Cellulase Loading Research Articles

Evaluation of efficient glucose release using sodium hydroxide and phosphoric acid as pretreating agents from the biomass of Sesbania grandiflora (L.) Pers.: A fast growing tree legume

Sesbania grandiflora (L.) Pers. is one of the fast growing tree legumes having the efficiency to produce around 50tha−1 above ground dry matters in a year. In this study, biomass of 2years old S. grandiflora was selected for the chemical composition, pretreatments and enzymatic hydrolysis studies. The stem biomass with a wood density of 3.89±0.01gmcm−3 contains about 38% cellulose, 12% hemicellulose and 28% lignin. Enzymatic hydrolysis of pretreated biomass revealed that phosphoric acid (H3PO4) pretreated samples even at lower cellulase loadings [1 Filter Paper Units (FPU)], could efficiently convert about 86% glucose, while, even at higher cellulase loadings (60FPU) alkali pretreated biomass could convert only about 58% glucose. The effectiveness of phosphoric acid pretreatment was also supported by X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM) and Fourier transform infrared spectroscopy (FTIR) analysis.

Process yield and economic trade-offs for enzymatic hydrolysis of alkaline pretreated corn stover

Response surface methodology was used to investigate the interaction of pH, temperature, and enzyme loadings on corn stover hydrolysis rates following soaking in aqueous ammonia pretreatment. Economic tradeoffs were estimated for cellulase and hemicellulase loadings under different hydrolysis conditions. Enzyme loadings had a more significant effect on rates than did pH or temperature. The effect of hydrolysis pH was independent of temperature and enzyme loadings, and the optimal pH for glucose and xylose yields were 4.5 and 4.3, respectively. Conducting hydrolysis at 50 °C rather than 37 °C enables either a 10% glucose yield increase, or a comparable yield with 40% and 65% reduction in cellulase and hemicellulase loadings, respectively. Although yield models showed that hydrolysis rates increase with higher enzyme loadings, economic models showed that optimal cellulase and hemicellulase loadings were as much as 47% and 23% lower, respectively, than the maximum loadings tested. Optimal enzyme loadings change with fluctuations in enzyme costs and ethanol price, but cellulase loadings were more sensitive to these changes than hemicellulase loadings. Enzyme loadings were also more sensitive to enzyme price at lower processing temperatures. Enzyme loadings can be adjusted to increase return based on enzyme costs, ethanol price, and process temperature.

Co-production of functional xylooligosaccharides and fermentable sugars from corncob with effective acetic acid prehydrolysis

A novel and green approach for the coproduction of xylooligosaccharides (XOS), in terms of a series of oligosaccharide components from xylobiose to xylohexose, and fermentable sugars was developed using the prehydrolysis of acetic acid that was fully recyclable and environmentally friendly, followed by enzymatic hydrolysis. Compared to hydrochloric acid and sulfuric acid, acetic acid hydrolysis provided the highest XOS yield of 45.91% and the highest enzymatic hydrolysis yield. More than 91% conversion of cellulose was achieved in a batch-hydrolysis using only a cellulase loading of 20FPU/g cellulose and even a high solid loading of 20% without any special strategies. The acetic acid pretreated corncob should be washed adequately before saccharification to achieve complete hydrolysis. Consequently, a mass balance analysis showed that 139.8g XOS, 328.1g glucose, 25.1g cellobiose, and 147.8g xylose were produced from 1000g oven dried raw corncob.

