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BACKGROUND: Lignin is an aromatic polymer deposited in secondary cell walls of higher plants to provide strength, rigidity, and hydrophobicity to vascular tissues. Due to its interconnections with cell wall polysaccharides, lignin plays important roles during plant growth and defense, but also has a negative impact on industrial processes aimed at obtaining monosaccharides from plant biomass. Engineering lignin offers a solution to this issue. For example, previous work showed that heterologous expression of a coliphage S-adenosylmethionine hydrolase (AdoMetase) was an effective approach to reduce lignin in the model plant Arabidopsis. The efficacy of this engineering strategy remains to be evaluated in bioenergy crops. RESULTS: We studied the impact of expressing AdoMetase on lignin synthesis in sorghum (Sorghum bicolor L. Moench). Lignin content, monomer composition, and size, as well as biomass saccharification efficiency were determined in transgenic sorghum lines. The transcriptome and metabolome were analyzed in stems at three developmental stages. Plant growth and biomass composition was further evaluated under field conditions. Results evidenced that lignin was reduced by 18% in the best transgenic line, presumably due to reduced activity of the S-adenosylmethionine-dependent O-methyltransferases involved in lignin synthesis. The modified sorghum features altered lignin monomer composition and increased lignin molecular weights. The degree of methylation of glucuronic acid on xylan was reduced. These changes enabled a ~20% increase in glucose yield after biomass pretreatment and saccharification compared to wild type. RNA-seq and untargeted metabolomic analyses evidenced some pleiotropic effects associated with AdoMetase expression. The transgenic sorghum showed developmental delay and reduced biomass yields at harvest, especially under field growing conditions. CONCLUSIONS: The expression of AdoMetase represents an effective lignin engineering approach in sorghum. However, considering that this strategy potentially impacts multiple S-adenosylmethionine-dependent methyltransferases, adequate promoters for fine-tuning AdoMetase expression will be needed to mitigate yield penalty.

SUMMARY: In grass cell walls, ferulic acid (FA) serves as an important cross‐linker between cell wall polymers, such as arabinoxylan (AX) and lignin, affecting the physicochemical properties of the cell walls as well as the utilization properties of grass lignocellulose for biorefinering. Here, we demonstrate that hydroxycinnamaldehyde dehydrogenase (HCALDH) plays a crucial role in the biosynthesis of the FA used for cell wall feruloylation in rice (Oryza sativa). Bioinformatic and gene expression analyses of aldehyde dehydrogenases (ALDHs) identified two rice ALDH subfamily 2C members, OsHCALDH2 (OsALDH2C2) and OsHCALDH3 (OsALDH2C3), potentially involved in cell wall feruloylation in major vegetative tissues of rice. CRISPR‐Cas9 genome editing of OsHCALDH2 and OsHCALDH3 revealed that the contents of AX‐bound ferulate were reduced by up to ~45% in the cell walls of the HCALDH‐edited mutants, demonstrating their roles in cell wall feruloylation. The abundance of hemicellulosic sugars including arabinosyl units on AX was notably reduced in the cell walls of the HCALDH‐edited mutants, whereas cellulose and lignin contents remained unaffected. In addition to reducing cell wall‐bound ferulate, the loss of OsHCALDH2 and/or OsHCALDH3 also partially reduced cell wall‐bound p‐coumarate and sinapate in the vegetative tissues of rice, whereas it did not cause detectable changes in the amount of γ‐oryzanol (feruloyl sterols) in rice seeds. Furthermore, the HCALDH‐edited mutants exhibited improved cell wall saccharification efficiency, both with and without alkaline pretreatment, plausibly due to the reduction in cell wall cross‐linking FA. Overall, HCALDH appears to present a potent bioengineering target for enhancing utilization properties of grass lignocellulose. [ABSTRACT FROM AUTHOR]

ABSTRACT Black rice (Oryza sativa) also known as forbidden rice, restricted only in northeast Asia, is now being cultivated in some Indian states and has a huge potential for producing vinegar being rich in health‐promoting characteristics. In the current study, enzymatic saccharification of black rice carried out using commercial amylases was optimized by response surface methodology (RSM) at 45°C with 5.5 pH. The optimized saccharified starch showed a Brix of 13.5°B with 10.937 g/100 mL and 8.602 g/100 mL of total sugars and reducing sugars, respectively. The saccharification validation of 2L showed a maximum of 14°B, 13.1 g/100 mL total sugars and 10.89 g/100 mL reducing sugars. The ethanolic fermentation of hydrolyzed starch was carried out using Saccharomyces cerevisiae KY069279, which led to 6.532% (v/v) ethanol at a fermentation efficiency of 93.11% and 0.47 g/g of ethanol productivity. The acetic acid fermentation was carried out post‐ethanol production using indigenous acetic acid bacteria, Acetobacter aceti AC1. The fermented vinegar showed 4.15% (w/v) volatile acidity at 64.7% fermentation efficiency and 0.38 g/g acetic acid productivity. The produced rice vinegar was found to be rich in antioxidants with a 77.8% DPPH scavenging activity at 50.93 µM concentration. The vinegar was found to be of standard quality with a mean sensory score of 7.92 ± 0.8 by a panel of 10 semitrained judges. [ABSTRACT FROM AUTHOR]

Increasing lignocellulosic feedstock for advanced biofuels can tackle the decarbonization of the transport sector. Dedicated biomass produced alongside food systems with low indirect land use change (iLUC) impact can broaden the feedstock availability, thus streamlining the supply chains. The objective of this study was the design and evaluation of advanced ethanol value chains for the Emilia‐Romagna region based on low iLUC feedstock. Two dedicated lignocellulosic crops (biomass sorghum and sunn hemp) were evaluated in double cropping systems alongside food crop residues (corn stover and wheat straw) as sources to simulate the value chains. A parcel‐level regional analysis was carried out, then the LocaGIStics2.0 model was used for the spatial design and review of the biomass delivery chain options regarding cost and greenhouse gas (GHG) emissions of the different feedstock mixes. Literature data on bioethanol production from similar feedstocks were used to estimate yields, process costs, and GHG emissions of a biorefinery process based on these biomasses. Within the chain options, GHG emissions were overly sensitive to cultivation input, mostly N‐fertilization. This considered, GHG emissions resulted similar across different feedstock with straw/stover (averaging 13 g CO2eq MJ−1 fuel), sunn hemp (14 g CO2eq MJ−1 fuel), and biomass sorghum (16 g CO2eq MJ−1 fuel). On the other hand, the bioethanol produced from biomass sorghum (608 € Mg−1 of bioethanol) was cheaper compared with straw (632 € Mg−1), sunn hemp (672 € Mg−1), and stover (710 € Mg−1). The bioethanol cost ranged from 0.0017 to 0.020 € MJ−1 fuel depending on the feedstock, with operations and maintenance impacting up to 90% of the final cost. In summary, a single bioethanol plant with an annual capacity of 250,000 Mg of biomass could replace from 5% to 7% of the Emilia‐Romagna's ethanol fuel consumption, depending on the applied sourcing scenario. [ABSTRACT FROM AUTHOR]

