Hydrolysis Of Biomass Research Articles

Enhanced tolerance of Clostridium tyrobutyricum to furan derivatives for efficient butyric acid production by overexpressing native transporter genes

Furan aldehydes generated during the hydrolysis of lignocellulosic biomass are toxic to Clostridium tyrobutyricum, severely inhibiting its butyric acid production. This study confirmed that overexpression of the ABC-transporter gene CtAT2 in C. tyrobutyricum significantly increased furfural tolerance (> 3.0 g/L) and butyrate production (13.8 %). Conversely, overexpression of the MFS-transporter gene CtMT1 enhanced tolerance to HMF (> 4.0 g/L). However, co-expression of CtAT2 and CtMT1 in C. tyrobutyricum failed to improve the strain’s tolerance to furfural and HMF. Using undetoxified corncob acid hydrolysate as the carbon source, the recombinant strain ATCC 25755/AT2 exhibited excellent performance in butyric acid fermentation due to its heightened furfural tolerance and improved butyrate production. ATCC 25755/AT2 demonstrated impressive butyrate production and productivity in fed-batch fermentation, reaching 29.8 g/L and 0.23 g/L·h, respectively. These values represented a 22.6 % increase in butyrate production and a 27.8 % increase in productivity compared to the wild-type strain.

Exploring the Utilization Potential of Spirogyra sp. Biomass for Ethanol Production: A Study on Saccharification Optimization and High-Temperature Ethanol Fermentation

Spirogyra sp. is one of the potential feedstocks for bioethanol production, owing to its high carbohydrate and low lignin contents. However, to date, its use has scarcely been reported, particularly in high-temperature ethanol fermentation. The present study investigated the use of Spirogyra biomass as a bioethanol feedstock by optimizing the conditions for biomass saccharification, followed by ethanol fermentation via thermotolerant yeasts, i.e., Saccahromyces cerevisiae DBKKU Y-53, Kluyveromyces marxianus DBKKU Y-102, and Pichia kudriazevii RZ8-1. The optimization of the algal biomass hydrolysis using response surface methodology (RSM) showed that a maximum total sugar production of 14.75 ± 0.13 g/L was attained using 2.67% (v/v) sulfuric acid, 7.97% (w/v) of biomass loading, and 20 min of hydrolysis time. The fermentation of Spirogyra sp. hydrolysate containing 20 g/L of total sugar at 37 °C showed that S. cerevisiae DBKKU Y-53, K. marxianus DBKKU Y-102, and P. kudriazevii RZ8-1 produced 4.05 ± 0.35 g/L, 4.48 ± 0.13 g/L, and 4.47 ± 0.19 g/L of ethanol, respectively. At 40 °C, lower ethanol production of 1.07 ± 0.47 g/L, 3.93 ± 0.24 g/L, and 3.97 ± 0.19 g/L, respectively, were observed. Nevertheless, P. kudriazevii RZ8-1 exhibited a promising potential for the further development of a high-temperature ethanol fermentation process.

Systematic identification of CAZymes and transcription factors in the hypercellulolytic fungus Penicillium funiculosum NCIM1228 involved in lignocellulosic biomass degradation

