Potential Enzyme Cost Reduction with the Addition of Surfactant during the Hydrolysis of Pretreated Softwood

The potential economic benefits of surfactants addition on enzymatic hydrolysis of steam-exploded lodgepole pine (SELP) and ethanol-pretreated lodgepole pine (EPLP) were investigated in this study. Free cellulase readsorption on fresh substrate was used to recover and recycle cellulase enzymes during the hydrolysis of SELP and EPLP substrate. Supplementing Tween 80 during the hydrolysis could facilitate enzyme recycling for EPLP substrate. A logarithmic correlation was established between surfactant concentration and free cellulase content after lignocellulosic hydrolysis, which was used to compute enzyme cost savings over various Tween 80 concentrations. A simple economic analysis of enzyme cost savings versus the cost of surfactant was undertaken. The results indicated that the addition of Tween 80 (priced at US $0.25/kg) during the hydrolysis of the EPLP substrate could save 60% of the total enzyme cost at concentrations in the 0.025% to 0.2% range. The addition of Tween for the hydrolysis of the SELP substrate significantly reduced the material cost by 24% per 1 gal of ethanol produced, and the ethanol production cost could be reduced by 8.6% with the addition of Tween and enzymes recycle for the hydrolysis of SELP substrate. A schematic concept of recycling enzyme and surfactant was also presented with a recirculation of process streams during hydrolysis. Further analysis indicated a 66% reduction in total enzyme cost could potentially be achieved under the concept.     

Hydrolysis of Ammonia-pretreated Sugar Cane Bagasse with Cellulase, β-Glucosidase, and Hemicellulase Preparations

Sugar cane bagasse consists of hemicellulose (24%) and cellulose (38%), and bioconversion of both fractions to ethanol should be considered for a viable process. We have evaluated the hydrolysis of pretreated bagasse with combinations of cellulase, β-glucosidase, and hemicellulase. Ground bagasse was pretreated either by the AFEX process (2NH₃: 1 biomass, 100 °C, 30 min) or with NH₄OH (0.5 g NH₄OH of a 28% [v/v] per gram dry biomass; 160 °C, 60 min), and composition analysis showed that the glucan and xylan fractions remained largely intact. The enzyme activities of four commercial xylanase preparations and supernatants of four laboratory-grown fungi were determined and evaluated for their ability to boost xylan hydrolysis when added to cellulase and β-glucosidase (10 filter paper units [FPU]: 20 cellobiase units [CBU]/g glucan). At 1% glucan loading, the commercial enzyme preparations (added at 10% or 50% levels of total protein in the enzyme preparations) boosted xylan and glucan hydrolysis in both pretreated bagasse samples. Xylanase addition at 10% protein level also improved hydrolysis of xylan and glucan fractions up to 10% glucan loading (28% solids loading). Significant xylanase activity in enzyme cocktails appears to be required for improving hydrolysis of both glucan and xylan fractions of ammonia pretreated sugar cane bagasse.     

Process Evaluation of Enzymatic Hydrolysis with Filtrate Recycle for the Production of High Concentration Sugars

Process simulation and lab trials were carried out to demonstrate and confirm the efficiency of the concept that recycling hydrolysate at low total solid enzymatic hydrolysis is one of the options to increase the sugar concentration without mixing problems. Higher sugar concentration can reduce the capital cost for fermentation and distillation because of smaller retention volume. Meanwhile, operation cost will also decrease for less operating volume and less energy required for distillation. With the computer simulation, time and efforts can be saved to achieve the steady state of recycling process, which is the scenario for industrial production. This paper, to the best of our knowledge, is the first paper discussing steady-state saccharification with recycling of the filtrate form enzymatic hydrolysis to increase sugar concentration. Recycled enzymes in the filtrate (15–30% of the original enzyme loading) resulted in 5–10% higher carbohydrate conversion compared to the case in which recycled enzymes were denatured. The recycled hydrolysate yielded 10% higher carbohydrate conversion compared to pure sugar simulated hydrolysate at the same enzyme loading, which indicated hydrolysis by-products could boost enzymatic hydrolysis. The high sugar concentration (pure sugar simulated) showed inhibition effect, since about 15% decrease in carbohydrate conversion was observed compared with the case with no sugar added. The overall effect of hydrolysate recycling at WinGEMS simulated steady-state conditions with 5% total solids was increasing the sugar concentration from 35 to 141 g/l, while the carbohydrate conversion was 2% higher for recycling at steady state (87%) compared with no recycling strategy (85%). Ten percent and 15% total solid processes were also evaluated in this study.     

