Cellulase Loading Research Articles

Batch and multi-step fed-batch enzymatic saccharification of Formiline-pretreated sugarcane bagasse at high solid loadings for high sugar and ethanol titers

Formiline pretreatment pertains to a biomass fractionation process. In the present work, Formiline-pretreated sugarcane bagasse was hydrolyzed with cellulases by batch and multi-step fed-batch processes at 20% solid loading. For wet pulp, after 144h incubation with cellulase loading of 10FPU/g dry solid, fed-batch process obtained ∼150g/L glucose and ∼80% glucan conversion, while batch process obtained ∼130g/L glucose with corresponding ∼70% glucan conversion. Solid loading could be further increased to 30% for the acetone-dried pulp. By fed-batch hydrolysis of the dried pulp in pH4.8 buffer solution, glucose concentration could be 247.3±1.6g/L with corresponding 86.1±0.6% glucan conversion. The enzymatic hydrolyzates could be well converted to ethanol by a subsequent fermentation using Saccharomices cerevisiae with ethanol titer of 60–70g/L. Batch and fed-batch SSF indicated that Formiline-pretreated substrate showed excellent fermentability. The final ethanol concentration was 80g/L with corresponding 82.7% of theoretical yield.

Sodium sulfite–formaldehyde pretreatment of mixed hardwoods and its effect on enzymatic hydrolysis

In this work, mixed hardwoods were pretreated by sodium sulfite–formaldehyde (SF). The effects of SF pretreatment on the chemical compositions and enzymatic hydrolysis of mixed hardwoods were investigated. SF pretreatment temperature had a significant effect on pulp yield and delignification, resulting in an increased efficiency of enzymatic hydrolysis. After 96h of enzymatic hydrolysis at the cellulase loading of 40FPU/g substrate, the yields of glucan and xylan on the basis of original wood were 37% and 11% for the pulp produced with 12% sulfite charge at 170°C for 2h. The total sugar recovery based on the sugar in original wood was 74%. These results indicate that sulfite–formaldehyde cooking is of great potential to be a pretreatment method for a greenfield mill to produce fuel ethanol from hardwood.

Sodium carbonate–sodium sulfite pretreatment for improving the enzymatic hydrolysis of rice straw

The performance of sodium carbonate–sodium sulfite (Na2CO3–Na2SO3) pretreatment on improving the enzymatic hydrolysis of rice straw was investigated. The results indicated that both Na2CO3 and Na2SO3 pretreatments are effective in improving the enzymatic digestibility of rice straw. A higher ratio of Na2SO3 in pretreatment liquor performed better at softening the rice straw through degradation of hemicellulose, delignification, and lignin sulfonation, while it was conserving more polysaccharides during the pretreatment than Na2CO3. The highest sugar recovery of pretreated rice straw, 74.5%, 82.7%, and 59.5% for total sugar, glucan, and xylan, respectively, was obtained at cellulase loading of 20FPU/g-cellulose after the pretreatment at 140°C, chemical charge of 12%, Na2CO3–Na2SO3 ratio of 0:1 (i.e., only Na2SO3 pretreatment). The corresponding delignification ratio of pretreated solid was 53.4%, and 91.4% of the polysaccharides were conserved. The sugar recovery of only Na2CO3 pretreatment at the ratio of 1:0 was 67.1%, 74.4%, and 53.7% for total sugar, glucan, and xylan, respectively, at 140°C, chemical charge of 12%, and enzyme loading of 20FPU/g-cellulose. Its corresponding delignification ratio was 40.3%, and polysaccharides conservation ratio was 88.9%.

Effects of sodium carbonate pretreatment on the chemical compositions and enzymatic saccharification of rice straw

The effects of sodium carbonate (Na2CO3) pretreatment on the chemical compositions and enzymatic saccharification of rice straw were investigated. The enzymatic digestibility of rice straw is enhanced after pretreatment since pretreated solids show significant delignification with high sugar availability. During pretreatment, an increasing temperature and Na2CO3 charge leads to enhanced delignification, whereas an increased degradation of polysaccharides as well, of which xylan acts more susceptible than glucan. The sugar recovery of enzymatic hydrolysis goes up rapidly with the total titratable alkali (TTA) increasing from 0% to 8%, and then it reaches a plateau. The highest sugar recovery of rice straw after pretreatment, 71.7%, 73.2%, and 76.1% for total sugar, glucan, and xylan, respectively, is obtained at 140°C, TTA 8% and cellulase loading of 20FPU/g-cellulose. In this condition, the corresponding delignification ratio of pretreated solid is 41.8%, while 95% of glucan and 76% of xylan are conserved.