BackgroundPenicillium funiculosum NCIM1228 is a filamentous fungus that was identified in our laboratory to have high cellulolytic activity. Analysis of its secretome suggested that it responds to different carbon substrates by secreting specific enzymes capable of digesting those substrates. This phenomenon indicated the presence of a regulatory system guiding the expression of these hydrolyzing enzymes. Since transcription factors (TFs) are the key players in regulating the expression of enzymes, this study aimed first to identify the complete repertoire of Carbohydrate Active Enzymes (CAZymes) and TFs coded in its genome. The regulation of CAZymes was then analysed by studying the expression pattern of these CAZymes and TFs in different carbon substrates—Avicel (cellulosic substrate), wheat bran (WB; hemicellulosic substrate), Avicel + wheat bran, pre-treated wheat straw (a potential substrate for lignocellulosic ethanol), and glucose (control).ResultsThe P. funiculosum NCIM1228 genome was sequenced, and 10,739 genes were identified in its genome. These genes included a total of 298 CAZymes and 451 TF coding genes. A distinct expression pattern of the CAZymes was observed in different carbon substrates tested. Core cellulose hydrolyzing enzymes were highly expressed in the presence of Avicel, while pre-treated wheat straw and Avicel + wheat bran induced a mixture of CAZymes because of their heterogeneous nature. Wheat bran mainly induced hemicellulases, and the least number of CAZymes were expressed in glucose. TFs also exhibited distinct expression patterns in each of the carbon substrates. Though most of these TFs have not been functionally characterized before, homologs of NosA, Fcr1, and ATF21, which have been known to be involved in fruiting body development, protein secretion and stress response, were identified.ConclusionsOverall, the P. funiculosum NCIM1228 genome was sequenced, and the CAZymes and TFs present in its genome were annotated. The expression of the CAZymes and TFs in response to various polymeric sugars present in the lignocellulosic biomass was identified. This work thus provides a comprehensive mapping of transcription factors (TFs) involved in regulating the production of biomass hydrolyzing enzymes.

New bio-based polyurethane (PU) foams synthesized using crude glycerol-based biopolyol and humin-based byproducts from biomass hydrolysis

Exploring novel applications for solid waste in industrial production reduces the production cost of polyurethane (PU) foams, which is of great significance for sustainable development. In this study, crude glycerol was utilized to synthesize bio-based polyols via a one-pot method. Around 45 % of the glycerol components present in crude glycerol underwent esterification or transesterification reactions, resulting in the formation of polyhydroxy compounds. The synthesized polyol had a hydroxyl number of 406 mg KOH/g, a viscosity of 1092 mPa·s, and an acid number of 1.9 mg KOH/g, rendering it suitable for the preparation of rigid polyurethane foams. Humin-based residues (FRR, referring to residues from hydrolysis of furfural residue, and GR, referring to residues from hydrolysis of glucose) were further incorporated for the first time to reinforce bio-based PU foams. The effects of various fillers content (0, 1 %, 3 %, 5 %, 7 %, and 10 %) on the mechanical properties, morphology, thermal insulation properties, and thermal stability of PU foams were investigated. At an FRR level of 7 %, the maximum compressive strength reached 0.1536 MPa, representing an increase of 28.30 %. Similarly, when GR levels were set at 5 %, the maximum compressive strength was found to be 0.1581 MPa, reflecting an increase of 32.08 %. The addition of GR filler had a greater impact on enhancing compression strength, potentially due to its spherical and uniform particle shape, which could facilitate better dispersion within the polyols. The PU foam synthesized in this study showed comparable performance to those using cellulose-based fillers. Furthermore, the thermal conductivity of all foams containing fillers was lower than 0.0350 W/(m·K). The thermal gravimetric analysis demonstrated that the addition of humins as filler effectively enhanced the stability of the foam. As indicated from FT-IR and XPS analysis, urethane bonds were successfully formed, while the hydroxyl group on the surface of humins potentially enhanced the consumption of the –NCO group. The foams produced in this study exhibited highly favorable thermal insulation properties, while also offering superior economic and environmental benefits.

Effect of microwave pretreatment on lignocellulosic degradation of corn cob

Lignocellulosic biomass is a widespread and renewable alternative to fossil resources from which valuable products such as biofuels and chemicals can be obtained. The usual biotechnological way of lignocellulose processing involves pre-treatment of biomass and enzymatic hydrolysis of complex carbohydrates (cellulose and hemicellulose) to simple sugars. The aim of this study was to examine the effect of microwave pretreatment on lignocellulosic degradation of corn cob. Biomass will be treated in a microwave oven (Bosch Serie 2, Germany) using hydrogen peroxide, under different conditions (pH and treatment time). After treatment, the solid fraction was subjected to enzymatic hydrolysis using the commercial enzyme CellicCTec2 (Novozymes, Denmark). The effect of the treatment was determined by determining the surface area of cellulose and lignin (based on the equation of Langmuir adsorption isotherm for adsorption of Congo red and Azure B, respectively), and the crystallinity index (iodine adsorption method). Enzymatic hydrolysis of biomass was monitored using spectrophotometric methods to determine the concentration of reducing sugars (DNS method), total hexose sugars (Antron method) and total pentose sugars (Orcinol method). The results of the treated samples were compared with the untreated ones in order to determine the most efficient treatment conditions for the degradation of lignocellulosic biomass.

