Saccharification Of Sugarcane Bagasse Research Articles

Cloning, expression and purification of cellobiohydrolase gene from Caldicellulosiruptor bescii for efficient saccharification of plant biomass

Anthropogenic activities have led to a drastic shift from natural fuels to alternative renewable energy reserves that demand heat-stable cellulases. Cellobiohydrolase is an indispensable member of cellulases that play a critical role in the degradation of cellulosic biomass. This article details the process of cloning the cellobiohydrolase gene from the thermophilic bacterium Caldicellulosiruptor bescii and expressing it in Escherichia coli (BL21) CondonPlus DE3-(RIPL) using the pET-21a(+) expression vector. Multi-alignments and structural modeling studies reveal that recombinant CbCBH contained a conserved cellulose binding domain III. The enzyme's catalytic site included Asp-372 and Glu-620, which are either involved in substrate or metal binding. The purified CbCBH, with a molecular weight of 91.8 kDa, displayed peak activity against pNPC (167.93 U/mg) at 65°C and pH 6.0. Moreover, it demonstrated remarkable stability across a broad temperature range (60–80°C) for 8 h. Additionally, the Plackett-Burman experimental model was employed to assess the saccharification of pretreated sugarcane bagasse with CbCBH, aiming to evaluate the cultivation conditions. The optimized parameters, including a pH of 6.0, a temperature of 55°C, a 24-hour incubation period, a substrate concentration of 1.5% (w/v), and enzyme activity of 120 U, resulted in an observed saccharification efficiency of 28.45%. This discovery indicates that the recombinant CbCBH holds promising potential for biofuel sector.

Enhanced Production of Clean Fermentable Sugars by Acid Pretreatment and Enzymatic Saccharification of Sugarcane Bagasse

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.

Exploring the potential of a new thermotolerant xylanase from Rasamsonia composticola (XylRc): production using agro-residues, biochemical studies, and application to sugarcane bagasse saccharification.

Xylanases from thermophilic fungi have a wide range of commercial applications in the bioconversion of lignocellulosic materials and biobleaching in the pulp and paper industry. In this study, an endoxylanase from the thermophilic fungus Rasamsonia composticola (XylRc) was produced using waste wheat bran and pretreated sugarcane bagasse (PSB) in solid-state fermentation. The enzyme was purified, biochemically characterized, and used for the saccharification of sugarcane bagasse. XylRc was purified 30.6-fold with a 22% yield. The analysis using sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed a molecular weight of 53kDa, with optimal temperature and pH values of 80°C and 5.5, respectively. Thin-layer chromatography suggests that the enzyme is an endoxylanase and belongs to the glycoside hydrolase 10 family. The enzyme was stimulated by the presence of K+, Ca2+, Mg2+, and Co2+ and remained stable in the presence of the surfactant Triton X-100. XylRc was also stimulated by organic solvents butanol (113%), ethanol (175%), isopropanol (176%), and acetone (185%). The Km and Vmax values for oat spelt and birchwood xylan were 6.7 ± 0.7mg/mL, 2.3 ± 0.59mg/mL, 446.7 ± 12.7µmol/min/mg, and 173.7 ± 6.5µmol/min/mg, respectively. XylRc was unaffected by different phenolic compounds: ferulic, tannic, cinnamic, benzoic, and coumaric acids at concentrations of 2.5-10mg/mL. The results of saccharification of PSB showed that supplementation of a commercial enzymatic cocktail (Cellic® CTec2) with XylRc (1:1 w/v) led to an increase in the degree of synergism (DS) in total reducing sugar (1.28) and glucose released (1.05) compared to the control (Cellic® HTec2). In summary, XylRc demonstrated significant potential for applications in lignocellulosic biomass hydrolysis, making it an attractive alternative for producing xylooligosaccharides and xylose, which can serve as precursors for biofuel production.

