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Background: White rot fungi produce various reactive oxygen species and ligninolytic enzymes for lignocellulose deconstruction. However, their interactions during the deconstruction of lignocellulosic structural barriers for efficient enzymatic saccharification remain unclear. Results: Herein, the extracellular enzyme activities and secretomic analysis revealed the sequential expression of hydroxyl radical (⋅OH) and manganese peroxidases (MnPs) for lignocellulose deconstruction by the white rot fungus Irpex lacteus. Subsequently, in vitro functional studies found that ⋅OH possessed the ability to disrupt the smooth surface structure of corn stover, resulting in increased enzymatic saccharification and cellulose accessibility. Purified recombinant MnPs from I. lacteus were able to cleave the β-O-4 bond in phenolic and non-phenolic lignin model dimers without the help of any mediators. Furthermore, the sequential pretreatment of corn stover with ⋅OH and MnP exhibited significant synergistic effects, increasing enzymatic saccharification and cellulose accessibility by 2.9-fold and 1.8-fold, respectively. Conclusions: These results proved for the first time the synergistic effects of ⋅OH and MnP pretreatment in improving the enzymatic saccharification and cellulose accessibility of corn stover. These findings also demonstrated the potential application of ⋅OH and MnP pretreatment for the efficient enzymatic saccharification of corn stover. [ABSTRACT FROM AUTHOR]

Saccharification of cellulosic material is crucial before utilizing it as the feedstock of fermentation processes. This study focuses on developing of robust catalysts for enzymatic hydrolysis of paper waste. Cellulase enzyme was immobilized on Fe3O4 magnetic nanoparticles (MNPs) via multi-covalent bonds. Therefore, the attachments of MNPs were performed by three approaches (3-aminopropyl)triethoxysilane (APTES) (Method I), polyethyleneimine (PEI) (Method II), and both monolayer molybdenum disulfide and PEI (MoS2-PEI) (Method III). The biocatalysts were characterized using Fourier transform infrared (FTIR) and thermogravimetric analysis (TGA). The immobilization yield and loading efficiency of cellulase were determined at 38.65% and 162 mg g−1 in Method I, 13.33% and 31 mg g−1 in Method II, and 14.38% and 33 mg g−1 in Method III. The specification of the biocatalysts was determined for the hydrolysis of filter paper, carboxymethylcellulose (CMC), microcrystalline cellulose, and cellobiose. The total cellulase activity was 2.77, 0.943, 1.38, and 2.02 µmolGlucose mgEnzyme−1 h−1for free-cellulase and the immobilized biocatalysts prepared by Method I, II, and III, respectively. The prepared biocatalyst in Method I maintained 54.54% of its original activity after five cycles, which was more robust than Method II (20.25%) and Method III (12.33%). Further studies were performed on the saccharification of paper waste. The results showed the biocatalyst obtained by Method II resulted in 5.8 folds of higher glucose than the free-cellulase. The highest glucose recovery of about 72.47% was achieved after 48 h using the immobilized biocatalyst prepared by Method II. [ABSTRACT FROM AUTHOR]

The presence of hemicellulose inhibits the enzymatic hydrolysis of lignocellulosic biomass. The purpose of this study is to investigate the effect of different hemicellulose fractions on the enzymatic hydrolysis and the way to eliminate the inhibiting effect caused by hemicellulose. Four kinds of hemicelluloses, namely, HXF, H15, H30, and H60, were first extracted from corn stover by ethanol fractional precipitation. The structures of hemicellulose samples were analyzed using Fourier transform infrared spectroscopy, 1H and 13C nuclear magnetic resonance, and high-performance ion chromatography. The results show that H30 has the strongest inhibition on the enzymatic hydrolysis of Avicel and corn stover, presenting inhibition ratio of 13.35% and 9.98%, respectively. The inhibition ratios of other hemicelluloses in Avicel and corn stover are 8–12% and 5–9%, respectively. However, the inhibiting effect caused by H30 is removed by adding hemicellulase, which even presents a 4.99% increase in the efficiency of enzymatic hydrolysis of corn stover. The corresponding glucose concentration reached 68.11 g/L. This research could help design effective processes to promote the enzymatic hydrolysis of lignocellulosic biomass. [ABSTRACT FROM AUTHOR]