Enhanced Enzymatic Hydrolysis of Lignocellulose by Optimizing Enzyme Complexes

To enhance the conversion of the cellulose and hemicellulose, the corncob pretreated by aqueous ammonia soaking was hydrolyzed by enzyme complexes. The saturation limit for cellulase (Spezyme CP) was determined as 15 mg protein/g glucan (50 filter paper unit (FPU)/g glucan). The accessory enzymes (β-glucosidase, xylanase, and pectinase) were supplemented to hydrolyze cellobiose (cellulase-inhibiting product), hemicellulose, and pectin (the component covering the fiber surfaces), respectively. It was found that β-glucosidase (Novozyme 188) loading of 1.45 mg protein/g glucan [30 cellobiase units (CBU)/g glucan] was enough to eliminate the cellobiose inhibitor, and 2.9 mg protein/g glucan (60 CBU/g glucan) was the saturation limit. The supplementation of xylanase and pectinase can increase the conversion of cellulose and hemicellulose significantly. The yields of glucose and xylose enhanced with the increasing enzyme loading, but the increasing trend became low at high loading. Compared with xylanase, pectinase was more effective to promote the hydrolysis of cellulose and hemicellulose. The supplementation of pectinase with 0.12 mg protein/g glucan could increase the yields of glucose and xylose by 7.5% and 29.3%, respectively.     

Relatively High-Substrate Consistency Hydrolysis of Steam-Pretreated Sweet Sorghum Bagasse at Relatively Low Cellulase Loading

Sweet sorghum bagasse (SSB) was steam pretreated in the conditions of 190 °C for 5 min to assess its amenability to the pretreatment and enzymatic hydrolysis. Results showed that pretreatment conditions were robust enough to pretreat SSB with maximum of 87% glucan and 72% xylan recovery. Subsequent enzymatic hydrolysis showed that the pretreated SSB at 2% substrate consistency resulted in maximum of 70% glucan-glucose conversion. Increasing substrate consistency from 2% to 16% led to a significant reduction in glucan conversion. However, the decrease ratio of glucan-glucose conversion was the minimum when the consistency increased from 2% to 12%. When the pretreated SSB consistency of 12% was applied for hydrolysis, increase in cellulase loading from 7.5 up to 20 filter paper units (FPU)/g glucan resulted only in 14% increase in glucan-glucose conversion compared to 20% increase with cellulase loading varying from 2.5 to 7.5 FPU/g glucan. More than 10 cellobiase units (CBU)/g glucan β-glucosidase supplementation had no noticeable improvement on glucan-glucose conversion. Additionally, supplementation of xylanase was found to significantly increase glucan-glucose conversion from 50% to 80% with the substrate consistency of 12%, when the cellulase and β-glucosidase loadings were at relatively low enzyme loadings (7.5 FPU/g and 10 CBU/g glucan). It appeared that residual xylan played a critical role in hindering the ease of hydrolysis of SSB. A proper xylanase addition was suggested to achieve a high hydrolysis yield at relatively high substrate consistency with relatively low enzyme loadings.     

