Hydrolysis Of Lignocellulosic Biomass Research Articles

Comparative Evaluation of Adsorption of Major Enzymes in a Cellulase Cocktail Obtained from Trichoderma reesei onto Different Types of Lignin.

Cellulase adsorption onto lignin decreases the productivity of enzymatic hydrolysis of lignocellulosic biomass. Here, adsorption of enzymes onto different types of lignin was investigated, and the five major enzymes—cellobiohydrolases (CBHs), endoglucanase (Cel7B), β-glucosidase (Cel3A), xylanase (XYNIV), and mannanase (Man5A)—in a cellulase cocktail obtained from Trichoderma reesei were individually analyzed through SDS-PAGE and zymogram assay. Lignin was isolated from woody (oak and pine lignin) and herbaceous (rice straw and kenaf lignin) plants. The relative adsorption of CBHs compared to the control was in the range of 14.15–18.61%. The carbohydrate binding motif (CBM) of the CBHs contributed to higher adsorption levels in oak and kenaf lignin, compared to those in pine and rice lignin. The adsorption of endoglucanase (Cel7B) by herbaceous plant lignin was two times higher than that of woody lignin, whereas XYNIV showed the opposite pattern. β-glucosidase (Cel3A) displayed the highest and lowest adsorption ratios on rice straw and kenaf lignin, respectively. Mannanase (Man5A) was found to have the lowest adsorption ratio on pine lignin. Our results showed that the hydrophobic properties of CBM and the enzyme structures are key factors in adsorption onto lignin, whereas the properties of specific lignin types indirectly affect adsorption.

Enzymatic hydrolysis of lignocellulosic biomass using a novel, thermotolerant recombinant xylosidase enzyme from Clostridium clariflavum: a potential addition for biofuel industry

The present study describes the cloning, expression, purification and characterization of the xylosidase gene (1650 bp) from a thermophilic bacterium Clostridium clariflavum into E. coli BL21 (DE3) using the expression vector pET-21a(+) for utilization in biofuel production. The recombinant xylosidase enzyme was purified to homogeneity by heat treatment and immobilized metal ion affinity chromatography. SDS-PAGE determined that the molecular weight of purified xylosidase was 60 kDa. This purified recombinant xylosidase showed its maximum activity at a temperature of 37 °C and pH 6.0. The purified recombinant xylosidase enzyme remains stable up to 90 °C for 4 h and retained 54.6% relative activity as compared to the control. The presence of metal ions such as Ca2+ and Mg2+ showed a positive impact on xylosidase enzyme activity whereas Cu2+ and Hg2+ inhibit its activity. Organic solvents did not considerably affect the stability of the purified xylosidase enzyme while DMSO and SDS cause the inhibition of enzyme activity. Pretreatment experiments were run in triplicate for 72 h at 30 °C using 10% NaOH. Saccharification experiment was performed by using 1% substrate (pretreated plant biomass) in citrate phosphate buffer of pH 6.5 loaded with 150 U mL−1 of purified recombinant xylosidase enzyme along with ampicillin (10 μg mL−1). Subsequent incubation was carried out at 50 °C and 100 rpm in a shaking incubator for 24 h. Saccharification potential of the recombinant xylosidase enzyme was calculated against both pretreated and untreated sugarcane bagasse and wheat straw as 9.63% and 8.91% respectively. All these characteristics of the recombinant thermotolerant xylosidase enzyme recommended it as a potential candidate for biofuel industry.

Bioconversion of Lignocellulosic Biomass into Value Added Products under Anaerobic Conditions: Insight into Proteomic Studies.