Reed sawdust is a kind of paper mill waste with high cellulose and hemicellulose content. To promote the rational use of resources, it is essential to make full use of waste resources and transform them into new values. In this work, reed sawdust was pretreated with liquid hot water (LHW) at 170 °C for 30 min. A total of 39.00 g/L glucose was obtained after enzymatic saccharification of cellulose at 50 °C, 20 FPU/g-reed sawdust cellulase, 25% (w/v) reed sawdust, in 5 replenishments. When the fermentation was performed 96 h, the medium contained xylo-oligosaccharides (XOS) 11.74 g/L and biomass 15.21 g/L, in which lipid was 4.14 g/L. After spray drying, feed additives containing 29.17% XOS and 10.29% docosahexaenoic acid (DHA) can be prepared. In particular, the hemicellulose and cellulose in reed sawdust are creatively used at the same time without separation, which greatly reduces the cost of purification in traditional processes and provides a new way for the high-value transformation of sawdust resources. [ABSTRACT FROM AUTHOR]

The main objective of this study was to develop a citric acid-catalyzed hydrothermal pretreatment to reach the maximum sugar recovery from soybean hull (SH), in the context of a biorefi nery concept. A central composite design (CCD) was used to optimize pretreatment conditions through the analysis of citric acid concentration, time, and temperature effects. Optimized conditions were defined as 3.56% w/w, 46.1 min, and 135.7 °C. From the solid fraction, 99.5% and 83.0% of glucose and xylose conversion, respectively, were obtained after enzymatic hydrolysis. Sugars were fermented successfully to L-lactic acid by Lactobacillus pentosus. In fact, SH hydrolysate presented no trace of inhibitor compounds, and glucose was totally consumed during fermentation. After 24 h of fermentation, a conversion rate of 0.724 g/g of sugars to lactic acid and 80.5% of the stoichiometric theoretical yield were reached. In addition, from the liquid fraction, 47.8 mg/g of xylooligosaccharides (XOS) was produced. This work demonstrates the potential of SH to be exploited as feedstock, with mild and efficient pretreatment process, for medium- to high-value bioproducts production in biorefineries. [ABSTRACT FROM AUTHOR]

Pretreatment is crucial for effective enzymatic saccharification of lignocellulose such as sugarcane bagasse (SCB). In the present study, SCB was pretreated with five kinds of heterogeneous Fenton‐like systems (HFSs), respectively, in which α‐FeOOH, α‐Fe2O3, Fe3O4, and FeS2 worked as four traditional heterogeneous Fenton‐like catalysts (HFCs), while FeVO4 worked as a novel HFC. The enzymatic reducing sugar conversion rate was then compared among SCB after different heterogeneous Fenton‐like pretreatments (HFPs), and the optimal HFS and pretreatment conditions were determined. The mechanism underlying the difference in saccharification efficiency was elucidated by analyzing the composition and morphology of SCB. Moreover, the ion dissolution characteristics, variation of pH and Eh values, H2O2 and hydroxyl radical (·OH) concentration of FeVO4 and α‐Fe2O3 HFSs were compared. The results revealed that the sugar conversion rate of SCB pretreated with FeVO4 HFS reached up to 58.25%, which was obviously higher than that under other HFPs. In addition, the surface morphology and composition of the pretreated SCB with FeVO4 HFS were more conducive to enzymatic saccharification. Compared with α‐Fe2O3, FeVO4 could utilize H2O2 more efficiently, since the dissolved Fe3+ and V5+ can both react with H2O2 to produce more ·OH, resulting in a higher hemicellulose and lignin removal rate and a higher enzymatic sugar conversion rate. It can be concluded that FeVO4 HFP is a promising approach for lignocellulose pretreatment. [ABSTRACT FROM AUTHOR]

In the present study, a sustainable pretreatment methodology combining liquid hot water and deep eutectic solvent is proposed for the efficient fractionation of hemicellulose, cellulose, and lignin from sugarcane bagasse, thereby facilitating the comprehensive utilization of both C5 and C6 sugars. The application of this combined pretreatment strategy to sugarcane bagasse led to notable enhancements in enzymatic saccharification and subsequent fermentation. Experiment results demonstrate that liquid hot water-deep eutectic solvent pretreatment yielded 85.05 ± 0.66 g·L−1 of total fermentable sugar (glucose: 60.96 ± 0.21 g·L−1, xylose: 24.09 ± 0.87 g·L−1) through enzymatic saccharification of sugarcane bagasse. Furthermore, fermentation of the pretreated sugarcane bagasse hydrolysate yielded 34.33 ± 3.15 g·L−1 of bioethanol. These findings confirm the effectiveness of liquid hot water-deep eutectic solvent pretreatment in separating lignocellulosic components, thus presenting a sustainable and promising pretreatment method for maximizing the valuable utilization of biomass resources. [ABSTRACT FROM AUTHOR]