Comparative analysis of Chrysoporthe cubensis exoproteomes and their specificity for saccharification of sugarcane bagasse

The phytopathogenic fungus Chrysoporthe cubensis is a relevant source of lignocellulolytic enzymes. This work aimed to compare the profile of lignocellulose-degrading proteins secreted by C. cubensis grown under semi-solid state fermentation using wheat bran (WB) and sugarcane bagasse (SB). The exoproteomes of the fungus grown in wheat bran (WBE) and sugarcane bagasse (SBE) were qualitative and quantitatively analyzed by liquid chromatography-electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS). Data are available via ProteomeXchange with identifier PXD046075. Label-free proteomic analysis of WBE and SBE showed that the fungus produced a spectrum of carbohydrate-active enzymes (CAZymes) with exclusive characteristics from each extract. While SBE resulted in an enzymatic profile directed towards the depolymerization of cellulose, the enzymes in WBE were more adaptable to the degradation of biomass rich in hemicellulose and other non-lignocellulosic polymers. Saccharification of alkaline pre-treated sugarcane bagasse with SBE promoted glucose release higher than commercial cocktails (8.11 g L−1), while WBE promoted the higher release of xylose (5.71 g L−1). Our results allowed an in-depth knowledge of the complex set of enzymes secreted by C. cubensis responsible for its high lignocellulolytic activity and still provided the identification of promising target proteins for biotechnological applications in the context of biorefinery.

Bioacetoin Production by Bacillus subtilis subsp. subtilis Using Enzymatic Hydrolysate of Lignocellulosic Biomass

Acetoin is an important bio-product useful in the chemical, food and pharmaceutical industries. Microbial fermentation is the major process for the production of bioacetoin, as the petroleum resources used in chemical methods are depleting day by day. Bioacetoin production using wild microorganisms is an easy, eco-friendly and economical method for the production of bioacetoin. In the present study, culture conditions and nutritional requirements were optimized for bioacetoin production by a wild and non-pathogenic strain of B. subtilis subsp. subtilis JJBS250. The bacterial culture produced maximum bioacetoin (259 mg L−1) using peptone (3%) and sucrose (2%) at 30 °C, 150 rpm and pH 7.0 after 24 h. Further supplementation of combinatorial nitrogen sources, i.e., peptone (1%) and urea (0.5%), resulted in enhanced titre of bioacetoin (1017 mg L−1) by the bacterial culture. An approximately 46.22–fold improvement in bioacetoin production was achieved after the optimization process. The analysis of samples using thin layer chromatography confirmed the presence of bioacetoin in the culture filtrate. The enzymatic hydrolysate was obtained by saccharification of pretreated rice straw and sugarcane bagasse using cellulase from Myceliophthora thermophila. Fermentation of the enzymatic hydrolysate (3%) of pretreated rice straw and sugarcane bagasse by the bacterial culture resulted in 210 and 473.17 mgL−1 bioacetoin, respectively. Enzymatic hydrolysates supplemented with peptone as a nitrogen source showed a two to four-fold improvement in the production of bioacetoin. Results have demonstrated the utility of wild type B. subtilis subsp. subtilis JJBS250 as a potential source for economical bioacetoin production by making use of renewable and cost-effective lignocellulosic substrate. Therefore, this study will help in the sustainable management of agricultural waste for the industrial production of bioacetoin, and in combating environmental pollution.

Production of cellulases from Amazonian fungi and their application in babassu cellulose hydrolysis

Two Amazonian fungi strains — Aspergillus sp. A1C2-06 and Talaromyces verruculosus A1C2-05 — were studied about their potential as cellulase producers and theis applications in the saccharification of sugarcane bagasse and babassu lignocellulosic biomass. For the cellulase production was studied the influence of cultivation parameters during the solid-state fermentation. The best initial pH were 3,0 and 7,0 for T. verruculosus and Aspergillus sp., respectively, and the optimum temperature was 35 °C for both strains. The ideal Mandel’ solution volume was 7,5 ml and 10,0 ml for T. verruculosus and Aspergillus sp., respectively. The optimal conditions during the hydrolysis of filter paper cellulose were determined, being 2,8 the optimal pH for both cellulases and the optimal temperature were 50 °C and 60 °C for Aspergillus sp and T. verruculosus, respectively. The cellulases from both Aspergillus sp and T. verruculosus were capable of hydrolyzing the lignocellulosic biomasses but in different degree of saccharification. The saccharification degree for both pretreated biomasses reached 80% when cellulases from T. verruculosus were applied for 72h, while the untreated biomasses reached only 60% for babassu and 50% for sugarcane bagasse, indicating that the removal of lignin has an important role in the process of biomass saccharification.