Production of biofuels and other value-added products from lignocellulose breakdown requires the coordinated metabolic activity of varied microorganisms. The increasing global demand for biofuels encourages the development and optimization of production strategies. Optimization in turn requires a thorough understanding of the microbial mechanisms and metabolic pathways behind the formation of each product of interest. Hydrolysis of lignocellulosic biomass is a bottleneck in its industrial use and often affects yield efficiency. The accessibility of the biomass to the microorganisms is the key to the release of sugars that are then taken up as substrates and subsequently transformed into the desired products. While the effects of different metabolic intermediates in the overall production of biofuel and other relevant products have been studied, the role of proteins and their activity under anaerobic conditions has not been widely explored. Shifts in enzyme production may inform the state of the microorganisms involved; thus, acquiring insights into the protein production and enzyme activity could be an effective resource to optimize production strategies. The application of proteomic analysis is currently a promising strategy in this area. This review deals on the aspects of enzymes and proteomics of bioprocesses of biofuels production using lignocellulosic biomass as substrate.

A novel AST2 mutation generated upon whole-genome transformation of Saccharomyces cerevisiae confers high tolerance to 5-Hydroxymethylfurfural (HMF) and other inhibitors.

Development of cell factories for conversion of lignocellulosic biomass hydrolysates into biofuels or bio-based chemicals faces major challenges, including the presence of inhibitory chemicals derived from biomass hydrolysis or pretreatment. Extensive screening of 2526 Saccharomyces cerevisiae strains and 17 non-conventional yeast species identified a Candida glabrata strain as the most 5-hydroxymethylfurfural (HMF) tolerant. Whole-genome (WG) transformation of the second-generation industrial S. cerevisiae strain MD4 with genomic DNA from C. glabrata, but not from non-tolerant strains, allowed selection of stable transformants in the presence of HMF. Transformant GVM0 showed the highest HMF tolerance for growth on plates and in small-scale fermentations. Comparison of the WG sequence of MD4 and GVM1, a diploid segregant of GVM0 with similarly high HMF tolerance, surprisingly revealed only nine non-synonymous SNPs, of which none were present in the C. glabrata genome. Reciprocal hemizygosity analysis in diploid strain GVM1 revealed AST2N406I as the only causative mutation. This novel SNP improved tolerance to HMF, furfural and other inhibitors, when introduced in different yeast genetic backgrounds and both in synthetic media and lignocellulose hydrolysates. It stimulated disappearance of HMF and furfural from the medium and enhanced in vitro furfural NADH-dependent reducing activity. The corresponding mutation present in AST1 (i.e. AST1D405I) the paralog gene of AST2, also improved inhibitor tolerance but only in combination with AST2N406I and in presence of high inhibitor concentrations. Our work provides a powerful genetic tool to improve yeast inhibitor tolerance in lignocellulosic biomass hydrolysates and other inhibitor-rich industrial media, and it has revealed for the first time a clear function for Ast2 and Ast1 in inhibitor tolerance.

Biological pretreatment of corn stover for enhancing enzymatic hydrolysis using Bacillus sp. P3

The biological pretreatment for the enzymatic hydrolysis of lignocellulosic biomasses depends exclusively on the effective pretreatment process. Herein, we report a significant enhancement of enzymatic saccharification obtained with corn stover using a bacterial strain Bacillus sp. P3. The hemicellulose removal from corn stover by the strain Bacillus sp. P3 was evaluated for enhancing subsequent enzymatic hydrolysis. Therefore, our study revealed that an alkaline-resistant xylanase as well as other enzymes produced by Bacillus sp. P3 in fermentation broth led to a substantially enhanced hemicellulose removal rate from corn stover within pH 9.36–9.68. However, after a 20-day pretreatment of corn stover by the strain P3, the glucan content was increased by 51% and the xylan content was decreased by 35%. After 72 h of saccharification using 20 U/g of commercial cellulase, the yield of reducing sugar released from 20-day pretreated corn stover was increased by 56% in comparison to the untreated corn stover. Therefore, the use of the strain P3 could be a promising approach to pretreat corn stover for enhancing the enzymatic hydrolysis process of industrial bioenergy productions.