The most abundant natural renewable resource in the world, lignocellulosic biomass (LCB), has the potential to be exploited as a substitute green feedstock for the synthesis of various chemicals, materials, and biofuels. The annual global production of 13 billion tonnes of LCB offers an opportunity to cater to the increasing energy and materials requirement of process industries and also restricts the discharge of greenhouse gases. Although LCB is enriched with valuable ingredients such as cellulose, lignin, and hemicellulose, its recalcitrant nature limits its efficient utilisation. These components of LCB are strongly interlinked with each other, which resists their isolation and conversion valorisation into useful products. To disrupt the complicated structure of LCB and to isolate the lignocellulosic components in pure form, pretreatment is a crucial process in the bio-refinery, ensuring the economic feasibility of downstream processes. This review provides an outline of the structure, composition, and various sources of LCB; and the necessity of the pretreatment. Moreover, this article provides an in-depth analysis of the underlying mechanisms, advantages, and limitations of various pretreatment methods, such as physical, chemical, biological, and physicochemical. Further, the impact of chemical pretreatment techniques on the physicochemical characteristics of the material that is extracted from the biomass is also covered in detail through the rigorous evaluation of performance metrics, including substrate digestibility, sugar yield, inhibitor production, and energy requirements. This review provides a balanced and comprehensive overview of the state-of-the-art pretreatment strategies and their impact on biomass valorisation that will be useful to the scientists, engineers, and policy makers interested in biomass conversion technologies. [ABSTRACT FROM AUTHOR]

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.

In this study, an attempt has been made to recover the lignin and enhance the sugar yield from the de-oiled Jatropha curcas waste (DJW) through alkaline hydrolysis and saccharification. The alkaline hydrolysis process was performed using sodium hydroxide (NaOH) for a solid loading rate of 10% w/v. The process parameters considered for the optimization study using RSM were — time (6.36–73.63 min), NaOH concentration (0.6137–12.38%), and temperature (85.68–156.3°C). The maximum kraft lignin (KL) recovered at the optimum conditions for the response was found to be 93.4%. The functional groups in the treated and untreated Jatropha curcas seed cake and the recovered lignin were studied using FTIR. The recovered kraft lignin has been characterized using 1H-NMR and FTIR. The enzymatic saccharification efficiency was estimated based on the release of glucose. Maximum glucose yield (%) was found to be 92.45% for NaOH pretreated DJW compared to the untreated biomass, for which the glucose yield was only 46.64%. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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Using Ionic Liquids (ILs) to convert lignocellulosic biomass (LCB) to high-value products has emerged as an environmentally benign process. The present study reports the potential of recovering reducing sugars and lignin from sugarcane bagasse (SCB) using two novel ILs, N, N-Diisopropylethylammonium hydrogen sulfate (IL-1) and N, N-Diisopropylethylammonium perchlorate (IL-2). The process parameters—reaction temperature, time, and IL concentration have been optimized using the Response surface methodology (RSM). At optimum reaction conditions, a reducing sugar yield of 53.4 ± 0.25% and 55.93 ± 0.7%, and lignin recovery of 28.92 ± 0.23% and 31.35 ± 0.45% were obtained for IL-1 and IL-2, respectively. The effect of ILs-based pretreatment on SCB was investigated by using Fourier-transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance (1H-NMR), powder X-ray diffraction (P-XRD), and scanning electron microscopy (SEM) analysis. Enzymatic hydrolysis of the IL-1 and IL-2 pretreated SCB has shown a 3.9 and 4.7-fold increase in saccharification efficiency compared to raw SCB. The compositional changes of ILs during recycling have been analyzed using 1H-NMR analysis. The IL reuse and recyclability studies demonstrate that both recycled ILs could be utilized till the fourth cycle. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The pretreatment and the enzymatic saccharification are the key steps in the extraction of fermentable sugars for further valorization of lignocellulosic biomass (LCB) to biofuels and value-added products via biochemical and/or chemical conversion routes. Due to low density and high-water absorption capacity of LCB, the large volume of water is required for its processing. Integration of pretreatment, saccharification, and co-fermentation has succeeded and well-reported in the literature. However, there are only few reports on extraction of fermentable sugars from LCB with high biomass loading (>10% Total solids-TS) feasible to industrial reality. Furthermore, the development of enzymatic cocktails can overcome technology hurdles with high biomass loading. Hence, a better understanding of constraints involved in the development of technology with high biomass loading can result in an economical and efficient yield of fermentable sugars for the production of biofuels and bio-chemicals with viable titer, rate, and yield (TRY) at industrial scale. The present review aims to provide a critical assessment on the production of fermentable sugars from lignocelluloses with high solid biomass loading. The impact of inhibitors produced during both pretreatment and saccharification has been elucidated. Moreover, the limitations imposed by high solid loading on efficient mass transfer during saccharification process have been elaborated. [ABSTRACT FROM AUTHOR]