Studies on the chromatographic fractionation of Trichoderma reesei cellulases by hydrophobic interaction

This work reports new studies on cellulases fractionation by hydrophobic interaction chromatography. The purification procedure for the Trichoderma reesei cellulase complex consists of gel permeation chromatography on Sephadex G-25M followed by an ultrafiltration step. The concentrated enzyme solution was then fractionated on Sepharose CL-6B modified by covalent immobilization of 1,4-butanediol diglycidyl ether. The influence of the mobile phase composition on the chromatographic behaviour of the T. reesei cellulase complex was investigated. By using 13% (w/v) ammonium sulphate in eluent buffer, a selective separation of beta-glucosidase with a two-fold increase in specific activity and a recovery of 60% cellobiase activity were obtained. Other commercial hydrophobic supports (octyl- and phenyl-Sepharose) were also tested and compared under the same conditions.     

Non-ionic Surfactants and Non-Catalytic Protein Treatment on Enzymatic Hydrolysis of Pretreated Creeping Wild Ryegrass

Our previous research has shown that saline Creeping Wild Ryegrass (CWR), Leymus triticoides, has a great potential to be used for bioethanol production because of its high fermentable sugar yield, up to 85% cellulose conversion of pretreated CWR. However, the high cost of enzyme is still one of the obstacles making large-scale lignocellulosic bioethanol production economically difficult. It is desirable to use reduced enzyme loading to produce fermentable sugars with high yield and low cost. To reduce the enzyme loading, the effect of addition of non-ionic surfactants and non-catalytic protein on the enzymatic hydrolysis of pretreated CWR was investigated in this study. Tween 20, Tween 80, and bovine serum albumin (BSA) were used as additives to improve the enzymatic hydrolysis of dilute sulfuric-acid-pretreated CWR. Under the loading of 0.1 g additives/g dry solid, Tween 20 was the most effective additive, followed by Tween 80 and BSA. With the addition of Tween 20 mixed with cellulase loading of 15 FPU/g cellulose, the cellulose conversion increased 14% (from 75 to 89%), which was similar to that with cellulase loading of 30 FPU/g cellulose and without additive addition. The results of cellulase and BSA adsorption on the Avicel PH101, pretreated CWR, and lignaceous residue of pretreated CWR support the theory that the primary mechanism behind the additives is prevention of non-productive adsorption of enzymes on lignaceous material of pretreated CWR. The addition of additives could be a promising technology to improve the enzymatic hydrolysis by reducing the enzyme activity loss caused by non-productive adsorption.     

Effects of ammonia fiber explosion treatment on activity of endoglucanase from Acidothermus cellulolyticus in transgenic plant

A critical parameter affecting the economic feasibility of lignocellulosic bioconversion is the production of inexpensive and highly active cellulase enzymes in bulk quantity. A promising approach to reduce enzyme costs is to genetically transform plants with the genes of these enzymes, thereby producing the desired cellulases in the plants themselves. Extraction and recovery of active proteins or release of active cellulase from the plants during bioconversion could have a significant positive impact on overall lignocellulose conversion economics. The effects of ammonia fiber explosion (AFEX) pretreatment variables (treatment temperature, moisture content, and ammonia loading) on the activity of plant-produced heterologous cellulase enzyme were individually investigated via heat treatmett or ammonia treatment. Finally, we studied the effects of all these variables in concert through the AFEX process. The plant materials included transgenic tobacco plants expressing E1 (endoglucanase from Acidothermus cellulolyticus). The E1 activity was measured in untreated and AFEX-treated tobacco leaves to investigate the effects of the treatment on the activity of this enzyme. The maximum observed activity retention in AFEX-treated transgenic tobacco samples compared with untreated samples was approx 35% (at 60°C, 0.5∶1 ammonia loading, and 40% moisture). Based on these findings, it is our opinion that AFEX pretreatment is not a suitable option for releasing cellulase enzyme from transgenic plants.     

Cellulase recovery via membrane filtration

A combined sedimentation and membrane filtration process was investigated for recycling cellulase enzymes in the biomass-to-ethanol process. In the first stage, lignocellulose particles longer than approx 50 μm were removed by means of sedimentation in an inclined settler. Microfiltration was then utilized to remove the remaining suspended solids. Finally, the soluble cellulase enzymes were recovered by ultrafiltration. The perm eate fluxes obtained in microfiltration and ultrafiltration were approx 400 and 80 L/(m²·h), respectively. A preliminary economic analysis shows that the cost benefit of enzyme recycling may be as much as 18 cents/gal of ethanol produced, provided that 75% of the enzyme is recycled in active form.