Effect of hemicellulase addition during enzymatic hydrolysis of switchgrass pretreated by soaking in aqueous ammonia

The focus of this study was to determine the effect of supplementing cellulase with hemicellulase during enzymatic hydrolysis of switchgrass pretreated by soaking in aqueous ammonia (SAA) under a range of conditions. SAA was performed using 15% aqueous ammonia for 8 or 24h at temperatures of 40 or 60°C. The combined effect of cellulase and hemicellulase loadings on glucose yield during enzymatic hydrolysis was modeled for each pretreatment condition. Glucose yields greater than 85% of theoretical were achieved for pretreatment at 40°C for 24h and for 60°C for 8h. Hemicellulase supplementation was not sufficient to achieve these glucose yields at lower severity SAA pretreatment. High severity SAA pretreatment also led to low yields despite improved delignification.

Integrated analysis of hydrothermal flow through pretreatment

BackgroundThe impact of hydrothermal flowthrough (FT) pretreatment severity on pretreatment and solubilization performance metrics was evaluated for three milled feedstocks (corn stover, bagasse, and poplar) and two conversion systems (simultaneous saccharification and fermentation using yeast and fungal cellulase, and fermentation by Clostridium thermocellum).ResultsCompared to batch pretreatment, FT pretreatment consistently resulted in higher XMG recovery, higher removal of non-carbohydrate carbon and higher glucan solubilization by simultaneous saccharification and fermentation (SSF). XMG recovery was above 90% for FT pretreatment below 4.1 severity but decreased at higher severities, particularly for bagasse. Removal of non-carbohydrate carbon during FT pretreatment increased from 65% at low severity to 80% at high severity for corn stover, and from 40% to 70% for bagasse and poplar.Solids obtained by FT pretreatment were amenable to high conversion for all of the feedstocks and conversion systems examined. The optimal time and temperature for FT pretreatment on poplar were found to be 16 min and 210°C. At these conditions, SSF glucan conversion was about 85%, 94% of the XMG was removed, and 62% of the non carbohydrate mass was solubilized. Solubilization of FT-pretreated poplar was compared for C. thermocellum fermentation (10% inoculum), and for yeast-fungal cellulase SSF (5% inoculum, cellulase loading of 5 and 10 FPU/g glucan supplemented with β-glucosidase at 15 and 30 U/g glucan). Under the conditions tested, which featured low solids concentration, C. thermocellum fermentation achieved faster rates and more complete conversion of FT-pretreated poplar than did SSF. Compared to SSF, solubilization by C. thermocellum was 30% higher after 4 days, and was over twice as fast on ball-milled FT-pretreated poplar.ConclusionsXMG removal trends were similar between feedstocks whereas glucan conversion trends were significantly different, suggesting that factors in addition to XMG removal impact amenability of glucan to enzymatic attack. Corn stover exhibited higher hydrolysis yields than bagasse or poplar, which could be due to higher removal of non-carbohydrate carbon. XMG in bagasse is more easily degraded than XMG in corn stover and poplar. Conversion of FT-pretreated substrates at low concentration was faster and more complete for C. thermocellum than for SSF.

Production of cellulases by solid state fermentation with Aspergillus terreus and enzymatic hydrolysis of mild alkali-treated rice straw

Rice straw was used as substrate for cellulase production by solid state fermentation with Aspergillus terreus. Substrate concentration, moisture ratio, inoculum size and initial pH were optimized using response surface methodology involving Box-Behnken design. The predicted filter paper activity under optimized parameters was 9.73U/g and the validated filter paper activity was 10.96U/g. Hydrolysis of the biomass pretreated with 0.125% to 1% NaOH for 24h at room temperature was performed using crude cellulase preparation. Treatment with 0.5% NaOH at room temperature for 24h was the most efficient treatment method for saccharification. Under the optimized conditions, rice straw yielded 676mg reducing sugars per gram of substrate at a cellulase loading of 9FPUg−1 substrate. The present study establishes the possibility of using mild alkali pretreated rice straw for the production of fermentable sugars with 74.19% efficiency which can be further utilized for the production of ethanol.

