Search

Parthenium hysterophorus (PH), often known as parthenium weed, is one of the seventh most devastating weeds in the world. The primary aim of the study was to produce and optimize fungal FPase enzymes under solid-state fermentation (SSF) conditions for application in enzymatic hydrolysis to produce bioethanol. The impact of various physiological factors was verified through a systematic approach, initially using the one-factor-at-a-time (OFAT) method. Further optimization was carried out using statistical tools by response surface methodology (RSM), selecting three variables, i.e., lactose concentration, peptone concentration, and pH level. The outcomes of this research demonstrated that the RSM-based model led to the optimization of enzyme activity, resulting in 4.08U/ml of FPase, with a lactose concentration of 1.62 %, a peptone concentration of 1.5 %, and a pH level of 6 being the ideal conditions. When employing the FPase enzyme for biomass hydrolysis, the maximum yield of reducing sugar, at 0.37 ± 0.01 g/g, was achieved within a 96-hour period, using enzyme and substrate levels of 30 FPU/100 ml and 3 % (w/v), respectively. Fermentation of enzymatic hydrolysate was carried out by Saccharomyces cerevisiae at 30 °C, pH 6.0, and 120 rpm, which resulted in 0.45g/g ethanol yield after 48 h. Overall, our findings reveal that PH biomass can be introduced into sugar-based biorefineries to produce biofuels and other valuable chemicals.

Abstract Background The polysaccharides in lignocellulosic biomass hold potential for production of biofuels and biochemicals. However, achieving efficient conversion of this resource into fermentable sugars faces challenges, especially when operating at industrially relevant high solid loadings. While it is clear that combining classical hydrolytic enzymes and lytic polysaccharide monooxygenases (LPMOs) is necessary to achieve high saccharification yields, exactly how these enzymes synergize at high solid loadings remains unclear. Results An LPMO-poor cellulase cocktail, Celluclast 1.5 L, was spiked with one or both of two fungal LPMOs from Thermothielavioides terrestris and Thermoascus aurantiacus, TtAA9E and TaAA9A, respectively, to assess their impact on cellulose saccharification efficiency at high dry matter loading, using Avicel and steam-exploded wheat straw as substrates. The results demonstrate that LPMOs can mitigate the reduction in saccharification efficiency associated with high dry matter contents. The positive effect of LPMO inclusion depends on the type of feedstock and the type of LPMO and increases with the increasing dry matter content and reaction time. Furthermore, our results show that chelating free copper, which may leak out of the active site of inactivated LPMOs during saccharification, with EDTA prevents side reactions with in situ generated H2O2 and the reductant (ascorbic acid). Conclusions This study shows that sustaining LPMO activity is vital for efficient cellulose solubilization at high substrate loadings. LPMO cleavage of cellulose at high dry matter loadings results in new chain ends and thus increased water accessibility leading to decrystallization of the substrate, all factors making the substrate more accessible to cellulase action. Additionally, this work highlights the importance of preventing LPMO inactivation and its potential detrimental impact on all enzymes in the reaction.

Polyhydroxyalkanoates (PHAs) are biodegradable polymers that can be produced from lignocellulosic biomass by microorganisms. Cheap and readily available raw material, such as corn stover waste, has the potential to lessen the cost of PHA synthesis. In this research study, corn stover is pretreated with NaOH under conditions optimized for high cellulose and low lignin with central composite design (CCD) followed by characterization using Fourier-transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA), and scanning electron microscopy (SEM). Design expert software performed further optimization of alkali pretreated corn stover for high total reducing sugar (TRS) enhancement using CCD using response surface methodology (RSM). The optimized condition by RSM produced a TRS yield of 707.19 mg/g. Fermentation using corn stover hydrolysate by Pseudomonas putida MTCC 2475 gave mcl-PHA detected through gaschromatography–tandemmassspectrometry (GC-MS/MS) and characterization of the PHA film by differential scanning calorimetry (DSC), FTIR, and nuclear magnetic resonance (NMR). Thus, this research paper focuses on using agriculture (stubble) waste as an alternative feedstock for PHA production.

Abstract Background High substrate concentrations and high sugar yields are important aspects of enzymatic saccharification of lignocellulosic substrates. The benefit of supporting the catalytic action of lytic polysaccharide monooxygenase (LPMO) through continuous aeration of slurries of pretreated softwood was weighed against problems associated with increasing substrate content (quantitated as WIS, water-insoluble solids, in the range 12.5–17.5%), and was compared to the beneficial effect on the saccharification reaction achieved by increasing the enzyme preparation (Cellic CTec3) loadings. Aerated reactions were compared to reactions supplied with N2 to assess the contribution of LPMO to the saccharification reactions. Analysis using 13C NMR spectroscopy, XRD, Simons’ staining, BET analysis, and SEM analysis was used to gain further insights into the effects of the cellulolytic enzymes on the substrate under different reaction conditions. Results Although glucose production after 72 h was higher at 17.5% WIS than at 12.5% WIS, glucan conversion decreased with 24% (air) and 17% (N2). Compared to reactions with N2, the average increases in glucose production for aerated reactions were 91% (12.5% WIS), 70% (15.0% WIS), and 67% (17.5% WIS). Improvements in glucan conversion through aeration were larger (55–86%) than the negative effects of increasing WIS content. For reactions with 12.5% WIS, increased enzyme dosage with 50% improved glucan conversion with 25–30% for air and N2, whereas improvements with double enzyme dosage were 30% (N2) and 39% (air). Structural analyses of the solid fractions revealed that the enzymatic reaction, particularly with aeration, created increased surface area (BET analysis), increased disorder (SEM analysis), decreased crystallinity (XRD), and increased dye adsorption based on the cellulose content (Simons' staining). Conclusions The gains in glucan conversion with aeration were larger than the decreases observed due to increased substrate content, resulting in higher glucan conversion when using aeration at the highest WIS value than when using N2 at the lowest WIS value. The increase in glucan conversion with double enzyme preparation dosage was smaller than the increase achieved with aeration. The results demonstrate the potential in using proper aeration to exploit the inherent capacity of LPMO in enzymatic saccharification of lignocellulosic substrates and provide detailed information about the characteristics of the substrate after interaction with cellulolytic enzymes.

Cellulosic ethanol has been gaining high attention due to its potential to reduce the greenhouse gas emission and cut down the world dependence on fossil fuels. Biorefinery approach for cellulosic ethanol has advantages due to its non-food competing status, natural abundance and benefit to decrease the combustion of agricultural wastes after harvesting seasons. Due to the recalcitrant structure of lignocellulose biomass, pretreatment and hydrolysis are critical to determine the economic viability of the process because they influence the conversion rate of fermentable sugars and, subsequently, final product i.e. ethanol. Therefore, the design for the process to compromise fermentation and upstream process is also essential. With all constraints exist when using harsh conditions during pretreatment, the recombinant engineered microorganisms have been developed and applied as biocatalysts during fermentation. To achieve the maximum production efficiency, different strategies of recombinant engineered microbes include expression optimization to modify the metabolic pathway, modification of secretion and transportation routes, improvement of stress tolerance, and utilization of both C5 and C6 sugars. This review provides the development and current status of cellulosic ethanol production via biorefining process by genetic engineered microbes with a focus on the technological aspects. The remaining challenges, perspective, and economical feasibility of the process are also discussed.

Pretreatment processes are essential for the preparation of biofuels from lignocellulosic feedstocks. Based on alkaline H2O2 pretreatment, photocatalytic alkaline H2O2 pretreatment (U-AHP) was investigated to examine the effects of reaction time, alkali concentration, and H2O2 concentration on the enzymatic digestion of corn stover. The optimum process conditions were determined by orthogonal tests: 1% NaOH, 2% H2O2, and reaction time of 8 h. Under these conditions, the lignin removal efficiency of UH-AHP was 90.2% and the saccharification yield was 94.7%. Furthermore, FT-IR, XRD, and SEM analyses showed that U-AHP pretreatment caused structural damage to the maize straw and increased the crystallinity of the cellulose, and it was speculated that the U-AHP pretreatment reaction was a complex mechanism, which might be a multiple synergistic reaction. This study shows that U-AHP pretreatment is a simple, green and effective method to promote lignin removal.