SACARIFICACIÓN ENZIMÁTICA DE BAGAZO DE CAÑA DE AZÚCAR PRETRATADO CON PEROXIDO DE HIDROGENO PARA LA PRODUCCIÓN DE BIOETANOL

<p><strong>Background.</strong> Sugarcane (<em>Saccharum officinarum</em> L.) is one of the main crops in Mexico; during the harvest 2021-2022, 54.6 million tons of raw milled sugarcane were industrialized, ranking sixth in production worldwide. However, during processing, several products and co-products are generated, including sugarcane bagasse, that is used to produce electricity by combustion, converting nearly 25% of its weight into greenhouse gases and generating environmental problems. For this reason, it is necessary to design processes aligned with the axes of sustainable development (environmental, social and economic) to promote valorization and diversification. <strong>Objective</strong>. To determine the effect of two pretreatments on the enzymatic saccharification of sugarcane bagasse for its subsequent conversion to bioethanol. <strong>Methodology.</strong> The production of enzymatic extracts by solid state fermentation (SSF) was carried out using sugarcane bagasse as support-substrate and autochthonous fungal strains as inoculum. For the enzymatic saccharification the sugarcane bagasse was pretreated with hydrogen peroxide and microwaves, for the hydrogen peroxide pretreatment the variables time (1, 3 and 6 h), ratio mL of peroxide per gram of bagasse (16. 6:1, 33.3:1 and 100:1), temperature (26, 50 and 70°C) and peroxide concentration (1, 3 and 5%); and for microwave pretreatment the variables microwave power (p30, p50 and p80) and time (5 and 10 min) were evaluated. For the enzymatic saccharification, enzymatic extracts produced by SSF and bagasse pretreated with hydrogen peroxide were used. Finally, fermentable sugars obtained from the enzymatic saccharification of sugarcane bagasse were used as a unique carbon source in alcoholic fermentation system. <strong>Results.</strong> The SSF results showed the presence of cellulase (2 U/g) and xylanase (61 U/g) activity. On the other hand, the results of pretreatment of sugarcane bagasse with hydrogen peroxide indicated that the best conditions were 100 mL of 3% hydrogen peroxide per gram of sugarcane bagasse at 50°C, while the best conditions of microwave pretreatment were 50p for 5 minutes. The enzymatic saccharification of sugarcane bagasse showed a maximum yield of reducing sugars when the sugarcane bagasse was pretreated with hydrogen peroxide, obtaining 0.41 g/g. Finally, the maximum ethanol concentration (4.32 g/L) was obtained after 48 hours of fermentation with commercial <em>Saccharomyces cerevisae</em>. <strong>Implications.</strong> The sugar agroindustry in Mexico is limited to the production of sucrose and ethanol, being necessary a sustainable productive diversification. <strong>Conclusions.</strong> The isolation of autochthonous fungal strains allowed the production of cellulases and xylanases by solid fermentation with adequate specificity to hydrolyze/saccharify pretreated sugarcane bagasse and its subsequent conversion to ethanol.</p>

Selective saccharification of crude glycerol pretreated sugarcane bagasse via fast pyrolysis: Reaction kinetics and life cycle assessment

Saccharification is a pivotal step in the conversion of lignocellulose to biofuels and chemicals. In this study, crude glycerol derived from biodiesel production was used in pretreatment to facilitate efficient and clean pyrolytic saccharification of sugarcane bagasse. Delignification, demineralization, destruction of lignin-carbohydrate complex structure, and cellulose crystallinity improvement in crude glycerol pretreated biomass could enhance levoglucosan producing reactions against competitive reactions, and therefore facilitate a kinetically controlled pyrolysis with apparent activation energy increased by 2-fold. Accordingly, selective levoglucosan production (44.4%) was promoted by 6-fold whilst light oxygenates and lignin monomers were limited to <25% in bio-oil. Owing to the high-efficiency saccharification, life cycle assessment suggested that the environmental impacts of the integrated process were less than those of typical acid pretreatment and petroleum-based processes, especially on the acidification (8-fold less) and global warming potential. This study provides an environmentally benign approach to efficient biorefinery and waste management.