Solvent-based delignification and decrystallization of wheat straw for efficient enzymatic hydrolysis of cellulose and ethanol production with low cellulase loadings

Several solvents and ionic liquids, namely formic acid (Formiline process), concentrated phosphoric acid (CPA), N-methylmorpholine-N-oxide (NMMO) and 1-allyl-3-methylimidazolium chloride ([AMIM]Cl), were used to pretreat wheat straw in order to increase cellulose digestibility for ethanol production. When being directly used to pretreat the raw wheat straw under corresponding optimized conditions, the improvement of cellulose hydrolyzability followed the order of CPA > Formiline > NMMO > [AMIM]Cl. However, when Formiline pretreated (delignified) substrates were further post-treated by the above cellulose solvents, the cellulose digestibility was significantly improved particularly with low cellulase loadings. Cellulose solvent post-treatment resulted in deconstruction of hydrogen-bond networking, alteration of cellulose polymorphs, decrease in crystallinity, depolymerization of cellulose chain with dramatic reduction in particle size, thus greatly increasing cellulose accessible surface area. CPA post-treatment showed the best efficacy. Semi-simultaneous scarification and fermentation (sSSF) of a CPA post-treated substrate obtained an ethanol concentration of 41.6 g L−1 with 91.2% of yield at a relatively low cellulase loading (5 FPU per g solid) within 24 h incubation. A biorefining process was proposed based on Formiline pretreatment coupled with CPA post-treatment to achieve a co-production of ethanol, furfural and high-purity lignin, which greatly increases the potential revenue of the process.

Saccharification of Acacia auriculiformis Benth. biomass for the production of glucose using phosphoric acid pretreatment

Acacia auriculiformis is an exotic plant species luxuriantly grown in different parts of India. Because of its drought tolerance potential and wood quality, it is widely grown as timber crops in some part of the country. In this study we have addressed the possible use of A.auriculiformis for production of glucose, an intermediate for the production of ethanol. Chemical composition of this species was carried out and it was found that this species contains about 34 % glucan. Using Cellulose –Solvent and Organic-solvent based Lignocellulose Fractionation pretreatment the biomass of the species was hydrolyzed at two different dosages of cellulase. It was found that even at low cellulase loadings [1 Filter Paper Unit (FPU)] about 10 International Units (IU) of β-glucosidase about 88 % glucan conversion took place. This study suggests the use of this species as a biofuel feedstock for next generation fuels.

Effect of cationic surfactant cetyltrimethylammonium bromide on the enzymatic hydrolysis of cellulose

Effect of cationic surfactants alkyltrimethylammonium bromide (CnTAB) with varied alkyl chain lengths on the enzymatic hydrolysis of Avicel and the surface charge of cellulase was investigated. Enzymatic hydrolysis of Avicel increased linearly from 42.1 to 61.4 % with the increase of the concentration of cetyltrimethylammonium bromide (C16TAB) logarithmically from 0.0001 to 0.01 mM, and reached a maximum value at the concentration of 0.01–0.03 mM. When the concentration was increased further, the cellulase solution became positively charged and the enzymatic hydrolysis of Avicel decreased rapidly. With the increasing alkyl chain length, CnTAB provided more proton and neutralized the negative charge of cellulase more obviously. Therefore, the required concentration of CnTAB could be less to enhance the enzymatic hydrolysis of Avicel. In addition, C16TAB could enhance enzymatic hydrolysis efficiency of corncob at high solid content from 35.0 to 56.3 %; C16TAB could reduce about 60 % of the cellulase loading in the enzymatic hydrolysis of corncob to obtain the same glucose yield. Effect of C16TAB on the enzymatic hydrolysis of typical pretreated softwood and hardwood was also investigated. This study laid the foundation for using CnTAB to recover cellulase, and provided the design direction for cellulase with higher activity and better stability by adjusting its hydrophilicity and chargeability.