Pretreatment processes are essential for the preparation of biofuels from lignocellulosic feedstocks. Based on alkaline H2O2 pretreatment, photocatalytic alkaline H2O2 pretreatment (U-AHP) was investigated to examine the effects of reaction time, alkali concentration, and H2O2 concentration on the enzymatic digestion of corn stover. The optimum process conditions were determined by orthogonal tests: 1% NaOH, 2% H2O2, and reaction time of 8 h. Under these conditions, the lignin removal efficiency of UH-AHP was 90.2% and the saccharification yield was 94.7%. Furthermore, FT-IR, XRD, and SEM analyses showed that U-AHP pretreatment caused structural damage to the maize straw and increased the crystallinity of the cellulose, and it was speculated that the U-AHP pretreatment reaction was a complex mechanism, which might be a multiple synergistic reaction. This study shows that U-AHP pretreatment is a simple, green and effective method to promote lignin removal.

Bamboo is considered a renewable energy bioresource for solving the energy crisis and climate change. Dendrocalamus branddisii (DB) was first subjected to sulfomethylation reaction at 95°C for 3 h, followed by Fenton oxidation pretreatment at 22°C for 24 h. The synergistic effect of combined pretreatment dramatically improved enzymatic digestibility efficiency, with maximum yield of glucose and ethanol content of 71.11% and 16.47 g/L, respectively, increased by 4.7 and 6.11 time comparing with the single Fenton oxidation pretreatment. It was found that the hydrophobicity of substrate, content of surface lignin, degree of polymerization, and specific surface area have significant effects on the increase of enzymatic saccharification efficiency. It also revealed that sulfomethylation pre-extraction can improve the hydrophilicity of lignin, leading to the lignin dissolution, which was beneficial for subsequent Fenton pretreatment of bamboo biomass. This work provides some reference for Fenton oxidation pretreatment of bamboo biomass, which can not only promote the utilization of bamboo in southwest China, but also enhances the Fenton reaction in the bamboo biorefinery.

Abstract Background To realize the full potential of softwood-based forest biorefineries, the bottlenecks of enzymatic saccharification of softwood need to be better understood. Here, we investigated the potential of lytic polysaccharide monooxygenases (LPMO9s) in softwood saccharification. Norway spruce was steam-pretreated at three different severities, leading to varying hemicellulose retention, lignin condensation, and cellulose ultrastructure. Hydrolyzability of the three substrates was assessed after pretreatment and after an additional knife-milling step, comparing the efficiency of cellulolytic Celluclast + Novozym 188 and LPMO-containing Cellic CTec2 cocktails. The role of Thermoascus aurantiacus TaLPMO9 in saccharification was assessed through time-course analysis of sugar release and accumulation of oxidized sugars, as well as wide-angle X-ray scattering analysis of cellulose ultrastructural changes. Results Glucose yield was 6% (w/w) with the mildest pretreatment (steam pretreatment at 210 °C without catalyst) and 66% (w/w) with the harshest (steam pretreatment at 210 °C with 3%(w/w) SO2) when using Celluclast + Novozym 188. Surprisingly, the yield was lower with all substrates when Cellic CTec2 was used. Therefore, the conditions for optimal LPMO activity were tested and it was found that enough O2 was present over the headspace and that the reducing power of the lignin of all three substrates was sufficient for the LPMOs in Cellic CTec2 to be active. Supplementation of Celluclast + Novozym 188 with TaLPMO9 increased the conversion of glucan by 1.6-fold and xylan by 1.5-fold, which was evident primarily in the later stages of saccharification (24–72 h). Improved glucan conversion could be explained by drastically reduced cellulose crystallinity of spruce substrates upon TaLPMO9 supplementation. Conclusion Our study demonstrated that LPMO addition to hydrolytic enzymes improves the release of glucose and xylose from steam-pretreated softwood substrates. Furthermore, softwood lignin provides enough reducing power for LPMOs, irrespective of pretreatment severity. These results provided new insights into the potential role of LPMOs in saccharification of industrially relevant softwood substrates.

Abstract Background The dense structure of cellulose lowers its reactivity and hinders its applications. Concentrated sulfuric acid is an ideal solvent to dissolve cellulose and thus has been used widely to treat cellulose. However, the changes of cellulose after reaction with concentrated sulfuric acid at near-limit S/L ratio and its effect on enzymatic saccharification still need further investigation. Results In this study, the interactions between cellulose (Avicel) and 72% sulfuric acid at very low acid loading conditions of 1:2 to 1:3 (S/L ratio) were studied for the enhanced production of glucose. The Avicel gradually transformed from cellulose I structure to cellulose II structure during the sulfuric acid treatment. Other physicochemical characteristics of Avicel also changed dramatically, such as the degree of polymerization, particle size, crystallinity index, and surface morphology. After acid treatment, both the yield and productivity of glucose from cellulose increased significantly under a very low enzyme loading of 5 FPU/g-cellulose. The glucose yields for raw cellulose and acid-treated (30 min) were 57% and 85%, respectively. Conclusion Low loadings of concentrated sulfuric acid were proven to be effective to break the recalcitrance of cellulose for enzymatic saccharification. A positive correlation between cellulose CrI and glucose yield was found for concentrated sulfuric acid-treated cellulose, which was opposite to previous reports. Cellulose II content was found to be an important factor that affects the conversion of cellulose to glucose.

A novel pretreatment strategy utilizing a combination of NaOH and 1-Butyl-3-methylimidazolium chloride ([Bmim]Cl) was proposed to enhance the enzymatic hydrolysis of abandoned Medium-density fiberboard (MDF). The synergistic effect of NaOH and [Bmim]Cl pretreatment significantly improved the glucose yield, reaching 445.8 mg/g within 72 h, which was 5.04 times higher than that of the untreated samples. The working mechanism was elucidated according to chemical composition, as well as FTIR, 13C NMR, XRD, and SEM analyses. The combined effects of NaOH and [Bmim]Cl led to lignin degradation, hemicellulose removal, the destruction and erosion of crystalline regions, pores, and an irregular microscopic morphology. In addition, by comparing the enzymatic hydrolysis sugar yield and elemental nitrogen content of untreated MDF samples, eucalyptus, and hot mill fibers (HMF), it was demonstrated that the presence of adhesives and additives in waste MDF significantly influences its hydrolysis process. The sugar yield of untreated MDF samples (88.5 mg/g) was compared with those subjected to hydrothermal pretreatment (183.2 mg/g), Ionic liquid (IL) pretreatment (406.1 mg/g), and microwave-assisted ionic liquid pretreatment (MWI) (281.3 mg/g). A long water bath pretreatment can reduce the effect of adhesives and additives on the enzymatic hydrolysis of waste MDF. The sugar yield produced by the combined pretreatment proposed in this study and the removal ability of adhesives and additives highlight the great potential of our pretreatment technology in the recycling of waste fiberboard.

This study evaluated the co-production of pigment and bacterial nanocellulose (BNC) from S. salsa biomass. The extraction of the beet red pigment reduced the salts and flavonoids contents by 82.7%–100%, promoting the efficiencies of enzymatic saccharification of the biomass and the fermentation of BNC from the hydrolysate. SEM analysis revealed that the extraction process disrupted the lignocellulosic fiber structure, and the chemical analysis revealed the lessened cellulase inhibitors, consequently facilitating enzymatic saccharification for 10.4 times. BNC producing strains were found to be hyper-sensitive to NaCl stress, produced up to 400.4% more BNC from the hydrolysate after the extraction. The fermentation results of BNC indicated that the LDU-A strain yielded 2.116 g/L and 0.539 g/L in ES-M and NES-M, respectively. In comparison to the control, the yield in ES-M increased by approximately 20.0%, while the enhancement in NES-M was more significant, reaching 292.6%. After conducting a comprehensive characterization of BNC derived from S. salsa through Scanning Electron Microscopy (SEM), Fourier Transform Infrared Spectroscopy (FTIR), X-ray Diffraction (XRD), and Thermogravimetric Analysis (TGA), the average fiber diameter distribution of these four BNC materials ranges from 22.23 to 33.03 nanometers, with a crystallinity range of 77%–90%. Additionally, they exhibit a consistent trend during the thermal degradation process, further emphasizing their stability in high-temperature environments and similar thermal properties. Our study found an efficient co-production approach of pigment and BNC from S. salsa biomass. Pigment extraction made biomass more physically and chemically digestible to cellulase, and significantly improved BNC productivity and quality.