Production of recombinant lytic polysaccharide monooxygenases and evaluation effect of its addition into Aspergillus fumigatus var. niveus cocktail for sugarcane bagasse saccharification

Lignocellulosic biomass is a promising alternative for producing biofuels, despite its recalcitrant nature. There are microorganisms in nature capable of efficiently degrade biomass, such as the filamentous fungi. Among them, Aspergillus fumigatus var. niveus (AFUMN) has a wide variety of carbohydrate-active enzymes (CAZymes), especially hydrolases, but a low number of oxidative enzymes in its genome. To confirm the enzymatic profile of this fungus, this study analyzed the secretome of AFUMN cultured in sugarcane bagasse as the sole carbon source. As expected, the secretome showed a predominance of hydrolytic enzymes compared to oxidative activity. However, it is known that hydrolytic enzymes act in synergy with oxidative proteins to efficiently degrade cellulose polymer, such as the Lytic Polysaccharide Monooxygenases (LPMOs). Thus, three LPMOs from the fungus Thermothelomyces thermophilus (TtLPMO9D, TtLPMO9H, and TtLPMO9O) were selected, heterologous expressed in Aspergillus nidulans, purified, and used to supplement the AFUMN secretome to evaluate their effect on the saccharification of sugarcane bagasse. The saccharification assay was carried out using different concentrations of AFUMN secretome supplemented with recombinant T. thermophilus LPMOs, as well as ascorbic acid as reducing agent for oxidative enzymes. Through a statistic design created by Design-Expert software, we were able to analyze a possible cooperative effect between these components. The results indicated that, in general, the addition of TtLPMO9D and ascorbic acid did not favor the conversion process in this study, while TtLPMO9O had a highly significant cooperative effect in bagasse saccharification compared to the control using only AFUMN secretome.

Biochemical characteristics of Myceliophthora thermophila recombinant β-glucosidase (MtBgl3c) applicable in cellulose bioconversion

The GH3 β-glucosidase gene of Myceliophthora thermophila (MtBgl3c) has been cloned and heterologously expressed in E. coli for the first time. This study highlights the important characteristics of recombinant MtBgl3c (rMtBgl3c) which make it a promising candidate in industrial applications. Optimization of the production of rMtBgl3c led to 28,000 U L−1. On purification, it has a molecular mass of ∼100 kDa. It is a broad substrate specific thermostable enzyme that exhibits pH and temperature optima at 5.0 and 55 °C, respectively. The amino acid residues Asp287 and Glu514 act as nucleophile and catalytic acid/base, respectively in the enzyme catalysis. Its low Km value (1.28 mM) indicates a high substrate affinity as compared to those previously reported. The rMtBgl3c displays a synergistic action with the commercial enzyme cocktail in the saccharification of sugarcane bagasse suggesting its utility in the cellulose bioconversion. Tolerance to solvents, detergents as well as glucose make this enzyme applicable in wine, detergent, paper and textile industries too.

Enhanced cellulosic ethanol production via fed-batch simultaneous saccharification and fermentation of sequential dilute acid-alkali pretreated sugarcane bagasse

This study reports high gravity fed-batch simultaneous saccharification and fermentation (FB-SSF) of sequentially pretreated sugarcane bagasse (SCB) for enhanced bioethanol by employing multiple inhibitor tolerant Kluyveromyces marxianusJKH5 C60. FB-SSF with intermittent feeding of SCB (total 20 % solid loading) and enzyme (total dose of 20 FPU/g) at 6 and 12 h resulted in superior bioethanol production at42 °C. Under optimizedlab-scaleFB-SSF, the maximum ethanoltiter, efficiency and productivities were73.4 ± 1.2 g/L,78 % and 3.0 g/L/h, respectively, after 72 h in presence of inhibitors (acetic acid, furfural, and vanillin at 3, 1, and 1 g/L, respectively). Furthermore, pentose rich dilute acid hydrolysate of SCB was subjected to fermentation by Pichia stipitis NCIM 3499, resulting in ethanol titer of 6.8 g/L. Overall ethanol yield during the developed process was 260.1 g/kg native SCB, which proves industrial potential of the developed bioethanol conversion process.

