Increased Saccharification Yield by Cellulases Coupling

In many cellulase preparations the enzymatic activity is not well balanced and the catalytic attack of the cellulose is limited. The purpose of this study was to investigate the possibility of simultaneously using two cellulases from distinct sources for improving the rate of hydrolysis as alternative to other costly techniques which require enzyme separation and purification

A comparative study on the hydrolysis of microcrystalline cellulose (Avicel), amorphous cellulose derivative (CMC) and native cellulose-olive husks, catalyzed by mixtures of cellulase complexes was performed in ultrafiltration and batch reactors. The experiments were carried out varying the percentages of Trickoderma viride and Aspergillus niger in the enzymatic preparation and the effect of enzyme composition on cellulose conversion and glucose selectivity was determined. The use of mixtures of these two enzymes improves cellulose saccharification in a different extent depending on the chemical and physical features of the cellulose and on the biomass particle size. The increase of glucose production is three times as big as with the use of the two enzymes separately. For the olive husks the optimum is attained for a T. viride to A. niger ratio of 2:1. An ultrafiltration-membrane reactor for the hydrolysis of the three substrates can be helpfully adopted since product inhibition is controlled and a higher glucose selectivity is reached in comparison With batch ones.     

Production and optimization of high grade cellulase from waste date seeds by Cellulomonas uda NCIM 2353 for biohydrogen production

Production of high grade cellulolytic enzymes from waste agricultural biomass would valorise these wastes to valuable products as well as avoid the pollution problems associated with landfilling of the biomass. In the present study, waste date palm (Phoenix dactylifera) seeds were valorised for cellulase production from Cellulomonas uda NCIM 2353 and for its subsequent usage in biohydrogen production. Optimization of key operational parameters such as date seed concentration, xylose, casein and initial media pH were performed using central composite design to obtain the maximum enzyme yield. The optimum values obtained were (g/L): date seed concentration 30.65, xylose concentration 0.55, casein 7.00 and pH 7.40 for a determination coefficient of 0.999. The results demonstrated a higher prediction accuracy of response surface methodology as the cellulase activity increased six fold (175.96 IU/mL) after optimization. The optimum pH and temperature of purified cellulase was 7 and 50 °C respectively where the enzyme retained nearly 80% of activity upto 180 min. Enzymatic hydrolysis studies showed that a high saccharification efficiency of 60.5% was obtained for acid pretreated sugarcane bagasse by the indigenous cellulase, equivalent to the performance of commercial cellulase. Further, the as-obtained reducing sugars were decomposed by Clostridium thermocellum to produce biohydrogen of maximum concentration 187.44 mmol/L at end of 24 h of fermentation. Results show that date seed substrate based cellulase protein can be employed for industrial processes of biohydrogen production.     

Conversion of paper sludge to ethanol by separate hydrolysis and fermentation (SHF) using Saccharomyces cerevisiae

The conversion of ethanol from paper sludge using the separate hydrolysis and fermentation (SHF) process with cellulase and Saccharomyces cerevisiae GIM-2 were investigated in this paper. Optimization strategy based on statistical experimental designs was employed to enhance degree of saccharification by enzymatic hydrolysis of paper sludge. Based on the Plackett-Burman design, hydrolysis time, substrate concentration and cellulase dosage were selected as the most significant variable on the degree of saccharification. Subsequently, the optimum combination of the selected factors was investigated by a Box-Behnken approach. A mathematical model was developed to show the effects of each factor and their combinatorial interactions on the degree of saccharification. The optimal conditions were hydrolysis time 82.7 h, substrate concentration 40.8 g L⁻¹ and cellulase dosage 18.1 FPU g⁻¹ substrate, and a degree of saccharification of 82.1% can be achieved. When hydrolysate was further fermented with S. cerevisiae GIM-2, the conversion rate of sugar to ethanol was 34.2% and the ethanol yield was 190 g kg⁻¹ of dry paper sludge, corresponding to an overall conversion yield of 56.3% of the available carbohydrates on the initial substrate. The results derived from this study indicate that the response surface methodology is a useful tool for optimizing the hydrolysis conditions to converse paper sludge to ethanol.     