Cellulolytic enzymes behavior in delignified green coconut residues and enzymatic hydrolysis with enzyme recovery

Lignocellulosic bioconversion to obtain fermentable sugars often requires high enzyme dosages, increasing the total production cost. This study investigated the adsorption, desorption, and recycling of cellulases in enzymatic hydrolysis experiments using pretreated green coconut shell as substrate. The green coconut shell (GCS) was subjected to different pretreatments including dilute acid, alkaline, and two-stage acid-alkaline pretreatments. Adsorption and desorption studies involving cellulases from Trichoderma reesei ATCC 26921 and Cellic Ctec2 (10 FPU/g, initial dosage) with 5% solid loading showed that alkaline and two-stage acid-alkaline pretreatments were more suitable for enzyme recycling, because they showed lower values of cellulase adsorption capacity (48 % and 69 %) and higher values of cellulase recovery by desorption without Tween 80 addition (reaching 50 % using two-stage acid-alkaline pretreated GCS). Cellulases bound to the solid residue and dissolved in the supernatant after enzymatic hydrolysis can be reused for a new enzymatic hydrolysis process without compromising the sugar yield results. Using GCS as a substrate, the addition of solid residue and the supernatant from the previous step of adsorption of cellulases achieved a satisfactory sugar yield g/g (73.0 % for LCM1 and 60.0 % for LCM3) using less enzyme (just using recycled enzyme with no new enzyme added to the system) than the control case. Thus, it was possible to recycle the cellulases in two successive hydrolysis cycles with reduced enzyme load to achieve the same reducing sugar yields (up to 84 %), enabling cheaper and more efficient hydrolysis of lignocellulosic biomass.

Sustainable Exploitation of Residual Cynara cardunculus L. to Levulinic Acid and n-Butyl Levulinate

Hydrolysis and butanolysis of lignocellulosic biomass are efficient routes to produce two valuable bio-based platform chemicals, levulinic acid and n-butyl levulinate, which find increasing applications in the field of biofuels and for the synthesis of intermediates for chemical and pharmaceutical industries, food additives, surfactants, solvents and polymers. In this research, the acid-catalyzed hydrolysis of the waste residue of Cynara cardunculus L. (cardoon), remaining after seed removal for oil exploitation, was investigated. The cardoon residue was employed as-received and after a steam-explosion treatment which causes an enrichment in cellulose. The effects of the main reaction parameters, such as catalyst type and loading, reaction time, temperature and heating methodology, on the hydrolysis process were assessed. Levulinic acid molar yields up to about 50 mol % with levulinic acid concentrations of 62.1 g/L were reached. Moreover, the one-pot butanolysis of the steam-exploded cardoon with the bio-alcohol n-butanol was investigated, demonstrating the direct production of n-butyl levulinate with good yield, up to 42.5 mol %. These results demonstrate that such residual biomass represent a promising feedstock for the sustainable production of levulinic acid and n-butyl levulinate, opening the way to the complete exploitation of this crop.

Enzymatic Hydrolysis of Lignocellulosic Biomass Using an Optimized Enzymatic Cocktail Prepared from Secretomes of Filamentous Fungi Isolated from Amazonian Biodiversity