Addition of expansin to cellulase enhanced bioethanol production

Abstract A metagenomic clone (pUC-189) that exhibited clear zone on CMC agar plate showed homology with glycosyl hydrolase (GH) family 12 endoglucanase. Purified recombinant cellulase (C18) exhibited the highest activity towards CMC followed by Avicel PH101 and waste paper which was confirmed by chromatography. Role of expansin (E11CB) was investigated to address the poor activity of C18 towards waste paper. The Langmuir isotherm for binding of the purified E11CB to waste paper showed a good fit (R 2 = 0.97). Recombinant E11CB protein bound to waste paper was detected in situ by UV fluorescence of [Ln(DPA) 3 ] 3− complex. Scanning electron microscopy confirmed that binding of E11CB transformed smooth microfibrils into rough and amorphic surface. Improvement in the enzymatic hydrolysis of pure cellulose and lignocelluloses was observed after treatment with purified E11CB at low cellulase loading. The enzymatic hydrolysate of waste cellulose paper was then used as the substrate for bioethanol production by Saccharomyces cerevisiae.

Implementation of Definite Screening Design in Optimization of In Situ Hydrolysis of EFB in Cholinium Acetate and Locally Produced Cellulase Combined System

Ionic liquids (ILs) have been found to be highly promising for lignocellulosic biomass pretreatment, due to their excellent abilities to dissolve biopolymers. However, they have found to inactivate enzymes. In previous studies, Cholinium-based ILs showed great compatibility with cellulases and thus were introduced for a combined-system. This study aims to find the optimum condition in order to achieve the maximum pretreatment-hydrolysis of empty fruit bunches (EFB). Definitive screening design (DSD) was employed with seven factors that are thought to impact the process including pretreatment temperature, pretreatment time, hydrolysis time, enzyme loading, particle size, biomass loading, and IL/buffer ratio. DSD offered several solutions for optimization in which they were experimentally tested. The maximum sugar concentration (77 g/L) was obtained at 90 °C, 70–116 min of treatment, 36–42 h of hydrolysis, 40–62 Unit/g cellulase loading, 34–35 %, w/v) biomass loading, 220–450 μ particle size, and 10 % (v/v) IL to buffer ratio, respectively. The subsequent optimization by response surface methodology (RSM) revealed the temperature for treatment can drop to 75 °C while fixing the pretreatment time at 100 min. Around 75 unit/g of cellulase and >22.0 % (w/v) of the biomass could be loaded to achieve a minimum of 70.0 ± 7.83 g/L of sugar, equivalent to 0.38 ± 0.08 g glucose/g and 0.48 ± 0.05 g total reducing sugar/g dry EFB. The locally produced cellulase (PKC-Cel) from Trichoderma reesei exhibited promising results in the single-step process and can be used as an efficient approach to be optimized for fermentation to bioethanol production.

Production of second generation ethanol using Eucalyptus dunnii bark residues and ionic liquid pretreatment

The production of ethanol using Eucalyptus dunnii bark pretreated with 1-butyl-3-methylimidazolium chloride ([BMIM]Cl) as ionic liquid (IL) was studied. In the present work high ethanol yields were obtained using the minimal quantity of IL necessary for wetting but not dissolving the lignocellulosic material. The best result obtained was 70% of ethanol yield using an IL:bark ratio of 5:1, with separate hydrolysis and fermentation (SHF) process, and a cellulase load of 50 FPU/gram of pretreated material.

Fermentative High-Titer Ethanol Production from Douglas-Fir Forest Residue Without Detoxification Using SPORL: High SO2 Loading at Low Temperature

Abstract This study evaluated high sulfur dioxide (SO2) loading in applying Sulfite Pretreatment to Overcome the Recalcitrance of Lignocelluloses (SPORL) to Douglas-fir forest residue (FS-10) for ethanol production through yeast fermentation. Three pretreatments were conducted at 140°C with a targeted total SO2 loading of 32 wt% on wood, or 80 g/L in the pretreatment liquor. Magnesium was used as the metal base with a targeted combined SO2 loading of 4.4 wt% on wood. A pretreatment duration of approximately 60 min was found sufficient to effectively remove the strong recalcitrance of FS-10 with ultra-low degradation of sugar to furans. Pretreatment mixing was performed through either a rotating digester or liquor circulation, and was found to have no significant effect on pretreatment performance. The ultra-low furan formation facilitated enzymatic saccharification and fermentation at total solids loading of 20% without detoxification. At cellulase loading of 35 mL/kg FS-10, a maximal ethanol yield of 322...