Mexico produces 1.27 million tons of pineapple and prickly pear, whose wastes account about 40%. Therefore, effective utilization of this lignocellulosic biomass is crucial. Our research emphasizes the need for efficient pretreatment to optimize enzyme efficacy and enhance saccharification processes. Particle size significantly affects the microbial breakdown of these biomasses. Sun-dried prickly pear peels with 1.84 mm particles showed a remarkable increase in xylanase and laccase activities, achieving up to 1431 ± 190 U/mL and 1165 ± 19 U/mL, respectively, a stark contrast to the lower enzymatic activities recorded in oven-dried counterparts. Surprisingly, untreated pineapple peels yielded the highest saccharification rates, suggesting that some traditional pretreatment practices might be redundant. Despite employing an enzymatic cocktail with high hydrolase activities, the saccharification of pineapple peels peaked at a modest 0.16 gRS/gbiomass, hinting at inefficiencies potentially due to high solid loading or inadequate enzyme cocktail components. Besides, this research proposes the sustainable use of the residual composites obtained after the biological processing of pretreated material. In this respect, a pathway to more cost-efficient and environmentally friendly industrial bioprocesses through valorizing lignocellulosic residues is suggested. [ABSTRACT FROM AUTHOR]

Endophytic fungi, as plant symbionts, produce an elaborate array of enzymes for efficient disintegration of lignocellulosic biomass into constituent monomeric sugars, making them novel source of lignocellulolytic CAZymes with immense potential in future biorefineries. The present study reports lignocellulolytic enzymes production potential of an endophytic halotolerant Penicillium oxalicum strain isolated from Citrus limon, under submerged and solid-state fermentation (SmF & SSF, respectively), in the presence and absence of salt (1 M NaCl). The comparative QTOF–LC/MS-based exoproteome analysis of the culture extracts unveiled differential expression of CAZymes, with the higher abundance of GH6 and GH7 family cellobiohydrolase in the presence of 1 M salt. The strain improvement program, employing cyclic mutagenesis and diploidization, was utilized to develop hyper-cellulase producing mutant strains of P. oxalicum. The enzyme production of the developed strain (POx-M35) was further enhanced through statistical optimization of the culture conditions utilizing glucose mix disaccharides (GMDs) as an inducer. This optimization process resulted in the lignocellulolytic cocktail that contained high titers (U/mL) of endoglucanase (EG) (146.16), cellobiohydrolase (CBHI) (6.99), β-glucosidase (β-G) (26.21), xylanase (336.05) and FPase (2.02 U/mL), which were 5.47-, 5.54-, 8.55-, 4.96-, and 4.39-fold higher when compared to the enzyme titers obtained in wild HP1, respectively. Furthermore, the lignocellulolytic cocktails designed by blending secretome produced by mutant POx-M35 with xylanases (GH10 and GH11) derived from Malbranchea cinnamomea resulted in efficient hydrolysis of unwashed acid pretreated (UWAP) rice straw slurry and mild alkali deacetylated (MAD) rice straw. This study underscores the potential of bioprospecting novel fungus and developing an improved strain for optimized production and constitution of lignocellulolytic cocktails that can be an important determinant in advancing biomass conversion technologies. [ABSTRACT FROM AUTHOR]

This study introduces a groundbreaking approach to the utilization of broken rice, an abundant and economical by-product of rice milling, to produce liquid glucose. Broken rice, often overlooked in resource utilization, emerges as a sustainable and cost-effective alternative sweetener in the food processing sector, marking a novel and impactful development. This research is focused on a novel synergy between saccharification time and enzyme concentrations, strategically influencing the efficiency, dextrose equivalent (DE), and water activity (aw) of liquid glucose derived from broken rice. Remarkably, we have achieved a substantial DE range of 40.39 to 55.83% and efficiencies ranging from 25.41 to 32.82%, all while maintaining aw values within the safe range of 0.60 to 0.75. The novelty of this study is the linear relationship between DE and the concentrations of α-amylase and glucoamylase during enzymatic hydrolysis. This finding empowers industries to precisely tailor liquid glucose properties to their specific processing needs, offering an economically viable alternative to conventional sucrose-based sweeteners. Advanced partial least-squares regression modelling was used to analyze the interplay of saccharification time, enzyme composition, and enzymatic digestibility parameters (aw, DE, efficiency). This model, known for its predictive accuracy, enables precise control of liquid glucose properties. Ice cream formulations with broken rice-derived liquid glucose and corn syrup were comparatively assessed. Encouragingly, critical parameters like pH, titrable acidity, meltdown characteristics, and total solids showed striking similarities. Sensory evaluations revealed high overall acceptability at 94.63%. This research taps into broken rice's potential and offers a sustainable approach to liquid glucose production. Future work can explore scalability and diverse applications of this innovative approach, transforming sweetener options in the food industry. [ABSTRACT FROM AUTHOR]

In a world where climate change and sustainability are at the forefront, innovative solutions are more pressing than ever. In the production process of distillates, the critical stage is the cooking of agave for thermal hydrolysis of fructans. This stage determines the efficiency of the process, incurs the highest production cost, and has a detrimental impact on the environment. In this study, we investigate the saccharification of agave fructans in fresh juice using fructanases produced in situ by Kluyveromyces marxianus as an alternative to traditional agave cooking. furthermore, we explore both sequential and simultaneous alcoholic fermentation processes using a native mixed culture to improve the overall yield and obtain a distillate with enjoyable sensory characteristics. The results show that saccharification and simultaneous fermentation allow for obtaining a fermented product containing an average of 7% alcohol. Gas chromatography and mass spectrometry analysis shows that the distillate is essentially an ethanol – water mixture with a broad profile and high concentration of volatile compounds. The composition of the distilled product provides a well-balanced sensory experience that is highly valued by consumers. Hence, for the first time, a novel, efficient, low-cost, and sustainable process for producing agave distillates has been successfully designed. [ABSTRACT FROM AUTHOR]

Starch, a crucial raw material, has been extensively investigated for biotechnological applications. However, its application in γ-polyglutamic acid (γ-PGA) production remains unexplored. Based on γ-PGA output of Bacillus subtilis SCP010-1, a novel asynchronous saccharification and fermentation process for γ-PGA synthesis was implemented. The results revealed that a starch concentration of 20%, α-amylase dosage of 75 U/g, liquefaction temperature of 72℃, and γ-PGA yield of 36.31 g/L was achieved. At a glucoamylase dosage of 100 U/g, saccharification 38 h at 60℃, the yield of γ-PGA increased to 48.88 g/L. The contents of total sugar, glucose, maltose and oligosaccharide in saccharified liquid were determined. Through batch fermentation of saccharified liquid in fermentor, the γ-PGA output was elevated to 116.08 g/L. This study can offer a potential cost reduction of 40%, which can be a promising advancement in industrial γ-PGA production. Moreover, our approach can be applied in other starch-based fermentation industries. [ABSTRACT FROM AUTHOR]

