e for ethanol cases and −542 to −432 kgCO2e for PLA cases. The lower GWP is also from using 1:1 phenoxyethanol: acid solution (1%). These GWP ranges are lower than conventional landfilling (2969 kgCO2e) and landfilling with landfill gas recovery (−28 kgCO2e).

Enhanced starch accumulation in Chlorella sorokiniana as sugar platform and the expression profiling of key regulatory proteins

Microalgae can accumulate considerable starch as a renewable sugar platform for biofuel production. In this study, Chlorella sorokiniana exhibited a significant increase in starch content from 3.0% to 49.5% DM under sulfur limitation (SL) conditions, and the lower the concentration of sulfur was, the more effective it was. Direct enzymatic hydrolysis of wet biomass resulted in a glucose yield of 97.1%, indicating its potential for fermentable sugar production. However, C. sorokiniana grew slowly, and cell division and biomass accumulation decreased under SL conditions, which could be explained by the decreased photosynthetic efficiency and protein content. The sulfur replenishment (SR) confirmed the rapid response of C. sorokiniana in starch accumulation to sulfur availability. Thus, we further investigated the expression profiling of key regulatory proteins under Control-SL-SR stages. The expression of AGPase A was strongly induced in the SL stage and subsequently downregulated during SR stage, suggesting its role as a key regulatory protein for enhanced starch accumulation. Additionally, downregulation of SBE and GBE expression likely contributed to structural modifications of the starch molecules. These proteins could serve as potential targets for metabolic engineering in further improving the starch production capacity in C. sorokiniana.

An integrated pretreatment strategy for enhancing enzymatic hydrolysis efficiency of poplar: Hydrothermal treatment followed by a twin-screw extrusion

To reduce the dependence on chemical inputs during biomass pretreatment, an integrated process on the basis of hydrothermal pretreatment followed by twin-screw extrusion for poplar was proposed. A comparative study was conducted on the pore structure, crystallinity, relative surface lignin concentration, cellulose accessibility, and enzymatic hydrolysis efficiency of the pretreated materials. Results suggested that the relative surface lignin concentration of fibers pretreated by hydrothermal treatment (conducted at 170 ℃ for 90 min) coupled with extrusion (HE) treatment was decreased compared to the sole hydrothermal (H) process, which had a positive impact on the subsequent enzymatic hydrolysis. Additionally, the HE process resulted in an increase in pore size by 34.4%, pore volume by 447.5%, and specific surface area by 93.5%, which enhanced the penetration and interaction of water into the fibers and thus increased cellulose accessibility by 143.2%. SEM and TEM observations revealed that fibers were separated and finely fibrillated, large amounts of fragmentary structure and cracks appeared on the fiber. In addition, the cell wall structure was deformed and damaged, when pretreated by HE process. Overall, this work showed that hydrothermal treatment followed by twin-screw extrusion was an achievable strategy for high fermentable sugar production of hardwood.

Semi-continuous subcritical hydrolysis of orange waste biomasses for integrated production of fermentable sugars and platform chemicals

Results for the obtaining of fermentable sugars (FSs) and platform chemicals (PCs) from the subcritical hydrolysis of orange wastes (peel and bagasse) are presented. Unlike what is typically reported in similar studies involving lignocellulosic biomasses, the hydrolysis process was performed in a semi-continuous configuration, which allowed the examination of how temperature (180 to 220 °C) and pressure (15 to 25 MPa) influenced the compounds’ kinetic profile. Furthermore, studies have been found that evaluated these points for such biomasses. The best yields of hydrolyzed solution were obtained for biomasses pretreated by soxhlet with hydrolysis conducted at 180 °C and 15 MPa (orange peel, OP) and 200 °C and 20 MPa (orange bagasse, OB), yielding 5.31 and 4.26 g (100 g biomass)−1, respectively. Among the FSs, xylose was the monosaccharide predominantly obtained for both biomasses: 21.12 g L−1 for OP pretreated with SCO2 (hydrolysis at 180 °C and 15 MPa); and 24.45 g L−1 for untreated OB (hydrolysis at 220 °C and 25 MPa). Regarding the PCs, furfural was the main compound obtained for both biomasses: 10.86 g L−1 for untreated OP and 7.33 g L−1 for OB pretreated with SCO2, with both hydrolysis conducted at 220 °C and 25 MPa.

Unlocking peach juice byproduct potential in food waste biorefineries: Phenolic compounds profile, antioxidant capacity and fermentable sugars

This work assesses an integrated pathway for the revalorization of peach byproduct (PB) within a biorefinery. PB was subjected to an oven-drying (OD) treatment for its evaluation as a storage treatment. It was compared to freeze-drying and untreated material in terms of antioxidant capacity (AOC), phenolic compounds (PC) profile and fermentable sugar production. OD reduced the water content to less than 15 % while preserving the bound hydrolysable polyphenols, which were the more abundant PC (≈64 %) with the highest AOC. Drying treatments hampered polysaccharide accessibility, but some enzyme preparations released 60–70 g/L of fermentable sugars at relatively high solids loading (10 %). This study proposes a novel enzyme-based strategy for the valorisation of fermentable sugars and antioxidant compounds from PB. The sugars can be fermented into several building blocks while the solid residue enriched in recalcitrant phenolic compounds and proteins could be used to develop novel functional products for food/feed sectors.