An extracellular glucose sensor for substrate-dependent secretion and display of cellulose-degrading enzymes.

The efficient hydrolysis of lignocellulosic biomass into fermentable sugars is key for viable economic production of biofuels and biorenewable chemicals from second-generation feedstocks. Consolidated bioprocessing (CBP) combines lignocellulose saccharification and chemical production in a single step. To avoid wasting valuable resources during CBP, the selective secretion of enzymes (independent or attached to the surface) based on the carbon source available is advantageous. To enable enzyme expression and secretion based on extracellular glucose levels, we implemented a G-protein-coupled receptor (GPCR)-based extracellular glucose sensor; this allows the secretion and display of cellulases in the presence of the cellulosic fraction of lignocellulose by leveraging cellobiose-dependent signal amplification. We focused on the glucose-responsiveness of the HXT1 promoter and engineered PHXT1 by changing its core to that of the strong promoter PTHD3 , increasing extracellular enzyme activity by 81%. We then demonstrated glucose-mediated expression and cell-surface display of the β-glucosidase BglI on the surface of Saccharomyces cerevisiae. The display system was further optimized by re-directing fatty acid pools from lipid droplet synthesis toward formation of membrane precursors via knock-out of PAH1. This resulted in an up to 4.2-fold improvement with respect to the baseline strain. Finally, we observed cellobiose-dependent signal amplification of the system with an increase in enzymatic activity of up to 3.1-fold when cellobiose was added.

Whole-Genome Sequence and Fermentation Characteristics of Enterobacter hormaechei UW0SKVC1: A Promising Candidate for Detoxification of Lignocellulosic Biomass Hydrolysates and Production of Value-Added Chemicals.

Enterobacter hormaechei is part of the Enterobacter cloacae complex (ECC), which is widespread in nature. It is a facultative Gram-negative bacterium of medical and industrial importance. We assessed the metabolic and genetic repertoires of a new Enterobacter isolate. Here, we report the whole-genome sequence of a furfural- and 5-hydroxymethyl furfural (HMF)-tolerant strain of E. hormaechei (UW0SKVC1), which uses glucose, glycerol, xylose, lactose and arabinose as sole carbon sources. This strain exhibits high tolerance to furfural (IC50 = 34.2 mM; ~3.3 g/L) relative to Escherichia coli DH5α (IC50 = 26.0 mM; ~2.5 g/L). Furfural and HMF are predominantly converted to their less-toxic alcohols. E. hormaechei UW0SKVC1 produces 2,3-butanediol, acetoin, and acetol, among other compounds of industrial importance. E. hormaechei UW0SKVC1 produces as high as ~42 g/L 2,3-butanediol on 60 g/L glucose or lactose. The assembled genome consists of a 4,833,490-bp chromosome, with a GC content of 55.35%. Annotation of the assembled genome revealed 4586 coding sequences and 4516 protein-coding genes (average length 937-bp) involved in central metabolism, energy generation, biodegradation of xenobiotic compounds, production of assorted organic compounds, and drug resistance. E. hormaechei UW0SKVC1 shows considerable promise as a biocatalyst and a genetic repository of genes whose protein products may be harnessed for the efficient bioconversion of lignocellulosic biomass, abundant glycerol and lactose-replete whey permeate to value-added chemicals.

Light-Driven Depolymerization of Cellulosic Biomass into Hydrocarbons.