The development of a cost-effective and environment-friendly conversion method to overcome the recalcitrance of biomass is a challenging task for enhancing the value-added potential of lignocellulosic compounds. In this study, a photo-enzyme composite catalyst by immobilizing laccase on Fe 3 O 4 /TiO 2 (denoted as LC-Fe 3 O 4 /TiO 2) was applied to the pretreatment and enzymatic saccharification of Spartina alterniflora Loisel (SAL) under the action of alternating light/dark cycles. The effect of the pretreatment temperature, pretreatment time, LC-Fe 3 O 4 /TiO 2 concentration, mass concentration of straw particles, and hydrolysis time on delignification efficiency and reducing sugar yield was investigated by a central composite design (CCD). Under the selected conditions (48 ℃, pretreatment time of 70 min, LC-Fe 3 O 4 /TiO 2 concentration of 4.9 mg/mL, mass concentration of straw particles of 22 % (w/v), hydrolysis time of 55 h), 92.53 % delignification and 129.85 mg/g reducing sugar yield were achieved. The enzymatic hydrolysis efficiency was higher compared to conventional methods. Moreover, the LC-Fe 3 O 4 /TiO 2 showed good recyclability and reusability. Developed LC-Fe 3 O 4 /TiO 2 particles have a high potential for use in biomass utilization. [Display omitted] • Laccase-Fe 3 O 4 /TiO 2 integrated catalyst (LC-Fe 3 O 4 /TiO 2) is first synthesized. • Effects of LC-Fe 3 O 4 /TiO 2 on pretreatment and saccharification is studied. • LC-Fe 3 O 4 /TiO 2 can enhance sugar yield under the alternation of light and dark. • LC-Fe 3 O 4 /TiO 2 could be efficiently reused up to 3 cycles. [ABSTRACT FROM AUTHOR]

Lignocellulosic mass consists of cellulose, hemicelluloses, and lignin. Although biomass promises to be efficiently used for biofuel production and many other value‐added products, lignin present in lignocellulosic biomass affects the hydrolysis of cellulose and hemicelluloses, making it necessary to develop techniques that provide better lignin removal efficiency and high cellulose hydrolysability. The current work aims to maximize lignin removal in sawdust and develop an understanding of the hydrolysis of pretreated biomass for sugar production. Different parameters such as solvent to solid ratio, temperature, and reaction time have been considered based on the design of experiment to understand the effect on the delignification and saccharification processes. After treating sawdust for 1.5 h, it was observed that a maximum of 85% lignin was removed at a temperature of 131°C and solid loading of 16 g. Subsequent hydrolysis of delignified sawdust at 131°C temperature, solvent to solid ratio 15, and 0.5 h resulted in a maximum reducing sugar production of 26.82 mg/mL. The study elucidated the optimum conditions for the effective processing of sawdust in terms of delignification and saccharification, leading to maximum benefits in lignin removal and sugar production. [ABSTRACT FROM AUTHOR]

Microalgae are a promising biomass source because of their capability to fixate CO2 very efficiently. In this study, the potential of Microchloropsis salina biomass as a feedstock for fermentation was explored, focusing on biomass hydrolysis by employing various mechanical and chemical cell disruption strategies in combination with enzymatic hydrolysis. Among the mechanical cell disruption methods investigated on a lab scale, namely ultrasonication, bead milling, and high-pressure homogenization, the most effective was bead milling using stainless-steel beads with a diameter of 2 mm. In this way, 87–97% of the cells were disrupted in 40 min using a mixer mill. High-pressure homogenization was also effective, achieving 86% disruption efficiency after four passes on a 30–200 L scale using biomass with 15% (w/w) solids content. Enzymatic hydrolysis of the disrupted cells using a mixture of cellulases and mannanases yielded up to 25% saccharification efficiency after 72 h. Acidic hydrolysis of undisrupted cells followed by enzymatic treatment yielded around 30% saccharification efficiency but was coupled with significant dilution of the resulting hydrolysate. Microalgal biomass hydrolysate produced was determined to have ~8.1 g L−1 sugars and 2.5% (w/w) total carbon, as well as sufficient nitrogen and phosphorus content as a fermentation medium. [ABSTRACT FROM AUTHOR]

In a world where climate change and sustainability are at the forefront, innovative solutions are more pressing than ever. In the production process of distillates, the critical stage is the cooking of agave for thermal hydrolysis of fructans. This stage determines the efficiency of the process, incurs the highest production cost, and has a detrimental impact on the environment. In this study, we investigate the saccharification of agave fructans in fresh juice using fructanases produced in situ by Kluyveromyces marxianus as an alternative to traditional agave cooking. furthermore, we explore both sequential and simultaneous alcoholic fermentation processes using a native mixed culture to improve the overall yield and obtain a distillate with enjoyable sensory characteristics. The results show that saccharification and simultaneous fermentation allow for obtaining a fermented product containing an average of 7% alcohol. Gas chromatography and mass spectrometry analysis shows that the distillate is essentially an ethanol – water mixture with a broad profile and high concentration of volatile compounds. The composition of the distilled product provides a well-balanced sensory experience that is highly valued by consumers. Hence, for the first time, a novel, efficient, low-cost, and sustainable process for producing agave distillates has been successfully designed.

Abstract Background Lignin is an aromatic polymer deposited in secondary cell walls of higher plants to provide strength, rigidity, and hydrophobicity to vascular tissues. Due to its interconnections with cell wall polysaccharides, lignin plays important roles during plant growth and defense, but also has a negative impact on industrial processes aimed at obtaining monosaccharides from plant biomass. Engineering lignin offers a solution to this issue. For example, previous work showed that heterologous expression of a coliphage S-adenosylmethionine hydrolase (AdoMetase) was an effective approach to reduce lignin in the model plant Arabidopsis. The efficacy of this engineering strategy remains to be evaluated in bioenergy crops. Results We studied the impact of expressing AdoMetase on lignin synthesis in sorghum (Sorghum bicolor L. Moench). Lignin content, monomer composition, and size, as well as biomass saccharification efficiency were determined in transgenic sorghum lines. The transcriptome and metabolome were analyzed in stems at three developmental stages. Plant growth and biomass composition was further evaluated under field conditions. Results evidenced that lignin was reduced by 18% in the best transgenic line, presumably due to reduced activity of the S-adenosylmethionine-dependent O-methyltransferases involved in lignin synthesis. The modified sorghum features altered lignin monomer composition and increased lignin molecular weights. The degree of methylation of glucuronic acid on xylan was reduced. These changes enabled a ~20% increase in glucose yield after biomass pretreatment and saccharification compared to wild type. RNA-seq and untargeted metabolomic analyses evidenced some pleiotropic effects associated with AdoMetase expression. The transgenic sorghum showed developmental delay and reduced biomass yields at harvest, especially under field growing conditions. Conclusions The expression of AdoMetase represents an effective lignin engineering approach in sorghum. However, considering that this strategy potentially impacts multiple S-adenosylmethionine-dependent methyltransferases, adequate promoters for fine-tuning AdoMetase expression will be needed to mitigate yield penalty.