Recombinant EXLX1 from Bacillus subtilis for enhancing enzymatic hydrolysis of corn stover with low cellulase loadings

BsEXLX1 protein from Bacillus subtilis has been proposed to have a structure that is similar to plant expansin. In this study, the recombinant BsEXLX1 protein was successfully expressed and purified in Escherichia coli BL21 (DE3). When the purified BsEXLX1 which contained the thioredoxin (Trx) protein was incubated with low-dose cellulases either simultaneously or sequentially, it showed a significant synergistic activity in corn stover hydrolysis. Furthermore, an even greater increase in the synergistic activity was obtained when cellulose was pretreated with BsEXLX1 followed by cellulase hydrolysis, and the synergistic activity was found as high as 1.5-fold greater than that when cellulose was treated simultaneously with the same concentrations of BsEXLX1 and cellulases. These results provided a feasible way for the potential application of BsEXLX1 in the efficient saccharification of cellulose materials for bioethanol production. Key word: Bacillus subtilis , BsEXLX1, cellulase, cellulose hydrolysis, bioethanol

Dilute Sulfuric Acid Pretreatment and Enzymatic Hydrolysis of Corn Stover into Fermentable Sugars

The pretreatment of corn stover with dilute sulfuric acid has been investigated by varying the acid concentration (0.5%-1.25%(w/w)) and the temperature (130-160°C). The pretreatment is aimed at improving enzymatic hydrolysis and increasing the fermentability of the biomass. Given the overall sugar yield, the most favourable pretreatment condition was performed with 0.75% sulfuric acid at 150°C for 30min and then with an enzyme loading of cellulase 15 FPU per gram of cellulose, and it resulted in a total of 49.74g glucose and xylose from 100g dry corn stover. The fiber physical feature, structure and property of pretreated residues were studied with Scanning Electron Microscope (SEM) and Fourier transform infrared spectroscopy. The SEM pictures indicated that the biomass structure was deformed and its fibers were exposed by the pretreatment. FTIR study showed that lignin and hemicellulose were partially removed during the diluted sulfuric acid pretreatment.

Aqueous ammonia pretreatment, saccharification, and fermentation evaluation of oil palm fronds for ethanol production

Oil palm fronds are the most abundant lignocellulosic biomass in Malaysia. In this study, fronds were tested as the potential renewable biomass for ethanol production. The soaking in aqueous ammonia pretreatment was applied, and the fermentability of pretreated fronds was evaluated using simultaneous saccharification and fermentation. The optimal pretreatment conditions were 7 % (w/w) ammonia, 80 °C, 20 h of pretreatment, and 1:12 S/L ratio, where the enzymatic digestibility was 41.4 % with cellulase of 60 FPU/g-glucan. When increasing the cellulase loading in the hydrolysis of pretreated fronds, the enzymatic digestibility increased until the enzyme loading reached 60 FPU/g-glucan. With 3 % glucan loading in the SSF of pretreated fronds, the ethanol concentration and yield based on the theoretical maximum after 12 and 48 h of the SSF were 7.5 and 9.7 g/L and 43.8 and 56.8 %, respectively. The ethanol productivities found at 12 and 24 h from pretreated fronds were 0.62 and 0.36 g/L/h, respectively.