Cellulose and hemicellulose are the main constituents of lignocellulosic biomass. Chemical derivatization of lignocellulosic biomass leads to a range of C5 and C6 organic compounds. These C5 and C6 compounds are valuable precursors (or fine chemicals) for developing sustainable chemical processes. Therefore, depolymerization of cellulose and hemicellulose is essential, leading to the development of various materials that have applications in biomaterial industries. However, most depolymerized processes for cellulose have limited success because of its structural quality: crystallinity, high hydrogen-bond networking, and mild solubility in organic and water. As a result, various chemical treatments, acidic (mineral or solid acids) and photocatalysis, have developed. One of the significant shortcomings of acidic treatment is that the requirement for high temperatures increases the commercial end cost (energy) and hampers product selectivity. For example, a catalyst with prolonged exposure to high temperatures damages the catalyst surface over time; therefore, it cannot be used for iterative cycles. Photocatalysts provide ample application to overcome such flaws as they do not require high temperatures to perform efficient catalysis. Various photocatalysts have shown efficient cellulosic biomass conversion into its C6 and C5 hydrocarbons and the production of hydrogen (as a green energy component). For example, TiO2-based photocatalysts are the most studied for biomass valorization. Herein, we discussed the feasibility of a photocatalyst with application to cellulosic biomass hydrolysis.

Carbon supported polyoxometalates as recyclable solid acid catalysts in aqueous reactions

Acidic Polyoxometalates have been identified as promising catalysts for the hydrolysis of lignocellulosic biomass. They possess a high Brønsted acidity but are soluble in water and most organic solvents, which impedes their recycling and thus industrial application. To develop a solid acid catalyst with both good activity and recyclability, phosphotungstic acid (PTA) was immobilized on activated carbon (AC) by equilibrium impregnation. Characterization of the catalyst materials revealed a stabilizing effect by oxygen functional groups on the support material by increasing the polarity of the support and enabling a chemical interaction between the heteropolyacid and the surface. PTA/AC was applied in the hydrolysis of the hemicellulose xylan as an aqueous model reaction, selectively yielding the biorefinery platform compound xylose with up to 74 % yield. The catalyst can be easily separated from the reaction system and possesses good reusability over 20 recycling runs with a low constant leaching of 3–4 % per run.

Single-Cell Oil Production by Engineered Ashbya gossypii from Non-Detoxified Lignocellulosic Biomass Hydrolysate

In this work, microbial lipid production from non-detoxified Eucalyptus bark hydrolysate (EBH) with oleaginous xylose-utilizing Ashbya gossypii strains was explored. The best producing strain from a set of engineered strains was identified in synthetic media mimicking the composition of the non-detoxified EBH (SM), the lipid profile was characterized, and yeast extract and corn steep liquor (CSL) were pinpointed as supplements enabling a good balance between lipid accumulation, biomass production, and autolysis by A. gossypii. The potential of the engineered A. gossypii A877 strain to produce lipids was further validated and optimized with minimally processed inhibitor-containing hydrolysate and high sugar concentration, and scaled up in a 2 L bioreactor. Lipid production from non-detoxified EBH supplemented with CSL reached a lipid titer of 1.42 g/L, paving the way for sustainable single-cell oil production within the concept of circular economy and placing lipids as an alternative by-product within microbial biorefineries.

Vine shoots pre-treatment strategies for improved hydrogen production and metabolites redistribution in Clostridium butyricum