Abstract The pretreatment and saccharification of dewaxed bagasse (DWB) has been investigated under various reaction conditions ranging 2000 to 3200 psi, at 70 ± 1 °C in supercritical carbon dioxide (SCC). This has been in attempt to transform the DWB into fermentable sugar and bioethanol in high yields. The effect of SCC mediated pretreatment and enzymatic hydrolysis on structural and morphological alterations in DWB has been ascertained through diverse analytical methods. The sugar has been released through cellulase (40 FPU/mL) mediated enzymatic hydrolysis of pretreated DWB in sodium acetate buffer (pH 4.7) within 1 h at SCC 2800 psi, 70 ± 1 °C. The released sugar was subsequently fermented in the presence of yeast (Saccharomyces crevices, 135 CFU) at 28 ± 1 °C over 72 h to afford the bioethanol. The SCC mediated process conducted in acetic acid:water media (1:1) at 2800 psi, 70 ± 1 °C over 6 h has afforded the pretreated DWB with maximum yield towards the production of fermentable sugar and bioethanol. The production of fermentable sugar and bioethanol has been electrochemically estimated through cyclic voltammetry (CV) and square wave voltammetry (SWV) over glassy carbon electrode in KOH (0.1 M). The electrochemical methods were found selective and in close agreement for estimation of the yields (%) of fermentable sugars and bioethanol. The yield (%) of fermentable sugar estimated from CV and SWV were 80.10 ± 5.34 and 79.00 ± 5.09 respectively. Whereas the yield (%) of bioethanol estimated from CV and SWV were 81.30 ± 2.78% and 78.6 ± 1.25% respectively. Present investigation delivers a SCC mediated green and sustainable method of pretreatment of DWB to afford the enhanced saccharification, to produce bioethanol in high yields.

The main aim of this study is to utilize municipal solid waste (MSW) and date molasses as a culture medium to reduce the production cost of enzymes. A novel cellulase-producing fungus, Trichoderma reesei Al-K1 149, was isolated from the date molasses. MSW and date molasses were processed and used as the substrate (1:1 ratio) in solid-state fermentation. The proximate composition of the substrate revealed that the MSW was enriched with cellulostic material and contributed about 33% of the available biomass. Elements such as Ca, K, S, P, Mg, Fe, Cu, and Na were found in the MSW. Fungal cellulase production was at its maximum after 96 h of incubation with the yields of β-glucanase (98 ± 3.9 U/gds), carboxymethyl cellulase (CMCase) (241 ± 12.8 U/gds), and filter paperase (FPase) (31.2 ± 3.1 U/gds). The combination of municipal solid waste and date molasses was found to be the best source of nitrogen and carbon for the biosynthesis of cellulase by T. reesei Al-K1 149. The optimal temperature and moisture content of the medium for cellulase production by T. reesei Al-K1 149 were 40 °C and 60%, respectively. The optimal pH and inoculum were 6.0 and 8% (v/w), respectively. The optimized culture condition was used to produce cellulases in a laboratory-scale tray reactor, and enzyme production was enhanced twofold compared to the unoptimized medium. The cellulolytic ability was tested in biomass saccharification with various plant materials (palm sawdust, palm leaves, palm fruit waste, and filter paper) and saccharified plant materials effectively. These findings revealed that the enzymes secreted by strain Al-K1 149 may have significant value for the industrial saccharification process. [ABSTRACT FROM AUTHOR]

Globally escalating ethanol demand necessitates the use of hybrid technologies integrating first- and second-generation biofuel feedstocks for achieving the futuristic targets of gasoline replacement with bioethanol. In present study, an optimized two-step sequential pre-treatment (first dilute alkali, then dilute acid) of Pine forest litter (PFL) was developed. Furthermore, the saccharification of pre-treated PFL was optimized through Response Surface Methodology using Box-Behnken Design, wherein 0.558 g/g of reducing sugar was released under the optimized conditions (12.5% w/v of biomass loading, 10 FPU/g of PFL enzyme loading, 0.15% v/v Tween-80 and 48 h incubation time). Moreover, during hydrolysate fermentation using Saccharomyces cerevisiae NCIM 3288 strain, 22.51 ± 1.02 g/L ethanol was produced. Remarkably, hydrophobic resin (XAD-4) treatment of PFL hydrolysate, significantly removed inhibitors (Furfural, 5-hydroxymethylfurfural and phenolics) and increased ethanol production to 27.38 ± 1.18 g/L. Furthermore, during fermentation of molasses supplemented PFL hydrolysate (total initial sugar: 100 ± 3.27 g/L), a maximum of 46.02 ± 2.08 g/L ethanol was produced with 0.482 g/g yield and 1.92 g/l/h productivity. These findings indicated that the integration of molasses to lignocellulosic hydrolysate, would be a promising hybrid technology for industrial ethanol production within existing bio-refinery infrastructure. [ABSTRACT FROM AUTHOR]

A novel pretreatment process based on prioritized microbes was developed to improve the yield of reducing sugars from red (Gracilaria corticata), brown (Sargassum wightii), and green (Ulva fasciata) seaweeds as a cheap, eco-friendly method for seaweed saccharification. Prioritization of six microbes from a collection of 24 microbes was initially done using a unique stepwise strategy considering different polysaccharides present in varied seaweed types. Final selection of three microbes was based on the release of reducing sugars from different seaweed groups in the saccharification process. The selected microbes significantly increased the release of reducing sugars compared to the control conditions in all three seaweed species, with significant differences (P<0.05) based on the media, microbes, seaweed species, processed condition, and days of hydrolysis. Factor analysis of mixed data indicated that microbes contributed to the maximum variability of the data. Vibrio parahaemolyticus caused the maximum biomass conversion ratio for reducing sugars from S. wightii (22.31 ± 0.65%) and U. fasciata (24.6 ± 1.28%) with an increment of 8.9% and 9.35%, respectively from control conditions. The maximum biomass conversion of G. corticata was 24.8 ± 0.51% following Bacillus amyloliquefaciens treatment with an increment of 6.39% from the control. Even though different combinations of three prioritized microbes produced better saccharification than the control conditions, individual use of prioritized microbes made a better release of reducing sugars. In brief, seaweed hydrolysis using the prioritized microbes of the present study can be applied to improve the saccharification process of seaweeds in an eco-friendly and less expensive platform. [ABSTRACT FROM AUTHOR]

The pretreatment and saccharification of dewaxed bagasse (DWB) has been investigated under various reaction conditions ranging 2000 to 3200 psi, at 70 ± 1 °C in supercritical carbon dioxide (SCC). This has been in attempt to transform the DWB into fermentable sugar and bioethanol in high yields. The effect of SCC mediated pretreatment and enzymatic hydrolysis on structural and morphological alterations in DWB has been ascertained through diverse analytical methods. The sugar has been released through cellulase (40 FPU/mL) mediated enzymatic hydrolysis of pretreated DWB in sodium acetate buffer (pH 4.7) within 1 h at SCC 2800 psi, 70 ± 1 °C. The released sugar was subsequently fermented in the presence of yeast (Saccharomyces crevices, 135 CFU) at 28 ± 1 °C over 72 h to afford the bioethanol. The SCC mediated process conducted in acetic acid:water media (1:1) at 2800 psi, 70 ± 1 °C over 6 h has afforded the pretreated DWB with maximum yield towards the production of fermentable sugar and bioethanol. The production of fermentable sugar and bioethanol has been electrochemically estimated through cyclic voltammetry (CV) and square wave voltammetry (SWV) over glassy carbon electrode in KOH (0.1 M). The electrochemical methods were found selective and in close agreement for estimation of the yields (%) of fermentable sugars and bioethanol. The yield (%) of fermentable sugar estimated from CV and SWV were 80.10 ± 5.34 and 79.00 ± 5.09 respectively. Whereas the yield (%) of bioethanol estimated from CV and SWV were 81.30 ± 2.78% and 78.6 ± 1.25% respectively. Present investigation delivers a SCC mediated green and sustainable method of pretreatment of DWB to afford the enhanced saccharification, to produce bioethanol in high yields. [ABSTRACT FROM AUTHOR]