The global cannabis (Cannabis sativa) market was 17.7 billion in 2019 and is expected to reach up to 40.6 billion by 2024. Canada is the 2nd nation to legalize cannabis with a massive sale of $246.9 million in the year 2021. Waste cannabis biomass is managed using disposal strategies (i.e., incineration, aerobic/anaerobic digestion, composting, and shredding) that are not good enough for long-term environmental sustainability. On the other hand, greenhouse gas emissions and the rising demand for petroleum-based fuels pose a severe threat to the environment and the circular economy. Cannabis biomass can be used as a feedstock to produce various biofuels and biochemicals. Various research groups have reported production of ethanol 9.2–20.2 g/L, hydrogen 13.5 mmol/L, lipids 53.3%, biogas 12%, and biochar 34.6% from cannabis biomass. This review summarizes its legal and market status (production and consumption), the recent advancements in the lignocellulosic biomass (LCB) pre-treatment (deep eutectic solvents (DES), and ionic liquids (ILs) known as "green solvents") followed by enzymatic hydrolysis using glycosyl hydrolases (GHs) for the efficient conversion efficiency of pre-treated biomass. Recent advances in the bioconversion of hemp into oleochemicals, their challenges, and future perspectives are outlined. A comprehensive insight is provided on the trends and developments of metabolic engineering strategies to improve product yield. The thermochemical processing of disposed-off hemp lignin into bio-oil, bio-char, synthesis gas, and phenol is also discussed. Despite some progress, barricades still need to be met to commercialize advanced biofuels and compete with traditional fuels. [ABSTRACT FROM AUTHOR]

This study evaluates the microwave pretreatment of sugarcane (Saccharum officinarum) trash and oil palm (Elaeis guineensis Jacq.) empty fruit bunch (OPEFB) to yield-reducing sugars using Al2(SO4)3 as the catalyst and water as the medium. Pretreatment using 1% oxalic acid was also carried out to compare the total reducing sugar yield for both biomasses. The effects of the pretreatment on the changes in functional groups, crystallinity, and surface morphology of the biomass are discussed. Microwave heating of sugarcane trash and OPEFB particles at 180 °C for 5 min with and without the addition of 100 and 200 µmol/g biomass of Al2(SO4)3 catalyst were performed, and the total reducing sugar yield from the pretreatment hydrolysate and the enzymatic hydrolysis was improved, recorded as 0.56 and 0.51 g/g of initial biomass, respectively, representing 88.8 and 81.6% of the theoretical yield. The use of 1% oxalic acid in the pretreatment of sugarcane trash and OPEFB yielded different results of total reducing sugar yield as the two biomasses behaved differently. [ABSTRACT FROM AUTHOR]

Parthenium hysterophorous, a widespread weed in India, contributes a substantial amount of lignocellulosic biomass. The key objective of this study is to evaluate the feasibility of producing xylanase enzyme from P. hysterophorus weed biomass using the fungus Aspergillus niger. The impact of various physiological factors was confirmed through a two-step approach: first, a one-factor-at-a-time (OFAT) investigation, and subsequently, employing the RSM-based CCD method in statistical design. This research revealed that the RSM-based model led to the optimization of enzyme activity, resulting in a value of 2098.08 IU/gds for xylanase. This was achieved with an incubation time of 4.5 days, a medium pH of 6, and a cultivation temperature of 32.5 °C. Additionally, a pretreatment involving 1% NaOH and a 30-min autoclave treatment was found to alter the chemical composition of lignocellulose substrates (cellulose 43.87% and xylan 28.7%), thereby enhancing the efficiency of enzymatic hydrolysis. Moreover, fermentable sugars were produced by autoclave-assisted alkali pretreatment (NaOH-1.0% w/v) at rates of 219.6 ± 2.05 mg/gds−1 by utilizing the crude xylanase from A. niger and 291.3 ± 1.2 mg/gds−1 from commercial xylanase enzyme. Our study revealed that P. hysterophorus served as a viable and affordable substrate for fermentable sugar liberation, and xylanase is a rate-limiting enzyme in enzymatic saccharification. [ABSTRACT FROM AUTHOR]

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.

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.