Improvement of wheat straw hydrolysis by cellulolytic blends of two Penicillium spp.

Co-culture of fungal strains Penicillium janthinellum EMS-UV-8 (E), Penicillium funiculosum strain P (P) and Aspergillus sp. strain G (G) and blending of their crude cellulase were evaluated for improvements in cellulase activities as well as for enhanced hydrolysis of dilute acid pretreated wheat straw (PWS). The blending of crude enzymes of P and E enhanced the hydrolysis of PWS more effectively due to synergism in cellulolytic enzyme activities. Here, three types of blends were made on the basis of equal FPUs, equal protein content or fixed volume containing different proportions of individual enzymes, the former blend hydrolyzed 42.6% of PWS due to the 98%,62%, 64% and 34% synergistic enhancement in endo-glucanase, cellulase (FPU), β-glucosidase and xylanase activities, respectively. Hydrolysis at 10% solid loading of PWS in roller bottle reactor with this blend further enhanced hydrolysis yield to 74% within 24 h, which was much better than the corresponding hydrolysis yields of individual (38.1% by E and 61.5% by P) or the commercial enzyme (62.3%). This study proved that synergistic blends of cellulases from two Penicillium spp. are cost-effective tools for efficient wheat straw hydrolysis for on-site biofuel production.     

Efficient cellulase-catalyzed saccharification of untreated paper sludge targeting for biorefinery

This aim of this study was to optimize the conditions for efficient saccharification of paper sludge (PS) considering PS cellulose concentration, buffer capacity, and cellulase activity from Acremonium cellulolyticus. PS is a cellulosic biomass, the residue of paper making industry, composed of 24.5% cellulose, 10.5% clay, and 65% water. Clay present in PS did not show any inhibitory effect on the saccharification process but influenced the buffer pH. Maleate buffer in the pH range 5.2–5.6 was suitable for the saccharification of PS cellulose. The maximum reducing sugar concentration that could be obtained from 75.6 kg m^?3 of PS cellulose was predicted to be 37.8 kg m^?3. This was experimentally confirmed to be 38.4 kg m^?3 under the optimal saccharification conditions at pH 5.2 (1.06 kmol m^?3 Maleate buffer) using Cellulase 20,000 FPU m^?3. Almost 100% saccharification yield (SY) was obtained at a low PS cellulose concentration of 15 kg m^?3, but this value decreased when the cellulose content increased. When PS was hydrolyzed with commercial cellulase GC220, the SY and glucose content were 54% and 60% of SY, respectively. The corresponding values with Cellulosin T2 were 63% and 75% of SY, respectively. A. cellulolyticus yielded SY and glucose content values almost 10% higher than those obtained with other cellulases.     

Enzymatic hydrolysis and fermentation of crushed whole sugar beets

Pectinase and cellulase enzymes were used for hydrolysis of whole sugar beets and the hydrolyzates were fermented with Escherichia coli KO11 and Saccharomyces cerevisiae via simultaneous saccharification and fermentation (SSF). Ethanol production rate was significantly higher for S. cerevisiae than for E. coli KO11. The combined effect of pectinase and cellulase loadings on ethanol production as well as residual galacturonic acid and arabinose concentrations were modeled for fermentations with S. cerevisiae. Ethanol yields of more than 92% were reached with moderate to high cellulase and pectinase loadings at 0.51 FPU g⁻¹ and 51 U g⁻¹ of dry biomass, respectively. Ethanol yields of 85% were achieved without any enzyme addition. However, addition of cellulase and pectinase enzymes increased effluent arabinose and galacturonic acid concentrations and reduced total suspended solids. This study demonstrated the yield potential of fermentation of crushed, whole sugar beets with or without the addition of cellulase and pectinase enzymes.     