Effect of Lignocellulosic Substrate and Commercial Cellulase Loading on Reducing Sugar Concentration for Ethanol Production

Fuel ethanol production from lignocellulosic biomass requires a relatively high initial sugar concentration in hydrolysate. Raising the substrate and enzyme concentration in the batch hydrolysis will normally help to heighten sugar concentration. In this study, dilute sulfuric acid pretreatment was used for chemical pretreatment of rice straw. The effect of rice straw and commercial cellulase (Accelerase 1000 TM ) loading were investigated. It was found that an increase in rice straw from 7.5 to 15.0 g/100 mL H2SO4 raised the reducing sugar concentration from 27.70 ± 1.72 to 58.42 ± 1.23 g/L. At the high level of rice straw up to 20.0 g/100 mL H2SO4 gave a slight increase in reducing sugar of 66.17 ± 0.00 g/L. On the contrary, the rice straw conversion decreased from 343.27 ± 1.29 to 286.22 ± 2.39 mg/g DS (Dry residue substrate) and holocellulose conversion slightly decreased from 47.02 ± 0.18 to 39.21 ± 0.33% (w/w) when the rice straw increased from 15.0 to 20.0 g/100 mL H2SO4. The reducing sugar concentration increased from 52.98 ± 0.73 g/L to 64.61 ± 0.53 g/L and rice straw conversion changed from 293.44 ± 5.12 to 414.16 ± 0.19 mg/g DS, when the use of Accellerase 1000 TM increased from 10 to 45 FPU/g DS. It also found that the large amount of Accelerase 1000 TM loading (50 and 55 FPU/g DS) did not improve the reducing sugar and rice straw conversion.

Ethanol production from palm pressed fiber by prehydrolysis prior to simultaneous saccharification and fermentation (SSF)

Abstract Palm pressed fiber (PPF) is a lignocellulosic material that has potential to be cheap substrate for ethanol production. In this study, the enzymatic hydrolysis of alkali pretreated PPF was investigated. It was found that the hydrolysis with cellulase (10 FPU g −1 of PPF) and β-glucosidase (10 U g −1 of PPF) gave the higher reducing sugar production than using cellulase alone. In addition, the optimum condition of simultaneous saccharification and fermentation (SSF) with 6 h of prehydrolysis for ethanol production was the PPF concentration of 100 kg m −3 with the enzyme loading of cellulase 6.0 FPU g −1 of PPF and β-glucosidase 3.0 U g −1 of PPF at pH of 5.0 and 35 °C. The ethanol concentration of 10.4 kg m −3 and ethanol yield of 192 g kg −1 of cellulose was obtained at the optimum condition in 24 h of SSF. Finally, the effect of prehydrolysis of pretreated PPF prior to SSF on the ethanol concentration was examined with batch and fed-batch mode of hydrolysis. It was found that prehydrolysis with the fed-batch mode did not improve ethanol yield. However, the fed-batch mode of prehydrolysis gave the highest ethanol production of 12.1 kg m −3 at 12 h of SSF process. Also, it was concluded that the advantages of fed-batch of prehydrolysis prior SSF were the less enzyme requirement and the increasing PPF loading for ethanol production.

Ethanol Production from Soybean Fiber, a Co‐product of Aqueous Oil Extraction, Using a Soaking in Aqueous Ammonia Pretreatment

AbstractThe effectiveness of soaking in aqueous ammonia (SAA) as a pretreatment method for the conversion of soybean fiber to ethanol via simultaneous saccharification and fermentation (SSF) was investigated. Insoluble fiber is a co‐product from oil and protein extraction using two‐stage, countercurrent, enzyme‐assisted, aqueous extraction processing of full‐fat soybean flakes (FFSF) and extruded FFSF. The fiber fractions were soaked in 15 wt% aqueous ammonia at 1:10 solid‐to‐liquid ratio. The effects of operating variables, including treatment times (6, 12, and 24 h), treatment temperatures (60 and 80 °C), and cellulase loadings (15 and 60 FPU/g‐glucan) on the degree of enzymatic hydrolysis were determined. The best SAA conditions were 80 °C for 12 h followed by an enzyme loading of 15 FPU/g‐glucan, which produced a 152‐mg/g glucose yield after 48 h of hydrolysis. This was 8.7 times the amount produced from the same fiber not pretreated with SAA. The glucose yield increased to 381 mg/g when fiber obtained from extruded FFSF was submitted to SAA. SAA (80 °C, 12 h) on extruded fiber subjected to SSF increased ethanol yield from 0.06 g of ethanol/g [40% of theoretical yield] (for non SAA pretreated fiber) to 0.25 g of ethanol/g [92% of theoretical yield]. The combination of extrusion and SAA was an efficient means for converting the fiber‐rich soybean fraction into ethanol.