Enzymatic digestion of lignocellulosic plant biomass is a key step in bio-refinery approaches for the production of biofuels and other valuable chemicals. However, the recalcitrance of this material in conjunction with its variability and heterogeneity strongly hampers the economic viability and profitability of biofuel production. To complement both academic and industrial experimental research in the field, we designed an advanced web application that encapsulates our in-house developed complex biophysical model of enzymatic plant cell wall degradation. PREDIG (https://predig.cs.hhu.de/) is a user-friendly, free, and fully open-source web application that allows the user to perform in silico experiments. Specifically, it uses a Gillespie algorithm to run stochastic simulations of the enzymatic saccharification of a lignocellulose microfibril, at the mesoscale, in three dimensions. Such simulations can for instance be used to test the action of distinct enzyme cocktails on the substrate. Additionally, PREDIG can fit the model parameters to uploaded experimental time-course data, thereby returning values that are intrinsically difficult to measure experimentally. This gives the user the possibility to learn which factors quantitatively explain the recalcitrance to saccharification of their specific biomass material.

AbstractBiological pretreatment of lignocellulosic biomass plays an important role for the enzymatic hydrolysis. In this study, a nitrogen-fixing and lignin-degrading strain of bacteria was isolated from an abandoned termite colony. Then, it was identified and named as R. ornithinolytica RS-1. To improve the degradation and enzymatic saccharification in corn stover, we used pretreatment with strain RS-1 to deplete the lignin, combined with a 2.5% mild NaOH pretreatment to further deplete the hemicellulose. After only seven days, lignin was degraded by the bacteria RS-1 with a 19% decrease, while the relative content of cellulose increased with 21%. Furthermore, the corn stover cellulose conversion was up to 48.58% by a two-stage process with 2.5% NaOH pretreatment. Meanwhile, significant lignin and hemicellulose removal were observed. Manganese peroxidase activity was highest on Day 3 with the value 181.0256 U/L and lignin peroxidase activity was highest on Day 5 with the value 37.473 U/L, respectively. Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR) and X-ray diffraction (XRD) analyses showed significant chemical structural changes after the combined pretreatment. Therefore, strain RS-1 provided an efficient strategy to enhance the enzymatic hydrolysis efficiency by pretreatment of corn stover.

Sulfite and kraft pulping are two principal methods of industrial delignification of wood. In recent decades, those have been considered as possibilities to pretreat recalcitrant wood lignocellulosics for the enzymatic hydrolysis of polysaccharides and the subsequent fermentation of obtained sugars to valuable bioproducts. Current work compares chemistry and technological features of two different cooking processes in the preparation of polysaccharide substrates for deep saccharification with P. verruculosum glycosyl hydrolases. Bleached kraft and sulfite pulps were subjected to hydrolysis with enzyme mixture of high xylanase, cellobiohydrolase, and β-glucosidase activities at a dosage of 10 FPU/g of dry pulp and fiber concentration of 2.5, 5, and 10%. HPLC was used to analyze soluble sugars after hydrolysis and additional acid inversion of oligomers to monosaccharides. Kraft pulp demonstrated higher pulp conversion after 48 h (74–99%), which mostly resulted from deep xylan hydrolysis. Sulfite-pulp hydrolysates, obtained in similar conditions due to higher hexose concentration (more than 50 g/L), had higher fermentability for industrial strains producing alcohols, microbial protein, or organic acids. Along with saccharification, enzymatic modification of non-hydrolyzed residues occurred, which led to decreased degree of polymerization and composition changes in two industrial pulps. As a result, crystallinity of kraft pulp increased by 1.3%, which opens possibilities for obtaining new types of cellulosic products in the pulp and paper industry. The high adaptability and controllability of enzymatic and fermentation processes creates prospects for the modernization of existing factories.

Introduction: A combination of deep eutectic solvent with ethanol was developed for pretreatment of Broussonetia papyrifera to effectively extract lignin and promote the subsequent enzymatic hydrolysis.Methods: In order to further explore the optimal conditions for enzymatic hydrolysis, a central composite design method was applied.Results and Discussion: The correlation between each factor and glucose yield was obtained, and the optimal conditions was 160°C, 60 min, the ratio of DES to E was 1/1 (mol/mol). The results showed that compared with control, the glucose yield increased by 130.67% under the optimal pretreatment conditions. Furthermore, the specific surface area of biomass was increased by 66.95%, and the content of xylan and lignin was decreased by 86.71% and 85.83%. The correlation between xylan/lignin removal and enzymatic hydrolysis showed that the removal of lignin facilitated the glucose yield more significantly than that of xylan. To further explore the lignin valorization, the structural and antioxidant analysis of recovered lignin revealed that high temperature was favorable for lignin with good antioxidant performance. This pretreatment is a promising method for separating lignin with high antioxidant activity and improving cellulose digestibility.

The role of lignin in enzymatic saccharification of cellulose involving lytic polysaccharide monooxygenase (LPMO) was investigated in experiments with the solid and liquid fractions of pretreated Norway spruce from a biorefinery demonstration plant using hydrothermal pretreatment and impregnation with sulfur dioxide. Pretreated biomass before and after enzymatic saccharification was characterized using HPAEC, HPLC, Py-GC/MS, 2D-HSQC NMR, FTIR, and SEM. Chemical characterization indicated that relatively harsh pretreatment conditions resulted in that the solid phase contained no or very little hemicellulose but considerable amounts of pseudo-lignin, and that the liquid phase contained a relatively high concentration (∼5 g/L) of lignin-derived phenolics. As judged from reactions continuously supplied with either air or nitrogen gas, lignin and lignin fragments from both the solid and the liquid phases efficiently served as reductants in LPMO-supported saccharification. When air was used to promote LPMO activity, the enzymatic conversion of cellulose after 72 h was 25% higher in reactions with pretreated solids and buffer, and 14% higher in reactions with pretreatment liquid and microcrystalline cellulose. Research in this area is useful for designing efficient saccharification steps in biochemical conversion of lignocellulosic biomass.

The extraction of bioactive compounds and cellulose saccharification are potential directions for the valorization of spent mushroom substrate (SMS). Therefore, investigating the suitability of different extraction methods for recovering bioactive compounds from SMS and how the extraction affects the enzymatic saccharification is of uppermost relevance. In this work, bioactive compounds were extracted from Pleurotus spp. SMS using four extraction methods. For Soxhlet extraction (SoE), a 40:60 ethanol/water mixture gave the highest extraction efficiency (EE) (69.9–71.1%) among the seven solvent systems assayed. Reflux extraction with 40:60 ethanol/water increased the extraction yield and EE compared to SoE. A shorter reflux time yielded a higher extraction of carbohydrates than SoE, while a longer time was more effective for extracting phenolics. The extracts from 240 min of reflux had comparable antioxidant activity (0.3–0.5 mM GAE) with that achieved for SoE. Ultrasound-assisted extraction (UAE) at 65 °C for 60 min allowed an EE (~82%) higher than that achieved by either reflux for up to 150 min or SoE. Subcritical water extraction (SWE) at 150 °C resulted in the best extraction parameters among all the tested methods. Vanillic acid and chlorogenic acid were the primary phenolic acids identified in the extracts. A good correlation between the concentration of caffeic acid and the antioxidant activity of the extracts was found. Saccharification tests revealed an enhancement of the enzymatic digestibility of SMS cellulose after the extraction of bioactive compounds. The findings of this initial study provide indications on new research directions for maximizing the recovery of bioactive compounds and fermentable sugars from SMS.

