Hydrolysis Of Lignocellulosic Biomass Research Articles

Techno‐Economic Analysis for Production of L‐Arabitol from L‐Arabinose

AbstractThe process design for synthesis of arabitol by hydrogenation of arabinose on a supported ruthenium catalyst is described. Aspen HYSYS software was used to design an arabitol production plant for subsequent generation of hydrogen through aqueous phase reforming (APR). The process design included hydrogen recycling requiring a flash drum and recompression. The total costs of arabitol were estimated to be substantially dependent on the feedstock costs and are closely related to arabinose feed cost. Feasible production of hydrogen using APR requires efficient extraction of hemicellulose from lignocellulosic biomass and subsequent hydrolysis.

Biodegradation of recalcitrant compounds and phthalates by culturable bacteria isolated from Liometopum apiculatum microbiota.

Liometopum apiculatum is a species of ants widely distributed in arid and semi-arid ecosystems where there is a relative food shortage compared with tropical ecosystems. L. apiculatum has established an ecological balance involving symbiotic interactions, which have allowed them to survive through mechanisms that are still unknown. Therefore, the aim of this study was to explore the metabolic potential of isolated bacteria from L. apiculatum using enzymatic activity assay and substrate assimilation. Results revealed a complex bacteria consortium belonging to Proteobacteria, Firmicutes, and Actinobacteria phylum. Most of the isolated bacteria showed activities associated with biopolymers degradation, from them Exiguobacterium and B. simplex showed the highest amylolytic activity (27 U/mg protein), while A. johnsonii and B. pumulis showed the highest cellulolytic and xylanolytic activities (1 and 2.9 U/mg protein, respectively). By other hand, some microorganisms such as S. ficaria, E. asburiae, P. agglomerans, A. johnsonii, S. rubidaea, S. marcescens, S. warneri, and M. hydrocarbonoxydans were able to grow up to 1000mg/L of phthalates esters. These results not only revealed the important contribution of the symbionts in L apiculatum ants feeding habits, but also have shown a promising source of enzymes with potential biotechnological applications such as lignocellulosic biomass hydrolysis and bioremediation processes.

Acid Hydrolysis of Lignocellulosic Biomass: Sugars and Furfurals Formation

Hydrolysis of lignocellulosic biomass is a crucial step for the production of sugars and biobased platform chemicals. Pretreatment experiments in a semi-continuous plant with diluted sulphuric acid as catalyst were carried out to measure the time-dependent formation of sugars (glucose, xylose, mannose), furfurals, and organic acids (acetic, formic, and levulinic acid) at different hydrolysis temperatures (180, 200, 220 °C) of one representative of each basic type of lignocellulose: hardwood, softwood, and grass. The addition of the acid catalyst is followed by a sharp increase in the sugar concentration. Xylose and mannose were mainly formed in the initial stages of the process, while glucose was released slowly. Increasing the reaction temperature had a positive effect on the formation of furfurals and organic acids, especially on hydroxymehtylfurfural (HMF) and levulinic acid, regardless of biomass type. In addition, large amounts of formic acid were released during the hydrolysis of miscanthus grass. Structural changes in the solid residue show a complete hydrolysis of hemicellulose at 180 °C and of cellulose at 200 °C after around 120 min reaction time. The results obtained in this study can be used for the optimisation of the hydrolysis conditions and reactor design to maximise the yields of desired products, which might be sugars or furfurals.

Cellulase and oxidative enzymes: new approaches, challenges and perspectives on cellulose degradation for bioethanol production.

Second-generation bioethanol is a sustainable energy source that can be produced from different renewable materials. However, there is a challenge we must overcome to significantly enhance bioethanol production: the hydrolysis of lignocellulosic biomass to fermentable sugars. Synergistic enzymes, such as endoglucanases, β-glucosidases, cellobiohydrolases, and, more recently, lytic polysaccharide monooxygenases and cellobiose dehydrogenases have been used with great success to hydrolyze pretreated biomass. Further advances in the field of second-generation bioethanol production will likely depend on an increased understanding of the interactions between enzymes and lignocellulosic substrates, the development of enzyme engineering, and the optimization of enzyme mixtures to enhance cellulose hydrolysis.