The use of lignocellulosic biomass (LCB) has emerged as one of the main strategies for generating renewable biofuels. For the efficient use of such feedstock, pre-treatments are essential. The hydrolysis of cellulose - major component of LCB - demands enzymatic cocktails with improved efficiency to generate fermentable sugars. In this scenario, lignocellulolytic fungi have enormous potential for the development of efficient enzyme platforms. In this study, two enzymatic cocktails were developed for hydrolysis of two lignocellulosic biomasses: industrial cellulose pulp and cassava peel. The solid biomass ratio in relation to the protein content of the enzyme cocktail was performed by experimental design. The optimized cocktail for the hydrolysis of cellulose pulp (AMZ 1) was composed, in protein base, by 43% of Aspergillus sp. LMI03 enzyme extract and 57% of T. reesei QM9414, while the optimal enzyme cocktail for cassava peel hydrolysis (AMZ 2) was composed by 50% of Aspergillus sp. LMI03 enzyme extract, 25% of the extract of P. citrinum LMI01 and 25% of T. reesei. The ratio between solids and protein loading for AMZ 1 cocktail performance was 52g/L solids and 30mg protein/g solids, resulting in a hydrolytic efficiency of 93%. For the AMZ 2 cocktail, the hydrolytic efficiency was 78% for an optimized ratio of 78g/L solids and 19mg protein/g solids. These results indicate that cocktails formulated with enzymatic extracts of P. citrinum LMI01, Aspergillus sp. LMI03, and T. reesei QM9414 are excellent alternatives for efficient hydrolysis of plant biomass and for other processes that depend on biocatalysis.

Two-Stage Fermentation of Lipomyces starkeyi for Production of Microbial Lipids and Biodiesel.

The high operating cost is currently a limitation to industrialize microbial lipids production by the yeast Lipomyces starkeyi. To explore economic fermentation technology, the two-stage fermentation of Lipomyces starkeyi using yeast extract peptone dextrose (YPD) medium, orange peel (OP) hydrolysate medium, and their mixed medium were investigated for seven days by monitoring OD600 values, pH values, cell growth status, C/N ratios, total carbon concentration, total nitrogen concentration, residual sugar concentration, lipid content, lipid titer, and fatty acids profiles of lipids. The results showed that two-stage fermentation with YPD and 50% YPD + 50% OP medium contributed to lipid accumulation, leading to larger internal lipid droplets in the yeast cells. However, the cells in pure OP hydrolysate grew abnormally, showing skinny and angular shapes. Compared to the one-stage fermentation, the two-stage fermentation enhanced lipid contents by 18.5%, 27.1%, and 21.4% in the flasks with YPD medium, OP medium, and 50%YPD + 50%OP medium, and enhanced the lipid titer by 77.8%, 13.6%, and 63.0%, respectively. The microbial lipids obtained from both one-stage and two-stage fermentation showed no significant difference in fatty acid compositions, which were mainly dominated by palmitic acid (33.36–38.43%) and oleic acid (46.6–48.12%). Hence, a mixture of commercial medium and lignocellulosic biomass hydrolysate could be a promising option to balance the operating cost and lipid production.

Role of Conformational Change and Glucose Binding Sites in the Enhanced Glucose Tolerance of Agrobacterium tumefaciens 5A GH1 β-Glucosidase Mutants.

β-Glucosidases are often inhibited by their reaction product glucose and a barrier to the efficient lignocellulosic biomass hydrolysis to glucose. We had previously reported the mutants, C174V, and H229S, with a nearly 2-fold increased glucose tolerance over the wild type (WT), H0HC94, encoded in Agrobacterium tumefaciens 5A (apparent Ki,Glc = 686 mM). We report our steady-state and time-resolved intrinsic fluorescence spectroscopy, circular dichroism, and isothermal titration calorimetry (ITC) studies to further understand increased glucose tolerance. Changes in the mutants' emission intensity and the differential change in quenching rate in the absence and presence of glucose reflect changes in protein conformation by glucose. Time-resolved lifetime and anisotropy measurements further indicated the microenvironment differences across solvent-exposed tryptophan residues and a higher hydrodynamic radius due to glucose binding, respectively. ITC measurements confirmed the increase of glucose binding sites in the mutants. The experiment results were supported by molecular dynamics simulations, which revealed significant variations in the glucose-protein hydrogen-bonding profiles. Protein structure network analysis of the simulated structures further indicates the mutants' conformation change than the WT. Computational studies also indicated additional glucose binding sites in mutants. Our results indicate the role of glucose binding in modulating the enzyme response to glucose.