This work deals with the use of vine shoots, a renewable, largely available, lacking of alternatives lignocellulosic material as a feedstock for hydrogen production. Physical pre-treatments by steam explosion (SE), chemical by organosolv (OS) and biological by laccase (LAC) were carried out in vine shoots to disrupt the cell fiber and increase the biomass hydrolysis and fermentation into hydrogen (H2). After SE, there was a slight decrease in cellulose and hemicellulose contents in biomass fibers, while a decrease in lignin content occurred after OS pre-treatment. There were no quantifiable changes after laccase pre-treatment, however the enzyme-substrate oxidative reactions were favorable for hydrolysis and fermentation since an increase in soluble sugars and H2 production was observed with LAC vine shoots as substrate. 300.1 mL H2/L were obtained from raw material vine shoots, while 649.4, 399.8 and 749.7 mL H2/L were obtained from biomass pre-treated by SE, OS and LAC, respectively. Furthermore, the hydrolysis of pre-treated biomass by addition of cellulase was evaluated to improve H2 production. Higher amounts of H2 were obtained from hydrolyzed biomass in relation to non-hydrolyzed ones (154.2%, 602.0% and 167.1% more with SE, OS and LAC hydrolyzed, respectively). In all cases, the mixed acid pathway was carried out by Clostridium butyricum, since acetic and butyric acids were produced.

Profiling of the β-glucosidases identified in the genome of Penicillium funiculosum: insights from genomics, transcriptomics, proteomics, and homology-modeling studies.

The enzymatic conversion of lignocellulosic biomass to bioethanol depends on efficient enzyme systems with β-glucosidase as one of the key components. In this study, we performed in-depth profiling of the various β-glucosidases present in the genome of the hypercellulolytic fungus Penicillium funiculosum using genomics, transcriptomics, proteomics, and molecular dynamics simulation approaches. Of the eight β-glucosidase genes identified in the P. funiculosum genome, three were predicted to be extracellular based on signal peptide prediction and abundance in the secretome. Among the three secreted β-glucosidases, two belonged to the GH3 family and one belonged to the GH1 family. Homology models of these proteins predicted a deep and narrow active site for the GH3 β-glucosidases (PfBgl3A and PfBgl3B) and a shallow open active site for the GH1 β-glucosidase (PfBgl1A). The enzymatic assays indicated that P. funiculosum-secreted proteins showed high β-glucosidase activities with prominent bands on the 4-methylumbelliferyl β-D-glucopyranoside zymogram. To understand the contributory effects of each of the three secreted β-glucosidases (PfBgls), the corresponding gene was deleted separately, and the effect of the deletion on the β-glucosidase activity of the secretome was examined. Although not the most abundant, PfBgl3A was found to be one of the most important β-glucosidases, as evidenced by a 42% reduction in β-glucosidase activity in the ΔPfBgl3A strain. Our results advance the understanding of the genetic and biochemical nature of all β-glucosidases produced by P. funiculosum and pave the way to design a superior biocatalyst for the hydrolysis of lignocellulosic biomass. IMPORTANCE Commercially available cellulases are primarily produced from Trichoderma reesei. However, external supplementation of the cellulase cocktail from this host with exogenous β-glucosidase is often required to achieve the desired optimal saccharification of cellulosic feedstocks. This challenge has led to the exploration of other cellulase-producing strains. The nonmodel hypercellulolytic fungus Penicillium funiculosum has been studied in recent times and identified as a promising source of industrial cellulases mainly due to its ability to produce a balanced concoction of cellulolytic enzymes, including β-glucosidases. Various genetic interventions targeted at strain improvement for cellulase production have been performed; however, the β-glucosidases of this strain have remained largely understudied. This study, therefore, reports profiling of all eight β-glucosidases of P. funiculosum via molecular and computational approaches. The results of this study provide useful insights that will establish the background for future engineering strategies to transform this fungus into an industrial workhorse.