In this research, the energy potential of switchgrass (SG) was analyzed to find promising directions for producing bioenergy from this biomass. The first direction is determining the thermal energy of bioethanol extracted from SG biomass after its pretreatment, enzymatic hydrolysis (saccharification), and fermentation of the resulting glucose. It was established that after a two-stage pretreatment of 1 ton of SG with dilute solutions of nitric acid and alkali, the largest amount of bioethanol can be extracted with an energy potential of 4.9 GJ. It is also shown that by the utilization of solid and liquid waste, the production cost of bioethanol can be reduced. On the other hand, the direct combustion of 1 ton of the initial SG biomass used as a solid biofuel provides an increased amount of thermal energy of 18.3 GJ, which is 3.7 times higher than the energy potential of the resulting bioethanol extracted from 1 ton of this biomass. Thus, if the ultimate goal is to obtain the maximum energy amount, then another direction for obtaining bioenergy from biomass should be implemented, namely, direct combustion, preferably after pelletizing. Studies have shown that fuel characteristics of SG pellets such as the gross thermal energy and density of thermal energy are lower than those of wood pellets, but they can be improved if the SG biomass is densified into pellets together with binders made from polymer waste. [ABSTRACT FROM AUTHOR]

Corn cobs consist primarily of a lignocellulosic material comprising hemicellulose, cellulose, and lignin in a crystalline state, which is resistant to microbial saccharification. Bioethanol production from corn cobs has rarely been attempted, especially using chemical pretreatment methods. The present study deals with the production and purification of fungal (Phanerochaete chrysosporium MTCC 787 and Pleurotus florida PAU 22‐01) extracellular ligninolytic enzymes – lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase (Lac) – followed by their utilization for the biological pretreatment of corn cobs along with saccharification using commercial cellulase. Crude LiP, MnP, and Lac demonstrated specific activity of 2.23, 2.1, and 2.63 U/mg, respectively. The one‐step purification of crude enzyme using diethyl amino ethyl (DEAE) cellulose ion exchange chromatography resulted in 11.3, 10.1 and 8.62‐fold purification of LiP, MnP and Lac activity, respectively, with corresponding specific activity of 25.1 U/mg (LiP), 21.2 U/mg (MnP) and 22.7 U/mg (Lac) in the partially purified ligninozymes. Using the latter, biological pretreatment of 2.5 g corn cobs in a reaction volume of 30 mL containing approximately 200 units of Lac, Lip and MnP enzymes (in phosphate buffer, pH 6) resulted in a maximum of 78.4% delignification with a saccharification efficiency of 97.1% using commercial cellulases. [ABSTRACT FROM AUTHOR]

Areca nut husk (ANH) holds promise as a viable biomass source for xylose production. Xylose is a precursor for various biochemicals. However, the recalcitrant nature of ANH makes saccharification more complex. To address this, lime and acid pretreatments were carried out to enhance the susceptibility of biomass to saccharification. Before this, a compositional analysis was conducted to determine the initial constituents of the feedstock. Saccharification was conducted under the following conditions: 2% (wV−1) substrate loading, 100 rpm agitation, and 30 °C hydrolysis temperature for 12 h hydrolysis time at pH 4.5 to 5.0. However, parameters like xylanase enzyme loading were varied to enhance the saccharification of the ANH. The results demonstrated that acid‐treated husk (ATH), lime‐treated husk (LTH), and raw husk (RH) achieved the highest yield (gg−1) of reducing sugar, approximately 90, 83, and 15%, respectively, at an enzyme loading of 15.0 IUg−1. Various analytical techniques, including Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR), zeta potential, thermogravimetric analysis (TGA), X‐ray diffraction (XRD), and field emission scanning electron microscopy (FESEM) were used to examine structural changes in the native, pretreated, and saccharified residues of ANH. The analysis revealed that a significant amount of partial crystalline and amorphous cellulose in the ANH biomass was hydrolyzed during the saccharification process. However, saccharification also led to the removal of amorphous substances, disruption of the crystalline structure, and conversion of crystalline regions into amorphous domains. [ABSTRACT FROM AUTHOR]

Sawdust, a major waste product of the forestry industry, is accumulating along the Lagos Lagoon in Lagos, Nigeria, without it being effectively managed. Besides its use in manufacturing sound-absorbing boards to reinforce concrete beams and for energy purposes, its potential as a renewable energy source and feedstock for bio-product development has not yet been realized. Cellulose, a glucose biopolymer and structural component of cellulose can be hydrolyzed by a hydrolytic enzyme known as cellulase. During the process, the enzyme breaks the B-1,4-glucosidic bond, which keeps the glucose units together, and by acting on this bond, numerous glucose units are released. As part of sawdust, the cellulose molecule is not freely available for the degradation action of the cellulase enzyme as it is strongly associated with lignin, which acts as bio-glue, keeping cellulose and hemicellulose together. Delignification is an effective technique that was used to make the sawdust from ten different trees along the Lagos Lagoon in Nigeria more susceptible to saccharification by cellulase isolated from the fungus Aspergillus niger. Delignified and non-delignified sawdust masses between 2 mg and 10 mg were incubated with the A. niger cellulase solution (2 mg.mL-1), whereafter, the amount of sugar produced by the cellulase action was determined. The percentage saccharification of each sawdust material was also linked with the amount of sugar produced during cellulase action. From these investigations was concluded that delignification increased sugar production when almost all the masses of different sawdust materials were degraded. It was also observed that the ratio of sawdust mass to enzyme concentration is an important variable that influences the effectiveness of the saccharification process. The percentage saccharification of the various sawdust materials was also determined, and it indicated that the highest percentage of saccharification was not obtained when the highest amount of sawdust was degraded, producing the highest amount of sugar. The saccharification of sawdust could contribute to the development of renewable energy sources and feedstock for bioproduct development. The process is, however, not that straightforward as variables such as the type of cellulase enzyme, pretreatment of the cellulose substrate, and optimizing of cellulase to cellulose ratio are a few that need to be optimized for the process to be effective in terms of glucose production. [ABSTRACT FROM AUTHOR]

