The impact of cell wall acetylation on corn stover hydrolysis by cellulolytic and xylanolytic enzymes

Analysis of variously pretreated corn stover samples showed neutral to mildly acidic pretreatments were more effective at removing xylan from corn stover and more likely to maintain the acetyl to xylopyranosyl ratios present in untreated material than were alkaline treatments. Retention of acetyl groups in the residual solids resulted in greater resistance to hydrolysis by endoxylanase alone, although the synergistic combination of endoxylanase and acetyl xylan esterase enzymes permitted higher xylan conversions to be observed. Acetyl xylan esterase alone did little to improve hydrolysis by cellulolytic enzymes, although a direct relationship was observed between the enzymatic removal of acetyl groups and improvements in the enzymatic conversion of xylan present in substrates. In all cases, effective xylan conversions were found to significantly improve glucan conversions achievable by cellulolytic enzymes. Additionally, acetyl and xylan removal not only enhanced the respective initial rates of xylan and glucan conversion, but also the overall extents of conversion. This work emphasizes the necessity for xylanolytic enzymes during saccharification processes and specifically for the optimization of acetyl esterase and xylanase synergies when biomass processes include milder pretreatments, such as hot water or sulfite steam explosion.     

Simultaneous saccharification and fermentation of pretreated hardwoods

A series of correlations was made between the performance of 15 wood species in simultaneous saccharification and fermentation (SSF) and their respective chemical compositions. A compelling inverse trend (p < 0.001) was demonstrated between the percent conversion of glucan to ethanol during SSF and the Klason lignin content of the wood samples before dilute acid pretreatment. No significant relationships were found between the glucan, xylan, and ash compositions of the native wood samples and ethanol yield. This observation is unique and provides a convenient predictor of biomass conversion efficiency.     

Can delignification decrease cellulose digestibility in acid pretreated corn stover?

It has previously been shown that the improved digestibility of dilute acid pretreated corn stover is at least partially due to the removal of xylan and the consequent increase in accessibility of the cellulose to cellobiohydrolase enzymes. We now report on the impact that lignin removal has on the accessibility and digestibility of dilute acid pretreated corn stover. Samples of corn stover were subjected to dilute sulfuric acid pretreatment with and without simultaneous (partial) lignin removal. In addition, some samples were completely delignified after the pretreatment step using acidified sodium chlorite. The accessibility and digestibility of the samples were tested using a fluorescence-labeled cellobiohydrolase (Trichoderma reesei Cel7A) purified from a commercial cellulase preparation. Partial delignification of corn stover during dilute acid pretreatment was shown to improve cellulose digestibility by T. reesei Cel7A; however, decreasing the lignin content below 5% (g g⁻¹) by treatment with acidified sodium chlorite resulted in a dramatic reduction in cellulose digestibility. Importantly, this effect was found to be enhanced in samples with lower xylan contents suggesting that the near complete removal of xylan and lignin may cause aggregation of the cellulose microfibrils resulting in decreased cellulase accessibility.     

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.     

Pretreatment of corn stover by soaking in aqueous ammonia

Soaking in aqueous ammonia (SAA) was investigated as a pretreatment method for corn stover. In this method, the feedstock was soaked in aqueous ammonia over an extended period (10–60 d) at room temperature. It was done without agitation at atmospheric pressure. SAA treatment removed 55–74% of the lignin, but retained nearly 100% of the glucan and 85% of the xylan. The xylan remaining in the corn stover after SAA treatment was hydrolyzed along with the glucan by xylanase present in the Spezyme CP enzyme. In the simultaneous saccharification and fermentation (SSF) test of SAA-treated corn stover, using S. cerevisiae (D₅A), an ethanol yield of 73% of theoretical maximum was obtained on the basis of the glucan content in the treated corn stover. The accumulation of xylose in the SSF appears to inhibit the cellulase activity on glucan hydrolysis, which limits the yield of ethanol. In the simultaneous saccharification and co-fermentation (SSCF) test, using recombinant E. coli (KO11), both the glucan and xylose were effectively utilized, resulting in on overall ethanol yield of 77% based on the glucan and xylan content of the substrate. When the SSCF process is used, the fact that the xylan fraction is retained during pretreatment is a desirable feature since the overall bioconversion can be carried out in a single step without separate recovery of xylose from the pretreatment liquid.     