     

Ammonia fiber explosion treatment of corn stover

Optimizing process conditions and parameters such as ammonia loading, moisture content of biomass, temperature, and residence time is necessary for maximum effectiveness of the ammonia fiber explosion process. Approximate optimal pretreatment conditions for corn stover were found to be temperature of 90°C, ammonia: dry corn stover mass ratio of 1∶1, moisture content of corn stover of 60% (dry weight basis), and residence time (holding at target temperature), of 5 min. Approximately 98% of the theoretical glucose yield was obtained during enzymatic hydrolysis of the optimal treated corn stover using 60 filter paper units (FPU) of cellulase enzyme/g of glucan (equal to 22 FPU/g of dry corn stover). The ethanol yield from this sample was increased up to 2.2 times over that of untreated sample. Lowering enzyme loading to 15 and 7.5 FPU/g of glucan did not significantly affect the glucose yield compared with 60 FPU, and any differences between effects at different enzyme levels decreased as the treatment temperature increased.     

Simultaneous saccharification and cofermentation of dilute-acid pretreated yellow poplar hardwood to ethanol using xylose-fermenting Zymomonas mobilis

Simultaneous saccharification and cofermentation (SSCF) was carried out at approximately 15% total solids using conditioned dilute-acid pretreated yellow poplar feedstock, an adapted variant of National Renewable Energy Laboratory (NREL) xylose-fermenting Zymomonas mobilis and either commercial or NREL-produced cellulase enzyme preparations. In 7 d, at a cellulase loading of 12 filter paper units pergram cellulose (FPU/g), the integrated system produced more than 3% w/v ethanol and achieved 54% conversion of all potentially available biomass sugars (total sugars) entering SSCF. A control SSCF employing Sigmacell cellulose and a commercial cellulase at an enzyme loading of 14 FPU/gachieved 65% conversion of total sugars to ethanol.     

Possibilities for recycling cellulases after use in cotton processing: part I: Effects of end-product inhibition,thermal and mechanical deactivation,and cellulase depletion by adsorption

Preliminary recycling experiments with cellulase enzymes after cotton treatments at 50 degrees C showed that activity remaining in the treatment liquors was reduced by about 80% after five recycling steps. The potential problems of end-product inhibition, thermal and mechanical deactivation, and the loss of some components of the cellulase complex by preferential and or irreversible adsorption to cotton substrates were studied. End-product inhibition studies showed that the build-up of cellobiose and glucose would be expected to cause no more than 40% activity loss after five textile treatment cycles. Thermal and mechanical treatments of cellulases suggested that the enzymes start to be deactivated at 60 degrees C and agitation levels similar to those used in textile processing did not cause significant enzyme deactivation. Analysis of cellulase solutions, by fast protein liquid chromatography, before and after adsorption on cotton fabrics, suggested that the cellobiohydrolase II (Cel6A) content of the cellulase complex was reduced, relative to the other components, by preferential adsorption. This would lead to a marked reduction in activity after several treatment cycles and top-up with pure cellobiohydrolase II would be necessary unless this component is easily recoverable from the treated fabric.     

Flow-Microcalorimetric Determination of Cellobiase Activity and Its Inhibition by Glucose

Abstract

A method for measuring cellobiase activity of the Trichoderma reesei CCF 1853 cellulase complex using a Thermal Activity Monitor and a flow - mix mode is described. The kinetic constant K_M and the linear dependence of dQ_max/dt (the maximum heat flow at the total saturation of enzyme with substrate) on the enzyme concentration were determined. The process of the end product inhibition of cellobiase activity by glucose has been observed too. The obtained results allow to determine the mechanism of the inhibition and an inhibition constant for glucose.

