Saccharification Of Pretreated Wheat Straw Research Articles

Bioprospecting CAZymes repertoire of Aspergillus fumigatus for eco-friendly value-added transformations of agro-forest biomass

BackgroundValorizing waste residues is crucial to reaching sustainable development goals and shifting from a linear fossil-based economy to a circular economy. Fungal cell factories, due to their versatility and robustness, are instrumental in driving the bio-transformation of waste residues. The present work isolated a potent strain, i.e., Aspergillus fumigatus (ZS_AF), from an ancient Złoty Stok gold mine, which showcased distinctive capabilities for efficient hydrolytic enzyme production from lignocellulosic wastes.ResultsThe present study optimized hydrolytic enzyme production (cellulases, xylanases, and β-glucosidases) from pine sawdust (PSD) via solid-state fermentation using Aspergillus fumigatus (ZS_AF). The optimization, using response surface methodology (RSM), produced a twofold increase with maximal yields of 119.41 IU/gds for CMCase, 1232.23 IU/gds for xylanase, 63.19 IU/gds for β-glucosidase, and 31.08 IU/gds for FPase. The secretome profiling validated the pivotal role of carbohydrate-active enzymes (CAZymes) and auxiliary enzymes in biomass valorization. A total of 77% of carbohydrate-active enzymes (CAZymes) were constituted by glycoside hydrolases (66%), carbohydrate esterases (9%), auxiliary activities (3%), and polysaccharide lyases (3%). The saccharification of pretreated wheat straw and PSD generated high reducing sugar yields of 675.36 mg/g and 410.15 mg/g, respectively.ConclusionThese findings highlight the significance of an efficient, synergistic, and cost-effective arsenal of fungal enzymes for lignocellulosic waste valorization and their potential to contribute to waste-to-wealth creation through solid-waste management. The utilization of Aspergillus fumigatus (ZS_AF) from an unconventional origin and optimization strategies embodies an innovative approach that holds the potential to propel current waste valorization methods forward, directing the paradigm toward improved efficiency and sustainability.

Production and properties of Irpex lacteus cellulase and xylanase

This study aimed to characterize the cellulolytic system of Irpex lacteus BCC 104 isolated from the forest of Georgia. The results obtained showed that the expression of endoglucanase and xylanase synthesis in this fungus is inducible in the presence of cellulose-containing growth substrates. Among them, crystalline cellulose appeared to be the best carbon source providing the highest endoglucanase (48.0 U/mL), total cellulase (7.6 U/mL), and xylanase (68.4 U/mL) activities. Supplementation of the Avicel-induced culture with 0.5 or 1% glycerol caused catabolite repression of the cellulase and xylanase formation by I.lacteus BCC 104. The enzyme synthesis resumed only after the depletion of easily metabolizable carbon source from the medium. The crude enzyme obtained after the cultivation of I. lacteus in the fermenter and used for saccharification of pretreated wheat straw (40 mg/mL, FPA load 20 U/g substrate) yielded 10.7 mg reducing sugars/mL (32.2% from the theoretically possible). CMCase and xylanase were purified 31- and 34-fold and characterized. The optimum pH for both enzyme activities was found to be 5.0; at pH 5-6, they retained 60% of their initial activity after incubation at 30°C for 48 h. CMCase expressed maximum activity at 50°C while xylanase – at 55°C. Half-inactivation periods of endoglucanase and xylanase during incubation at 60°C were 60 min and 150 min, respectively.

Production of a β-Glucosidase-Rich Cocktail from Talaromyces amestolkiae Using Raw Glycerol: Its Role for Lignocellulose Waste Valorization.

As β-glucosidases represent the major bottleneck for the industrial degradation of plant biomass, great efforts are being devoted to discover both novel and robust versions of these enzymes, as well as to develop efficient and inexpensive ways to produce them. In this work, raw glycerol from chemical production of biodiesel was tested as carbon source for the fungus Talaromyces amestolkiae with the aim of producing enzyme β-glucosidase-enriched cocktails. Approximately 11 U/mL β-glucosidase was detected in these cultures, constituting the major cellulolytic activity. Proteomic analysis showed BGL-3 as the most abundant protein and the main β-glucosidase. This crude enzyme was successfully used to supplement a basal commercial cellulolytic cocktail (Celluclast 1.5 L) for saccharification of pretreated wheat straw, corroborating that even hardly exploitable industrial wastes, such as glycerol, can be used as secondary raw materials to produce valuable enzymatic preparations in a framework of the circular economy.