Optimization of prehydrolysis time and substrate feeding to improve ethanol production by simultaneous saccharification and fermentation of furfural process residue

Ethanol is a very important industrial chemical. In order to improve ethanol productivity using Saccharomyces cerevisiae in fermentation from furfural process residue, we developed a process of simultaneous saccharification and fermentation (SSF) of furfural process residue, optimizing prehydrolysis cellulase loading concentration, prehydrolysis time, and substrate feeding strategy. The ethanol concentration obtained from the optimized process was 19.3g/L, corresponding 76.5% ethanol yield, achieved by running SSF for 48h from 10% furfural process residue with prehydrolysis at 50°C for 4h and cellulase loading of 15FPU/g furfural process residue. For higher ethanol concentrations, fed-batch fermentation was performed. The optimized fed-batch process increased the ethanol concentration to 37.6g/L, 74.5% yield, obtained from 10% furfural process residue with two additions of 5% substrate at 12 and 24h.

Microwave-assisted Dilute Acid Pretreatment and Enzymatic Hydrolysis of Sago Palm Bark

Maximizing the amount of monomeric sugar yield from lignocellulosic materials requires an effective pretreatment process and identification of an optimal enzyme loading for cost-effectiveness. In this work, a microwave-diluted sulfuric acid pretreatment was applied prior to enzymatic hydrolysis of sago palm bark (SPB). Characterization of the solid fraction was completed before and after the pretreatment process. Analysis of SPB ash showed a presence of 6.8% silica. There was a 32% reduction in lignin content, an increased crystallinity from 29% to 47%, and clear damage and fragmentation to the surface structure of SPB after the pretreatment. Inhibitors were not detectable in the liquor after the microwave-acid pretreatment. The enzymatic hydrolysis of SPB was employed by adding 6 to 42 FPU/g of cellulase and 50 U/g of β-glucosidase to identify the optimal cellulase loading at fixed β-glucosidase loading. The maximum total monomeric sugar yield and total reducing sugar (using DNS method) at 77 mg/g and 378 mg/g were achieved using 24 FPU/g of cellulose, respectively. Thus, this enzyme loading can be recommended for further microwave-acid pretreatment and enzymatic hydrolysis of SPB.

Improved enzymatic saccharification of steam exploded cotton stalk using alkaline extraction and fermentation of cellulosic sugars into ethanol

Cotton stalk, a widely available and cheap agricultural residue lacking economic alternatives, was subjected to steam explosion in the range 170–200°C for 5min. Steam explosion at 200°C and 5min led to significant hemicellulose solubilization (71.90±0.10%). Alkaline extraction of steam exploded cotton stalk (SECOH) using 3% NaOH at room temperature for 6h led to 85.07±1.43% lignin removal with complete hemicellulose solubilization. Besides, this combined pretreatment allowed a high recovery of the cellulosic fraction from the biomass. Enzymatic saccharification was studied between steam exploded cotton stalk (SECS) and SECOH using different cellulase loadings. SECOH gave a maximum of 785.30±8.28mg/g reducing sugars with saccharification efficiency of 82.13±0.72%. Subsequently, fermentation of SECOH hydrolysate containing sugars (68.20±1.16g/L) with Saccharomyces cerevisiae produced 23.17±0.84g/L ethanol with 0.44g/g yield.