Forage maize is a versatile crop extensively utilized for animal nutrition in agriculture and holds promise as a valuable resource for the production of fermentable sugars in the biorefinery sector. Within this context, the carbohydrate fraction of the lignocellulosic biomass undergoes deconstruction during ruminal digestion and the saccharification process. However, the cell wall's natural resistance towards enzymatic degradation poses a significant challenge during both processes. This so-called biomass recalcitrance is primarily attributed to the presence of lignin and ferulates in the cell walls. Consequently, maize varieties with a reduced lignin or ferulate content or an altered lignin composition can have important beneficial effects on cell wall digestibility. Considerable efforts in genetic improvement have been dedicated towards enhancing cell wall digestibility, benefiting agriculture, the biorefinery sector and the environment. In part I of this paper, we review conventional and advanced breeding methods used in the genetic improvement of maize germplasm. In part II, we zoom in on maize mutants with altered lignin for improved digestibility and biomass processing. [ABSTRACT FROM AUTHOR]

The experiment was conducted during 2023 at Bioenergy Laboratory, Department of RBEE, College of Agricultural Engineering and Technology, CCSHAU, Hisar, Haryana, India. Compositional changes in sugarcane bagasse subjected to varying concentrations of sodium hydroxide (0.3% to 1.2%), revealing significant increases in glucan content (from 37.13% to 53.81%) alongside decreases in xylan, acidinsoluble lignin, acid-soluble lignin, ash, and other extractives. These changes were validated using microscopic technique SEM, confirming the efficacy of the pretreatment process. The utilization of a customized cellulase cocktail derived from Trichoderma harzianum and Trichoderma viride holds significant promise in enhancing the saccharification from alkali-pretreated sugarcane bagasse. This study investigates the synergistic effects of cellulase enzymes produced by these fungi on the hydrolysis of lignocellulosic biomass. The enzymatic hydrolysis process is optimized by varying enzyme dosages, reaction conditions, and incubation times to maximize the release of fermentable sugars. Results indicate a substantial improvement in saccharification efficiency with the customized cellulase cocktail, highlighting its potential for sustainable biofuel production. The pretreated sugarcane bagasse, when saccharified with Trichoderma harzianum and Trichoderma viride individually, released 254.43 mg g-1 and 325.53 mg g-1 of reducing sugars, respectively, after 40 h of incubation. In contrast, the combined enzymatic cocktail achieved a substantial increase in glucose yields (345.12 mg g-1) at 40 h, showcasing the synergistic effect of the combined enzymatic activity. This research contributes to advancing bioconversion technologies for utilizing lignocellulosic biomass resources efficiently and economically, thus addressing key challenges in sustainable energy production. [ABSTRACT FROM AUTHOR]

In order to improve the process feasibility and cost function of bioethanol production, higher titres of biomass saccharifying cellulases need to be produced on commercial scale. Diverse lignocellulosic substrates including abundantly accessible paddy straw can be utilized as carbonaceous substrates for the production of cellulose hydrolysing enzymes. This study aims to meliorate milled and sequential acid/alkali pre-treated paddy straw as inducer-substrates for the synthesis of fungal secretomes from Penicillium mallochii repertoires under solid state and liquid shake flask fermentation (SSF and LSF, respectively). The analysis of enzymatic activities of the respective secretomes reinforced the cellulolytic potential of P. mallochii where the maximum cellulase production (Filter paper cellulase: 76.43, Carboxymethyl cellulase: 130.29, Avicelase: 18.6 and β-glucosidase: 83.59 U L−1) was exhibited under LSF conditions using pre-treated paddy straw as the inducer. The disorganisation of the cellulosic structure via hydrogen bond disruption, as indicated by FTIR analysis, after acid/alkali pretreatment allowed a better establishment of fungus on the substrate, thereby facilitating higher cellulase production. A maximum ethanol content of 11.63 g L−1 was obtained at 48 h of simultaneous saccharification and fermentation using pre-hydrolysed paddy straw at 10% (w/v) solid loading and a cellulase dosage of 25 FPU gds−1. These results affirm the utilization of paddy straw for cellulase and ethanol production in an integrated bioprocess that can further be explored and optimized for scale-up studies to cater industrial applications. [ABSTRACT FROM AUTHOR]

The effective recovery of the immobilized enzymes using magnetic carriers has led to growing interest in this technology. The objective of this research was to evaluate the efficiency of immobilized laccase on magnetized multiwall carbon nanotubes (m-MWCNTs) in terms of stability and reusability. Laccases were efficiently adsorbed onto magnetized multiwall carbon nanotubes (m-MWCNTs) synthesized using water. The concentration of 7 mg laccase/mL was found to be ideal for immobilization. The optimal activity of both free and immobilized laccases was observed at pH 5, while for the latter, the optimal temperature was shifted from 40 to 50 °C. Compared to the free laccase, the immobilized laccase exhibited a greater range of stability at more extreme temperatures. At the fourth cycle of reactions, the immobilized laccase exhibited more than 60% relative activity in terms of reusability. Based on the fourier-transform infrared spectroscopy (FTIR) peak at 2921 cm−1, saccharification of paddy straw using immobilized laccase verified lignin degradation. The easy recovery of the immobilized laccase on m-MWCNTs lends credence to its potential use in biomass hydrolysis. [ABSTRACT FROM AUTHOR]

In this research, the energy potential of switchgrass (SG) was analyzed to find promising directions for producing bioenergy from this biomass. The first direction is determining the thermal energy of bioethanol extracted from SG biomass after its pretreatment, enzymatic hydrolysis (saccharification), and fermentation of the resulting glucose. It was established that after a two-stage pretreatment of 1 ton of SG with dilute solutions of nitric acid and alkali, the largest amount of bioethanol can be extracted with an energy potential of 4.9 GJ. It is also shown that by the utilization of solid and liquid waste, the production cost of bioethanol can be reduced. On the other hand, the direct combustion of 1 ton of the initial SG biomass used as a solid biofuel provides an increased amount of thermal energy of 18.3 GJ, which is 3.7 times higher than the energy potential of the resulting bioethanol extracted from 1 ton of this biomass. Thus, if the ultimate goal is to obtain the maximum energy amount, then another direction for obtaining bioenergy from biomass should be implemented, namely, direct combustion, preferably after pelletizing. Studies have shown that fuel characteristics of SG pellets such as the gross thermal energy and density of thermal energy are lower than those of wood pellets, but they can be improved if the SG biomass is densified into pellets together with binders made from polymer waste.

Abstract Background Lignin and xylan are important determinants of cell wall structure and lignocellulosic biomass digestibility. Genetic manipulations that individually modify either lignin or xylan structure improve polysaccharide digestibility. However, the effects of their simultaneous modifications have not been explored in a similar context. Here, both individual and combinatorial modification in xylan and lignin was studied by analysing the effect on plant cell wall properties, biotic stress responses and integrity sensing. Results Arabidopsis plant co-harbouring mutation in FERULATE 5-HYDROXYLASE (F5H) and overexpressing Aspergillus niger acetyl xylan esterase (35S:AnAXE1) were generated and displayed normal growth attributes with intact xylem architecture. This fah1-2/35S:AnAXE1 cross was named as hyper G lignin and hypoacetylated (HrGHypAc) line. The HrGHypAc plants showed increased crystalline cellulose content with enhanced digestibility after chemical and enzymatic pre-treatment. Moreover, both parents and HrGHypAc without and after pre-treating with glucuronyl esterase and alpha glucuronidase exhibited an increase in xylose release after xylanase digestion as compared to wild type. The de-pectinated fraction in HrGHypAc displayed elevated levels of xylan and cellulose. Furthermore, the transcriptomic analysis revealed differential expression in cell wall biosynthetic, transcription factors and wall-associated kinases genes implying the role of lignin and xylan modification on cellular regulatory processes. Conclusions Simultaneous modification in xylan and lignin enhances cellulose content with improved saccharification efficiency. These modifications loosen cell wall complexity and hence resulted in enhanced xylose and xylobiose release with or without pretreatment after xylanase digestion in both parent and HrGHypAc. This study also revealed that the disruption of xylan and lignin structure is possible without compromising either growth and development or defense responses against Pseudomonas syringae infection.