Optimization of enzymatic hydrolysis of pretreated rice straw and ethanol production

Cellulase, Tween 80, and β-glucosidase loading were studied and optimized by response surface methodology to improve saccharification. Microwave alkali-pretreated rice straw used as substrate for onsite enzyme production by Aspergillus heteromorphus and Trichoderma reesei. The highest enzymatic hydrolysis (84%) was obtained from rice straw at crude enzyme loading of 10 FPU/gds of cellulase, 0.15% Tween 80, and 100 international unit/g dry solids of β-glucosidase activities. Enzymatic hydrolyzate of pretreated rice straw was used for ethanol production by Saccharomyces cerevisiae, Scheffersomyces stipitis, and by co-culture of both. The yield of ethanol was 0.50, 0.47, and 0.48 g(p)/g(s) by S. cerevisiae, S. stipitis, and by co-culture, respectively, using pretreated rice straw hydrolyzate. The co-culture of S. cerevisiae and S. stipitis produced 25% more ethanol than S. cerevisiae alone and 31% more ethanol than S. stipitis alone. During anaerobic fermentation 65.08, 36.45, and 50.31 μmol/ml CO(2) released by S. cerevisiae, S. stipitis, and by co-culture, respectively. The data indicated that saccharification efficiency using optimized crude enzyme cocktail was good, and enzymatic hydrolyzate could be fermented to produce ethanol.

Enzymatic Saccharification and Ethanol Fermentation of Reed Pretreated with Liquid Hot Water

Reed is a widespread-growing, inexpensive, and readily available lignocellulosic material source in northeast China. The objective of this study is to evaluate the liquid hot water (LHW) pretreatment efficiency of reed based on the enzymatic digestibility and ethanol fermentability of water-insoluble solids (WISs) from reed after the LHW pretreatment. Several variables in the LHW pretreatment and enzymatic hydrolysis process were optimized. The conversion of glucan to glucose and glucose concentrations are considered as response variables in different conditions. The optimum conditions for the LHW pretreatment of reed area temperature of 180°C for 20min and a solid-to-liquid ratio of 1 : 10. These optimum conditions for the LHW pretreatment of reed resulted in a cellulose conversion rate of 82.59% in the subsequent enzymatic hydrolysis at 50°C for 72 h with a cellulase loading of 30 filter paper unit per gram of oven-dried WIS. Increasing the pretreatment temperature resulted in a higher enzymatic digestibility of the WIS from reed. Separate hydrolysis and fermentation of WIS showed that the conversion of glucan to ethanol reached 99.5% of the theoretical yield. The LHW pretreatment of reed is a suitable method to acquire a high recovery of fermentable sugars and high ethanol conversion yield.

Deactivation of individual cellulase components

Deactivation extents of cellobiohydrolase, endoglucanase, and a total cellulase mixture (Spezyme CP) were studied independently as functions of incubating time and mixing intensity. It was found that the decrease in total cellulase activity was more strongly related to deactivation of cellobiohydrolase 1 (CBH1) than endoglucanase. The mass-averaged shear in orbiting flasks at 50, 150, and 250 rpm was quantified by computational fluid dynamics and was two-orders smaller than shear in typical stirred tanks. Endoglucanase activity did not change significantly with mixing speed, but CBH1 and total cellulase activities were 10–25% higher at 250 rpm compared to the lower speeds after a 24-h incubation. Total deactivation due to mechanical mixing (∼20%) may be too low to account for all the rate reduction during cellulose hydrolysis. Thermal deactivation was independent of enzyme concentration while deactivation due to mechanical stress decreased when cellulase loading increased over 0.15 filter paper unit/ml.