A novel staggered hybrid SSF approach for efficient conversion of cellulose/hemicellulosic fractions of corncob into ethanol

The following study reports bioconversion of corncob into ethanol using hybrid approach for co-utilization of dilute acid hydrolysate (pentose rich stream) and hexose rich stream obtained by enzymatic saccharification employing commercial cellulase Cellic CTec2 as well as in-house cellulase preparations derived from Malbranchea cinnamomea, Scytalidium thermophilium and a recombinant Aspergillus strain. Acid hydrolysis (1% H₂SO₄) of corncob at 1:15 solid liquid ratio led to removal of 80.5% of hemicellulosic fraction. The solid glucan rich fraction (63.5% glucan, 8.3% pentosans and 27.9% lignin) was hydrolysed at 10% substrate loading rate with different enzymes for 72 h at 50 °C resulting in release of 732 and 535 (mg/g substrate) total sugars by Cellic CTec2 and M. cinnamomea derived enzymes, respectively. The fermentation of enzyme hydrolysate with co-culture of Saccharomyces cerevisiae and Pichia stipitis added in sequential manner resulted in 3.42 and 2.50% (v/v) ethanol in hydrolysate obtained from commercial Cellic CTec2 and M. cinnamomea, respectively. Employing a hybrid approach, where dilute acid hydrolysate stream was added to solid residue along with enzyme Cellic CTec2 during staggered simultaneous saccharification and fermentation at substrate loading rate of 15% resulted in 252 g ethanol/kg corncob.     

Fed batch enzymatic saccharification of newspaper cellulosics improves the sugar content in the hydrolysates and eventually the ethanol fermentation by Saccharomyces cerevisiae

The newspaper is comprised of (w w^?1) holocellulose (70.0%) with substantial amount of lignin (16.0%). Bioconversion of the carbohydrate component of newspaper to sugars by enzymatic saccharification, and its fermentation to ethanol was investigated. Of various enzymatic treatments using cellulase, xylanase and laccase, cellulase enzyme system was found to deink the newspaper most efficiently. The saccharification of deinked paper pulp using enzyme cocktail containing exoglucanase (20 U g^?1), β-glucosidase (60 U g^?1) and xylanase (80 U g^?1) resulted in 59.8% saccharification. Among additives, 1% (v v^?1) Tween 80 and 10 mol m^?3 CoCl₂ improved the enzymatic hydrolysis of newspaper maximally, releasing 14.64 g L^?1 sugars. The fed batch enzymatic saccharification of the newspaper increased the sugar concentration in hydrolysate from 14.64 g L^?1 to 38.21 g L^?1. Moreover, the batch and fed batch enzymatic hydrolysates when fermented with Saccharomyces cerevisiae produced 5.64 g L^?1 and 14.77 g L^?1 ethanol, respectively.     

Optimization of cellulases production by Trichoderma citrinoviride on marc of Artemisia annua and its application for bioconversion process

The production of cellulases by Trichoderma citrinoviride fermented on marc of Artemisia annua, and bioconversion of the same marc by produced cellulase system was studied. The effects of pretreatments, substrate concentration, particle size, initial pH, temperature and concentration of the medium components on production of FPase, endoglucanase and β-glucosidase were monitored and comparatively evaluated. Among the three pretreatment processes, alkali hydrolysis with autoclaving was found to be most suitable for production of all the three enzymes. Optimum production of FPase, endoglucanase and β-glucosidase was obtained at 96 h, 96 h and 72 h of fermentation period, respectively. Substrate concentration of 1% with particle size between 200 μm and 475 μm gave the higher yields. Higher production of all the three enzymes was obtained with initial pH value of 5.5, temperature of 28 °C and 75% of mineral salt solution. Partially purified enzyme system obtained by optimized fermentation procedure, was applied for saccharification. Forty six percent of saccharification was noticed after 48 h of incubation on alkali hydrolyzed and autoclaved substrate which was 3.26 fold more than that of unpretreated substrate.     