Differential \u03b2-glucosidase expression as a function of carbon source availability in Talaromyces amestolkiae: a genomic and proteomic approach

BackgroundGenomic and proteomic analysis are potent tools for metabolic characterization of microorganisms. Although cellulose usually triggers cellulase production in cellulolytic fungi, the secretion of the different enzymes involved in polymer conversion is subjected to different factors, depending on growth conditions. These enzymes are key factors in biomass exploitation for second generation bioethanol production. Although highly effective commercial cocktails are available, they are usually deficient for β-glucosidase activity, and genera like Penicillium and Talaromyces are being explored for its production.ResultsThis article presents the description of Talaromyces amestolkiae as a cellulase-producer fungus that secretes high levels of β-glucosidase. β-1,4-endoglucanase, exoglucanase, and β-glucosidase activities were quantified in the presence of different carbon sources. Although the two first activities were only induced with cellulosic substrates, β-glucosidase levels were similar in all carbon sources tested. Sequencing and analysis of the genome of this fungus revealed multiple genes encoding β-glucosidases. Extracellular proteome analysis showed different induction patterns. In all conditions assayed, glycosyl hydrolases were the most abundant proteins in the supernatants, albeit the ratio of the diverse enzymes from this family depended on the carbon source. At least two different β-glucosidases have been identified in this work: one is induced by cellulose and the other one is carbon source-independent. The crudes induced by Avicel and glucose were independently used as supplements for saccharification of slurry from acid-catalyzed steam-exploded wheat straw, obtaining the highest yields of fermentable glucose using crudes induced by cellulose.ConclusionsThe genome of T. amestolkiae contains several genes encoding β-glucosidases and the fungus secretes high levels of this activity, regardless of the carbon source availability, although its production is repressed by glucose. Two main different β-glucosidases have been identified from proteomic shotgun analysis. One of them is produced under different carbon sources, while the other is induced in cellulosic substrates and is a good supplement to Celluclast in saccharification of pretreated wheat straw.

Investigating the enzyme-lignin binding with surfactants for improved saccharification of pilot scale pretreated wheat straw

In this study, commercial surfactants have been investigated at economically viable dosage to enhance the enzymatic saccharification of pretreated wheat straw at high solid loadings. Twenty one surfactants were evaluated with pilot scale pretreated wheat straw and mechanism of surfactant action has been elucidated. One surfactant has improved the saccharification of dilute acid wheat straw (DAWS) by 26.4% after 24h and 23.1% after 48h while, steam exploded wheat straw (SEWS) saccharification was increased by 51.2% after 24h and 36.4% after 48h at 10% solid loading. At 20% solid loading, about 31% increase in yield was obtained on DAWS and about 55% on SEWS after 48h. Further, lignin was isolated from pretreated wheat straws and characterized which revealed that SEWS derived lignin was more hydrophobic than DAWS lignin. This investigation suggests that surfactant supplementation during saccharification is an effective strategy to achieve higher saccharification yield.

Production of Cellulase for Ethanol Fermentation from Pretreated Wheat Straw

Effect of different media compositions on cellulase production was observed using a strain of Trichoderma viride. Medium with yeast extract as nitrogen source was found the best from the other sources used. Then two modes of fermentation solid state and submerged were compared for the enhanced cellulase production. Better cellulase activity was observed in submerged fermentation than the solid state fermentation. Effect of mash size and agitation was also studied. Small mash size with agitation showed higher cellulase activity than the large size pretreated wheat straw. Bacillus cellulosilyticus was also used for the cellulase production. Trichoderma produced enhanced activities of cellulase enzyme (20.738 ± 0.006 IU). Scarification of pretreated wheat straw released maximum sugar up to 16.1 g/L, after hydrolysis in 48 h using indigenously produced enzyme. The optimum conditions for the saccharification of pretreated wheat straw were 5 and 30 °C for pH and temperature, respectively in 48 h. The yield of ethanol was observed 10.4 g/L in saccharified wheat straw based medium.