Two-Stage Fractionation of Corn Stover Using Aqueous Ammonia and Hot Water

Hot water and aqueous ammonia fractionation of corn stover were used to separate hemicellulose and lignin and improve enzymatic digestibility of cellulose. A two-stage approach was used: The first stage was designed to recover soluble lignin using aqueous ammonia at low temperature, while the second stage was designed to recover xylan using hot water at high temperature. Specifically, the first stage employed a batch reaction using 15 wt.% ammonia at 60 °C, in a 1:10 solid:liquid ratio for 8 h, while the second stage employed a percolation reaction using hot water, 190–210 °C, at a 20 ml/min flow rate for 10 min. After fractionation, the remaining solids were nearly pure cellulose. The two-stage fractionation process achieved 68% lignin purity with 47% lignin recovery in the first stage, and 78% xylan purity, with 65% xylan recovery in the second stage. Two-stage treatment enhanced the enzymatic hydrolysis of remaining cellulose to 96% with 15 FPU/g of glucan using commercial cellulase enzymes. Enzyme hydrolyses were nearly completed within 12–24 h with the remaining solids fraction.     

Impact of Pretreatment with Dilute Sulfuric Acid Under Moderate Temperature on Hydrolysis of Corn Stover with Two Enzyme Systems

Pretreatment of corn stover with dilute sulfuric acid at moderate temperature was investigated, and glucan digestibility by Cellic CTec2 and Celluclast on the pretreated biomass was compared. Pretreatments were carried out from 60 to 180 min at the temperature from 105 to 135 °C, with acid concentrations ranging from 0.5 to 2 % (w/v). Significant portion of xylan was removed during pretreatment, and the glucan digestibility by CTec2 was significantly better than that by Celluclast in all cases. Analysis showed that glucan digestibility by both two enzymes correlated directly with the extent of xylan removal in pretreatment. Confidence interval was built to give a more precise range of glucan conversion and to test the significant difference among pretreatment conditions. Response surface model was built to obtain the optimal pretreatment condition to achieve high glucan conversion after enzymatic hydrolysis. Considering the cost and energy savings, the optimal pretreatment condition of 1.75 % acid for 160 min at 135 °C was determined, and glucan conversion can achieve the range from 72.86 to 76.69 % at 95 % confidence level after enzymatic hydrolysis, making total glucan recovery up to the range from 89.42 to 93.25 %.     

Potential of Agricultural Residues and Hay for Bioethanol Production

Production of bioethanol from agricultural residues and hays (wheat, barley, and triticale straws, and barley, triticale, pearl millet, and sweet sorghum hays) through a series of chemical pretreatment, enzymatic hydrolysis, and fermentation processes was investigated in this study. Composition analysis suggested that the agricultural straws and hays studied contained approximately 28.62-38.58% glucan, 11.19-20.78% xylan, and 22.01-27.57% lignin, making them good candidates for bioethanol production. Chemical pretreatment with sulfuric acid or sodium hydroxide at concentrations of 0.5, 1.0, and 2.0% indicated that concentration and treatment agent play a significant role during pretreatment. After 2.0% sulfuric acid pretreatment at 121 degrees C/15 psi for 60 min, 78.10-81.27% of the xylan in untreated feedstocks was solubilized, while 75.09-84.52% of the lignin was reduced after 2.0% sodium hydroxide pretreatment under similar conditions. Enzymatic hydrolysis of chemically pretreated (2.0% NaOH or H2SO4) solids with Celluclast 1.5 L-Novozym 188 (cellobiase) enzyme combination resulted in equal or higher glucan and xylan conversion than with Spezyme(R) CP- xylanase combination. The glucan and xylan conversions during hydrolysis with Celluclast 1.5 L-cellobiase at 40 FPU/g glucan were 78.09 to 100.36% and 74.03 to 84.89%, respectively. Increasing the enzyme loading from 40 to 60 FPU/g glucan did not significantly increase sugar yield. The ethanol yield after fermentation of the hydrolyzate from different feedstocks with Saccharomyces cerevisiae ranged from 0.27 to 0.34 g/g glucose or 52.00-65.82% of the theoretical maximum ethanol yield.     