Simultaneous saccharification and fermentation (SSF) of Jatropha curcas shells: utilization of co-products from the biodiesel production process

Jatropha curcas has great potential as an oil crop for use in biodiesel applications, and the outer shell is rich in lignocellulose that may be converted to ethanol, giving rise to the concept of a biorefinery. In this study, two dilute pretreatments of 0.5% H(2)SO(4) and 1.0% NaOH were performed on Jatropha shells with subsequent simultaneous saccharification and fermentation (SSF) of the pretreated water-insoluble solids (WIS) to evaluate the effect of inhibitors in the pretreatment slurry. A cellulase loading of 15 FPU/g WIS, complimented with an excess of cellobiase (19.25 U/g), was used for SSF of either the washed WIS or the original slurry to determine the effect of inhibitors. Ethanol and glucose were monitored during SSF of 20 g of pretreated biomass. The unwashed slurry showed to have a positive effect on SSF efficiency for the NaOH-pretreated biomass. Maximum efficiencies of glucan conversion to ethanol in the WIS were 40.43% and 41.03% for the H(2)SO(4)- and NaOH-pretreated biomasses, respectively.

Evaluating kinetics of enzymatic saccharification of lignocellulose by fractal kinetic analysis

The effects of impacting factors, including cellulase loading, operation temperature, product glucose inhibition, and high solid pretreated biomass loading were examined systemically on the enzymatic saccharification of lignocellulose (dilute acid pretreated corn stover) in the presence and absence of tri-block copolymer L64 (also referred to polymeric nonionic surfactant). The complex kinetics of enzymatic saccharification of cellulose were subjected to fractal kinetic analysis based on a fractal kinetic model, which is described with fractal kinetic parameters of the rate constant and fractal exponent. The results indicate that glucose inhibition including high lignocellulose loading is indexed by decreasing rate constant while lignin inhibition and high operation temperature is indexed by increasing fractal exponent. The effect of a nonionic surfactant on the enzymatic saccharification of lignocellulose mainly contributed to the elimination of lignin inhibition by decreasing the corresponding fractal exponent. However, the effect of the nonionic surfactant on cellulase activity and stability was very limited.

Combination of hot compressed water treatment and wet disk milling for high sugar recovery yield in enzymatic hydrolysis of rice straw

Rice straw has attracted significant interest in Japan as a potential raw material for biorefineries. Combination of hot-compressed water treatment (HCWT) and wet disk milling (WDM) was investigated to improve the enzymatic digestibility of rice straw and enhance sugar recovery yield. Rice straw, cut to <3mm, was autoclaved at 121, 135, and 150°C for 60min, and subsequently treated by wet disk milling. WDM with HCWT at 135°C for 60min produced maximum xylose and glucose yields of 79% and 90%, respectively, at 10FPU/g-substrate cellulase loading. Autoclaving at 150°C leaked a 35% arabinose effluence in the liquid phase. Hydrolysis via WDM with HCWT required a lower enzyme loading (5FPU/g-substrate) than either pretreatment process in isolation for >70% xylose and 80% glucose yield. Economical analysis indicate that enzymes cost for ethanol production is reduced by 19–67% by WDM with HCWT.

Effect of Thermo-Chemical Pretreatment on Bioethanol Production from Corncobs

Currently, ethanol has gained significance since it is substantially used as an alternative biofuel. It can be produced by agro-industrial residuals such as corncobs. In ethanol production, hemicellulose and lignin are inhibitors of the enzymatic hydrolysis between cellulase and cellulose. They must be removed during in the pretreatment stage. In this research H2SO4 and NaOH were used as pretreatment solution at high temperature. The temperature were varied for 120 to 170 °C. The result showed that the optimal pretreatment condition of corncobs was using 2% (v/v) NaOH for 24 hr digestion in 1% (v/v) H2SO4 and then heating at 170 °C for 5 min. At this condition, the cellulose content of corncobs increased from 66.52% (untreated) to 92.25% (pretreated). Enzyme digestion of pretreated corncobs at an optimum condition yielded the reducing sugar level of 8.74 g/L. The optimum conditions were achieved at cellulase loading of 10 FPU/g and digestion time of 4 hr. Finally, the reducing sugar solution of 5 g/L was fermented with Zymomonas mobilis TISTR405 at 30°C and pH 5.0 for 48 hr, yielding ethanol of 1.8 g/l or 35.93% of the theoretical ethanol yield.