Extrusion process to enhance the pretreatment effect of ionic liquid for improving enzymatic hydrolysis of lignocellulosic biomass

Extrusion process using 1-ethyl-3-methylimidazolium acetate ([EMIM]Ac) in co-solvent, dimethyl sulfoxide (DMSO), was conducted to improve the pretreatment effect on the enzymatic saccharification of pussy willow (Salix gracilistyla Miq.). The use of a twin screw extruder capable of continuous processing increased the solid loading to 50% in [EMIM]Ac and its co-solvent DMSO. The water-soluble fraction was increased by increasing the [EMIM]Ac amount in co-solvent and the extrusion temperature but decreasing the screw rotating speed and solid loading. The surface morphologies of the extruded product showed remarkable disruption of the cell wall structures and fibrillation with increasing amounts of [EMIM]Ac in co-solvent. With increasing [EMIM]Ac amount in co-solvent and extrusion temperature and with decreasing screw rotating speed and solid loading, the crystalline structure of cellulose was more disrupted, resulting in the increase in saccharification yield. The highest yield of glucose and xylose was 99.0% and 99.5%, respectively, from the pretreated products using only [EMIM]Ac under solid loading of 15% at 160 °C extrusion temperature and 5 rpm screw rotating speed. The initial saccharification rate was faster in the pretreated product precipitated in alcohol and acetone than in water.

Caulobacter crescentus β-Xylosidase II Is Highly Tolerant to Inhibitors Present in Fermentative Processes Involving Lignocellulosic Biomass

More stable enzymes with improved properties are in constant demand for various biotechnological processes. In this work, it was found that the activity of recombinant purified β-xylosidase II (CcXynB2) from the bacterium Caulobacter crescentus is increased 62% by 5 mM KCl, likely due to the presence of K+ ions. CcXynB2 activity was measured in the presence of various compounds that have been described as inhibitors of lignocellulosic biomass hydrolysis and fermentation. CcXynB2 was found to be 61% more tolerant than 200 mM ethanol over a 48-h incubation at 37 °C. The specific activity of CcXynB2 was determined in the presence of phenol, hydroxymethylfurfural, ferulic, acetic, and coumaric acids; arabinose, glucose, xylose, and pectin. After 48 h, CcXynB2 activity in the presence of these compounds was found to be equal to 100% or higher than CcXynB2 activity in the absence of these compounds. When inhibitors were used in combination, CcXynB2 retained 67% of its initial activity over 48 h at 37 °C. Enzymatic hydrolysis of hemicellulose from corncobs was performed with CcXynB2 alone or with CcXynB2 in conjunction with recombinant xylanase and β-glucosidase-β-xylosidase-α-arabinosidase from C. crescentus producing reducing sugars. The immobilized CcXynB2 was more active than the soluble enzyme at both temperatures tested, 37 and 50 °C. In addition, immobilized CcXynB2 retained most of the enzyme activity incubated at 50 °C than at 37 °C, maintaining more than 70% of its initial activity after 1 h of incubation. In general, the CCXynB2 has potential to be applied in a bioprocess since it showed robust resistance in presence of many chemical compounds that may be generated during fermentation steps and physicochemical pre-treatment of biomass if considering a simultaneous saccharification fermentation process in a biorefinery.

Developing Clostridium diolis as a biorefinery chassis by genetic manipulation

Clostridium diolis can efficiently utilize various inexpensive, renewable resources such as crude glycerol and lignocellulosic biomass hydrolysate to produce bulk chemicals and fuels. However, its study has been impeded by the lack of efficient plasmids electro-transformation techniques. In this study, an efficient electroporation protocol for C. diolis was developed and two replicons functional in C. diolis were identified. After optimizing parameters, the electro-transformation efficiency was enhanced from 5 to 692 transformants/ug DNA. Moreover, metabolic engineering of C. diolis was performed as proof of concept for the first time. By simply overexpressing heterologous genes based on the replicable plasmids, the strain was engineered to improve productions of diol (1,3-propanediol) and n-alcohol (butanol), and to enable butyl acetate synthesis in vivo, respectively under different culture conditions. This work represented a milestone of breeding C. diolis using metabolic engineering, and paved the way for studying C. diolis on the molecular level.