Perennial grasses could be utilized as potential biofuel feedstocks and many of the members still remain unexplored. Pennisetum polystachion is a perennial grass which grows in marginal lands, and the present study aims to understand the plausibility of using it as a tropical bioenergy crop based on an eco-friendly pretreatment. The lignocellulosic content of P. polystachion was determined and 50% 1-ethyl, 3-methylimidazolium acetate ([EMIM][Ac]) pretreatment resulted in delignification rate comparable to pure [EMIM][Ac] in whole biomass. Glucan and xylan digestibility improved to 31.5–59.2% and 52.8–85.5%, respectively, after pretreatment. Glucan yield and total fermentable sugar recovery increased to 48.2–53.8% and 49.4–82.0%, respectively, in the treated sample. Extensive removal of cell wall–bound ferulic acid (62.5–81.7%) and p-coumaric acid (79.5–93.8%) was also observed. FTIR and XRD showed a marked decrease in lignin, hemicellulose and crystalline cellulose in pretreated biomass. Morphological changes observed through FESEM showed disruption of compact biomass structure, indicating disintegration efficiency of pretreatment. Fermentation of the enzymatic hydrolysate from pretreated samples using Saccharomyces cerevisiae at 24 h yielded 73.6, 63.5, and 47.9 mg/g of ethanol in leaf, whole biomass and culm, respectively. The findings of the current study revealed that P. polystachion can be utilized as a potential feedstock for biofuel production. [ABSTRACT FROM AUTHOR]

The current energy scenario has encouraged the replacement of carbon sources with renewable sources. Second‐generation (2G) ethanol production from lignocellulosic material or agroindustrial biomass involves a three‐step process consisting of pretreatment, saccharification and fermentation. Enzymatic saccharification is the most critical step for recovering fermentable sugars from biomass. Many factors such as pretreatment, enzyme load, pH, temperature, substrate and surfactant concentrations, and reaction time, among others, can affect biomass saccharification. In this sense, different design of experiment (DoE) tools together with response surface methodology (RSM) have been used as a viable strategy to determine how factors influence saccharification and to optimize this process. The strategy involves fewer experiments, less time and lower cost. This review summarizes how DoE and RSM tools have been used to optimize biomass saccharification. Moreover, an overview of the main experimental design tools used in this field is provided. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Biodiesel production currently follows a first-generation model using edible oils as raw materials. Such a production model is unsustainable, considering that it is limited by the high cost of edible oils, competes with the food sector, and is linked to deforestation and other environmental threats. Changing the raw material base to non-edible oils provides an opportunity to increase the sustainability of the biodiesel industry and to avoid conflicts with food production. Processing non-edible oilseeds for extracting the oil to be used for producing biodiesel generates large amounts of residues, such as de-oiled cakes, seed husks, and fruit shells and pods as well as plant stems and leaves resulting from pruning and other agronomy practices. Most of those residues are currently disposed of by burning or used in a suboptimal way. Bioconversion following the sugar platform route, anaerobic digestion, or enzyme production provides means for upgrading them to advanced biofuels and high-added value products. Bioconversion of plant biomass, including oilseed residues, requires pretreatment to enhance their susceptibility to enzymes and microorganisms. This review provides an outlook on bioconversion approaches applicable to different residues of oilseed-bearing plant species. Recent reports on the pretreatment of non-edible oilseed residues for enhancing their bioconversion through either the sugar platform route or anaerobic digestion are critically discussed. This review is based on an exhaustive Web of Science search performed in January–May 2023. [ABSTRACT FROM AUTHOR]

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.

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 FROM AUTHOR]

In this study, a total utilization system consisting of deep eutectic solvent (DES) treatment, separation, acetone extraction, enzymatic saccharification, and grinder treatment was developed and evaluated to utilize the cellulose and lignin in moso bamboo effectively. The efficient removal of hemicellulose and lignin through a high reaction temperature and a long reaction time via DES treatment allowed to obtain the solid residue with a cellulose-rich residue fraction. The cellulose ratio in the solid residue was higher than 75% at 150 °C for 3 h. The enzyme saccharification and the preparation of the lignocellulose nanofiber through a grinder treatment were carried out to utilize the solid residue effectively. The DES treatment significantly increased the saccharification and glucose ratio, with respective maximum values of 75.5% and 0.271 g-glucose/g-bamboo were obtained at 120 °C for 6 h, respectively. These values are 10 times higher than those of untreated bamboo. The specific tensile strength and specific Young's modulus of lignocellulose nanofiber obtained from solid residue at 150 °C for 3 h were 131 MPa/(g/cm3) and 8.88 GPa/(g/cm3), respectively. These values are higher than those of commercial cellulose nanofiber (BiNFi-s), which are 110 MPa/(g/cm3) and 5.91 GPa/(g/cm3), respectively. This result shows that the DES treatment is a relatively gentle treatment and a pretreatment method capable of delignification while preserving the cellulose nanofiber length for obtaining a lignocellulose nanofiber with high mechanical strengths. In addition, since the precipitate has a lignin-rich extracted fraction from the supernatant of the DES-treated bamboo, it has low-molecular-weight lignin. Therefore, it can be used as a raw material of epoxy resin. [ABSTRACT FROM AUTHOR]