Production of xylitol from acidic hydrolysates of lignocellulosic biomass by catalytic hydrogenation over a Ni–Ru/C catalyst

Acid hydrolysis and hydrogenation reactions were capable to convert xylose obtained from biomass materials using heterogeneous catalysts based on Ni and Ru. Xylose was extracted from sugar cane bagasse using dilute acid sulfuric solutions, and then xylitol was obtained through xylose catalytic hydrogenation. A bimetallic catalyst (Ni 10.0 wt.% and Ru 1.0 wt.%) was prepared by the wet impregnation method, supporting the metallic phase in activated carbon. Sugarcane bagasse was delignified in a three-step pretreatment (acid, basic and organic extractions, respectively), allowing effective hydrolysis of the resulting holocellulose. The acid hydrolysis process for xylose extraction was more effective at the highest concentration of sulfuric acid in the study (2.5% v/v), obtaining a xylose-rich hydrolysate with concentration over 10 g L−1. For the xylose hydrogenation process, conversion of xylose and selectivity to xylitol was near 90 % after 1 h of reaction. Kinetic evolutions were evaluated through a mathematical model in terms of operating time and temperature, providing hydrogenation activation energy values of the order of 40.76 kJ mol−1.

Exploring Proteomes of Robust Yarrowia lipolytica Isolates Cultivated in Biomass Hydrolysate Reveals Key Processes Impacting Mixed Sugar Utilization, Lipid Accumulation, and Degradation

ABSTRACTYarrowia lipolytica is an oleaginous yeast exhibiting robust phenotypes beneficial for industrial biotechnology. The phenotypic diversity found within the undomesticated Y. lipolytica clade from various origins illuminates desirable phenotypic traits not found in the conventional laboratory strain CBS7504 (or W29), which include xylose utilization, lipid accumulation, and growth on undetoxified biomass hydrolysates. Currently, the related phenotypes of lipid accumulation and degradation when metabolizing nonpreferred sugars (e.g., xylose) associated with biomass hydrolysates are poorly understood, making it difficult to control and engineer in Y. lipolytica. To fill this knowledge gap, we analyzed the genetic diversity of five undomesticated Y. lipolytica strains and identified singleton genes and genes exclusively shared by strains exhibiting desirable phenotypes. Strain characterizations from controlled bioreactor cultures revealed that the undomesticated strain YB420 used xylose to support cell growth and maintained high lipid levels, while the conventional strain CBS7504 degraded cell biomass and lipids when xylose was the sole remaining carbon source. From proteomic analysis, we identified carbohydrate transporters, xylose metabolic enzymes, and pentose phosphate pathway proteins stimulated during the xylose uptake stage for both strains. Furthermore, we distinguished proteins involved in lipid metabolism (e.g., lipase, NADPH generation, lipid regulators, and β-oxidation) activated by YB420 (lipid maintenance phenotype) or CBS7504 (lipid degradation phenotype) when xylose was the sole remaining carbon source. Overall, the results relate genetic diversity of undomesticated Y. lipolytica strains to complex phenotypes of superior growth, sugar utilization, lipid accumulation, and degradation in biomass hydrolysates.IMPORTANCE Yarrowia lipolytica is an important industrial oleaginous yeast due to its robust phenotypes for effective conversion of inhibitory lignocellulosic biomass hydrolysates into neutral lipids. While lipid accumulation has been well characterized in this organism, its interconnected lipid degradation phenotype is poorly understood during fermentation of biomass hydrolysates. Our investigation into the genetic diversity of undomesticated Y. lipolytica strains, coupled with detailed strain characterization and proteomic analysis, revealed metabolic processes and regulatory elements conferring desirable phenotypes for growth, sugar utilization, and lipid accumulation in undetoxified biomass hydrolysates by these natural variants. This study provides a better understanding of the robust metabolism of Y. lipolytica and suggests potential metabolic engineering strategies to enhance its performance.