The procedure is completely general in nature and is applicable to other enzymatic systems.     

A Novel Stepwise Recovery Strategy of Cellulase Adsorbed to the Residual Substrate after Hydrolysis of Steam Exploded Wheat Straw

Cellulase distribution between residual substrate and supernatant in the process of enzymatic hydrolysis of steam-exploded wheat straw was investigated. Subsequently, a novel stepwise recovery strategy with three successive steps was adopted to recover cellulase adsorbed to the residual substrate. The results showed that cellulase protein in the supernatant increased as the hydrolysis time increased. When hydrolysis ended, the cellulase remaining on the residual substrate accounted for 33–42% of the original added cellulase according to the different cellulase loading. To obtain the maximum cellulase recovery rate, the residual substrate was dealt with in three successive steps: washed with sodium acetate buffer (step 1), shaken with sodium acetate buffer (step 2), and then treated with 0.0015 mol/L, pH 10 Ca(OH)₂ (step 3). The total cellulase protein recovered by the three steps reached 96.70–98.14%. The enzyme activity of cellulase recovered by the first two steps was kept well. The ratios of the specific activity between the recovered cellulase and the original were 89–96%, which was by far higher than that using step 3 (the value was 48% ∼ 56%).     

Characterization and performance of immobilized amylase and cellulase

The performance of cellulase and amylase immobilized on siliceous supports was investigated. Enzyme uptake onto the support depended on the enzyme source and immobilization conditions. For amylase, the uptake ranged between 20 and 60%, and for cellulase, 7–10%. Immobilized amylase performance was assessed by batch kinetics in 100–300 g/L of corn flour at 65°C. Depending on the substrate and enzyme loading, between 40 and 60% starch conversion was obtained. Immobilized amylase was more stable than soluble amylase. Enzyme samples were preincubated in a water bath at various temperatures, then tested for activity. At 105°C, soluble amylase lost ∼55% of its activity, compared with ∼30% loss for immobilized amylase. The performance of immobilized cellulase was evaluated from batch kinetics in 10 g/L of substrate (shredded wastepaper) at 55°C. Significant hydrolysis of the wastepaper was also observed, indicating that immobilization does not preclude access to and hydrolysis of insoluble cellulose.     

Ammonia fiber expansion pretreatment and enzymatic hydrolysis on two different growth stages of reed canarygrass

Plant materials from the vegetative growth stage of reed canarygrass and the seed stage of reed canarygrass are pretreated by ammonia fiber expansion (AFEX) and enzymatically hydrolyzed using 15 filter paper units (FPU) cellulase/g glucan to evaluate glucose and xylose yields. Percent conversions of glucose and xylose, effects of temperature and ammonia loading, and hydrolysis profiles are analyzed to determine the most effective AFEX treatment condition for each of the selected materials. The controls used in this study were untreated samples of each biomass material. All pretreatment conditions tested enhanced enzyme digestibility and improved sugar conversions for reed canarygrass compared with their untreated counterparts. Based on 168 h hydrolysis results using 15 FPU Spezyme CP cellulase/g glucan the most effective AFEX treatment conditions were determined as: vegetative growth stage of reed canarygrass--100 degrees C, 60% moisture content, 1.2:1 kg ammonia/kg of dry matter (86% glucose and 78% xylose) and seed stage of reed canarygrass--100 degrees C, 60% moisture content, 0.8:1 kg ammonia/kg of dry matter (89% glucose and 81% xylose). Supplementation by commercial Multifect 720 xylanase along with cellulase further increased both glucose and xylose yields by 10-12% at the most effective AFEX conditions.     