Paenibacillus barengoltzii A1_50L2 as a Source of Plant Cell Wall Degrading Enzymes and Its Use on Lignocellulosic Biomass Hydrolysis

Lignocellulosic residues daily generated as a by-product of economic activities worldwide, stand out as potential feedstock to biorefineries. Industrial processes based on their use still face a challenge in the development of low-cost enzymatic mixtures resistant to inhibitors produced during pre-treatment step and efficient in the complete deconstruction of different lignocellulosic wastes. In the present work, Paenibacillus barengoltzii A1_50L2 isolated from bovine rumen was studied as a source of plant cell wall degrading enzymes. In addition, the natural produced enzymatic cocktail was characterized and applied to the hydrolysis of lignocellulosic substrates. P. barengoltzii grew in liquid media containing cellulose, sugarcane bagasse and wheat bran, as carbon source producing endo-1,4-β-xylanases, pectinases, mannanases, endo-1,4-β-glucanases, exo-1,4-β-glucanases and β-glucosidases, with highest values of endo-1,4-β-xylanase activity. Six isoforms of endo-1,4-β-xylanase (45 to 116 kDa), three of endo-1,4-β-glucanase (40 to 66 kDa) and a single band of mannanase (62 kDa) were secreted by the bacterium during growth on wheat bran, all enzymes presented maximal activity in pH ranging from 4 up to 8 and temperature from 45 up to 60 °C. Endo-1,4-β-xylanases, endo-1,4-β-glucanases, exo-1,4-β-glucanases, pectinases, mannanases and β-glucosidases produced by P. barengoltzii are tolerant to lignin-derived compounds showing a minimal residual activity of 61%. Ferulic, gallic, tannic and cinnamic acids enhanced activities of endo-1,4-β-glucanases/endo-1,4-β-xylanases, mannanase and pectinase, respectively. P. barengoltzii enzymatic cocktail (PbEC) hydrolyzes sugarcane bagasse, banana stem and corncob with hydrolysis values comparable to those obtained for the commercial blend Viscozyme®. The addition of PbEC to Viscozyme® increases the hydrolysis rate of sugarcane bagasse, banana stem and corncob in three to four times.

Xylose utilization in Saccharomyces cerevisiae during conversion of hydrothermally pretreated lignocellulosic biomass to ethanol.

With growing interest in alternative fuels to minimize carbon and particle emissions, research continues on the production of lignocellulosic ethanol and on the development of suitable yeast strains. However, great diversities and continued technical advances in pretreatment methods for lignocellulosic biomass complicate the evaluation of developed yeast strains, and strain development often lags industrial applicability. In this review, recent studies demonstrating developed yeast strains with lignocellulosic biomass hydrolysates are compared. For the pretreatment methods, we highlight hydrothermal pretreatments (dilute acid treatment and autohydrolysis), which are the most commonly used and effective methods for lignocellulosic biomass pretreatment. Rather than pretreatment conditions, the type of biomass most strongly influences the composition of the hydrolysates. Metabolic engineering strategies for yeast strain development, the choice of xylose-metabolic pathway, adaptive evolution, and strain background are highlighted as important factors affecting ethanol yield and productivity from lignocellulosic biomass hydrolysates. A comparison of the parameters from recent studies demonstrating lignocellulosic ethanol production provides useful information for future strain development.

Bioethanol production estimated from volatile compositions in hydrolysates of lignocellulosic biomass by deep learning

The cell growth and ethanol production from hydrolysates of various types were estimated from the volatile composition of lignocellulosic biomass by deep neural network (DNN) and the significant compositions estimated by asymmetric autoencoder-decoder (AAE). A six-layer DNN achieved good accuracy with learning and validation losses-0.033 and 0.507, respectively-and estimated overall time courses of yeast growth and ethanol fermentation. The AAE decoded the volatile compositions and represented the features of significant inhibitors via nonlinear dimensionality reduction, which was partly different from those using partial least squares regression reported previously. It revealed the significant features of hydrolysates for bioethanol production, which are lost in conventional approaches. The approach using DNN and AAE is, therefore, useful for bioethanol fermentation and other bioproductions using raw materials.