Dilute nitric acid pretreatment of Ulva biomass for production of fermentable sugars and its validation using Design of Experiments (DoE)

Dilute nitric acid pretreatment of Ulva biomass for production of fermentable sugars and its validation using Design of Experiments (DoE)

Exploring the use of supercritical carbon dioxide in enzymatic hydrolysis of cellulosic substrate

Enzymes are critical components for production of fermentable sugars in lignocellulosic biorefineries. Their activity could be influenced by various reaction parameters. Herein, a formulated cellulolytic enzyme cocktail was immobilized into mesoporous aerogel material and used under high pressure environment of supercritical CO2 (scCO2). The cocktail was immobilized at different enzyme loads (36.0 to 144.0 mg enzyme/g aerogel) using sol-gel entrapment method and scCO2 drying. The immobilized cocktail at 144.0 mg/g aerogel enzyme load retained a residual yield of >50.0 % after fourth cycle of reuse compared to first cycle under atmospheric pressure. Sugar yield by the cocktail aerogel was below 10 wt% in second cycle under scCO2 due to enzyme leaching. Additionally, surface area and porous volume of reused aerogels was decreased over the reuse cycles. The study suggests that scCO2 could be employed for aerogel preparation after optimizing the immobilization process for pure enzymes rather than crude enzymes.

Non-targeted discovery of high-value bio-products in Nicotiana glauca L: a potential renewable plant feedstock

The evaluation of plant-based feedstocks is an important aspect of biorefining. Nicotiana glauca is a solanaceous, non-food crop that produces large amounts of biomass and is well adapted to grow in suboptimal conditions. In the present article, compatible sequential solvent extractions were applied to N. glauca leaves to enable the generation of enriched extracts containing higher metabolite content comparing to direct leaf extracts. Typically, between 60 to 100 metabolite components were identified within the fractions. The occurrence of plant fatty acids, fatty acid alcohols, alkanes, sterols and terpenoids was detected by gas liquid chromatography–mass spectrometry (GC–MS) and metabolite identification was confirmed by comparison of physico-chemical properties displayed by available authentic standards. Collectively, co-products such waxes, oils, fermentable sugars, and terpenoids were all identified and quantified. The enriched fractions of N. glauca revealed a high level of readily extractable hydrocarbons, oils and high value co-products. In addition, the saccharification yield and cell wall composition analyses in the stems revealed the potential of the residue material as a promising lignocellulosic substrate for the production of fermentable sugars. In conclusion a multifractional cascade for valuable compounds/commodities has been development, that uses N. glauca biomass. These data have enabled the evaluation of N. glauca material as a potential feedstock for biorefining.Graphical

Novel strategy to elevate solid state fermentation to produce alkilophilic endoglucanase using date waste feedstocks and peapod extract based nutrient media and expired probiotic strain: Application in fermentable sugar production

Solid waste generation and its impactful management are among the most raised environmental concerns seeking sustainable alternatives in the form of value addition. In this study, a novel strategy has been presented towards enhanced bacterial endoglucanase (EG) production under solid state fermentation (SSF). The EG was produced using the waste probiotic juice of the bacteria Lactobacillus casei strain Shirota on a substrate of waste date pulp and seeds in co-fermentation mode. Additionally, peapod extract has been applied as a nutrient medium to maintain moisture in place of commercial media in SSF. Further, for enhancement in enzyme production, the impact of incubation temperature and pH of the SSF medium have been studied to obtain optimum enzyme activity. Under optimum conditions, bacteria produced 17.2 U/mL EG at a temperature of 37 °C and a pH of 8.0. The raw EG showed pH and thermal stability up to 5 h at 60 °C and pH 8.0. Furthermore, the enzymatic hydrolysis of date pulp released 67.71 g/L fermentable glucose obtained in 24 h of the reaction at 60 °C and pH 8.0. This work may have great significance in the areas of solid waste management and microbe-mediated value addition of waste biomass in the context of hydrolytic enzyme production for biorefinery applications.