This study aimed to explore the viability of converting potato peel waste into bioethanol using a custom-produced multi-enzyme preparation. Various pretreatment approaches were employed on the potato peels, including thermal, chemical, and thermo-chemical methods. These methods involved boiling for 30 and 60 min, steaming at different pressures and durations, and applying different concentrations of chemicals, including H2SO4, HNO3, CH3COOH, HCl, NaOH, Ca(OH)2, KOH, NH3, and H2O2, either individually or in combination with steam treatment. The pretreated potato peels were subsequently subjected to enzymatic hydrolysis using a crude multi-enzyme cocktail obtained from solid-state fermentation of wheat bran by a naturally occurring strain of Aspergillus niger P-19. This enzyme cocktail consisted of cellulases, hemicellulases, pectinase, and amylases. The most effective pretreatment combination involved the use of 3% H2SO4 followed by steam treatment under pressure, and enzymatic hydrolysis utilizing the crude multi-enzyme preparation. This combination resulted in the highest concentration of reducing sugars (141.04 ± 12.31 g/L), with a carbohydrate conversion rate of 98.49% when a substrate loading of 20% was used. As a result, an ethanol yield of 43.2 ± 3.82 g/L, representing 21.6% based on dry matter, was achieved. Furthermore, supplementing the medium with peptone, (NH4)(H2PO4), and ZnSO4 at a concentration of 0.1% w/v each, along with solid loadings of 22% and 24%, respectively, achieved yield improvements of 51.67 g/L and 54.75 g/L. However, the maximum productivity of 23.4% was observed with a 22% loading, compared to a yield of 22.8% with a 24% solid loading, based on dry matter. [ABSTRACT FROM AUTHOR]

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.

Summary: Trees constitute promising renewable feedstocks for biorefinery using biochemical conversion, but their recalcitrance restricts their attractiveness for the industry. To obtain trees with reduced recalcitrance, large‐scale genetic engineering experiments were performed in hybrid aspen blindly targeting genes expressed during wood formation and 32 lines representing seven constructs were selected for characterization in the field. Here we report phenotypes of five‐year old trees considering 49 traits related to growth and wood properties. The best performing construct considering growth and glucose yield in saccharification with acid pretreatment had suppressed expression of the gene encoding an uncharacterized 2‐oxoglutarate‐dependent dioxygenase (2OGD). It showed minor changes in wood chemistry but increased nanoporosity and glucose conversion. Suppressed levels of SUCROSE SYNTHASE, (SuSy), CINNAMATE 4‐HYDROXYLASE (C4H) and increased levels of GTPase activating protein for ADP‐ribosylation factor ZAC led to significant growth reductions and anatomical abnormalities. However, C4H and SuSy constructs greatly improved glucose yields in saccharification without and with pretreatment, respectively. Traits associated with high glucose yields were different for saccharification with and without pretreatment. While carbohydrates, phenolics and tension wood contents positively impacted the yields without pretreatment and growth, lignin content and S/G ratio were negative factors, the yields with pretreatment positively correlated with S lignin and negatively with carbohydrate contents. The genotypes with high glucose yields had increased nanoporosity and mGlcA/Xyl ratio, and some had shorter polymers extractable with subcritical water compared to wild‐type. The pilot‐scale industrial‐like pretreatment of best‐performing 2OGD construct confirmed its superior sugar yields, supporting our strategy. [ABSTRACT FROM AUTHOR]

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 FROM AUTHOR]

Harum Manis mango leaves are considered lignocellulosic (LC) biomass that could contribute to the production of second generation (2G) bioethanol. However, pretreatment should be conducted to overcome the recalcitrance of LC biomass prior to bioethanol production. Hence, this study aims to elucidate the structural changes of the biomass of Harum Manis mango leaves after single-stage and two-stage chemical pretreatment. The Harum Manis mango leaves were treated with only acid, only alkaline, acid-alkaline, or alkaline-acid process. One per cent (v/v) sulphuric acid and 1% (w/v) sodium hydroxide were used as the acid and alkaline hydrolysing agent in the four approaches of pretreatment, respectively. Consequently, the pretreated biomass was enzymatically hydrolysed to produce reducing sugar followed by the conversion into bioethanol through yeast fermentation. Acid-alkaline pretreatment yielded the highest holocellulose composition of 95.26%, with the highest delignification effect of 86.97 ± 1.26%. Furthermore, Fourier transform infrared (FTIR) spectroscopy and X-ray powder diffraction (XRD) validated the acid-alkaline pretreated biomass to be the most feasible substrate for bioethanol production in this study. Finally, the maximum fermentable sugar content and bioethanol concentration of 415.02 ± 7.01 mg/g and 1.57 ± 0.06 mg/ml, respectively, were obtained. [ABSTRACT FROM AUTHOR]