Abstract Background Pretreatment is a critical step required for efficient conversion of woody biomass into biofuels and platform chemicals. Fungal pretreatment is regarded as one of the most promising technology for woody biomass conversion but remains challenging for industrial application. The exploration of potential fungus strain with high efficient delignification and less processing time for woody biomass pretreatment will be valuable for development of biorefinery industry. Here, a newly isolated white-rot basidiomycete Peniophora incarnate T-7 was employed for poplar wood pretreatment. Results The chemical component analysis showed that cellulose, hemicellulose and lignin from poplar wood declined by 16%, 48% and 70%, respectively, after 7 days submerged fermentation by P. incarnate T-7. Enzymatic saccharification analysis revealed that the maximum yields of glucose and xylose from 7 days of P. incarnate T-7 treated poplar wood reached 33.4% and 27.6%, respectively, both of which were enhanced by sevenfold relative to the untreated group. Fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM), X-ray diffraction (XRD) and pyrolysis gas chromatography–mass spectrometry (Py-GC/MS) characterization confirmed that lignocellulosic structure of poplar wood was largely broken by P. incarnate T-7, including delignification and de-crystalline of cellulose. Meanwhile, lignin component of poplar wood was selectively degraded by P. incarnate T-7, and G-type unit of lignin was preferentially attacked by the strain. Furthermore, quantitative proteomic analysis revealed that a considerable amount of lignocellulolytic enzymes were detected in the secretory proteins of P. incarnate T-7, especially with high abundance of lignin-degrading enzymes and hemicellulases. Combination of quantitative proteomic with transcriptomic analysis results showed that most of those lignocellulolytic enzymes were highly upregulated on poplar wood substrate compared to glucose substrate. Conclusions This study showed that P. incarnate T-7 could selectively delignify poplar wood by submerged fermentation with short time of 7 days, which greatly improved its enzymatic saccharification efficiency. Our results suggested that P. incarnate T-7 might be a promising candidate for industrial woody biomass pretreatment.

Abstract Background Salix (willow) species represent an important source of bioenergy and offer great potential for producing biofuels. Salix spp. like many hardwoods, produce tension wood (TW) characterized by special fibres (G-fibres) that produce a cellulose-rich lignin-free gelatinous (G) layer on the inner fibre cell wall. Presence of increased amounts of TW and G-fibres represents an increased source of cellulose. In the present study, the presence of TW in whole stems of different Salix varieties was characterized (i.e., physical measurements, histochemistry, image analysis, and microscopy) as a possible marker for the availability of freely available cellulose and potential for releasing d-glucose. Stem cross sections from different Salix varieties (Tora, Björn) were characterized for TW, and subjected to cellulase hydrolysis with the free d-glucose produced determined using a glucose oxidase/peroxidase (GOPOD) assay. Effect of cellulase on the cross sections and progressive hydrolysis of the G-layer was followed using light microscopy after staining and scanning electron microscopy (SEM). Results Tension wood fibres with G-layers were developed multilaterally in all stems studied. Salix TW from varieties Tora and Björn showed fibre G-layers were non-lignified with variable thickness. Results showed: (i) Differences in total % TW at different stem heights; (ii) that using a 3-day incubation period at 50 °C, the G-layers could be hydrolyzed with no apparent ultrastructural effects on lignified secondary cell wall layers and middle lamellae of other cell elements; and (iii) that by correlating the amount of d-glucose produced from cross sections at different stem heights together with total % TW and density, an estimate of the total free d-glucose in stems can be derived and compared between varieties. These values were used together with a literature value (45%) for estimating the contribution played by G-layer cellulose to the total cellulose content. Conclusions The stem section-enzyme method developed provides a viable approach to compare different Salix varieties ability to produce TW and thus freely available d-glucose for fermentation and biofuel production. The use of Salix stem cross sections rather than comminuted biomass allows direct correlation between tissue- and cell types with d-glucose release. Results allowed correlation between % TW in cross sections and entire Salix stems with d-glucose production from digested G-layers. Results further emphasize the importance of TW and G-fibre cellulose as an important marker for enhanced d-glucose release in Salix varieties.

The sustainability and economic viability of the bioethanol production process from lignocellulosic biomass depend on efficient and effective pretreatment of biomass. Traditional pretreatment strategies implicating the use of mineral acids, alkalis, and organic solvents release toxic effluents and the formation of inhibitory compounds posing detrimental effects on the environment and interfering with the enzymatic saccharification process, respectively. Ionic liquids (ILs) as green solvents were used to overcome this issue, but the deep eutectic solvent as an emerging class of ionic liquids performed better in terms of making the process environmentally and economically viable. The green solvent-based pretreatment strategy applied in the current research was levulinic, acid-based natural deep eutectic solvent (NADES). Three different hydrogen bond acceptors (HBAs)—acetamide, betaine, and choline chloride—in combination with levulinic acid as hydrogen bond donor (HBD) in (HBD: HBA) molar ratio 2:1, were screened for biomass pretreatment. The best deep eutectic solvent was levulinic acid: choline chloride in an optimized molar ratio of 1:0.5, resulting in 91% delignification. The physicochemical parametric optimization of saccharification exhibited maximum enzymatic hydrolysis of 25.87% with 125 mg of pretreated sawdust via simultaneous addition of three thermostable cellulases [i.e., endo-1,4-β-D-glucanase (240 U), exo-1,4-β-D-glucanase (180 U), and β-glucosidase (320 U)] for 5 h of incubation at 75°C. The reducing sugar slurry obtained from the saccharified biomass was then added to a fermentation medium for bioethanol production, and a maximum of 11.82% of production was obtained at 30°C, 72 h, and 180 rpm using a 2.5% 24 h old Saccharomyces cerevisiae seed culture. The current study revealed that the levulinic-based deep eutectic solvent exhibited remarkable delignification, which led to the efficient enzymatic hydrolysis of sawdust and hence bioethanol production. Furthermore, it will prospect new avenues in bioethanol production using a deep eutectic solvent. Deep eutectic solvent overcame the issues posed by ionic liquids: toxicity, expensive and complex preparation, and non-biodegradability.

Upscaling of biomass pretreatment from laboratory scale to a bench-scale reactor is one of the important steps in the application of the pretreatment for pilot or commercial scale. This study reports the optimization of pretreatment conditions, namely reaction temperature and time, by one factor at a time (OFAT) method for the enhancement of enzymatic saccharification of oil palm empty fruit bunch (OPEFB). OPEFB was pretreated using high temperature-pressure steam reactor with different reaction temperatures (160, 170, 180, 190, 200 °C) and times (10, 20, 30, 40, 50 min). The effectiveness of the pretreatment was determined based on chemical compositions of raw OPEFB and OPEFB pulp and sugar production from enzymatic saccharification of the OPEFB pulp. Solubilized components from OPEFB, such as glucose, xylose, formic acid, acetic acid, 5-hydroxymethyl furfural (HMF), and furfural in the hydrolysate that generated during steam pretreatment were also determined. Pretreatment at 180°C for 20 min provides the highest sugar yields (97.30% of glucose yield per initial cellulose and 88.86% of xylose yield per initial hemicellulose). At the optimum condition, 34.9% of lignin and 30.75% of hemicellulose are successfully removed from the OPEFB and resulted in 3.43 delignification selectivity. The relationship between severity factor and by-products generated and the sugars obtained after enzymatic saccharification are discussed. The pulp of OPEFB at the optimum condition was also characterized for its morphological characteristic by scanning electron microscopy (SEM) and crystallinity by X-ray diffractometry (XRD). These pulp characteristics are then compared with those of the raw OPEFB. The steam pretreatment causes some fiber disruptions with more defined and opened structures and increases the crystallinity index (CrI) by 2.9% compared to the raw OPEFB.

Many countries have their biofuel policy programs in place as part of their overall strategy to achieve sustainable development. Among biofuels, bioethanol as a promising alternative to gasoline is of substantial interest. However, there is limited availability of a sufficient quantity of bioethanol to meet demands due to bottlenecks in the present technologies to convert non-edible feedstocks, including lignocelluloses. This review article presents and critically discusses the recent advances in the pretreatment of lignocellulosic biomass, with a focus on the use of green solvents, including ionic liquids and deep eutectic solvents, followed by enzymatic saccharification using auxiliary proteins for the efficient saccharification of pretreated biomass. Different techniques used in strain improvement strategies to develop hyper-producing deregulated lignocellulolytic strains are also compared and discussed. The advanced techniques employed for fermentation of mixed sugars contained in lignocellulosic hydrolysates for maximizing bioethanol production are summarized with an emphasis on pathway and transporters engineering for xylose assimilation. Further, the integration of different steps is suggested and discussed for efficient biomass utilization and improved ethanol yields and productivity.