Co-Fermentation of Glucose–Xylose–Cellobiose–XOS Mixtures Using a Synthetic Consortium of Recombinant Saccharomyces cerevisiae Strains

The efficient conversion of cellulosic sugars is vital for the economically viable production of biofuels/biochemicals from lignocellulosic biomass hydrolysates. Based on comprehensive screening, Saccharomyces cerevisiae RC212 was chosen as the chassis strain for multiple integrations of heterologous β-glucosidase and β-xylosidase genes in the present study. The resulting recombinant BLN26 and LF1 form a binary synthetic consortium, and this co-culture system achieved partial fermentation of four sugars (glucose, xylose, cellobiose, and xylo-oligosaccharides). Then, we developed a ternary S. cerevisiae consortium consisting of LF1, BSGIBX, and 102SB. Almost all four sugars were efficiently fermented to ethanol within 24 h, and the ethanol yield is 0.482 g g−1 based on the consumed sugar. To our knowledge, this study represents the first exploration of the conversion of mixtures of glucose, xylose, cellobiose, and xylo-oligosaccharides by a synthetic consortium of recombinant S. cerevisiae strains. This synthetic consortium and subsequent improved ones have the potential to be used as microbial platforms to produce a wide array of biochemicals from lignocellulosic hydrolysates.

Heterologous expression and characterization of salt-tolerant β-glucosidase from xerophilic Aspergillus chevalieri for hydrolysis of marine biomass.

A salt-tolerant exo-β-1,3-glucosidase (BGL_MK86) was cloned from the xerophilic mold Aspergillus chevalieri MK86 and heterologously expressed in A. oryzae. Phylogenetic analysis suggests that BGL_MK86 belongs to glycoside hydrolase family 5 (aryl-phospho-β-D-glucosidase, BglC), and exhibits D-glucose tolerance. Recombinant BGL_MK86 (rBGL_MK86) exhibited 100-fold higher expression than native BGL_MK86. rBGL_MK86 was active over a wide range of NaCl concentrations [0%-18% (w/v)] and showed increased substrate affinity for p-nitrophenyl-β-D-glucopyranoside (pNPBG) and turnover number (kcat) in the presence of NaCl. The enzyme was stable over a broad pH range (5.5-9.5). The optimum reaction pH and temperature for hydrolysis of pNPBG were 5.5 and 45°C, respectively. rBGL_MK86 acted on the β-1,3-linked glucose dimer laminaribiose, but not β-1,4-linked or β-1,6-linked glucose dimers (cellobiose or gentiobiose). It showed tenfold higher activity toward laminarin (a linear polymer of β-1,3 glucan) from Laminaria digitata than laminarin (β-1,3/β-1,6 glucan) from Eisenia bicyclis, likely due to its inability to act on β-1,6-linked glucose residues. The β-glucosidase retained hydrolytic activity toward crude laminarin preparations from marine biomass in moderately high salt concentrations. These properties indicate wide potential applications of this enzyme in saccharification of salt-bearing marine biomass.

The green novel approach in hydrolysis of pistachio shell into xylose by microwave-assisted high-pressure CO2 /H2 O.

Pistachio shell is a valuable lignocellulosic biomass because almost 90% of its hemicellulose fraction is xylan, which can be converted into high value-added compounds such as xylooligosaccarides, xylose, xylitol and furfural. The present study represents a green and novel approach to produce xylose from lignocellulosic biomass. Microwave-assisted high-pressure CO2 /H2 O hydrolysis (MW-HPCO2 ) comprising a combination never previously used was performed to produce xylose from pistachio shell. Response surface methodology with a Box-Behnken design was implemented to optimize microwave-assisted high-pressure CO2 /H2 O hydrolysis (MW-HPCO2 ). The effect of temperature, time and liquid-to-solid ratio was studied in the ranges of 180-210 °C, 10-30 min and 5-30 mL g-1 , respectively. A maximum xylose yield of 61.39% and minimum degradation compounds (5-hydroxymethyl furfural and furfural) of 11.07% were attained under reaction conditions of 190 °C, 30 min and 18 mL g-1 . The results showed that hydrolysis temperature, time and liquid-to-solid ratio had a strong influence on the xylose yield, as well as on the formation of degradation compounds. MW-HPCO2 significantly increased accessibility to cellulose-derived products in the subsequent enzymatic hydrolysis. The results of the present study reveal that MW-HPCO2 can be a promising green technique for the hydrolysis of lignocellulosic biomass. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Production of fermentable glucose from bioconversion of cellulose using efficient microbial cellulases produced from water hyacinth waste