An integrative process for bio-ethanol production employing SSF produced cellulase without extraction

Bio-ethanol production from pretreated biomass in a single vessel was investigated in which cellulase production via SSF by Penicillium janthinellum EMS-UV-8, hydrolysis of biomass and ethanol fermentation was carried out sequentially. In this study, feasibility of using whole fermented matter with enzyme, fungal mycelia, spores and residual substrate, for the saccharification of pretreated wheat straw or avicel and its further fermentation to ethanol was investigated. Whole fermented matter produced during cellulase production via solid-state fermentation and extracted cellulase was compared for hydrolysis efficiency toward cellulosic biomass. Ethanol fermentation by Kluveromyces marxianus MTCC 4136 was investigated for both the above cases and no significant difference was observed. We could obtain similar titers of ethanol by both methods. Employing the whole fermented matter offered advantages over using extracted enzyme by reducing enzyme extraction step and thereby reducing economic constraint.

Enzymatic saccharification of pretreated wheat straw: Comparison of solids-recycling, sequential hydrolysis and batch hydrolysis

In the enzymatic hydrolysis of lignocellulose materials, the recycling of the solid residue has previously been considered within the context of enzyme recycling. In this study, a steady state investigation of a solids-recycling process was made with pretreated wheat straw and compared to sequential and batch hydrolysis at constant reaction times, substrate feed and liquid and enzyme consumption. Compared to batch hydrolysis, the recycling and sequential processes showed roughly equal hydrolysis yields, while the volumetric productivity was significantly increased. In the 72h process the improvement was 90% due to an increased reaction consistency, while the solids feed was 16% of the total process constituents. The improvement resulted primarily from product removal, which was equally efficient in solids-recycling and sequential hydrolysis processes. No evidence of accumulation of enzymes beyond the accumulation of the substrate was found in recycling. A mathematical model of solids-recycling was constructed, based on a geometrical series.

Inhibition of cellulase, β‐glucosidase, and xylanase activities and enzymatic hydrolysis of dilute acid pretreated wheat straw by acetone‐butanol‐ethanol fermentation products

Simultaneous saccharification and fermentation has been regarded as a promising process for bioconversion of lignocellulosic biomass to acetone‐butanol‐ethanol (ABE); however, there exits potential inhibition of cellulase, β‐glucosidase, and xylanase by the fermentation product(s) due to the combination of enzymatic hydrolysis and microbial fermentation in one vessel. The effects of ABE fermentation products including acetone, butanol, ethanol, acetic acid, and butyric acid, individually and in a combination, on the cellulase, β‐glucosidase, and xylanase activities and enzymatic saccharification of dilute acid pretreated wheat straw (WS) were studied. The experimental results showed that butanol and mixture of inhibitor compounds strongly depressed cellulolytic and hemicellulolytic activities, and no synergistic inhibiting effect between inhibitors on enzyme activities was observed. Saccharification of pretreated WS was mainly affected by butanol and temperature. Moreover, preincubation of enzyme solutions with inhibitors mixture for 72 h decreased cellulase and xylanase activities by 15–25% at the three temperatures examined (30, 37, and 50°C), and a significant decrease (64%) in β‐glucosidase activity was found at 50°C. © 2013 American Institute of Chemical Engineers Environ Prog, 33: 497–503, 2014

Production of a cellulolytic enzyme system in mixed-culture solid-state fermentation of soybean hulls supplemented with wheat bran