Mechanical deconstruction of lignocellulose cell walls and their enzymatic saccharification

Laboratory mechanical softwood pulps (MSP) and commercial bleached softwood kraft pulps (BSKP) were mechanically fibrillated by stone grinding with a SuperMassColloider^®. The extent of fibrillation was evaluated by SEM imaging, water retention value (WRV) and cellulase adsorption. Both lignin content and mechanical treatment significantly affected deconstruction and enzymatic saccharification of fibrillated MSP and BSKP. Fibrillation of MSP and BSKP cell walls occurs rapidly and then levels off; further fibrillation has only limited effect on cell wall breakdown as measured by water retention value and cellulase adsorption. Complete (100 %) saccharification can be achieved at cellulase loading of 5 FPU/g glucan for BSKP after only 15 min fibrillation with energy input of 0.69 MJ/kg. However, the presence of lignin in MSP affects the extent of fibrillation producing fibrils mainly above 1 μm. Lignin binds nonproductively to cellulases and blocks cellulose thereby reducing its accessibility. As a result, the cellulose saccharification efficiency of MSP fibrils (6 h of fibrillation, energy input of 13.33 MJ/kg) was only 55 % at same cellulase loading of 5 FPU/g glucan.     

Enhancement of Enzymatic Hydrolysis and Klason Lignin Removal of Corn Stover Using Photocatalyst-Assisted Ammonia Pretreatment

Photocatalyst-assisted ammonia pretreatment was explored to improve lignin removal of the lignocellulosic biomass for effective sugar conversion. Corn stover was treated with 5.0–12.5 wt.% ammonium hydroxide, two different photocatalysts (TiO₂ and ZnO) in the presence of molecular oxygen in a batch reactor at 60 °C. Various solid-to-liquid ratios (1:20–1:50) were also tested. Ammonia pretreatment assisted by TiO₂-catalyzed photo-degradation removed 70 % of Klason lignin under the optimum condition (12.5 % ammonium hydroxide, 60 °C, 24 h, solid/liquid?=?1:20, photocatalyst/biomass?=?1:10 with oxygen atmosphere). The enzymatic digestibilities of pretreated corn stover were 85 % for glucan and 75 % for xylan with NH₃-TiO₂-treated solid and 82 % for glucan and 77 % for xylan with NH₃-ZnO-treated solid with 15 filter paper units/g-glucan of cellulase and 30 cellobiase units/g-glucan of β-glucosidase, a 2–13 % improvement over ammonia pretreatment alone.     

Inhibition Effects of Dilute-Acid Prehydrolysate of Corn Stover on Enzymatic Hydrolysis of Solka Floc

Dilute-acid pretreatment liquor (PL) produced at NREL through a continuous screw-driven reactor was analyzed for sugars and other potential inhibitory components. Their inhibitory effects on enzymatic hydrolysis of Solka Floc were investigated. When the PL was mixed into the enzymatic hydrolysis reactor at 1:1 volume ratio, the glucan and xylan digestibility decreased by 63% and 90%, respectively. The tolerance level of the enzyme for each inhibitor was determined. Of the identified degradation components, acetic acid was found to be the strongest inhibitor for cellulase activity, as it decreased the glucan yield by 10% at 1 g/L. Among the sugars, cellobiose and glucose were found to be strong inhibitors to glucan hydrolysis, whereas xylose is a strong inhibitor to xylan hydrolysis. Xylo-oligomers inhibit xylan digestibility more strongly than the glucan digestibility. Inhibition by the PL was higher than that of the simulated mixture of the identifiable components. This indicates that some of the unidentified degradation components, originated mostly from lignin, are potent inhibitors to the cellulase enzyme. When the PL was added to a simultaneous saccharification and co-fermentation using Escherichia coli KO11, the bioprocess was severely inhibited showing no ethanol formation or cell growth.     