The economic production of cellulase enzymes for various industrial applications is one of the major research areas. A number of broad industrial applications, for example, in cellulosic biomass hydrolysis for simple sugars such as glucose and subsequent biofuel production, make these enzyme systems the third most demanding enzymes. Nevertheless, due to their production on commercial substrates, cellulases fall into the category of costly enzymes. Therefore, the goal of the present work is to evaluate the enhancement of cellulase production and its utilization in the enzymatic hydrolysis of biomass using low-cost cellulosic substrate, which is abundant and widely available. In this context, waste biomasses of water hyacinth (WH), including leaves and stems, have been used as feedstock to produce cellulases via solid-state fermentation (SSF) in the current study, which improves its production as well as activity. Furthermore, the impact of process parameters like temperature and pH has been investigated for improved cellulase production. At optimum concentration using 10 g of feedstock, 22 IU/gds of FP, 92 IU/gds of BGL, and 111 IU/gds of EG have been noticed in day 5 of SSF. Herein, 40 °C has been identified as the optimum temperature for cellulase production, whereas 50-55 °C has been recorded as the optimum reaction temperature for cellulase enzyme activity. Additionally, pH 5.5 has been identified as the optimum pH for cellulase enzyme production, whereas this enzyme was thermally stable (55 °C) at pH 5.0 up to 3.5 h. Further, the cellulosic biomass hydrolysis of WH leaves via an optimized crude enzyme has been performed, and this could release 24.34 g/L of glucose in 24 h of the reaction. The current findings may have potential for developing cellulases for mass-scale production using WH-based waste bioresources for numerous biorefinery applications.

A novel mineral-acid free biphasic deep eutectic solvent/γ-valerolactone system for furfural production and boosting the enzymatic hydrolysis of lignocellulosic biomass

The failure of hemicellulose valorization in a deep eutectic solvent (DES) pretreatment has become a bottleneck that challenges its further development. To address this issue, this study developed a DES/GVL (γ-valerolactone) biphasic system for effective hemicellulose-furfural conversion, enhanced cellulose saccharification and lignin isolation. The results indicated that the biphasic system could significantly improve the lignin removal (as high as 89.1%), 86.0% higher than the monophasic DES, accompanied by ∼100% hemicellulose degradation. Notably, the GVL in the biphasic solvent restricted the condensation of hemicellulose degradation products, which as a result generated large amount of furfural in the pretreatment liquid with a yield of 68.6%. With the removal of hemicellulose and lignin, cellulose enzymatic hydrolysis yield was boosted and reached near 100%. This study highlighted that the novel DES/GVL is capable of fractionating the biomass and benefiting their individual utilization, which could provide a new biorefinery configuration for a DES pretreatment.

A combined liquid chromatography – trapped ion mobility – tandem high-resolution mass spectrometry and multivariate analysis approach for the determination of enzymatic reactivity descriptors in biomass hydrolysates

Intermediate products such as oxygenated compounds may interfere with bioconversion kinetics of lignocellulosic biomass into bioethanol. This work presents a multidimensional approach, based on liquid chromatography (LC), trapped ion mobility spectrometry (TIMS), tandem high-resolution mass spectrometry (HRMS/MS), and multivariate analysis, for the identification of enzymatic reactivity descriptors in 22 industrial biomass samples, called hydrolysates. The first part of the study is dedicated to the improvement of the chemical diversity assessment of the hydrolysates through an original three-dimensional Van Krevelen diagram displaying the double bond equivalent (DBE) as third dimension. In a second part, the evaluation of data by multivariate data analysis allowed the discrimination of sample according to the biomass type and the level of enzymatic reactivity. In the last part, a potential descriptor of low enzymatic reactivity was selected and used in a case study. An in-depth structural analysis was performed on the feature annotated as carbohydrate derivative. Considering the intricate fragmentation spectrum exhibited by the selected feature, trapped ion mobility was employed to enhance separation prior to the HRMS/MS experiments. This final step improved data interpretation and increased the identification confidence level leading to the characterization of xylotriose, 3,5-dimethoxy-4-hydroxybenzaldehyde and 4-hydroxy-3-methoxy-cinnamaldehyde. This is the first study to present an untargeted multidimensional approach for the identification of enzymatic hydrolysis inhibitors in industrial hydrolysate samples.