Stochastic optimization of enzymatic hydrolysis of lignocellulosic biomass

Uncertainties in feedstock composition and kinetic parameters impact the performance of enzymatic hydrolysis of lignocellulosic biomass. Operating strategies need to be accordingly modified to achieve higher yield of glucose. Therefore, stochastic optimization and optimal control problems were formulated to maximize glucose concentration and minimize variance. The optimal temperature for maximization of glucose concentration reduced in the presence of uncertainties. For uncertainties in kinetic parameters, the optimal temperature reduced from 323.34 K to 317.07 K for a batch time of 12 h, and the glucose concentration increased by 7.7% when the stochastic optimal temperature was used instead of deterministic temperature. Similarly, for the objective of minimization of variance in glucose concentration at the end of the batch, stochastic optimization reduced the variance by 89% as compared to deterministic optimization. Stochastic optimal control resulted in up to 60% improvement in mean glucose concentration in comparison to the deterministic optimal control.

Metabolic engineering of Escherichia coli to produce succinate from woody hydrolysate under anaerobic conditions.

It is of great economic interest to produce succinate from low-grade carbon sources, e.g., lignocellulosic biomass hydrolysate, which mainly contains glucose and xylose. Inactivation of the glucose uptake system PtsG was evaluated for succinate production from xylose-rich feedstocks. Strains with integration of succinate production modules into the chromosome of Escherichia coli were then constructed. These strains have better succinate production performance from xylose-rich feedstocks than strain FZ560 harboring pHL413KF1. Glucose utilization was enhanced in FZ661T by manipulation of the gal operon to allow efficient use of the high-concentration glucose in woody biomass hydrolysate. Up to 906.7mM (107.0g/L) succinate was produced from mixed sugars in fed-batch fermentation and more than 461.7mM (54.5g/L) succinate was produced from woody hydrolysate in a batch fermentation. In this study, FZ661T was able to produce succinate from woody hydrolysate in minimal medium efficiently, making it attractive for industrial applications in succinate production.

Recombinant xylose-fermenting yeast construction for the co-production of ethanol and cis,cis-muconic acid from lignocellulosic biomass

New exogenous cis,cis-muconic acid biosynthetic pathway genes were expressed in Saccharomyces cerevisiae. The xylose isomerase gene from Bacteroides valgutus and pentose phosphate pathway genes from S. cerevisiae were overexpressed in the yeast strain. The strain was further modified by the overexpression of gene Aro1 (with a stop codon of AroE) and a feedback-resistant Aro4opt mutant gene from S. cerevisiae. Under oxygen-limited conditions, it produced 65 mg/L cis,cis-muconic acid from xylose. Co-fermentation of 88 g/L glucose and 50 g/L xylose generated 54 g/L ethanol and 248 mg/L cis,cis-muconic acid. Under aerobic conditions, muconic acid titer reached 424 mg/L. With the supplement of 1 g/L catechol, 1286 mg/L muconic acid was produced. Fermentation of an oil palm empty fruit bunch hydrolysate resulted in 31.3 g/L ethanol and 53.4 mg/L muconic acid. This is the first report on the production of muconic acid from lignocellulosic biomass hydrolysate using a recombinant xylose-fermenting yeast.

Insertional tagging of the Scheffersomyces stipitis gene HEM25 involved in regulation of glucose and xylose alcoholic fermentation.

Amid known microbial bioethanol producers, the yeast Scheffersomyces (Pichia) stipitis is particularly promising in terms of alcoholic fermentation of both glucose and xylose, the main constituents of lignocellulosic biomass hydrolysates. However, the ethanol yield and productivity, especially from xylose, are still insufficient to meet the requirements of a feasible industrial technology; therefore, the construction of more efficient S. stipitis ethanol producers is of great significance. The aim of this study was to isolate the insertional mutants of S. stipitis with altered ethanol production from glucose and xylose and to identify the disrupted gene(s). Mutants obtained by random insertional mutagenesis were screened for their growth abilities on solid media with different sugars and for resistance to 3-bromopyruvate. Of more than 1,300 screened mutants, 17 were identified to have significantly changed ethanol yields during the fermentation. In one of the best fermenting strains (strain 4.6), insertion was found to occur within the ORF of a homolog to theSaccharomyces cerevisiae gene HEM25 (YDL119C), encoding a mitochondrial glycine transporter required for heme synthesis. The role of HEM25 in heme accumulation, respiration, and alcoholic fermentation in the yeast S. stipitis was studied using strain 4.6, the complementation strain Comp-a derivative from the 4.6 strain with expression of the WT HEM25 allele and the deletion strain hem25Δ. As hem25Δ produced lower amounts of ethanol than strain 4.6, we assume that the phenotype of strain 4.6 may be caused not only by HEM25 disruption but additionally by some point mutation.