Characterization of glycoside hydrolase family 11 xylanase from Streptomyces sp. strain J103; its synergetic effect with acetyl xylan esterase and enhancement of enzymatic hydrolysis of lignocellulosic biomass

BackgroundXylanase-containing enzyme cocktails are used on an industrial scale to convert xylan into value-added products, as they hydrolyse the β-1,4-glycosidic linkages between xylopyranosyl residues. In the present study, we focused on xynS1, the glycoside hydrolase (GH) 11 xylanase gene derived from the Streptomyces sp. strain J103, which can mediate XynS1 protein synthesis and lignocellulosic material hydrolysis.ResultsxynS1 has an open reading frame with 693 base pairs that encodes a protein with 230 amino acids. The predicted molecular weight and isoelectric point of the protein were 24.47 kDa and 7.92, respectively. The gene was cloned into the pET-11a expression vector and expressed in Escherichia coli BL21(DE3). Recombinant XynS1 (rXynS1) was purified via His-tag affinity column chromatography. rXynS1 exhibited optimal activity at a pH of 5.0 and temperature of 55 °C. Thermal stability was in the temperature range of 50–55 °C. The estimated Km and Vmax values were 51.4 mg/mL and 898.2 U/mg, respectively. One millimolar of Mn2+ and Na+ ions stimulated the activity of rXynS1 by up to 209% and 122.4%, respectively, and 1 mM Co2+ and Ni2+ acted as inhibitors of the enzyme. The mixture of rXynS1, originates from Streptomyces sp. strain J103 and acetyl xylan esterase (AXE), originating from the marine bacterium Ochrovirga pacifica, enhanced the xylan degradation by 2.27-fold, compared to the activity of rXynS1 alone when Mn2+ was used in the reaction mixture; this reflected the ability of both enzymes to hydrolyse the xylan structure. The use of an enzyme cocktail of rXynS1, AXE, and commercial cellulase (Celluclast® 1.5 L) for the hydrolysis of lignocellulosic biomass was more effective than that of commercial cellulase alone, thereby increasing the relative activity 2.3 fold.ConclusionThe supplementation of rXynS1 with AXE enhanced the xylan degradation process via the de-esterification of acetyl groups in the xylan structure. Synergetic action of rXynS1 with commercial cellulase improved the hydrolysis of pre-treated lignocellulosic biomass; thus, rXynS1 could potentially be used in several industrial applications.

Mechanisms of bio-additives on boosting enzymatic hydrolysis of lignocellulosic biomass

Expensive cellulase is one of the major obstacles hinders large-scale biorefining of lignocellulosic biomass. The cheap and biodegradable additives sophorolipid and whey protein were found to boost enzymatic hydrolysis, their mechanisms were clarified firstly in this study. Results showed that the effects of these additives on enhancing enzymatic hydrolysis were positively correlated with substrate content; when the solid dosage was 20% (w/v), the presence of sophorolipid and whey protein increased glucose yield by 17.8% and 11.9%, respectively; this could be attributed to sophorolipid favor to alleviate the non-productive adsorption between undesired substrates and enzymes caused by hydrophobic and electrostatic forces, and the ability of whey protein to block the site of enzyme adsorption of lignin; high shear and temperature conditions accelerate the inactivation of cellulase, and the addition of sophorolipid and whey protein reduced the inactivation rate by 7.8% and 13.6%, respectively, under enzymatic hydrolysis conditions.

Hydrolysis of Lignocellulosic Biomass in Hot-Compressed Water with Supercritical Carbon Dioxide.