Abstract We estimated the effect of residual lignin and pulp swelling on the rate of enzymatic saccharification to increase production of ethanol from acid sulfite pulp (SP) by means of enzymatic treatment. The resolution ratio of hardwood (Acacia mearnsii) SP after the enzymatic treatment was lower compared to softwood (Larix leptolepis) SP even though lignin content of hardwood SP was lower. The pyrolysis–gas chromatography/mass spectrometry (Py-GC/MS) analysis revealed that the residual lignin in hardwood SP could more easily adsorb enzyme compared to softwood SP, and the residual lignin in hardwood SP should interfere with the binding of the enzyme to cellulose. The beating treatment of pulp increased the swelling of pulp. The enzymatic saccharification rate was increased by the beating treatment. On the other hand, the delignification treatment was more effective than the beating treatment at enhancing enzymatic saccharification of both hardwood and softwood SPs. We found that the delignification process should be considered a high-priority technique for enhancing enzymatic saccharification of SP.

Abstract Photosynthetic microorganisms are considered excellent feedstock for biofuel production in developing biomass production technologies. A study was conducted to evaluate ethanol production with the sequential enzymatic saccharification and fermentation of Arthrospira platensis (Spirulina) biomass with the metabolically engineered Escherichia coli strain MS04. A. platensis was cultivated semicontinuously in an open raceway pond, and the carbohydrate content was determined to be as high as 40%. The enzymatic saccharification was designed to release the maximum amount of glucose. After 40 h of enzymatic saccharification, 27 g L−1 of monosaccharides was obtained. These slurries were fermented with ethanologenic bacteria, achieving 12.7 g L−1 ethanol after 9 h of fermentation, which corresponds to 92% conversion yield of the glucose content in the hydrolysate, 0.13 g of ethanol per 1 g of Spirulina biomass and a volumetric productivity of 1.4 g of ethanol L−1 h−1. Therefore, we conclude that it is possible, in a short time, to obtain a high ethanol yield corresponding to 160 L per ton of dry biomass with a high productivity.

Biochemical conversion of lignocellulosic feedstocks to advanced biofuels and other bio-based commodities typically includes physical diminution, hydrothermal pretreatment, enzymatic saccharification, and valorization of sugars and hydrolysis lignin. This approach is also known as a sugar-platform process. The goal of the pretreatment is to facilitate the ensuing enzymatic saccharification of cellulose, which is otherwise impractical due to the recalcitrance of lignocellulosic feedstocks. This review focuses on hydrothermal pretreatment in comparison to alternative pretreatment methods, biomass properties and recalcitrance, reaction conditions and chemistry of hydrothermal pretreatment, methodology for characterization of pretreatment processes and pretreated materials, and how pretreatment affects subsequent process steps, such as enzymatic saccharification and microbial fermentation. Biochemical conversion based on hydrothermal pretreatment of lignocellulosic feedstocks has emerged as a technology of high industrial relevance and as an area where advances in modern industrial biotechnology become useful for reducing environmental problems and the dependence on fossil resources.

Due to awareness of global warming and the devastation of environmental resources, the management of agricultural residues after each harvesting season has been integrated into the biorefining process to increase its value and mitigate environmental pollution caused by burning or combustion. This research focuses on the process development to utilize agricultural biomass residues for renewable energy production in the form of bioethanol. The study employed organosolv pretreatment with sulfuric acid as a catalyst to promote the enzymatic conversion of corn husk into reducing sugars. To determine the optimal conditions for the process, a one-factor-at-a-time method was initially employed to assess the influence of temperature (80-140 ºC), time (40-60 min), and sulfuric acid concentration (0.01-0.5% w/w). Subsequently, Response Surface Methodology (RSM) was conducted based on the Box-Behnken design (BBD) to identify the optimal pretreatment conditions. The predicted optimal pretreatment conditions were found to be 135.4 ºC, 57 min, and 0.46% w/w, resulting in a reducing sugar yield of 20.69% with a margin of error of 1.2%. Additionally, biomass composition analysis and Fourier Transform Infrared spectroscopy (FTIR) were performed to decipher the mechanism of organosolv pretreatment on enzymatic saccharification. This study demonstrated the potential of corn husk as an alternative raw material for the production of value-added products like bioethanol. The obtained reducing sugars serve as vital substrates for the fermentation process required to produce bioethanol as an alternative fuel to meet the target of sustainable development goals (SDGs).

Inhibitors formed during pretreatment impair lignocellulose bioconversion by making enzymatic saccharification and microbial fermentation less efficient, but conditioning of slurries and hydrolysates can improve fermentability and sometimes also enzymatic digestibility. Conditioning of pretreated softwood using four industrial reducing agents (sodium sulfite, sodium dithionite, sodium borohydride, and hydrogen) was compared with standard methods, such as overliming and treatment with activated charcoal. A dosage of approx. 1 mM sulfur oxyanion (sulfite or dithionite) per percent water-insoluble solids (WIS) in the slurry was found to result in good fermentability. Treatment of 10–20% WIS slurries with 15 mM sulfur oxyanion under mild reaction conditions (23°C, pH 5.5) resulted in sulfonation of the solid phase and saccharification improvements of 18–24% for dithionite and 13–16% for sulfite. Among the different conditioning methods studied, treatment of slurries with sodium sulfite was superior with respect to cost-efficient improvement of fermentability. Treatments of slurry or pretreatment liquid with 15 mM sulfite or dithionite resulted in 58–76% reduction of the content of formaldehyde. The comparison indicates that conditioning of pretreated biomass using sulfur oxyanions warrants further attention.

Abstract Background Salix spp. are high-productivity crops potentially used for lignocellulosic biofuels such as bioethanol. In general, pretreatment is needed to facilitate the enzymatic depolymerization process. Biomass resistance to degradation, i.e., biomass recalcitrance, is a trait which can be assessed by measuring the sugar released after combined pretreatment and enzymatic hydrolysis. We have examined genetic parameters of enzymatic sugar release and other traits related to biorefinery use in a population of 286 natural Salix viminalis clones. Furthermore, we have evaluated phenotypic and genetic correlations between these traits and performed a genomewide association mapping analysis using a set of 19,411 markers. Results Sugar release (glucose and xylose) after pretreatment and enzymatic saccharification proved highly variable with large genetic and phenotypic variations, and chip heritability estimates (h 2) of 0.23–0.29. Lignin syringyl/guaiacyl (S/G) ratio and wood density were the most heritable traits (h 2 = 0.42 and 0.59, respectively). Sugar release traits were positively correlated, phenotypically and genetically, with biomass yield and lignin S/G ratio. Association mapping revealed seven marker–trait associations below a suggestive significance threshold, including one marker associated with glucose release. Conclusions We identified lignin S/G ratio and shoot diameter as heritable traits that could be relatively easily evaluated by breeders, making them suitable proxy traits for developing low-recalcitrance varieties. One marker below the suggestive threshold for marker associations was identified for sugar release, meriting further investigation while also highlighting the difficulties in employing genomewide association mapping for complex traits.

One of the major concerns for utilizing ionic liquid on an industrial scale is the cost involved in the production. Despite its proven pretreatment efficiency, expenses involved in its usage hinder its utilization. A better way to tackle this limitation could be overcome by studying the recyclability of ionic liquid. The current study has applied the Box–Behnken design (BBD) to optimize the pretreatment condition of rice straw through the usage of 1-ethyl-3-methylimidazolium acetate (EMIM-Ac) as an ionic liquid. The model predicted the operation condition with 5% solid loading at 128.4 °C for 71.83 min as an optimum pretreatment condition. Under the optimized pretreatment condition, the necessity of the best anti-solvent was evaluated among water, acetone methanol, and their combinations. The study revealed that pure methanol is the suitable choice of anti-solvent, enhancing the highest sugar yield. Recyclability of EMIM-Ac coupled with anti-solvent was conducted up to five recycles following the predicted pretreatment condition. Fermentation studies evaluated the efficacy of recycled EMIM-Ac for ethanol production with 89% more ethanol production than the untreated rice straw even after five recycles. This study demonstrates the potential of recycled ionic liquid in ethanol production, thereby reducing the production cost at the industrial level.