Sweet sorghum bagasse (SSB) is the resultant waste after extraction of sugar-rich juice from the stalks during bioethanol production. As a typical biomass, the SSB consists of moisture, unspent soluble sugar, and abundant lignocellulosic component (cellulose, hemicelluloses and lignin). The undisposed SSB can inevitably cause the environmental pollution and resource waste. Hence, the effective storage of SSB is necessary for the year-round stable operation of biofuel plants. Among them, ensiling (wet storage) can be an efficient technology available for the SSB preservation and utilization, which enable the SSB to be processed all year around. Moreover, ensiling can also act as a pretreatment strategy, due to the benefits for the cell wall degradation and the improvement of biomass bioconversion for subsequent processing. In this study, the potential of ensiling pretreatment was investigated to fortify with Lactobacillus plantarum inoculant and cellulolytic enzymes on the improvement of ensiling quality to modulate the performance of enzymatic saccharification of SSB. The SSB were ensiled with no additives (CK), Lactobacillus plantarum (L), cellulolytic enzyme (E), and a combination of L and E (LE) for 21 days. The dynamic changes of nutrient composition, lignocellulosic components, and ensiling fermentation characteristics were investigated, and then dynamic evolution of the bacterial community structure was analyzed by high-throughput sequencing at HiSeq2500 platform. Ensiling quality was comprehensively evaluated by membership function method, in order to screen the suitable silages additives in consideration of enzymatic hydrolysis performance. The results showed that the addition of Lactobacillus plantarum inoculant and cellulolytic enzymes either alone or in combination was facilitated the lactic acid fermentation to reduce the fermentation losses, as evidenced by the higher content of crude protein, starch, neutral detergent fiber, acid detergent fiber, hemicellulose and cellulose components than that of un-ensiled SSB. The content of dry matter, crude protein and water-soluble carbohydrates in silages with additives were higher than those in the CK, but the content of holocellulose lower. Furthermore, the content of dry matter in the E silages, the content of crude protein in the L silages, and the content of water-soluble carbohydrates in the LE silages were the highest in all silages, respectively. The pH value of all silages significantly decreased to below 4.2, accompanied by the ratio of lactic Acid (LA) and total organic acids (TOA) higher than 0.65 and the ratio of lactic acid (LA) and acetic acid (AA) higher than 2 during the whole ensiling for 21 days. The content of LA and AA in the L silages were significantly higher than those of silages at the CK (P<0.05), the ratio of LA/TOA and LA/AA in the E silages were significantly higher than those of silages at the CK(P<0.05). The membership function analysis indicated that the silages at the E group shared the highest comprehensive scores at 21 days. The bacterial community structure showed that the Proteobacteria was the main bacteria in all silages during ensiling fermentation. At 21 days of ensilage, Enterobacteriaceae, pantoea and leuconostoc were the main species in the silages at genus level. ACE, Chao1 and Shannon index in the E, L and LE silages were significantly lower than those of CK group (P<0.05). It infers that the ensiling pretreatment with the additives was effectively reduced the microbial richness and diversity. Principal Coordinate Analysis (PCA) was constructed using the relative abundance of bacteria at the genus level, indicating the outstanding separation in the SSB before and after ensiling pretreatment. After ensiling pretreatment, the yield of reducing sugar in all SSB silages significantly increased, compared with the un-ensiled SSB. Consequently, the reducing sugar yield in the E silages increased by 117%. The highest net income of E silages fortified with cellulolytic enzyme was almost three-fold that of un-ensiled SSB using cost-benefit analysis. In conclusion, the ensiling pretreatment fortified by cellulolytic enzymes can be expected to serve as a cost-effective, eco-friendly and tech-feasible strategy for the preservation and pretreatment of SSB biomass. Especially, cellulolytic enzyme can be used to modulate the ensiling pretreatment performance. Therefore, the ensiling quality and the biodegradation performance of SSB silage can be effectively improved for the bioenergy utilization of sweet sorghum bagasse. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Polyhydroxybutyrate (PHB) is considered a potential substitute for conventional, non-biodegradable petroleum-based plastics. However, high production cost has been a major drawback to the commercialization of PHB. The use of low-cost lignocellulosic agricultural residues such as corn stover, together with an effective pretreatment method, can reduce production costs. In this study, optimization of pretreatment methods for corn stover - namely, steam explosion and sequential steam explosion-dilute acid pretreatment - was done to maximize the concentration of reducing sugars in the hydrolysate obtained after enzymatic saccharification. Response surface methodology (RSM)-designed experiments showed that steam explosion temperature had the highest impact on reducing sugar production for both pretreatment methods. Optimum conditions for steam explosion pretreatment were 220.9 °C steam explosion temperature and 11.2 min residence time, yielding a hydrolysate with 9.67 g/L (64.50% yield) of reducing sugars after saccharification. For the sequential pretreatment, 9.14 g/L of reducing sugars (60.93% yield) was produced from the optimum conditions of 224.8 °C steam explosion temperature, 144.2 °C dilute acid temperature, 43.8 min reaction time, and 5% w/v acid concentration. Dilute acid pretreatment significantly decreased the reducing sugar yield after the steam explosion by 5.5%, probably due to the degradation of reducing sugars, making steam explosion sufficient in the pretreatment of corn stover. More importantly, PHB was produced using the hydrolysate from steam-exploded corn stover, with a maximum concentration of 1.81 g/L after 24 h of bacterial fermentation. Therefore, the steam explosion was proven effective in producing sugar-rich hydrolysates from corn stover for PHB production. [ABSTRACT FROM AUTHOR]