Oxidative Lime Pretreatment of Dacotah Switchgrass

Oxidative lime pretreatment increases the enzymatic digestibility of lignocellulosic biomass primarily by removing lignin. In this study, recommended pretreatment conditions (reaction temperature, oxygen pressure, lime loading, and time) were determined for Dacotah switchgrass. Glucan and xylan overall hydrolysis yields (72 h, 15 FPU/g raw glucan) were measured for 105 different reaction conditions involving three different reactor configurations (very short term, short term, and long term). The short-term reactor was the most productive. At the recommended pretreatment condition (120 °C, 6.89 bar O₂, 240 min), it achieved an overall glucan hydrolysis yield of 85.2 g glucan hydrolyzed/100 g raw glucan and an overall xylan yield of 50.1 g xylan hydrolyzed/100 g raw xylan. At this condition, glucan oligomers (1.80 g glucan recovered/100 g glucan in raw biomass) and xylan oligomers (25.20 g xylan recovered/100 g xylan in raw biomass) were recovered from the pretreatment liquor, which compensate for low pretreatment yields.     

Pretreatment of corn stover by low-liquid ammonia recycle percolation process

A pretreatment method using aqueous ammonia was investigated with the intent of minimizing the liquid throughput. This process uses a flow-through packed column reactor (or percolation reactor). In comparison to the ammonia recycle percolation (ARP) process developed previously in our laboratory, this process significantly reduces the liquid throughput to one reactor void volume in packed bed (2.0–4.7 mL of liquid/g of corn stover) and, thus, is termed low-liquid ARP (LLARP). In addition to attaining short residence time and reduced energy input, this process achieves 59–70% of lignin removal and 48–57% of xylan retention. With optimum operation of the LLARP to corn stover, enzymatic digestibilities of 95, 90 and 86% were achieved with 60, 15, and 7.5 filter paper units/g of glucan, respectively. In the simultaneous saccharification and fermentation test of the LLARP samples using Saccharomyces cerevisiae (NREL-D₅A), an ethanol yield of 84% of the theoretical maximum was achieved with 6% (w/v) glucan loading. In the simultaneous saccharification and cofermentation (SSCF) test using recombinant Escherichia coli (KO11), both the glucan and xylan in the solid were effectively utilized, giving an overall ethanol yield of 109% of the theoretical maximum based on glucan, a clear indication that the xylan content was converted into ethanol. The xylooligomers existing in the LLARP effluent were not effectively hydrolyzed by cellulase enzyme, achieving only 60% of digestibility. SSCF of the treated corn stover was severely hampered when the substrate was supplemented with the LLARP effluent, giving only 56% the overall yield of ethanol. The effluent appears to significantly inhibit cellulase and microbial activities.     

Pretreatment of corn stover by soaking in aqueous ammonia at moderate temperatures

Soaking in aqueous ammonia at moderate temperatures was investigated as a method of pretreatment for enzymatic hydrolysis as well as simultaneous saccharification and cofermentation (SSCF) of corn stover. The method involves batch treatment of the feedstock with aqueous ammonia (15-30 wt%) at 40-90 degrees C for 6-24 h. The optimum treatment conditions were found to be 15 wt% of NH(3), 60 degrees C, 1:6 of solid-to-liquid ratio, and 12 h of treatment time. The treated corn stover retained 100% glucan and 85% of xylan, but removed 62% of lignin. The enzymatic digestibility of the glucan content increased from 17 to 85% with 15 FPU/g-glucan enzyme loading, whereas the digestibility of the xylan content increased to 78%. The treated corn stover was also subjected to SSCF test using Spezyme-CP and recombinant Escherichia coli (KO11). The SSCF of the soaking in aqueous ammonia treated corn stover resulted in an ethanol concentration of 19.2 g/L from 3% (w/v) glucan loading, which corresponds to 77% of the maximum theoretical yield based on glucan and xylan.     

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.     

Pretreatment of sugarcane bagasse by acidified aqueous polyol solutions

Pretreatments of sugarcane bagasse by three high boiling-point polyol solutions were compared in acid-catalysed processes. Pretreatments by ethylene glycol (EG) and propylene glycol solutions containing 1.2 % H₂SO₄ and 10 % water at 130 °C for 30 min removed 89 % lignin from bagasse resulting in a glucan digestibility of 95 % with a cellulase loading of ~20 FPU/g glucan. Pretreatment by glycerol solution under the same conditions removed 57 % lignin with a glucan digestibility of 77 %. Further investigations with EG solutions showed that increases in acid content, pretreatment temperature and time, and decrease in water content improved pretreatment effectiveness. A good linear correlation of glucan digestibility with delignification was observed with R² = 0.984. Bagasse samples pretreated with EG solutions were characterised by scanning electron microscopy, Fourier transform infrared spectroscopy and X-ray diffraction, which confirmed that improved glucan enzymatic digestibility is mainly due to delignification and defibrillation of bagasse. Pretreatment by acidified EG solutions likely led to the formation of EG-glycosides. Up to 36 % of the total lignin was recovered from pretreatment hydrolysate, which may improve the pretreatment efficiency of recycled EG solution.     