Pretreatment and enzymatic saccharification of new phytoresource for bioethanol production from halophyte species

In this work, new halophyte plant biomass is proposed as raw material for bioenergy production. The bioconversion process of lignocellulosic materials into ethanol needs a pretreatment before enzymatic hydrolysis of vegetal material to increase the yield of fermentable sugars. Various mechanical and chemical pre-treatment processes were investigated in an attempt to facilitate the solubilization of large fraction of hemicelluloses.The fungi Trichoderma spp., Aspergillus niger, Penicillium italicum and Fusarium spp. have been developed on the halophyte plant, Juncus maritimes, producing polysaccharides hydrolases activities, i.e. endoglucanasecatalyses cellulose hydrolysis, and beta-glucosidasecatalyses hydrolysis of terminal non-reducing residues in beta-glucosides. The saccharification was carried out with an enzymatic preparation extracted from filamentous fungus Trichoderma spp. Experimental Doehlert design was performed to optimize the reducing sugars concentration. High sugar yields (8.5 g/L) were obtained using a small amount of the extracted enzyme without stirring.The highest enzymatic activities of endoglucanase and beta-glucosidases were produced by Trichoderma spp. at 22.12 and 0.07 U/mg, respectively.     

Characterization of a Trichoderma atroviride strain isolated from switchgrass bales and its use to saccharify ammonia-pretreated switchgrass for biobutanol production

The feedstock-specific enzyme systems for saccharification of biofuel feedstocks like switchgrass may potentially provide better enzymatic systems for production of second-generation biofuels. One strategy to develop these enzyme systems could be to harness the microorganisms growing naturally on specific feedstocks. This study presents the isolation and screening of fungal cultures from switchgrass bales for saccharification of ammonia-pretreated switchgrass for subsequent biobutanol production. The best performing fungal isolate during screening was identified through Sanger sequencing of its ITS region to be a unique strain of Trichoderma atroviride and further characterized for production of an enzyme system for saccharification of ammonia pretreated switchgrass. The maximum FPase, CMCase and xylanase activity produced by T. atroviride CUA1 were 0.25 fpu/mL, 0.18 IU/mL and 5.8 IU/mL, respectively. T. atroviride CUA1 also produced considerable amount of β-glucosidase activity. This isolate was used to produce an enzyme system to convert switchgrass to soluble sugars that were then fermented to butanol, ethanol, acetate and butyrate. Glucose was the major product of hydrolysis of ammonia-pretreated switchgrass performed using the enzyme system produced by the isolate. This fungus may be useful for the hydrolysis for the bioenergy crop of switchgrass to overcome this rate-limiting step in the overall conversion of biomass to biofuels.     

Enhancing saccharification of Agave tequilana bagasse by oxidative delignification and enzymatic synergism for the production of hydrogen and methane

Agave tequilana bagasse is a suitable lignocellulosic residue for energy production. However, the presence of lignin and the heterogeneous structure of hemicellulose may hinder the availability of polysaccharides. In this work, the pretreatment of A. tequilana bagasse with alkaline hydrogen peroxide (AHP) followed by enzymatic saccharification with hemicellulases and cellulases was assessed for the removal of lignin and extraction of fermentable sugars, respectively. Results of the AHP pretreatment indicated that it is possible to attain up to 97% delignification and recover 88% of cellulose and hemicellulose after only 1.5 h of treatment. Regarding the saccharification process, the total sugar yield and productivity were both increased by 2-fold using an enzymatic mixture (cellulases + hemicellulases) compared to single enzyme hydrolysis (cellulases), evidencing synergism. Further evaluation of the hydrolyzates as substrate for hydrogen and methane production, resulted in yields 1.5 and 3.6-times (215.14 ± 13 L H₂ and 393.4 ± 13 L CH₄ per kg bagasse, respectively) superior to those obtained with hydrolyzates of non-pretreated bagasse processed with a single enzyme. Overall, using AHP pretreatment and subsequent hydrolysis with enzymatic mixtures improves the saccharification of A. tequilana bagasse enhancing the production of hydrogen and methane.     