BACKGROUND: The conversion of lignocellulose to sugar is part of the process of lignocellulose biorefining and, therefore, is crucial for effective lignocellulose utilization. RESULTS: In this study, alkali and acidic pretreatments were carried out to deconstruct corn stalk (CS) material for efficient subsequent enzymatic saccharification. Most xylan and lignin can be removed from CS material by acid and alkali pretreatments, respectively, as verified by XRD (X‐ray diffractometer) and FT‐IR (Fourier Transform Infrared Spectroscopy) analyses. Fractal‐like kinetics were assessed to analyze the effect of enzyme loading on the glucose and xylose sugar yields from the two pretreated CS materials, with fractal dimension considered as a dominant factor in rate constant decrease. Fractal dimension analysis demonstrated that the alkali pretreated CS was more easily hydrolyzed that the acid pretreated CS. For glucan hydrolysis, the effect of enzyme loading on the rate constant of the alkali pretreated CS was marginally higher than that of the acid pretreated CS. For xylan hydrolysis, enzyme loading was shown to significantly improve the rate constant of the alkali pretreated CS compared to the acid pretreated CS. Furthermore, the developed fractal‐like model exhibits high prediction accuracy for sugar yield at high enzyme loadings. CONCLUSION: The application of the fractal‐like theory was quite successful for studying enzymatic kinetics. © 2022 Society of Chemical Industry (SCI). [ABSTRACT FROM AUTHOR]

The conversion of plant‐based lignocellulosic materials into alternative fuels entails various pretreatment strategies (chemical, physico‐chemical or enzymatic) to boost the release of sugar constituents from biomass. This investigation reports the influence of different pretreatment techniques such as acid, alkali and steam treatment followed by enzymatic hydrolysis in single and (or) consolidated system, for releasing fermentable reducing sugars from sugarcane bagasse (SB) and corn cobs (CC) (major agricultural residues). Alkaline pretreatment facilitated maximal lignin removal, 83.2 and 69.4% from CC and SB, respectively, thereby expediting enzymatic hydrolysis by in‐house xylanases produced from indigenously isolated Aspergillus tubingensis strains, AUMS60 (Xyn60) and AUMS64 (Xyn64A, B). The major operating conditions for enzymatic treatment of raw and pretreated residues were set at a substrate loading of 2% (w/v), an enzyme loading of 100–500 U g−1 substrate and incubation at 40°C, 140 rpm for 72 h. Treatment with Xyn60 and Xyn64A, B resulted in 81.4% (72 h) and 75.9% (24 h) saccharification of delignified SB and CC, respectively. Scanning electron micrographs and Fourier transform infrared spectra corroborated structural deformations induced by physico‐chemical pretreatment processes, thereby validating the maximal enzymatic hydrolysis of delignified substrates. The analysis of enzyme hydrolysis products suggested higher saccharification of hemicellulosic fraction as compared with cellulose. The observations of the present study signify that the thermotolerant in‐house enzymes show great potential in the saccharification of lignocelluloses within a short span of time and may open a new vista for biomass valorization in the bioenergy sector. © 2022 Society of Chemical Industry and John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]