Characterization of a GH5 endoxylanase from Penicillium funiculosum and its synergism with GH16 endo-1,3(4)-glucanase in saccharification of sugarcane bagasse

The production of second-generation fuels from lignocellulosic residues such as sugarcane bagasse (SCB) requires the synergistic interaction of key cellulose-degrading enzymes and accessory proteins for their complete deconstruction to useful monomeric sugars. Here, we recombinantly expressed and characterized unknown GH5 xylanase from P. funiculosum (PfXyn5) in Pichia pastoris, which was earlier found in our study to be highly implicated in SCB saccharification. The PfXyn5 has a molecular mass of ~ 55 kDa and showed broad activity against a range of substrates like xylan, xyloglucan, laminarin and p-nitrophenyl-β-d-xylopyranoside, with the highest specific activity of 0.7 U/mg against xylan at pH 4.5 and 50 °C. Analysis of the degradation products of xylan and SCB by PfXyn5 showed significant production of xylooligosaccharides (XOS) with a degree of polymerization (DP) ranging from two (DP2) to six (DP6), thus, suggesting that the PfXyn5 is an endo-acting enzyme. The enzyme synergistically improved the saccharification of SCB when combined with the crude cellulase cocktail of P. funiculosum with a degree of synergism up to 1.32. The PfXyn5 was further expressed individually and simultaneously with a notable GH16 endoglucanase (PfEgl16) in a catabolite-derepressed strain of P. funiculosum, PfMig188, and the saccharification efficiency of the secretomes from the resulting transformants were investigated on SCB. The secretome of PfMig188 overexpressing Xyn5 or Egl16 increased the saccharification of SCB by 9% or 7%, respectively, over the secretome of PfMig188, while the secretome of dual transformant increased SCB saccharification by ~ 15% at the same minimal protein concentration.

Wastes recycling of non-sterile cellulosic ethanol production from low-temperature pilot-scale enzymatic saccharification of alkali-treated sugarcane bagasse

High energy consumption has been one of bottlenecks for bioconversion of lignocellulose into biofuels and biochemicals. A self-designed pilot-scale device with recycling of black liquor (BL) and waste washing water (WWW) was constructed to conduct low-temperature pretreatment and high-solid enzymatic hydrolysis of sugarcane bagasse (SCB) at ordinary atmospheric pressure. Results showed that enzymatic hydrolysis of WWW-washed BL-WWW-NaOH-treated SCB at 30% solid concentration for 72 h achieved 91.59 g/L glucose with glucan conversion of 70.94%. The pretreatment unit shared only 19.68%–22.43% of total energy consumption and 8.32%–8.81% of total cost. The non-sterile enzymatic hydrolysate could be directly fermented by BL-adapted yeasts to maximally produce 44.82 g/L ethanol at 48 h, while the sterile hydrolysate could not be fermented. 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one, furancarboxaldehyde and 5-hydroxymethylfurfural might be the dominant inhibitors in the sterile enzymatic hydrolysate for ethanol production. Finally, a low-emission strategy for cellulosic ethanol production with low energy consumption was proposed.

Effect of alkaline combined with ultrasonic pretreatment to enhance enzymatic saccharification of sugar cane bagasse and Enset fiber (Ensete ventricosum)

Effect of alkaline combined with ultrasonic pretreatment to enhance enzymatic saccharification of sugar cane bagasse and Enset fiber (Ensete ventricosum)

Characterization of a novel recombinant halophilic β-glucosidase of Trichoderma harzianum derived from Hainan mangrove