Improving the enzymatic hydrolysis of dilute acid pretreated wheat straw by metal ion blocking of non-productive cellulase adsorption on lignin

Eleven salts were selected to screen the possible metal ions for blocking the non-productive adsorption of cellulase onto the lignin of dilute acid pretreated wheat straw. Mg2+ was screened finally as the promising candidate. The optimal concentration of MgCl2 was 1mM, but the beneficial action was also dependent on pH, hydrolysis time and cellulase loading. Significant improvement of glucan conversion (19.3%) was observed at low cellulase loading (5FPU/g solid). Addition of isolated lignins, tannic acid and lignin model compounds to pure cellulose hydrolysis demonstrated that phenolic hydroxyl group (Ph-OH) was the main active site blocked by Mg2+. The interaction between Mg2+ and Ph-OH of lignin monomeric moieties followed an order of p-hydroxyphenyl (H)>guaiacyl (G)>syringyl (S). Mg2+ blocking made the lignin surface less negatively charged, which might weaken the hydrogen bonding and electrostatically attractive interaction between lignin and cellulase enzymes.

Production of bioethanol as useful biofuel through the bioconversion of water hyacinth (Eichhornia crassipes).

Water hyacinth (Eichhornia crassipes) represents a promising candidate for fuel ethanol production in tropical countries because of their high availability and high biomass yield. Bioconversion of such biomass to bioethanol could be wisely managed through proper technological approach. In this work, pretreatment of water hyacinth (10 %, w/v) with dilute sulfuric acid (2 %, v/v) at high temperature and pressure was integrated in the simulation and economic assessment of the process for further enzymatic saccharification was studied. The maximum sugar yield (425.6 mg/g) through enzymatic saccharification was greatly influenced by the solid content (5 %), cellulase load (30 FPU), incubation time (24 h), temperature (50 °C), and pH (5.5) of the saccharifying medium. Central composite design optimized an ethanol production of 13.6 mg/ml though a mixed fermentation by Saccharomyces cerevisiae (MTCC 173) and Zymomonas mobilis (MTCC 2428). Thus the experiment imparts an economic value to water hyacinths that are cleared from choking waterways.

A novel approach for intensification of enzymatic depolymerization of carboxymethyl cellulose using ultrasonic and ultraviolet irradiations

The present work investigates the application of ultrasonic (US) and ultraviolet (UV) irradiations operated individually or in combination for intensification of enzymatic depolymerization of aqueous solution of carboxymethyl cellulose (CMC) present as sodium salt. The effect of different operating parameters such as reaction temperature, enzyme loading, UV and US power dissipation on the extent of depolymerization have been studied. The effectiveness of treatment approach has been analyzed on the basis of kinetic rate constant, limiting intrinsic viscosity and the time required for the desired extent of intrinsic viscosity reduction. The kinetic rate constant has been found to increase with an increase in the temperature and cellulase loading. The extent of depolymerization increased from 20% to 58.5% with an increase in the operating power of UV from 8W to 32W (irradiation time as 180min) whereas the increase was from 74% to 87% for an increase in ultrasonic power from 60W to 120W (irradiation time of 120min). In the presence of US (60W)+UV (16W)+enzyme (0.05%), the maximum extent of depolymerization of CMC has been observed as 99% with treatment time of 18min. The effect of ultrasonic irradiation on viscosity reduction was established to be more pronounced than the ultraviolet irradiations. The obtained results from FTIR spectra clearly established that no changes in the chemical structure were observed for the treated CMC using combined ultrasound, ultraviolet irradiations with enzyme and native CMC. The work has conclusively established, for the first time, that the combination of ultraviolet and ultrasonic irradiation in presence of cellulase enzyme gives maximum viscosity reduction in CMC polysaccharides as compared to individual approaches.

Batch-based enzymatic saccharification of sweet sorghum bagasse

ABSTRACTTo improve enzymatic digestibility and sugar concentration, sweet sorghum bagasse was pretreated with alkali and liquid hot water, and then subjected to fed-batch enzymatic hydrolysis. Scanning electron microscopy assay suggested that different pretreatment methods resulted in different composition and structure of residues; these changes had a significant influence on cellulose hydrolysis. Fresh substrate was pretreated and then added at different amounts during the first 48 h to yield a final dry matter content of 30% (w/v). For liquid hot water pretreatment, a maximal glucose concentration of 95.71 g/L, corresponding to 52.85% xylan removal, was obtained with the sweet sorghum bagasse pretreated at 184°C for 18 min. NaOH soaking at ambient conditions removed lignin up to 60%, and the subsequent hydrolysis with cellulase loading of less than 10 FPU/g DM, and substrate supplementation every few hours yield the high glucose and xylose concentrations of 114.89L and 29.93 g/L, respectively after 144 h.