Biomass pretreatment for the production of second-generation (2G) ethanol and biochemical products is a challenging process. The present study investigated the synergistic efficiency of purified carboxymethyl cellulase (CMCase), β-glucosidase, and xylanase from Aspergillus fumigatus JCM 10253 in the hydrolysis of alkaline-pretreated sugarcane bagasse (SCB). The saccharification of pretreated SCB was optimised using a combination of CMCase and β-glucosidase (C + β; 1:1) and addition of xylanase (C + β + xyl; 1:1:1). Independent and dependent variables influencing enzymatic hydrolysis were investigated using response surface methodology (RSM). Hydrolysis using purified CMCase and β-glucosidase achieved yields of 18.72 mg/mL glucose and 6.98 mg/mL xylose. Incorporation of xylanase in saccharification increased the titres of glucose (22.83 mg/mL) and xylose (9.54 mg/mL). Furthermore, characterisation of SCB biomass by scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy respectively confirmed efficient structural disintegration and revealed the degree of crystallinity and spectral characteristics. Therefore, depolymerisation of lignin to produce high-value chemicals is essential for sustainable and competitive biorefinery development. [ABSTRACT FROM AUTHOR]

Bacillus sp. PM06, previously isolated from sugarcane waste pressmud, could produce dual enzymes α-amylase and cellulase. The isolate's crude enzymes were purified homogeneously using ammonium sulfate precipitation followed by High Quaternary amine anion exchange chromatography. Purified enzymes revealed the molecular weights of α-amylase and cellulase as 55 and 52 kDa, with a purification fold of 15.4 and 11.5, respectively. The specific activity of purified α-amylase and cellulase were 740.7 and 555.6 U/mg, respectively. It demonstrated a wide range of activity from pH 5.0 to 8.5, with an optimum pH of 5.5 and 6.4 for α-amylase and cellulase. The optimum temperature was 50 °C for α-amylase and 60 °C for cellulase. The kinetic parameters of purified α-amylase were 741.5 ± 3.75 µmol/min/mg, 1.154 ± 0.1 mM, and 589 ± 3.5/(smM), using starch as a substrate. Whereas cellulase showed 556.3 ± 1.3 µmol/min/mg, 1.78 ± 0.1 mM, and 270.9 ± 3.8/(smM) of Vmax,Km, Kcat/Km, respectively, using carboxymethyl cellulose (CMC) as substrate. Among the various substrates tested, α-amylase had a higher specificity for amylose and CMC for cellulase. Different inhibitors and activators were also examined. Ca2+ Mg2+, Co2+, and Mn2+ boosted α-amylase and cellulase activities. Cu2+ and Ni2+ both inhibited the enzyme activities. Enzymatic saccharification of wheat bran yielded 253.61 ± 1.7 and 147.5 ± 1.0 mg/g of reducing sugar within 12 and 24 h of incubation when treated with purified α-amylase and cellulase. A more significant amount of 397.7 ± 1.9 mg/g reducing sugars was released from wheat bran due to the synergetic effect of two enzymes. According to scanning electron micrograph analysis, wheat bran was effectively broken down by both enzymes. [ABSTRACT FROM AUTHOR]

Lignocellulosic biomass represents an excellent alternative to fossil fuels in terms of both energy production and raw material usage for a plethora of daily-use products. Organosolv pretreatment is a fractionation technique able to separate lignocellulosic biomass into individual streams of cellulose, hemicellulose, and lignin under controlled conditions. Sawdust, the by-product of sawmill processing of Picea abies wood, was the subject of our investigation in this work. The aim was to evaluate the effects of different parameters of the organosolv process of spruce sawdust on the yield of components and how this affects the enzymatic saccharification of cellulose. Sixteen distinct pretreatments were performed with ethanol concentrations of 50 and 60% v/v at 180 and 200 °C for 15 and 30 min. Half of the pretreatments contained 1% sulfuric acid as a catalyst, while the other half were acid-free. Thereafter, the effects of different variables on the yield of products were assessed and compared to determine the ideal pretreatment condition. The results showed that cellulose-rich pulps, with cellulose content as high as 55% were generated from an initial mass of 37.7% spruce sawdust with the reactor operating at 180 °C for 30 min using 60% ethanol and 1% sulfuric acid. With the pretreatments performed with the catalyst at 200 °C, hemicellulose was almost entirely removed from the pulps obtained. The recovered hemicellulose fraction was composed mainly of monomers achieving up to 10 g/100 g of biomass. Delignification values of up to 65.7% were achieved with this pretreatment technique. Fractionated lignin presented low levels of sugar and ashes contamination, with values as low as 1.29% w/w. Enzymatic saccharification of the pretreated pulps yielded 78% cellulose hydrolysis, with glucose release higher than 0.54 g/g of biomass, indicating the potential of the pulps to be applied in a fermentation process. [ABSTRACT FROM AUTHOR]

In the current study, we compared the production of extracellular lignocellulose degrading enzymes and bioethanol from the spent mushroom substrate (SMS) of Calocybe indica and Volvariella volvacea. From SMS at different stages of the mushroom development cycle, ligninolytic and hydrolytic enzymes were analysed. The activities of lignin-degrading enzymes, including lignin peroxidase (LiP), laccase, and manganese peroxidase (MnP) were maximal in the spawn run and primordial stages, while hydrolytic enzymes including xylanase, cellobiohydrolase (CBH), and carboxymethyl cellulase (CMCase) showed higher activity during fruiting bodies development and at the end of the mushroom growth cycle. SMS of V. volvacea showed relatively lower ligninase activity than the SMS of C. indica, but had the maximum activity of hydrolytic enzymes. The enzyme was precipitated with acetone and further purified with the DEAE cellulose column. The maximum yield of reducing sugars was obtained after hydrolysis of NaOH (0.5 M) pretreated SMS with a cocktail of partially purified enzymes (50% v/v). After enzymatic hydrolysis, the total reducing sugars were 18.68 ± 0.34 g/l (SMS of C. indica) and 20.02 ± 0.87 g/l (SMS of V. volvacea). We observed the highest fermentation efficiency and ethanol productivity (54.25%, 0.12 g/l h) obtained from SMS hydrolysate of V. volvacea after 48 h at 30 ± 2 °C, using co-culture of Saccharomyces cerevisiae MTCC 11,815 and Pachysolen tannophilus MTCC 1077. [ABSTRACT FROM AUTHOR]