Fast-growing broad-leaf tree species can serve as feedstocks for production of bio-based chemicals and fuels through biochemical conversion of wood to monosaccharides. This conversion is hampered by the xylan acetylation pattern. To reduce xylan acetylation in the wood, the Hypocrea jecorina acetyl xylan esterase (HjAXE) from carbohydrate esterase (CE) family 5 was expressed in hybrid aspen under the control of the wood-specific PtGT43B promoter and targeted to the secretory pathway. The enzyme was predicted to deacetylate polymeric xylan in the vicinity of cellulose due to the presence of a cellulose-binding module. Cell-wall-bound protein fractions from developing wood of transgenic plants were capable of releasing acetyl from finely ground wood powder, indicative of active AXE present in cell walls of these plants, whereas no such activity was detected in wild-type plants. The transgenic lines grew in height and diameter as well as wild-type trees, whereas their internodes were slightly shorter, indicating higher leaf production. The average acetyl content in the wood of these lines was reduced by 13%, mainly due to reductions in di-acetylated xylose units, and in C-2 and C-3 mono-acetylated xylose units. Analysis of soluble cell wall polysaccharides revealed a 4% reduction in the fraction of xylose units and an 18% increase in the fraction of glucose units, whereas the contents of cellulose and lignin were not affected. Enzymatic saccharification of wood from transgenic plants resulted in 27% higher glucose yield than for wild-type plants. Brunauer–Emmett–Teller (BET) analysis and Simons’ staining pointed toward larger surface area and improved cellulose accessibility for wood from transgenic plants compared to wood from wild-type plants, which could be achieved by HjAXE deacetylating xylan bound to cellulose. The results show that CE5 family can serve as a source of enzymes for in planta reduction of recalcitrance to saccharification.

The pretreatment method is one of the challenging steps in the production of biofuel through the biorefinery process that unlocks the recalcitrant nature of lignocellulosic biomass. Ionic liquid pretreatment gained attention for being highly effective to improve the enzymatic saccharification of the biomass, however its high cost hinders its industrial application. In this study, the combined effect of ionic liquid 1- ethyl-3-methylimidazolium acetate (EMIM-Ac) with inorganic salts (NaCl and KCl) was used for the pretreatment of rice straw. Optimization of pretreatment was conducted based on Response Surface Methodology and sugar yields obtained by EMIM-Ac+NaCl (160 °C, 88.7 min, 7.6%wt) and EMIMAc+ KCl (160 °C, 68.2 min, 12.5%wt) were 670.7 and 392.9 mg/g-biomass, respectively. The effect of combined pretreatment on ethanol production was analyzed after 48h fermentation. The results showed that the ethanol yield from pretreated samples with EMIM-Ac+NaCl (0.72%) and EMIM+KCl (0.76%) was increased by 2.18 and 2.25 fold times, respectively, compared to untreated sample (0.33%). This combined effect of inorganic salts and ionic liquid significantly removed the lignin during pretreatment, while maintaining efficient enzymatic saccharification of rice straw. Thus, this cost-effective combined chemical method may be an alternative strategy for increasing cellulosic ethanol production.

Brown rice is nutritionally superior to white rice, yet oil rancidity can be problematic during processing and storage regarding sensory attributes. Germinating brown rice is known to generally increase some health-promoting compounds. In response to increasing the consumption of plant-based beverages, we sprouted unstabilized brown rice, using green technologies and saccharification enzymes for value-added beverages. ‘Rondo’ paddy rice was dehulled, sorted and germinated, and beverages were produced and compared against non-germinated brown and white brewers rice beverages. The preliminary germinated brown rice beverage contained significantly higher concentrations of total lipids, diacylglycerols, triacylglycerols, free sterols, phytosterol esters and oryzanols than both non-germinated brown and white rice beverages. White rice beverages had significantly higher free fatty acids. Significant lipid losses occurred during sieving, yet novel germinated brown rice beverages contained appreciable levels of valuable health-beneficial lipids, which appeared to form natural emulsions. Further pilot plant investigations should be scaled-up for pasteurization and adjusted through emulsification to ameliorate sieving losses.

A lot of recent research in the biomass sector is focusing on how to improve the efficiency of biomass resources. Pretreatment of biomass resources is a novel approach and has gained a lot of attention in the last decade. A review of modern methods and the latest technologies of enhancing the enzymatic saccharification of sugarcane bagasse are presented in this work. This paper looks at the very recent developments in this field. Use of such advanced methods as coupling ionic liquid pretreatment with supercritical fluids and ultrasound irradiation is taking us swiftly towards the ultimate goal which is achieving 100% yield at minimum cost with no adverse environmental effects. However, Optimum process conditions for these methods are yet to be discovered. There is a need to optimize the processes and learn completely about the reaction mechanism in order to shift from lab scale to pilot scale and ultimately to the industrial level. An effort is made to report the latest work and because of it, this paper contains about 95% citations from papers within the last five years. In the end, useful recommendations are given in the conclusion section.

Abstract Background The lignin can compete for binding cellulase enzymes with cellulose fibers and decrease the accessibility of enzymes to carbohydrates. The competitive adsorption of cellulase to lignin mainly depended on the chemical structure of lignin. The post-pretreatment can decrease the lignin content and modify the lignin structure of pretreated substrates, which reduced the lignin inhibition on enzymatic saccharification. Therefore, the post-treatment by modifying the lignin structure would attract considerable attention for weakening the cellulase–lignin interactions. Results Three modified lignins, including sulfonated lignin (SL), oxidized lignin (OL), and carboxylated lignin (CL), were prepared from alkali lignin (AL) and their structures and physicochemical properties were characterized using FTIR, NMR, XPS analysis, zeta potential, and contact angle, respectively. Compared to AL, three modified lignin preparations exhibited the decrease in contact angle by 61–70% and phenolic hydroxyls content by 17–80%, and an obvious increase of negative charges by about 21–45%. This was mainly due to the drop of condensation degree and the incorporation of carboxylic and sulfonic acid groups into modified lignins. Langmuir adsorption isotherms showed that the affinity strength between cellulase and modified lignins significantly reduced by 54–80%. Therefore, the 72 h hydrolysis yield of Avicel with SL, OL, and CL was 48.5, 51.3, and 49.4%, respectively, which was increased 8–15.3% than that of Avicel with AL, 44.5%. In the enzymatic hydrolysis of bamboo biomass, the glucose yield at 5 d was 38.5% for AS-P. amarus, 15.4% for AO-P. amarus and 21.4% for AC-P. amarus, respectively, which were 1.4–3.5 times of alkali pretreated P. amarus. Conclusions The post-treatment can weaken the nonproductive adsorption between lignin and cellulase proteins and improve the enzymatic saccharification efficiency. This study will provide a conceptual combination of pretreatment technologies and post-pretreatment by modifying lignin structure for reducing the cellulase–lignin interaction.

Abstract Background To increase the production of bioethanol, a two-step process based on hydrothermal and dilute alkaline treatment was applied to reduce the natural resistance of biomass. However, the process required a large amount of water and a long operation time due to the solid/liquid separation before the alkaline treatment, which led to decrease the pure economic profit for production of bioethanol. Therefore, four one-step processes based on order of hydrothermal and alkaline treatment have been developed to enhance concentration of glucose of wheat straw by enzymatic saccharification. The aim of the present study was to systematically evaluated effect for different one-step processes by analyzing the physicochemical properties (composition, structural change, crystallinity, surface morphology, and BET surface area) and enzymatic saccharification of the treated substrates. Results In this study, hemicelluloses and lignins were removed from wheat straw and the morphologic structures were destroyed to various extents during the four one-step processes, which were favorable for cellulase absorption on cellulose. A positive correlation was also observed between the crystallinity and enzymatic saccharification rate of the substrate under the conditions given. The surface area of the substrate was positively related to the concentration of glucose in this study. As compared to the control (3.0 g/L) and treated substrates (11.2–14.6 g/L) obtained by the other three one-step processes, the substrate treated by one-step process based on successively hydrothermal and alkaline treatment had a maximum glucose concentration of 18.6 g/L, which was due to the high cellulose concentration and surface area for the substrate, accompanying with removal of large amounts of lignins and hemicelluloses. Conclusions The present study demonstrated that the order of hydrothermal and alkaline treatment had significant effects on the physicochemical properties and enzymatic saccharification of wheat straw. The one-step process based on successively hydrothermal and alkaline treatment is a simple operating and economical feasible method for the production of glucose, which will be further converted into bioethanol.