Lactic acid production from cellulosic material by synergetic hydrolysis and fermentation

The hydrolysis process on corncob residue was catalyzed synergetically by the cellulase from Trichoderma reesei and the immobilized cellobiase. The feedback inhibition to cellulase reaction caused by the accumulation of cellobiose was eliminated efficiently. The hydrolysis yield of corncob residue was 82.5%, and the percentage of glucose in the reducing sugar reached 88.2%. The glucose in the cellulosic hydrolysate could be converted into lactic acid effectively by the immobilized cells of Lactobacillus delbrueckii. When the enzymatic hydrolysis of cellulose and the fermentation of lactic acid were coupled together, no glucose was accumulated in the reaction system, and the feedback inhibition caused by glucose was also eliminated. Under the batch process of synergetic hydrolysis and lactic acid fermentation with 100 g/L of cellulosic substrate, the conversion efficiency of lactic acid from cellulose and the productivity of lactic acid reached 92.4% and 0.938 g/(L·h), respectively. By using a fed-batch technique, the total concentration of cellulosic substrate and lactic acid in the synergetic process increased to 200 and 107.5 g/L, respectively, whereas the dosage of cellulase reduced from 20 to 15 IU/g of substrate in the batch process. The results of the bioconversion of renewable cellulosic resources were significant.     

Improved Cellulase Production by <Emphasis Type="Italic">Trichoderma reesei </Emphasis>RUT C30 under SSF Through Process Optimization

The 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 includes 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). The current study investigated the production of cellulase by Trichoderma reesei RUT C30 on wheat bran under SSF. Process parameters important in cellulase production were identified by a Plackett and Burman design and the parameters with significant effects on enzyme production were optimized for maximal yield using a central composite rotary design (CCD). Higher initial moisture content of the medium had a negative effect on production whereas incubation temperature influenced cellulase production positively in the tested range. Optimization of the levels of incubation temperature and initial moisture content of the medium resulted in a 6.2 fold increase in production from 0.605 to 3.8 U/gds of cellulase. The optimal combination of moisture and temperature was found to be 37.56% and 30 °C, respectively, for maximal cellulase production by the fungus on wheat bran.     

Endocellulase and exocellulase activities of two Streptomyces strains isolated from a forest soil

Two Streptomyces strains, M7a and M23, from a Brazilian forest soil were evaluated for the cellulase production of their superna tants after growth in a microcrystalline cellulose medium, using carboxy methylcellulose and filter paper as substrates at different temperatures and pH values. Endoglucanase and exoglucanase activities were compared to a commercial Trichoderma reesei cellulase using fluorogenic conjugated substrates Similar specific activities were observed for the enzyme preparations of strain M23 and T. reesei. For M7a the activities were about seven times higher than those obtained for T. reesei. Extracellular or cell-associated cellobiase activities were not detected in both strains.

     

Effect of Bovine Serum Albumin (BSA) on Enzymatic Cellulose Hydrolysis

Bovine serum albumin (BSA) was added to filter paper during the hydrolysis of cellulase. Adding BSA before the addition of the cellulase enhances enzyme activity in the solution, thereby increasing the conversion rate of cellulose. After 48 h of BSA treatment, the BSA adsorption quantities are 3.3, 4.6, 7.8, 17.2, and 28.3 mg/g substrate, each with different initial BSA concentration treatments at 50 °C; in addition, more cellulase was adsorbed onto the filter paper at 50 °C compared with 35 °C. After 48 h of hydrolysis, the free-enzyme activity could not be measured without the BSA treatment, whereas the remaining activity of the filter paper activity was approximately 41 % when treated with 1.0 mg/mL BSA. Even after 96 h of hydrolysis, 25 % still remained. Meanwhile, after 48 h of incubation without substrate, the remaining enzyme activities were increased 20.7 % (from 43.7 to 52.7 %) and 94.8 % (from 23.3 to 45.5 %) at 35 and 50 °C, respectively. Moreover, the effect of the BSA was more obvious at 35 °C compared with 50 °C. When using 15 filter paper cellulase units per gram substrate cellulase loading at 50 °C, the cellulose conversion was increased from 75 % (without BSA treatment) to ≥90 % when using BSA dosages between 0.1 and 1.5 mg/mL. Overall, these results suggest that there are promising strategies for BSA treatment in the reduction of enzyme requirements during the hydrolysis of cellulose.