Chemical-free saturated steam pretreatment of oil palm empty fruit bunch for improved enzymatic saccharification

Oil palm empty fruit bunch (EFB) as the main lignocellulosic residue from the oil palm industry serves as suitable feedstock for enzymatic saccharification, yielding fermentable sugars. Due to complex inter-association between hemicellulose, cellulose and lignin within EFB lignocellulosic matrix, different saturated steam conditions at 140-230 °C, 0.3-2.3 MPa was chosen as a pretreatment strategy, targeted to hydrolyze hemicellulose and reduce crystallinity of cellulose to enhance enzymatic saccharification which need no chemical requirement and potential for energy recovery. As a result, SEM analysis of EFB morphology indicated that the enhanced silica body removal from 190-230 °C of saturated steam pretreatment correspondingly led to substantial 82% removal of hemicellulose. Consequently, enzymatic saccharification yielded between 0.621-0.637 g glucose per g EFB, equivalent to 93-95% of cellulose saccharification by Acremonium cellulase on EFB treated with saturated steam at 210-230 °C. Notably, a short duration pretreatment of EFB at 210 °C for 2 min was proved sufficient for high fermentable sugar production. The potential energy estimated for employing saturated steam pretreatment of EFB in a palm oil mill was sufficient by 2.2 x 1012 kJ/year.

Insights into the cellulose degradation for the production of fermentable sugars by Alkalispirochaeta odontotermitis JC202T

Insights into the cellulose degradation for the production of fermentable sugars by Alkalispirochaeta odontotermitis JC202T

Production of fermentable sugar, ethanol, D-lactic acid, and biochar from starch-rich traditional Chinese medicine decoction residues

Production of fermentable sugar, ethanol, D-lactic acid, and biochar from starch-rich traditional Chinese medicine decoction residues

Natural lignin modulators improve bagasse saccharification of sugarcane and energy cane in field trials

AbstractThe burgeoning cellulosic ethanol industry necessitates advancements in enzymatic saccharification, effective pretreatments for lignin removal, and the cultivation of crops more amenable to saccharification. Studies have demonstrated that natural inhibitors of lignin biosynthesis can enhance the saccharification of lignocellulose, even in tissues generated several months post‐treatment. In this study, we applied daidzin (a competitive inhibitor of coniferaldehyde dehydrogenase), piperonylic acid (a quasi‐irreversible inhibitor of cinnamate 4‐hydroxylase), and methylenedioxy cinnamic acid (a competitive inhibitor of 4‐coenzyme A ligase) to 60‐day‐old crops of two conventional Brazilian sugarcane cultivars and two energy cane clones, bred specifically for enhanced biomass production. The resultant biomasses were evaluated for lignin content and enzymatic saccharification efficiency without additional lignin‐removal pretreatments. The treatments amplified the production of fermentable sugars in both the sugarcane cultivars and energy cane clones. The most successful results softened the most recalcitrant lignocellulose to the level of the least recalcitrant of the biomasses tested. Interestingly, the softest material became even more susceptible to saccharification.

Integrated production of humic-like acid, fulvic-like acid, and fermentable sugars from industrial xylooligosaccharides manufacturing waste residues via hydrothermal pretreatment

Commercial humic substances (HS) are usually extracted from non-renewable resources such as lignite and coal. In this study, artificial HS (mainly humic-like acid (HLA) and fulvic-like acid (FLA)) was produced from renewable biomass-alkaline xylooligosaccharides manufacturing waste residue (XOR) of corncob via hydrothermal pretreatment (HP), as well as fermentable sugars were coproduced. The HLA yield was highest at 1.5% and the content of FLA was 9.6 g/L after HP at 210 °C for 8 h. The glucose yield of HP-pretreated XOR reached 98.8% at CTec 2 dosages of 20 FPU/g glucan. Structural characterization analysis proved that the structures and compositions of HLA were similar to those of commercial humic acid. The results of mass balance indicated that 100 g of XOR yielded 1.5 g of HLA, 9.6 g of FLA, and 60.4 g of monosaccharides (46.9 g of glucose and 13.5 g of xylose). Therefore, this work provides a promising strategy for valorization of lignocellulosic biomass in producing HLA, FLA, and fermentable sugars.

Effect of ozone pretreatment of flax fiber processing waste under less water environment: Physicochemical property evaluation and digestibility metrics

Compared with the commonly used pretreatment such as organosolv, diluted acid/alkali, etc., gas phase pretreatment got the advantages of low water consumption, low reaction temperature/ time, and could degraded cellulose to smaller chains without much cellulose loss. Thus, this study pretreated flax fiber manufacturing processing waste (FFPW) with gas phase ozone for producing fermentable sugar. Experimental results showed that FFPW got about 32.5% lower lignin (13.0%) and 59.0% higher cellulose/hemicellulose ratio (3.66) compared with most of the commonly used lignocellulose materials; which make it suitable for fermentable sugar production. Gas phase pretreatment got 31.9% higher delignification rate than that under liquid phase. Under the optimal reaction condition (extra water content 79.7%, pH 3.2, ozone feed speed 101.4 mg/L/min, 21.8 min, room temperature), the FFPW get lignin content of 3.6%, cellulose content of 73.8%, digestibility of 96.1% (solid concentration 0.02 g/mL). Under high solid concentration hydrolysis (solid concentration 0.4 g/mL), ozone pretreated FFPW released 168.0 g/L glucose, which was about 10.0% higher than that of the commonly used lignocellulose materials.