Auto-catalyzed hydrothermal pretreatment (A-HTP) and sulfuric-acid-catalyzed hydrothermal pretreatment (SA-HTP) were applied to quinoa stalks in order to reduce their recalcitrance towards enzymatic saccharification. Prior to pretreatment, quinoa stalks were extracted with either water or a 50:50 (v/v) ethanol–water mixture for removing saponins. Extraction with water or aqueous ethanol, respectively, led to removal of 52 and 75% (w/w) of the saponins contained in the raw material. Preliminary extraction of quinoa stalks allowed for a lower overall severity during pretreatment, and it led to an increase of glucan recovery in the pretreated solids (above 90%) compared with that of non-extracted quinoa stalks (73–74%). Furthermore, preliminary extraction resulted in enhanced hydrolysis of hemicelluloses and lower by-product formation during pretreatment. The enhancement of hemicelluloses hydrolysis by pre-extraction was more noticeable for SA-HTP than for A-HTP. As a result of the pretreatment, glucan susceptibility towards enzymatic hydrolysis was remarkably improved, and the overall conversion values were higher for the pre-extracted materials (up to 83%) than for the non-extracted ones (64–69%). Higher overall conversion was achieved for the aqueous ethanol-extracted quinoa stalks (72–83%) than for the water-extracted material (65–74%).

Banana is a major fruit crop throughout the world with abundant lignocellulose in the pseudostem and rachis residues for biofuel production. In this study, we collected a total of 11 pseudostems and rachis samples that were originally derived from different genetic types and ecological locations of banana crops and then examined largely varied edible carbohydrates (soluble sugars, starch) and lignocellulose compositions. By performing chemical (H2SO4, NaOH) and liquid hot water (LHW) pretreatments, we also found a remarkable variation in biomass enzymatic saccharification and bioethanol production among all banana samples examined. Consequently, this study identified a desirable banana (Refen1, subgroup Pisang Awak) crop containing large amounts of edible carbohydrates and completely digestible lignocellulose, which could be combined to achieve the highest bioethanol yields of 31–38% (% dry matter), compared with previously reported ones in other bioenergy crops. Chemical analysis further indicated that the cellulose CrI and lignin G-monomer should be two major recalcitrant factors affecting biomass enzymatic saccharification in banana pseudostems and rachis. Therefore, this study not only examined rich edible carbohydrates for food in the banana pseudostems but also detected digestible lignocellulose for bioethanol production in rachis tissue, providing a strategy applicable for genetic breeding and biomass processing in banana crops.

Abstract Background Ionic liquids (ILs) draw attention as green solvents for pretreatment of lignocellulose before enzymatic saccharification. Imidazolium-based ILs with different anionic constituents ([HSO4]−, [Cl]−, [MeCO2]−) were compared with regard to pretreatment of wood from aspen and spruce. The objective was to elucidate how the choice of anionic constituent affected the suitability of using the IL for pretreatment of hardwood, such as aspen, and softwood, such as spruce. The investigation covered a thorough analysis of the mass balance of the IL pretreatments, the effects of pretreatment on the cell wall structure as assessed by fluorescence microscopy, and the effects of pretreatment on the susceptibility to enzymatic saccharification. Torrefied aspen and spruce were included in the comparison for assessing how shifting contents of hemicelluloses and Klason lignin affected the susceptibility of the wood to IL pretreatment and enzymatic saccharification. Results The glucose yield after IL pretreatment increased in the order [Cl]−

The National Renewable Energy Laboratory (NREL) developed low severity Deacetylation and Mechanical Refining (DMR) process under atmospheric pressure, generates highly fermentable, low toxicity sugar syrups and highly reactive lignin streams. The dilute alkali deacetylation step saponifies acetyl groups from the hemicellulosic fraction of biomass into a black liquor waste stream that also contains solubilized acetic, ferulic, p-coumaric acids, lignin-carbohydrate complexes (LCCs), oligosaccharides, and solubilized lignin. Valorization of the soluble components in the deacetylation black liquor waste stream requires characterization of the black liquor. Our analyses show that glucan, xylan, and lignin were three main components in black liquor and the oligosaccharides in the black liquor were mainly dimers and trimers with arabinofuranose branch groups mainly on the xylooligomers. GPC chromatograms showed that the black liquor also contained oligomeric lignin moieties with molecular weights ranging between 1,800 and ~10,000 Da. Enzymatic saccharification of the black liquor was conducted based on the compositional and structural information. Incorporation of commercial cellulase and hemicellulase enzyme preparations with various accessary enzymes showed up to 57% hydrolysis of the xylooligosaccharides in the black liquor to monomeric sugars, which could be used to improve biorefinery carbon utilization in biological fermentations to improve end product fuel or bioproduct volumes per metric ton of biomass processed. This is so far the first study about characterizing DMR black liquor and exploring breaking down the oligosaccharides in black liquor for potential use.

Commonly, the agricultural waste, i.e. lignocellulosic biomass is disposed through combustion causing air pollution with production of PM2.5 and PM10 particles. However, it has been found that these biomasses can be used as source for the production of biofuels and other valuable biochemicals. Though deconstruction of lignocellulosic biomass is challenging due to its complex structure. In this study, rice straw (RS) was pretreated using potassium permanganate (KMnO4) to enhance the enzymatic saccharification efficiency. The study was carried out by varying the operational factors in pretreatment, including temperature (30-90°C), time (30-360 min) and concentration of KMnO4 (0.5-3.0, % w/v), respectively, based on Box-Behnken design (BBD). Through multi-regression analysis of the experimental data obtained after pretreatment, the optimum conditions were determined. The optimum conditions for temperature, time and potassium permanganate concentration were 48.09°C, 360 min, and 1.36% w/v, respectively. The saccharifications of pretreatment and untreated rice straw were carried out using Cellic Ctec2. The reducing sugar was determined by using DNS method and the yields of the untreated and pretreated RS were 32.38 and 49.011 mg/mL, respectively. The results showed that the sugar for pretreated RS were 1.51 fold times higher compared to untreated RS. Therefore, this work illustrates the pretreatment efficiency for KMnO4 to enhance the reducing sugar yield during saccharification, which can be used for biofuel and value-added product productions.

Most of the agricultural wastes in developing countries are disposed of by on-site combustion leading to unmanaged environmental pollutions. Conversion of agricultural wastes to value-added products, such as bioethanol and biogas, is a promising method to reduce agro-waste after harvesting seasons. In this study, Citrus maxima peels (Pomelo peels) was selected to be converted to reducing sugars, which could be a raw material to produce other value-added products. To promote enzymatic hydrolysis reactions, pomelo peels were pretreated with sodium hydroxide by variations of three pretreatment parameters, including temperature (50-100 ºC), time (0.5-6 h), and concentration of NaOH (0.5-3.0 M). Box-Behnken design (BBD) was applied in Response Surface Methodology (RSM) to determine the optimized pretreatment condition and to find the relationship between pretreatment factors and reducing sugar yields. The predicted optimal pretreatment condition was determined to be at 94.28 ºC, 4.5h, 2.17M with reducing sugar yield of 98.9 mg/g of dried pomelo peels. The results clearly showed that reducing sugar yields obtained from pretreated pomelo peels were 1.87 folds higher than untreated biomass (52.81 mg/g of pomelo peels). Therefore, this study demonstrated the potential of pomelo peels to be used as an alternative raw material for value-added products rather than being a landfill or causal agent of pollution.