Solid-state fermentation of soybean hulls supplemented with wheat bran using a co-culture of Trichoderma reesei and Aspergillus oryzae was performed. Three parameters — initial moisture content, incubation temperature, and initial pH — were optimized in culture flasks using response surface methodology. Parameter optimization was carried out with respect to filter paper activity and β-glucosidase activity in the culture. Temperature of 30 °C, pH of 5, and moisture content of 70% were found to be optimum. Optimized parameters were used for laboratory scale-up in static tray fermenters. The maximum filter paper activity of 10.7 FPU/g-ds and β-glucosidase of 10.7 IU/g-ds were obtained after 96-h incubation period in static tray fermenters in agreement with optimized activities at shake flask level. The results of static tray fermentation also highlighted the importance of mixed-culture fermentation. Both enzyme activities and volumetric productivities of enzyme produced were significantly higher in mixed-culture fermentation as compared to mono-culture static tray fermentation. Expression profile of cellulase system was characterized using SDS-PAGE and it indicated the presence of all the five major activities corresponding to β-glucosidase, CBH I, CBH II, EG I and xylanase. Enzyme broth was centrifuged and concentrated in an ultrafiltration cell. The concentrate was used for enzymatic saccharification of pretreated wheat straw and the potential of an indigenously developed enzyme concoction was reported in terms of saccharification efficiency. Pretreatment using both acid and alkali was carried out, and differences in sugar yield due to differences in composition as a result of pretreatment were reported. Results showed that alkali treatment generated higher sugars as compared to acid pretreatment. This was due to lignin removal and concentration of the cellulosic fraction. Present work showed that solid-state fermentation in a static tray bioreactor is a valuable technique for producing a system of enzymes with balanced activities that can efficiently saccharify lignocellulosic biomass like wheat straw.

Saccharification of pretreated wheat straw and corn stover using cellulolytic enzymes from Trichoderma viride and Aspergillus niger

Saccharification of pretreated wheat straw and corn stover using cellulolytic enzymes from Trichoderma viride and Aspergillus niger

Liquefaction of lignocellulose at high‐solids concentrations

To improve process economics of the lignocellulose to ethanol process a reactor system for enzymatic liquefaction and saccharification at high-solids concentrations was developed. The technology is based on free fall mixing employing a horizontally placed drum with a horizontal rotating shaft mounted with paddlers for mixing. Enzymatic liquefaction and saccharification of pretreated wheat straw was tested with up to 40% (w/w) initial DM. In less than 10 h, the structure of the material was changed from intact straw particles (length 1-5 cm) into a paste/liquid that could be pumped. Tests revealed no significant effect of mixing speed in the range 3.3-11.5 rpm on the glucose conversion after 24 h and ethanol yield after subsequent fermentation for 48 h. Low-power inputs for mixing are therefore possible. Liquefaction and saccharification for 96 h using an enzyme loading of 7 FPU/g.DM and 40% DM resulted in a glucose concentration of 86 g/kg. Experiments conducted at 2%-40% (w/w) initial DM revealed that cellulose and hemicellulose conversion decreased almost linearly with increasing DM. Performing the experiments as simultaneous saccharification and fermentation also revealed a decrease in ethanol yield at increasing initial DM. Saccharomyces cerevisiae was capable of fermenting hydrolysates up to 40% DM. The highest ethanol concentration, 48 g/kg, was obtained using 35% (w/w) DM. Liquefaction of biomass with this reactor system unlocks the possibility of 10% (w/w) ethanol in the fermentation broth in future lignocellulose to ethanol plants.

Enzymic saccharification of pretreated wheat straw

Studies of pretreatment of wheat and its subsequent saccharification by Trichoderma reesei cellulases are reported. Steam explosion was found to be the most effective of the pretreatment methods tested. Data are presented describing the effect of enzyme and substrate concentration on the rate and degree of hydrolysis. Significant inhibition of the cellulases was observed when sugar concentrations were 6% or higher. This inhibition increased when glucose and ethanol were present simultaneously. Adsorption of enzymes to the substrate was followed during a 24-h hydrolysis period. An initial rapid and extensive adsorption occurred, followed by a short desorption period that was followed in turn by a further increased adsorption peaking after 3 h. Intermediate removal of hydrolysate, particularly in combination with a second addition of enzyme, clearly improved the yield of saccharification compared to an uninterrupted hydrolysis over a 24-h period. Thus, a 74% yield of reducing sugars was obtained. Furthermore, an increase in the amount of recoverable enzymes was observed under these conditions. Evidence is presented that suggests that a countercurrent technique, whereby free enzymes in recovered hydrolysate are adsorbed onto new substrate, may provide a means of recirculating dissolved enzymes.