Enhanced saccharification of SO2 catalyzed steam-exploded corn stover by polyethylene glycol addition

Corn stover is a renewable, low cost and abundant feedstock in China. Its effective utilization is crucial for providing bioenergy, releasing environmental pollution and increasing farmers’ income. This aim of this study was to obtain the efficient saccharification of SO₂ catalyzed steam-exploded corn stover (SSECS) by polyethylene glycol (PEG) addition. According to the results, adding PEG6000 could lower the enzyme loading by 33.3%. With 20% solid loading, the highest glucose concentration of 102 g L⁻¹ and 91.3% saccharification yield were obtained using 30 CBU (g glucan)⁻¹ ??-glucosidase and 10 FPU (g glucan)⁻¹ cellulase in presence of PEG6000. In addition, protein and enzyme activities assays in the supernatants revealed that PEG could facilitate the desorption of enzyme protein from lignocellulose. These indicated that PEG addition not only can enhance enzymatic saccharification at high substrate concentration, but also can improve enzyme recycling by reducing the enzyme activity loss caused by adsorption during the hydrolysis.     

Biochemical conversion of sugarcane bagasse into bioproducts

The ground sugarcane bagasse conversions were examined through chemical treatment methods employing soaking in aqueous ammonia (SAA), and ethyl-hydro-oxides (EHOs). To characterize a chemical treatment method, both generated solvent based extract and pulp were examined. The generated pulps were evaluated through chemical composition and enzymatic saccharification. The enzyme mixtures were investigated including Trichoderma reesei Rut C-30 originated cellulase, T. reesei Rut C-30 originated cellulase with external added β-glucosidase, Accellerase^® 1500, and Cellic^® CTec2. The physiochemical effects of chemical treatments on the structural-chemical properties of treated-bagasse were also analyzed at high substrate enzymatic saccharification. The substrate loadings (using both SAA-treated and EHOs-treated bagasse) of 125, 150, 175, 200, and 225 g L⁻¹ were examined during enzymatic saccharification process. The generated phenolic compounds were characterized based on density, antioxidant activity, and anticancer activity. All findings are discussed in relation to developing a self-sustainable integrated biorefinery.     

Feasibility of reed for biobutanol production hydrolyzed by crude cellulase

The aim of this study was to efficiently utilize reed for both cellulase and biobutanol production. The unprocessed cellulase blend produced under solid-state fermentation using reed as the substrate showed a similar reducing sugar yield using Whatman filter paper to the commercial enzyme blend (38.61%). Organosolv pretreatment method could efficiently reduce hemicellulose (29.3%–14.6%) and lignin (17.2%–14.1%) content and increase cellulose content (42.5%–62.3%) from reed. Enzymatic hydrolysis of organosolv-pretreated reed using the crude cellulase with enzyme loading of 25 FPU/g reed, 20% solid content at 50 °C and pH 5.5 resulted in a reed hydrolysate containing 40.01 g/L glucose and 3.55 g/L xylose after 72 h. Fermentation of the hydrolysate medium by Clostridium acetobutylicum produced 9.07 and 14.24 g/L of biobutanol and ABE with yield of 0.21 g/g and 0.33 g/g, respectively. This study proved that crude cellulase complex produced under solid state fermentation and organsolv pretreatment can efficiently provide reed hydrolysate that can be converted to biobutanol without any commercial cellulase usage.     

Covalent immobilization of enzymes and yeast: Towards a continuous simultaneous saccharification and fermentation process for cellulosic ethanol

The immobilization of enzymes and yeast cells is a key factor for establishing a continuous process of cellulosic ethanol production, which can combine the benefits of a separated hydrolysis and fermentation process and a simultaneous saccharification and fermentation process. This paper investigates the use of cellulase enzyme and yeast cell immobilization under a flow regime of ethanol production from soluble substrates such as cellobiose and carboxymethyl cellulose. The immobilization was achieved by incubating enzymes and yeast cells on polystyrene surfaces which had been treated by nitrogen ion implantation. The saccharification by immobilized enzymes and the fermentation by immobilized yeast cells were conducted in two separate vessels connected by a pump. During the experiments, glucose concentrations were always maintained at low levels which potentially reduce product inhibition effects on the enzymes. Covalent immobilization of enzymes and yeast cells on the plasma treated polymer reduces loss by shear flow induced detachment. The potential for continuous flow production of ethanol and the influence of daughter yeast cells in the circulating flow on the immobilized enzyme activity are discussed.     