Comparison of dilute acid and alkali pretreatments in production of fermentable sugars from bamboo: Effect of Tween 80

As a fast growing woody grass with high sugar contents, bamboo is a potential material for the production of bio-ethanol, and the saccharification of bamboo has been extensively investigated. However, the role of surfactants on the pretreatment and enzymatic hydrolysis of bamboo has not been clearly elucidated. In this work, effects of aqueous ammonia (SAA), dilute acid (DA), and NaOH pretreatments on the chemical composition, structural features, and enzymatic hydrolysis of bamboo were compared and the effects of Tween 80 on the pretreatments and hydrolysis were explored. NaOH pretreatment showed strong delignification and gave the highest sugar recovery, followed by severe SAA pretreatment (70°C, 72h). Total reducing sugars from bamboo with NaOH pretreated bamboo samples were 42.3g per 100g dry biomass. With the addition of 3% (w/w) Tween 80, the removal of lignin increased from 9.3% and 54.2% to 13.5% and 57.9% after DA and NaOH pretreatments, respectively. The effectiveness of Tween 80 in enzymatic hydrolysis of different pretreated bamboo was high at a cellulase loading less than 20FPU/g DM. Addition of Tween 80 both before and after enzymatic saccharification increased hydrolysis yield of bamboo compared to the hydrolysis without Tween 80. The removal of xylan in bamboo by xylanase could further improve the effectiveness of Tween 80, suggesting the importance of the combination of xylanase and Tween 80 in enzymatic hydrolysis of bamboo by cellulases.

Effect of magnesium chloride on fractionation and enzymatic digestibility of cattails

ABSTRACTThe effect of magnesium chloride (a Lewis acid) on the fractionation of aquatic plant cattail was studied using a Dionex accelerated solvent extractor varying salt concentration (0–0.4 M), treatment temperature (140–180°C), and residence time (5–15 min). MgCl2 treatment enhanced xylan degradation and delignification, and up to 33.8% of total xylan was directly hydrolyzed to the xylose monomer. By removing xylan and lignin, the MgCl2 treatment effectively increased enzymatic digestibility of cattail cellulose and xylan. When cattails were treated with the 0.4 M MgCl2 at 180°C for 15 min and subsequently hydrolyzed for 48 h with a cellulase loading of 15 FPU/g glucan, the highest glucose yield of 61.7% and the highest total xylose yield of 90.6% were reached, respectively. Application of Lewis acids as a novel biomass fractionation approach will require lower xylanase loading for hydrolysis, and have lower corrosion to equipment compared to other acid pretreatment processes.

Simultaneous Saccharification and Fermentation of Banana (Musa cuminata) Peel for Bioethanol Production

Banana (Musa acuminata) peel is discarded after the fruit processing process. Banana peel biomass can be used as raw material to produce bioethanol. In this research, banana peel was fermented by simultaneous saccharification and fermentation using Saccharomyces cerevisiae. Production of bioethanol was determined and the effects of various operation conditions which included different parameters in fermentation process: fermentation time, temperature, medium pH, concentration of yeast, enzyme loading, and shaking period. peel was cut, pretreated with H2SO4 2% and simultaneous hydrolyzed and fermented. Ethanol production was greatest (over 10,5%) when the initial pH of fermentation medium was adjusted to 4.5, 5% yeast supplement (8x107 cfu/ml), 7% cellulase (100 IU, v/v) loading, 24 hours and 37oC incubating,. The results illustrated that the simultaneous accharification and fermentation (SSF) of banana peel with S.cerevisiae has potential for industrial application.