This study investigated the decomposition behavior of bamboo under hydrothermal and hydrolysis conditions with H2O/CO2 in a semicontinuous-flow reactor at 9.8 MPa. At 255 °C, with and without CO2, xylan in bamboo completely decomposed into xylo-oligosaccharide (XOD). The yield of glucan degradation products with CO2 was significantly higher compared with that under the hydrothermal reaction (25.7 vs 14.9 wt %, respectively). The reaction rate of glucan decomposition with CO2 was slightly higher than the rate of hydrothermal reaction (kH2O/CO2/kH2O = 1.3). Increasing the fluid velocity of the hydrothermal reaction (3–10 mL/min) significantly accelerated the solubilization rate, but the ultimate yield of the soluble fraction was unchanged. The ultimate yield of the soluble fraction was slightly affected by physical effects. Hydrolysis with CO2 under severe conditions exhibited effective degradation of glucan. The catalytic activity of the H2O/CO2 system under hydrolysis can be explained by the system’s chemical effect.

Bioreactor and Bioprocess Design Issues in Enzymatic Hydrolysis of Lignocellulosic Biomass

Saccharification of lignocellulosic biomass is a fundamental step in the biorefinery of second generation feedstock. The physicochemical and enzymatic processes for the depolymerization of biomass into simple sugars has been achieved through numerous studies in several disciplines. The present review discusses the development of technologies for enzymatic saccharification in industrial processes. The kinetics of cellulolytic enzymes involved in polysaccharide hydrolysis has been discussed as the starting point for the design of the most promising bioreactor configurations. The main process configurations—proposed so far—for biomass saccharification have been analyzed. Attention was paid to bioreactor configurations, operating modes and possible integrations of this operation within the biorefinery. The focus is on minimizing the effects of product inhibition on enzymes, maximizing yields and concentration of sugars in the hydrolysate, and reducing the impact of enzyme cost on the whole process. The last part of the review is focused on an emerging process based on the catalytic action of laccase applied to lignin depolymerization as an alternative to the consolidated physicochemical pretreatments. The laccases-based oxidative process has been discussed in terms of characteristics that can affect the development of a bioreactor unit where laccases or a laccase-mediator system can be used for biomass delignification.

Cellulolytic enzymes production guided by morphology engineering

Endoglucanase and xylanase are critical enzymes for liquefaction and enzyme hydrolysis of high solids lignocellulosic biomass to facilitate its transport and production of desired derived products. Here is reported how combinations of different spore concentrations and pH influence microbial morphology, and how this may be used to direct expression and secretion of enzymes by Aspergillus niger. While xylanase production is not affected by A. niger morphology changes, endoglucanase production is enhanced under conditions of lower stress and by morphology that results in pellets. β-glucosidase production is enhanced under dispersed morphology, which results in up to fourfold increase of this enzyme production under the tested experimental conditions. A morphologic scale (Y) is proposed based on a form factor that considers the size and frequency of each morphology class, and that points to conditions that result in high selectivity for either endoglucanase or β-glucosidase production. An equation proposed to relate enzyme activity to morphology provides a useful tool for tuning enzyme production of A. niger, where morphology is a first indication of relative enzyme activities in a fermentation broth.

Optimisation of xylanases production by two Cellulomonas strains and their use for biomass deconstruction

One of the main distinguishing features of bacteria belonging to the Cellulomonas genus is their ability to secrete multiple polysaccharide degrading enzymes. However, their application in biomass deconstruction still constitutes a challenge. We addressed the optimisation of the xylanolytic activities in extracellular enzymatic extracts of Cellulomonas sp. B6 and Cellulomonas fimi B-402 for their subsequent application in lignocellulosic biomass hydrolysis by culture in several substrates. As demonstrated by secretomic profiling, wheat bran and waste paper resulted to be suitable inducers for the secretion of xylanases of Cellulomonas sp. B6 and C. fimi B-402, respectively. Both strains showed high xylanolytic activity in culture supernatant although Cellulomonas sp. B6 was the most efficient xylanolytic strain. Upscaling from flasks to fermentation in a bench scale bioreactor resulted in equivalent production of extracellular xylanolytic enzymatic extracts and freeze drying was a successful method for concentration and conservation of the extracellular enzymes, retaining 80% activity. Moreover, enzymatic cocktails composed of combined extra and intracellular extracts effectively hydrolysed the hemicellulose fraction of extruded barley straw into xylose and xylooligosaccharides.Key points• Secreted xylanase activity of Cellulomonas sp. B6 and C. fimi was maximised.• Biomass-induced extracellular enzymes were identified by proteomic profiling.• Combinations of extra and intracellular extracts were used for barley straw hydrolysis.