Backgroundβ-glucosidase is an important biomass-degrading enzyme and plays a vital role in generating renewable biofuels through enzymatic saccharification. In this study, we analyzed the transcriptome of Trichoderma harzianum HTASA derived from Hainan mangrove and identified a new gene encoding β-glucosidase Bgl3HB. And the biochemically characterization of β-glucosidase activity was performed.ResultsBgl3HB showed substantial catalytic activity in the pH range of 3.0–5.0 and at temperatures of 40 ℃-60 ℃. The enzyme was found quite stable at 50 ℃ with a loss of only 33.4% relative activity after 240 min of heat exposure. In addition, all tested metal ions were found to promote the enzyme activity. The β-glucosidase activity of Bgl3HB was enhanced by 2.12-fold of its original activity in the presence of 5 M NaCl. Surprisingly, Bgl3HB also showed a remarkable ability to hydrolyze laminarin compared to other measured substrates. Enzyme efficiency was examined in the sugarcane bagasse saccharification processes, in which Bgl3HB with 5 M NaCl worked better supplementing Celluclast 1.5L than the commercial Novozyme 188 ascertained it as an admirably suited biocatalyst for the utilization of agricultural waste. In this work, this is the first report of a halophilic β-glucosidase from Trichoderma harzianum, and represents the β-glucosidase with the highest known NaCl activation concentration. And adding 5 M NaCl could enhance saccharification performance even better than commercial cellulase.ConclusionsThese results show that Bgl3HB has great promise as a highly stable and highly efficient cellulase with important future applications in the industrial production of biofuels.

Multicopper oxidase enzymes from Chrysoporthe cubensis improve the saccharification yield of sugarcane bagasse

The genome of phytopathogenic fungus Chrysoporthe cubensis has distinct groups of multicopper oxidase (MCO) coding genes, including new laccases, that could have the potential for biotechnological applications. Here, the complete genomic arsenal of MCOs from C. cubensis was identified and characterized. Laccases were induced, purified from the fungal crude extract, and these enzymes' potential to improve the sugarcane bagasse saccharification was tested. In silico genomic analysis revealed 13 MCOs classified as fungal ferroxidases; ascomycete laccases; laccases/ferroxidases; fungal pigments MCOs, and ascorbate oxidases. The C. cubensis cultivation in different lignocellulosic biomass yielded enzymatic extracts more efficiently for sugarcane bagasse saccharification than commercial preparations. The higher laccase activity (4.88 U g-1) was detected in the crude extract of the fungus cultivated on wheat bran and orange peel (3:1), and MCOs with laccase activity were purified from this protein extract. Mass spectrometry using MALDI-TOF/TOF identified two MCOs with 53.4 kDa and 74 kDa. The mixture containing the purified laccases oxidized 91% of the phenolic compounds produced from the alkaline pretreatment of sugarcane bagasse and allowed the improved saccharification yields. These enzymes are promising for application in biomass hydrolysis, especially in the detoxification of phenolic compounds.

The lytic polysaccharide monooxigenases secreted by the fungus Chrysoporthe cubensis: Genomic analysis and impact on sugarcane bagasse saccharification

This work aimed to identify and evaluate the impact of oxidoreductases secreted by Chrysoporthe cubensis COAD 3356 on the saccharification of sugarcane bagasse. Genomic analyses focusing on lytic polysaccharide monooxygenases from Auxiliary Activity family 9 (LPMO9s) indicated fourteen putative LPMO9-coding genes in the C. cubensis genome. The predicted proteins were clustered with other LPMO9s based on sequence similarity. We evaluated if the clusters correlate with LPMO9 function, such as substrate specificity and oxygenation regioselectivity, and with evolutionary features (phylo-specific proteins and the presence of genes coding for LPMO9 redox partners in fungal genomes). There was no apparent connection between the LPMO9 sequences and the evaluated functional properties. On the other hand, we detected clusters containing phylo-specific LPMO9, and a cluster enriched in LPMO9s from fungi with no genes coding for cellobiose dehydrogenase (CDH). Proteomic analysis using LC-MS/MS revealed the presence of seven LPMO9s in C. cubensis exoproteome, in addition to flavin-containing oxidoreductases. Saccharification of alkaline pretreated sugarcane bagasse in aerobic and anaerobic conditions using C. cubensis exoproteome fractions, which contained different LPMO9s, indicated that the CcLPMO9C and CcLPMO9D are better fitted than other LPMO9s secreted by C. cubensis for sugarcane bagasse saccharification.