New insights into transport capability of sugars and its impact on growth from novel mutants of Escherichia coli.

The fast-growing capability of Escherichia coli strains used to produce industrially relevant metabolites relies on their capability to transport efficiently glucose or potential industrial feedstocks such as sucrose or xylose as carbon sources. E. coli imports extracellular glucose into the periplasmic space across the outer membrane porins: OmpC, OmpF, and LamB. As the internal membrane is an impermeable barrier for sugars, the cell employs several primary and secondary active transport systems, and the phosphoenolpyruvate (PEP)-sugar phosphotransferase (PTS) system for glucose transport. PTS:glucose is the preferred system by E. coli to transport and phosphorylate the periplasmic glucose; nevertheless, PTS imposes a strict metabolic control mechanism on the preferential consumption of glucose over other carbon sources in sugar mixtures such as glucose and xylose resulting from the hydrolysis of lignocellulosic biomass, by the carbon catabolite repression. In this contribution, we summarize the major sugar transport systems for glucose and disaccharide transport, the exhibited substrate plasticity, and their impact on the growth of E. coli, highlighting the relevance of PTS in the control of the expression of genes for the transport and catabolism of other sugars as xylose. We discuss the strategies developed by evolved mutants of E. coli during adaptive laboratory evolution experiments to overcome the nutritional stress condition imposed by inactivation of PTS as a strategy for the selection of fast-growing derivatives in glucose, xylose, or mixtures of glucose:xylose. This approach results in the recruitment of other primary and secondary active transporters, demonstrating relevant sugar plasticity in derivative-evolved mutants. Elucidation of the molecular and biochemical basis of sugar-transport substrate plasticity represents a consistent approach for sugar-transport system engineering for the design of efficient E. coli derivative strains with improved substrate assimilation for biotechnological purposes.

Enzymatic hydrolysis of lignocellulosic biomass from pineapple leaves by using endo-1,4-xylanase: Effect of pH, temperature, enzyme loading and reaction time

Pineapple leaves; is one of lignocellulosic materials that composed of about 80% of carbohydrate containing lignin, cellulose and hemicellulose This waste still retains a considerable amount of soluble sugars that can be transformed into valuable fermentable sugars. This research was conducted to study the effect of different pH, temperature, enzyme loading and reaction time in order to identify the best working conditions to produce the highest yield of sugar. Prior to enzymatic hydrolysis, the pineapple leaves were subjected to hydrothermal pretreatment in order to obtain the hydrolysate liquid. Enzymatic hydrolysis was then carried out by using endo-1,4-xylanase and the reducing sugar was analyzed by using dinitrosalicylic (DNS) method. The result has shown that the highest reducing sugar concentration of 70.9 mg/L was obtained both at pH 5.5 and 50 °C. In addition, the best enzyme loading and reaction time were recorded at 0.5% (w/v) and 45 min that resulted 60.1 mg/L and 72. 0 mg/L respectively.

Simplifying levulinic acid conversion towards a sustainable biomass valorisation

In the range of biomass valorisation, hydroconversion of levulinic acid (one of the products of the hydrolysis of lignocellulosic biomass) was tested for the first time using nanosized Ni3N and Ni0catalysts, with very promising results alongside high stability.

Isolation of xylose-assimilating yeasts and optimization of xylitol production by a new Meyerozyma guilliermondii strain.