Real-Time Measurement of Cellobiose and Glucose Formation during Enzymatic Biomass Hydrolysis

Enzymatic hydrolysis of lignocellulosic biomass for biofuel production relies on complex multi-enzyme ensembles. Continuous and accurate measurement of the released key products is crucial in optimizing the industrial degradation process and also investigating the activity and interaction between the involved enzymes and the insoluble substrate. Amperometric biosensors have been applied to perform continuous cellobiose measurements during the enzymatic hydrolysis of pure cellulose powders. The oxygen-sensitive mediators used in these biosensors restricted their function under physiological or industrial conditions. Also, the combined measurements of the hydrolysis products cellobiose and glucose require a high selectivity of the biorecognition elements. We employed an [Os(2,2′-bipyridine)2Cl]Cl-modified polymer and cellobiose dehydrogenase to fabricate a cellobiose biosensor, which can accurately and specifically detect cellobiose even in the presence of oxygen and the other main product glucose. Additionally, a glucose biosensor was fabricated to simultaneously measure glucose produced from cellobiose by β-glucosidases. The cellobiose and glucose biosensors work at applied potentials of +0.25 and +0.45 V versus Ag|AgCl (3 M KCl), respectively, and can selectively detect their substrate. Both biosensors were used in combination to monitor the hydrolysis of pure cellulose of low crystallinity or industrial corncob samples. The obtained results correlate with the high-performance liquid chromatography pulsed amperometric detection analysis and demonstrate that neither oxygen nor the presence of redox-active compounds from the lignin fraction of the corncob interferes with the measurements.

Investigating the effects of substrate morphology and experimental conditions on the enzymatic hydrolysis of lignocellulosic biomass through modeling

BackgroundUnderstanding how the digestibility of lignocellulosic biomass is affected by its morphology is essential to design efficient processes for biomass deconstruction. In this study, we used a model based on a set of partial differential equations describing the evolution of the substrate morphology to investigate the interplay between experimental conditions and the physical characteristics of biomass particles as the reaction proceeds. Our model carefully considers the overall quantity of cellulase present in the hydrolysis mixture and explores its interplay with the available accessible cellulose surface.ResultsExploring the effect of various experimental and structural parameters highlighted the significant role of internal mass transfer as the substrate size increases and/or the enzyme loading decreases. In such cases, diffusion of cellulases to the available cellulose surface limits the rate of glucose release. We notably see that increasing biomass loading, while keeping enzyme loading constant should be favored for both small- (R < 300 mu m) and middle-ranged (300 < R < 1000 mu m) substrates to enhance enzyme diffusion while minimizing the use of enzymes. In such cases, working at enzyme loadings exceeding the full coverage of the cellulose surface (i.e. eI>1) does not bring a significant benefit. For larger particles (R > 1000 mu m), increases in biomass loading do not offset the significant internal mass transfer limitations, but high enzyme loadings improve enzyme penetration by maintaining a high concentration gradient within the particle. We also confirm the well-known importance of cellulose accessibility, which increases with pretreatment.ConclusionsBased on the developed model, we are able to propose several design criteria for deconstruction process. Importantly, we highlight the crucial role of adjusting the enzyme and biomass loading to the wood particle size and accessible cellulose surface to maintain a strong concentration gradient, while avoiding unnecessary excess in cellulase loading. Theory-based approaches that explicitly consider the entire lignocellulose particle structure can be used to clearly identify the relative importance of bottlenecks during the biomass deconstruction process, and serve as a framework to build on more detailed cellulase mechanisms.