Lignocellulosic cellulose serves as a key source for bioethanol production. The efficient conversion of cellulose relies on three major cellulase components. High cost of cellulases and the need for a single microbe that produces all cellulase components in the right proportion and quantities are a few challenges in bioethanol production from cellulose. This investigation is an attempt in developing an enzyme cocktail involving recombinant thermostable cellulases (rMtEgl, rMtCel6A and rMtBgl3c) of the thermophilic mold Myceliophthora thermophila and testing their applicability in the conversion of agro residues to ethanol for the first time. The most effective pretreatment method for paddy straw and sugarcane bagasse was optimized. Pretreatment of sugarcane bagasse with sodium chlorite and acetic acid resulted in a 5.5-fold increase in total reducing sugar liberation, while, in paddy straw, total reducing sugar release increased by 9-fold, when biomass was treated with NaOH and microwaves compared to untreated biomass. The inclusion of recombinant enzymes in the enzyme cocktail supported 80–90% saccharification of pretreated paddy straw and sugarcane bagasse, which is 2-fold higher than that achieved using commercial enzyme mix alone. The ethanol production levels of 55.8 and 37.0 g/L, with the fermentation efficiencies of 80 and 76%, were attained from the pre-treated paddy straw and sugarcane bagasse hydrolysates, respectively. An appropriate blend of each enzyme component and pretreatment method tailored for the specific biomass is crucial for efficient biofuel production. [ABSTRACT FROM AUTHOR]

Background: Lignin and xylan are important determinants of cell wall structure and lignocellulosic biomass digestibility. Genetic manipulations that individually modify either lignin or xylan structure improve polysaccharide digestibility. However, the effects of their simultaneous modifications have not been explored in a similar context. Here, both individual and combinatorial modification in xylan and lignin was studied by analysing the effect on plant cell wall properties, biotic stress responses and integrity sensing. Results: Arabidopsis plant co-harbouring mutation in FERULATE 5-HYDROXYLASE (F5H) and overexpressing Aspergillus niger acetyl xylan esterase (35S:AnAXE1) were generated and displayed normal growth attributes with intact xylem architecture. This fah1-2/35S:AnAXE1 cross was named as hyper G lignin and hypoacetylated (HrGHypAc) line. The HrGHypAc plants showed increased crystalline cellulose content with enhanced digestibility after chemical and enzymatic pre-treatment. Moreover, both parents and HrGHypAc without and after pre-treating with glucuronyl esterase and alpha glucuronidase exhibited an increase in xylose release after xylanase digestion as compared to wild type. The de-pectinated fraction in HrGHypAc displayed elevated levels of xylan and cellulose. Furthermore, the transcriptomic analysis revealed differential expression in cell wall biosynthetic, transcription factors and wall-associated kinases genes implying the role of lignin and xylan modification on cellular regulatory processes. Conclusions: Simultaneous modification in xylan and lignin enhances cellulose content with improved saccharification efficiency. These modifications loosen cell wall complexity and hence resulted in enhanced xylose and xylobiose release with or without pretreatment after xylanase digestion in both parent and HrGHypAc. This study also revealed that the disruption of xylan and lignin structure is possible without compromising either growth and development or defense responses against Pseudomonas syringae infection. [ABSTRACT FROM AUTHOR]

The lignocellulolytic accessory enzyme, Feruloyl esterase C (FE_5DR), encoded in the genome of thermotolerant Myceliophthora verrucosa was successfully cloned and heterologously expressed in Pichia pastoris. The expressed FE_5DR was purified using UNOsphere™ Q anion exchange chromatography column, exhibiting a homogeneous band of ~ 39 kDa. Its optimum temperature was determined to be 60 °C, with an optimal pH of 6.0. Additionally, the enzyme activity of FE_5DR was significantly enhanced by preincubation in a buffer containing Mg2+, Cu2+ and Ca2 metal ions. Enzyme kinetic parameters, computed from double reciprocal Lineweaver–Burk plots, yielded observed Vmax and Km values of 0.758 U/mg and 0.439 mM, respectively. Furthermore, the potential of custom-made cocktails comprising FE_5DR and benchmark cellulase derived from the developed mutant strain of Aspergillus allahabadii MAN 40, as well as the biorefinery-relevant lignocellulolytic enzyme Cellic CTec 3, resulted in improved saccharification of unwashed acid pretreated (UWAP) rice straw slurry and mild alkali deacetylated (MAD) rice straw when compared to benchmark MAN 40 and Cellic CTec 3. [ABSTRACT FROM AUTHOR]

The hydrolysis and biotransformation of lignocellulose, i.e., biorefinery, can provide human beings with biofuels, bio-based chemicals, and materials, and is an important technology to solve the fossil energy crisis and promote global sustainable development. Biorefinery involves steps such as pretreatment, saccharification, and fermentation, and researchers have developed a variety of biorefinery strategies to optimize the process and reduce process costs in recent years. Lignocellulosic hydrolysates are platforms that connect the saccharification process and downstream fermentation. The hydrolysate composition is closely related to biomass raw materials, the pretreatment process, and the choice of biorefining strategies, and provides not only nutrients but also possible inhibitors for downstream fermentation. In this review, we summarized the effects of each stage of lignocellulosic biorefinery on nutrients and possible inhibitors, analyzed the huge differences in nutrient retention and inhibitor generation among various biorefinery strategies, and emphasized that all steps in lignocellulose biorefinery need to be considered comprehensively to achieve maximum nutrient retention and optimal control of inhibitors at low cost, to provide a reference for the development of biomass energy and chemicals. [ABSTRACT FROM AUTHOR]

Sugarcane bagasse (SCB), an agro-industrial byproduct generated by a sugar mill, holds a substantial carbohydrate content of around 70 wt.%, comprising cellulose and hemicellulose. Saccharification plays a pivotal role in the conversion of SCB into second-generation (2G)-ethanol and valuable compounds, which is significantly aided by thermochemical pretreatments. In this study, SCB underwent diluted sulfuric acid pretreatment (2% H2SO4, 80 rpm, 200 °C, 20 min), resulting in the removal of 77.3% of the xylan. The hemicellulosic hydrolysate was analyzed to identify the sugars and degraded products acting as microbial inhibitors. The acid hydrolysate showed a xylose yield of 68.0% (16.4 g/L) and a yield of 3.8 g/L of acetic acid. Afterward, the hemicellulosic hydrolysate was concentrated 2.37 times to obtain a xylose-rich stream (39.87 g/L). The sequential detoxification, employing calcium oxide and activated carbon, removed the inhibitory compounds, including acetic acid, while preserving the xylose at 38.10 g/L. The enzymatic saccharification of cellulignin at 5% and 10% of the total solids (TSs) yielded comparable reducing sugar (RS) yields of 47.3% (15.2 g/L) and 47.4% (30.4 g/L), respectively, after 96 h, employing a 10 FPU/g enzyme loading of Cellic® CTec3 (Novozymes Inc. Parana, Brazil). In summary, these findings outline an integrated green chemistry approach aimed at addressing the key challenges associated with pretreatment, concentration, detoxification, and enzymatic hydrolysis to produce fermentable sugars. [ABSTRACT FROM AUTHOR]

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