Enzyme saccharification of pretreated brewers spent grains (BSG) was investigated, aiming at maximising glucose production. Factors investigated were; variation of the solids loadings at different cellulolytic enzyme doses, reaction time, higher energy mixing methods, supplementation of the cellulolytic enzymes with additional enzymes (and cofactors) and use of fed-batch methods. Improved slurry agitation through aerated high-torque mixing offered small but significant enhancements in glucose yields (to 53 ± 2.9 g/L and 45% of theoretical yield) compared to only 41 ± 4.0 g/L and 39% of theoretical yield for standard shaking methods (at 15% w/v solids loading). Supplementation of the cellulolytic enzymes with additional enzymes (acetyl xylan esterases, ferulic acid esterases and α-L- arabinofuranosidases) also boosted achieved glucose yields to 58 – 69 ± 0.8 - 6.2 g/L which equated to 52 - 58% of theoretical yield. Fed-batch methods also enhanced glucose yields (to 58 ± 2.2 g/L and 35% of theoretical yield at 25% w/v solids loading) compared to non-fed-batch methods. From these investigations a novel enzymatic saccharification method was developed (using enhanced mixing, a fed-batch approach and additional carbohydrate degrading enzymes) which further increased glucose yields to 78 ± 4.1 g/L and 43% of theoretical yield when operating at high solids loading (25% w/v).

The effectiveness of microwave-assisted sodium hydroxide pretreatments to enhance the saccharification performance of pineapple waste was evaluated. Microwave alkali pretreatments for short exposure times (up to 60 s) significantly improved the yield of the enzymatic hydrolysis compared with non-pretreated waste. The greatest increase of fermentable (35.7%) and total sugars (33.5%) was obtained at 6.375 W/g for 5 s. However, longer exposure times resulted in sugar degradation and released fermentation inhibitors, such as phenols or hydroxymethylfurfural (HMF), as a consequence of thermal degradation. Nevertheless, the obtained phenols values were not sufficient to inhibit subsequent fermentation. Scanning electron microscope (SEM) images confirmed that applying microwaves for short exposure times promoted structural changes that improved enzymatic hydrolysis. By contrast, an increase in the severity of the treatment led to a compacted structure, which hindered access to enzymes and consequently reduced the release of sugars into the medium.

Synergistic effect of cellulase and hemicellulase (xylanase) was evaluated because lignocellulosic material is a heterogeneous complex of cellulose and hemicellulose. Various effects of HTec2 addition on enzymatic saccharification and fermentation were evaluated using two different substrates such as corn stover and rice straw. Corn stover and rice straw were pretreated by the LMAA (low-moisture anhydrous ammonia) method at the preselected same conditions (90 °C, 120 h, moisture content = 50%, NH3 loading = 0.1 g NH3/g). It was observed that the enzymatic saccharification yield of pretreated corn stover (76.4% for glucan digestibility) was higher than that of pretreated rice straw (70.9% for glucan) using CTec2 cellulase without HTec2 addition. Glucan digestibility of pretreated corn stover was significantly increased from 76.4% to 91.1% when the HTec2/CTec2 (v/v) increased from 0 to 10. However, it was interesting that the ethanol production was decreased from 89.9% to 76.3% for SSF and 118.0% to 87.9% for SSCF at higher HTec2/CTec2. As the glucan loading increased from 2.0% to 7.0%, the ethanol yields of both SSF and SSCF were decreased from 96.3% to 88.9% and from 116.6% to 92.4%, respectively. In addition, the smallest inoculum size (optical density of 0.25) resulted in the highest ethanol production (20.5 g/L).

The enzymatic hydrolysis of cellulose is inhibited by non-productive adsorption of cellulases to lignin, and that is particularly problematic with lignin-rich materials such as softwood. Although conventional surfactants alleviate non-productive adsorption, using biosurfactants in softwood hydrolysis has not been reported. In this study, the effects of four biosurfactants, namely horse-chestnut escin, Pseudomonas aeruginosa rhamnolipid, and saponins from red and white quinoa varieties, on the enzymatic saccharification of steam-pretreated spruce were investigated. The used biosurfactants improved hydrolysis, and the best-performing one was escin, which led to cellulose conversions above 90%, decreased by around two-thirds lignin inhibition of Avicel hydrolysis, and improved hydrolysis of pretreated spruce by 24%. Red quinoa saponins (RQS) addition resulted in cellulose conversions above 80%, which was around 16% higher than without biosurfactants, and it was more effective than adding rhamnolipid or white quinoa saponins. Cellulose conversion improved with the increase in RQS addition up to 6 g/100 g biomass, but no significant changes were observed above that dosage. Although saponins are known to inhibit yeast growth, no inhibition of Saccharomyces cerevisiae fermentation of hydrolysates produced with RQS addition was detected. This study shows the potential of biosurfactants for enhancing the enzymatic hydrolysis of steam-pretreated softwood.

We report on the pretreatment of poplar wood with three different 1-ethyl-3-methylimidazolium ionic liquids, [EMim][OAc], [EMim][MeSO3], and [EMim][HSO4], at varying water contents from 0–40 wt% at 100 °C. The performance was evaluated by observing the lignin and hemicellulose removal, as well as enzymatic saccharification and lignin yield. The mechanism of pretreatment varied between the ionic liquids studied, with the hydrogen sulfate ionic liquid performing delignification and hemicellulose hydrolysis more effectively than the other solvents across the investigated water content range. The acetate ionic liquid produced superior glucose yield at low water contents, while the hydrogen sulfate ionic liquid performed better at higher water contents and produced a recoverable lignin. The methanesulfonate ionic liquid did not introduce significant fractionation or enhancement of saccharification yield under the conditions used. These findings help distinguish the roles of anion hydrogen bonding, solvent acidity, and water content on ionic liquid pretreatment and can aid with anion and water content selections for different applications.

The improvement of the saccharification of rice straw is one of the strategies to reduce the sophisticated pretreatment that results in high cost and is unfriendly to the environment. We explored the cell wall features in tetraploid rice and highlighted the enhanced saccharification of tetraploid with large biomass. Results showed that lignin content and S/G ratio reduced to 17.09% and 0.37, respectively, in tetraploid straw by the determination of the pyGC-MS method. After the pretreatment, the cellulose crystallinity index decreased from 63.22% to 57.65% in tetraploid straw, which is lower than that of pretreated diploid straw. Surface topological analysis of SEM images indicated that tetraploid straw was more susceptible to the pretreatment. Tetraploid straw showed a strong advantage in the process of enzymatic hydrolysis. The enzyme efficiency reached the highest value of 77.60%, and the rate of enzyme reaction was improved to make the reaction saturated earlier than conventional rice. We concluded that the high saccharification has resulted from the alteration of lignin and cellulose in tetraploid rice. Our research provides an improved green feedstock for bioenergy, and the tetraploid rice straw shows the potential utilization value in bioethanol production.

Enzymatic saccharification of Laminaria japonica seaweed biomass was optimized by four independent factors (enzyme dose, hydrolysis time, pH, and temperature) using response surface methodology (RSM). To confirm the significance of the quadratic model, an analysis of variance (ANOVA) was performed, and the F-value of 8.76 showed that the regression model was highly significant (≤0.1%). In the accuracy study, average recoveries were in the range of 97.00% to 98.32%. The optimum experimental conditions were an enzyme dose of 8.2%, a hydrolysis time of 26 h, a pH of 4.1, and a temperature of 43 °C. Temperature was the most important factor in the enzymatic saccharification. A relatively low temperature and short hydrolysis time were shown to improve the yield of reducing sugars.

Ethanol production from lignocellulosic biomasses raises a global interest because it represents a good alternative to petroleum-derived energies and reduces the food versus fuel conflict generated by first generation ethanol. In this study, alkaline-acid pretreated brewers’ spent grain (BSG) was evaluated for ethanol production after enzymatic hydrolysis with commercial enzymes. The obtained hydrolysate containing a glucose concentration of 75 g/L was adopted, after dilution up to 50 g/L, for fermentation by the strain Saccharomyces cerevisiae NRRL YB 2293 selected as the best producer among five ethanologenic microorganims. When the hydrolysate was supplemented with yeast extract, 12.79 g/L of ethanol, corresponding to 0.28 g of ethanol per grams of glucose consumed (55% efficiency), was obtained within 24 h, while in the non-supplemented hydrolysate, a similar concentration was reached within 48 h. The volumetric productivity increased from 0.25 g/L·h in the un-supplemented hydrolysate to 0.53 g/L h in the yeast extract supplemented hydrolysate. In conclusion, the strain S. cerevisiae NRRL YB 2293 was shown able to produce ethanol from BSG. Although an equal amount of ethanol was reached in both BSG hydrolysate media, the nitrogen source supplementation reduced the ethanol fermentation time and promoted glucose uptake and cell growth.