Multistep Structural and Chemical Evaluation of Sugarcane Baggase, Pretreated With Alkali for Enhancing the Enzymatic Saccharification by Cellulase and Xylanase of the Pseudomonas sp. CVB-10 (MK443365) and Bacillus paramycoides T4 (MN370035) Mix-Culture System

The enzymatic saccharification of sugarcane bagasse was significantly increased by alkali pretreatment under mild conditions. The effectiveness of different concentrations of alkali and acid pretreatment of sugarcane bagasse for improving the enzymatic saccharification of lignocellulose has been evaluated. The sugarcane bagasse was characterized to contain 39.52% celluloses, 25.63% hemicelluloses, and 30.36% lignin. After that, sugarcane bagasse was pretreated with 5 and 10% of H2SO4 and NaOH at 121°C for 60 min. FTIR, XRD, and SEM analyses also showed significant molecular and surface structure changes of the sugarcane bagasse with 10% NaOH. Maximum saccharification was 489.5 mg/g from 10% NaOH pretreatment followed by 322.75, 301.25, and 276.6 mg/g from 10% H2SO4, 5% NaOH, and 5% H2SO4, respectively, which were 55.1, 32.0, 27.1, and 20.6 times higher than the that of the control. Cellulase and xylanase produced by Pseudomonas sp. CVB-10 (MK443365) and Bacillus paramycoides T4 (MN370035) were used to hydrolyze the pretreated bagasse, and the optimal condition was determined to be 30 h of the enzymatic reaction with the 3:1 ratio of enzymes under the temperature of 55°C, pH 5.0, and substrate concentration of 3%, leading to celluloses and hemicelluloses conversion in the enzymatic hydrolysis/saccharification that is more proficient.

Pretreatment and Enzymatic Saccharification of Water Hyacinth, Sugarcane Bagasse, Maize Straw, and Green Coconut Shell Using an Organosolv Method with Glycerol and FeCl3

A method to pretreatment of biomasses using glycerol as a green solvent was developed. Optimization of organosolv pretreatment was performed using a 23 factorial design, investigating synergism among the variables particle size (from &lt; 0.85 to &gt; 2.0 mm), FeCl3 concentration (0.025-0.175 mol L–1), and temperature (160-220 °C). Although the effects of the variables differed according to the type of lignocellulose, it was nonetheless possible to find an optimal condition in common (&lt; 0.85 mm; 0.025 mol L–1; 220 °C), which was economically and environmentally sustainable, employing a small amount of catalyst. The biomasses pretreated using this combination of factors exhibited enzymatic digestibility exceeding 93% after 48 h, reflecting efficient reduction of recalcitrance as a result of high removal of hemicellulose (ca. 90%), provided by the use of FeCl3, together with substantial delignification. There was low formation of inhibitors in the hydrolysate, indicating that it could be directly fermented. The lignin removed to liquors could be easily recovered by centrifugation. Tests carried out under the optimal condition revealed that after the pretreatment the biomass could be directly used in enzymatic hydrolysis, without washing and wet. Furthermore, after simple treatment, crude glycerol was as effective as commercial glycerol.

Development of multiple inhibitor tolerant yeast via adaptive laboratory evolution for sustainable bioethanol production

The present research work aimed at developing robust yeast cell factory via adaptive laboratory evolution (ALE) for improved cellulosic bioethanol production. Kluyveromyces marxianus JKH5, a newly isolated thermotolerant ethanologenic yeast, was engineered by serial passaging for 60 generations in medium supplemented with gradually higher concentration of inhibitors (acetic acid, furfural, and vanillin) that are generated during dilute acid pretreatment. The improved strain K. marxianus JKH5 C60, showed 3.3-fold higher specific growth rate, 56% reduced lag phase and 80% enhanced fermentation efficiency at 42 °C in comparison to parent strain in inhibitor cocktail comprising medium. Bioethanol production by simultaneous saccharification and fermentation of sequential dilute acid-alkali pretreated sugarcane bagasse in presence of inhibitors, resulted in ethanol titre and yield, respectively, 54.8 ± 0.9 g/L and 0.40 g/g. The adapted yeast can be used to ferment unwashed pretreated biomass, thereby, reducing overall cost, time, and wastewater generation, hence making bioethanol production sustainable.