Establishment of rumen-mimic bacterial consortia: A functional union for bio-hydrogen production from cellulosic bioresource

The study aimed to establish stable rumen-mimic bacterial consortia as a functional union for simultaneous saccharification and fermentation from cellulosic bioresource. The consortia was constructed by repeated-batch culture with ruminal microflora and napiergrass at 38 °C. The major bacterial composition of batch culture was monitored by 16S rRNA gene-targeted denaturing gradient gel electrophoresis (DGGE). The result showed that a stable consortia constituted by ruminal microflora was formed, and the consortia includes bacterial strains such as Clostridium xylanolyticum, Clostridium papyrosolvens, Clostridium beijerinckii, Ruminococcus sp., Ethanoligenens harbinense, and Desulfovibrio desulfuricans. The Clostridium genus was showed as the dominant population in the system and contributed to the biohydrogen production. During each eight days incubation period, the functional consortia could degrade an average of 27% hemicellulose and 2% cellulose from napiergrass biomass. While the increasing of the reducing sugars and their converting to biohydrogen gas productivity were also observed. The time course profile for cellulytic enzymes showed that the hydrolysis of complex lignocellulosic material may occur through the ordered actions of xylenase and cellulase activities.     

Cellulase production using biomass feed stock and its application in lignocellulose saccharification for bio-ethanol production

A major constraint in the enzymatic saccharification of biomass for ethanol production is the cost of cellulase enzymes. Production cost of cellulases may be brought down by multifaceted approaches which include the use of cheap lignocellulosic substrates for fermentation production of the enzyme, and the use of cost efficient fermentation strategies like solid state fermentation (SSF). In the present study, cellulolytic enzymes for biomass hydrolysis were produced using solid state fermentation on wheat bran as substrate. Crude cellulase and a relatively glucose tolerant BGL were produced using fungi Trichoderma reesei RUT C30 and Aspergillus niger MTCC 7956, respectively. Saccharification of three different feed stock, i.e. sugar cane bagasse, rice straw and water hyacinth biomass was studied using the enzymes. Saccharification was performed with 50 FPU of cellulase and 10 U of β-glucosidase per gram of pretreated biomass. Highest yield of reducing sugars (26.3 g/L) was obtained from rice straw followed by sugar cane bagasse (17.79 g/L). The enzymatic hydrolysate of rice straw was used as substrate for ethanol production by Saccharomyces cerevisiae. The yield of ethanol was 0.093 g per gram of pretreated rice straw.     

Effect of the type and concentration of cellulose and temperature on metabolite formation by a fermentative thermophilic consortium

In this study, we hypothesized that anaerobic biodegradation of cellulose is influenced by cellulose type and concentration, temperature, and their interactions. Cellulose biodegradation by an anaerobic consortium was tested in thermophilic batch experiments that combined cellulase action, hydrolysis, and fermentation. Initially, the main constituents in the inocula were Thermoanaerobacter, Clostridium, and Acetivibrio spp. Four types of cellulose and a range of concentrations were used as feedstock with pathways involving hydrolysis and glycolysis to produce H₂, CO₂, acetate, and ethanol. Long fibrous cellulose, two types of microcrystalline cellulose, and filter paper squares were tested at several concentrations between 2 and 20 g/l as substrates. The yields ranged between 0.1 and 2.9 mmol H₂ and 0.7–2.6 mmol ethanol per g cellulose. The rates ranged between 0.01 and 0.2 mmol H₂, 0.03–0.2 mmol CO₂, and 0.01–0.05 mmol ethanol per g cellulose·h. Statistical analyses indicated that the rates and yields of metabolite production were influenced by two-way interactions between the temperature, type, and concentration of cellulose. The results suggest that two-way interactions between experimental variables may impact the outcomes in cellulose bioconversion studies.