Production of xylitol from lignocellulosic biomass is of interest to modern biorefineries, because this biomass should be processed into a spectrum of chemicals (bio-based products) and not only energy. The isolation of new yeast strains capable of efficiently converting xylose into xylitol and withstanding inhibitors released from biomass hydrolysis can contribute to making its production feasible in biorefineries. Forty-three out of 128 yeast strains isolated from the gut of Passalidae beetles were capable of assimilating xylose as the sole carbon source. Meyerozyma guilliermondii UFV-1 was selected due to its ability to grow and ferment D-xylose in a synthetic medium. This yeast assimilated the broad range of sugars present in lignocellulosic biomass hydrolysates, such as xylose, raffinose, cellobiose, rhamnose, arabinose, and glucose. Its optimum growth conditions were pH 8.0 and a temperature of 30 °C. In concentrations of 0.07 mol/Lacetic acid, 0.05 mol/L 5-hydroximethylfurfural, and 0.04 mol/L furfural, M. guilliermondii UFV-1 did not grow. Maximum xylitol production in aerobiosis and hypoxia were 51.88 and 27.73 g/L, respectively. Under aerobic condition, xylose concentration and agitation rate were the factors which were statistically significant, while only the agitation rate was significant in hypoxia. We fitted a response surface (RS) that estimated the best agitation rate (113.33 rpm) and xylose concentration (90 g/L) for maximum xylitol production in aerobiosis. Therefore, M. guilliermondii UFV-1 displays potential for being used for xylitol production in biorefineries.

Production of formic acid from oil palm empty fruit bunch via dilute acid hydrolysis by response surface methodology

Formic acid is the simplest carboxylic acid which has myriad applications in food, textiles, agriculture, pharmaceutics, and chemicals. Formic acid can be synthesized from hydrolysis of lignocellulosic biomass. Oil palm empty fruit bunch (OPEFB) is lignocellulosic biomass produced by oil palm industries to which cellulose content has potential for conversion into formic acid by hydrolysis. We investigated effects of three parameters in acid-catalysed hydrolysis reaction of OPEFB such as reaction time (20, 40, 60 minutes), temperature (140, 160, 180 °C), and H2SO4 concentration (0.3; 0.5; 0.7 M) and optimized them to obtain maximum formic acid concentration by using response surface methodology. Microwave assisted alkaline pretreatment of OPEFB sample under microwave radiation at 840 Watt for 9 minutes with 2% NaOH concentration was done prior to hydrolysis. The pretreatment was effective to reduce lignin content of OPEFB from 28.9% to 7.6%. The highest actual formic acid concentration we obtained from the experiment was 2725 ppm at 180 °C, 60 minutes reaction time, and 0.5 M H2SO4. While according to the polynomial model, the optimal condition for obtaining maximum formic acid concentration of 2890.673 ppm was at 180 °C, reaction time of 60 minutes, and 0.3 M H2SO4.

Stable cellulase immobilized on graphene oxide@CMC-g-poly(AMPS-co-AAm) hydrogel for enhanced enzymatic hydrolysis of lignocellulosic biomass

This study indicated tailoring efficient polymer-enzyme bioconjugates with superb stability and activity for practical utilization of cellulase enzyme in hydrolyzing lignocellulosic biomass. To this goal, a dual crosslinking (DC) strategy was presented to synthesize novel 3D networks of carboxymethyl cellulose grafted copolymers of 2-acrylamido-2methyl propane sulfonate and acrylamide (CMC-g-poly(AMPS-co-AAm)) hydrogels. Graphene oxide (GO) nano-sheets were utilized as nano-filler and physical cross-linker making H-bondings between polymeric chains to prepare GO@CMC-g-poly(AMPS-co-AAm) networks. The GO content effects on the performance of as-synthesized architectures in conjugation to a model enzyme (PersiCel1) were examined. PersiCel1 immobilization on the GO reinforced hydrogels resulted in noticeable retaining near 60 % of its maximum activity at 90 °C, along with the remarkable enhancement of its specific activity and storage stability. Compared with the free PersiCel1, the immobilized enzyme on the GO containing hydrogels showed 154.8 % increase in conversion of alkalin-treated sugar beet pulp, while the PersiCel1/neat-Hydrogel indicated an increment of 66.7 %, under the same conditions.