Agronomic and environmental aspects of organo-mineral fertilizers produced with a by-product of the intermediate process of tanning

IntroductionThe by-product of the intermediate process of tanning (BPIPT) can be used in the manufacturing of environmentally-friendly organo-mineral fertilizers (OMF). However, the presence of potentially toxic elements (e.g., chromium, Cr), can hinder the use of BPIPT in agriculture. This study aimed: i) to evaluate soil chemical and biological properties following the application of fertilizers produced with a BPIPT, in contrast to other OMF produced with traditional organic matrices; and ii) to assess the impacts of such products on wheat growth and nutrition.MethodsSamples (0-0.2 m) of two Oxisols [Red-Yellow Latosol (LVAd) and Red Latosol (LVd)] were used in this study, consisting of two experiments (with five treatments and five replicates) using different OMF, in a sequence: 1) incubation of fertilizers in soil samples for 30 days, and 2) wheat cultivation (30 days) after the fertilizer incubation. The treatments consisted of an arrangement of mineral fertilizers based on nitrogen, phosphorus, and potassium (NPK fertilizers), associated with OMF based on BPIPT (OM-IPT and OM-IPT+S) or commercial manure (OM-CM and OM-CM+S), with or without sulfur (S), and a control treatment. Elemental availability in soils and microbial attributes were determined after the incubation of the OMF in the soils. After wheat cultivation, plant biomass, nutritional composition, β-glucosidase activity, and fluorescein diacetate hydrolysis (FDA) were measured.Results and discussionThe application of BPIPT-derived OMF in the present study increased available Cr fraction contents in both soils. However, OM-IPT caused low soil acidification, enhanced wheat growth and nutrition, and stimulated microbial activity in soils (FDA and β-glucosidase), thus evidencing the agronomic and environmental benefits of this OMF and their potential to contribute to a cleaner, more sustainable agriculture.

Coriolus versicolor laccase-based inorganic protein hybrid synthesis for application in biomass saccharification to enhance biological production of hydrogen and ethanol

In this study, a bio-friendly inorganic protein hybrid-based enzyme immobilization system using partially purified Coriolus versicolor laccase (CvLac) was successfully applied to biomass hydrolysis for the enhancement of sugar production aimed at generating biofuels. After four days of incubation, the maximum CvLac production was achieved at 140 U/mg of total protein in the presence of inducers such as copper and wheat bran after four days of incubation. Crude CvLac immobilized through inorganic protein hybrids such as nanoflowers (NFs) using zinc as Zn3(PO4)2/CvLac hybrid NFs (Zn/CvLac-NFs) showed a maximum encapsulation yield of 93.4% and a relative activity of 265% compared to free laccase. The synthesized Zn/CvLac-NFs exhibited significantly improved activity profiles and stability compared to free enzymes. Furthermore, Zn/CvLac-NFs retained a significantly high residual activity of 96.2% after ten reuse cycles. The saccharification of poplar biomass improved ∼2-fold in the presence of Zn/CvLac-NFs, with an 8-fold reduction in total phenolics compared to the control. The Zn/CvLac-NFs treated biomass hydrolysate showed high biological hydrogen (H2) production and ethanol conversion efficiency of up to 2.68 mol/mol of hexose and 79.0% compared to the control values of 1.27 mol of H2/mol of hexose and 58.4%, respectively. The CvLac hybrid NFs are the first time reported for biomass hydrolysis, and a significant enhancement in the production of hydrogen and ethanol was reported. The synthesis of such NFs based on crude forms of diverse enzymes can potentially be extended to a broad range of biotechnological applications.