Cellulase production of Trichoderma reesei rut C 30 using steam-pretreated spruce

Various techniques are available for the conversion of lignocellulosics to fuel ethanol. During the last decade processes based on enzymatic hydrolysis of cellulose have been investigated more extensively, showing good yield on both hardwood and softwood. The cellulase production of a filamentous fungi, Trichoderma reesei Rut C30, was examined on carbon sources obtained after steam pretreatment of spruce. These materials were washed fibrous steam-pretreated spruce (SPS), and hem icellulose hydrolysate. The hemicellulose hydrolysate contained, besides water-soluble carbohydrates, lignin and sugar degradation products, which were formed during the pretreatment and proved to be inhibitory to microorganisms. Experiments were performed in a 4-L laboratory fermentor. The hydrolytic capacity of the produced enzyme solutions was compared with two commercially available enzyme preparations, Celluclast and logen Cellulase, on SPS, washed SPS, and Solka Floc cellulose powder. There was no significant difference among the different enzymes produced by T. reesei Rut C30. However, the conversion of cellulose using these enzymes was higher than that obtained with logen or Celluclast cellulases using steam-pretreated spruce as substrate.     

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.     

Production of Cellulase from Kraft Paper Mill Sludge by Trichoderma Reesei Rut C-30

Paper mill sludge is a solid waste material generated from pulping and papermaking operations. Because of high glucan content and its well-dispersed structure, paper mill sludges are well suited for bioconversion into value-added products. It also has high ash content originated from inorganic additives used in papermaking, which causes hindrance to bioconversion. In this study, paper mill sludges from Kraft process were de-ashed by a centrifugal cleaner and successive treatment by sulfuric acid and sodium hydroxide, and used as a substrate for cellulase production. The treated sludge was the only carbon source for cellulase production, and predominantly inorganic nutrients were used as the nitrogen source for this bioprocess. The cellulase enzyme produced from the de-ashed sludge exhibited cellulase activity of 8 filter paper unit (FPU)/mL, close to that obtainable from pure cellulosic substrates. The yield of cellulase enzyme was 307 FPU/g glucan of de-ashed sludge. Specific activity was 8.0 FPU/mg protein. In activity tests conducted against the corn stover and α-cellulose, the xylanse activity was found to be higher than that of a commercial cellulase. Relatively high xylan content in the sludge appears to have induced high xylanase production. Simultaneous saccharification and fermentation (SSF) was performed using partially de-ashed sludge as the feedstock for ethanol production using Sacharomyces cerevisiae and the cellulase produced in-house from the sludge. With 6% (w/v) glucan feed, ethanol yield of 72% of theoretical maximum and 24.4 g/L ethanol concentration were achieved. These results were identical to those of the SSF using commercial cellulases.     

Xylanase supplementation on enzymatic saccharification of dilute acid pretreated poplars at different severities

Three pairs of solid substrates from dilute acid pretreatment of two poplar wood samples were enzymatically hydrolyzed by cellulase preparations supplemented with xylanase. Supplementation of xylanase improved cellulose saccharification perhaps due to improved cellulose accessibility by xylan hydrolysis. Total xylan removal directly affected enzymatic cellulose saccharification. Furthermore, xylan removal by pretreatment and xylanase are indifferent to enzymatic cellulose saccharification. However, more enzymatic xylose and glucose yields were obtained for a substrate with lower xylan content after a severer pretreatment at the same xylanase dosage. The effectiveness of xylanase at increased dosages depended on the substrates structure or accessibility. High xylanase dosages were more effective on well pretreated substrates than on under-pretreated substrates with high xylan content. The application sequence of xylanase and cellulase affected cellulose saccharification. This effect varied with substrate accessibility, perhaps due to competition between xylanase and cellulase binding to the substrate.