High-solids Enzymatic Hydrolysis Research Articles

The Effect of Xylan Removal on the High-Solid Enzymatic Hydrolysis of Sugarcane Bagasse

The Effect of Xylan Removal on the High-Solid Enzymatic Hydrolysis of Sugarcane Bagasse

Optimization of high-solid enzymatic hydrolysis of two-step alkaline and dilute acid-pretreated sugarcane bagasse at low enzyme loadings by response surface methodology

Optimization of high-solid enzymatic hydrolysis of two-step alkaline and dilute acid-pretreated sugarcane bagasse at low enzyme loadings by response surface methodology

High-solids enzymatic hydrolysis of biomass: Hydrodynamics and reaction kinetics integration via numerical modeling

This study presents a novel computational fluid dynamics (CFD) model to investigate important aspects of the complex high-solids enzymatic hydrolysis (HSEH) process. The uniqueness of this CFD model lies in integrating the biochemical reaction taking place in the secondary phase and the corresponding mass transfer of the products from the secondary phase to the non-Newtonian primary phase, while dual axial impellers blend the multiphase system. The distribution of the reactants and products in the non-Newtonian primary phase affects the overall conversion of glucan to glucose, which, in turn, affects the commercial deployment of these systems for the production of renewable sugars. We investigated the effect of slurry viscosity on insoluble and soluble solids distribution, the impact of initial insoluble solids loading on total solids distribution, and varying the initial chemical composition of the insoluble solids on the total solids distribution. The comprehensive CFD model results show that variations in the chemical composition of the insoluble solids and the solids loading can each have a pronounced effect on the distribution of solids. This behavior would then affect the rate and extent of conversion of insoluble solids to soluble solids. Thus, the comprehensive CFD model can account for the interactions between independent variables, facilitating the design of small and large-scale reactors, while improving the conversion of insoluble solids to soluble solids. This novel CFD model thus represents the combined effects of key factors that influence HSEH in a realistic process environment.

Efficient high-solids enzymatic hydrolysis of corncobs by an acidic pretreatment and a fed-batch feeding mode

Corncob is an abundant and renewable resource that could be enzymatically hydrolyzed to fermentable sugar. A major impediment in corncob utilization is the low hydrolysis efficiency at high-solids content. This study attempted different pretreatment methods and fed-batch modes to achieve a 25% solids content hydrolysis with high yields. Natural corncobs were compared with acid-treated and acid-alkali-treated corncobs in terms of kinetics parameters, conversion rate and glucose titer. By feeding in batches, a “low amount and high frequency” mode (10%-3%-3%-3%-3%-3%, every 5 h) was confirmed to be optimal for a 25% high-solids hydrolysis system with a cellulase loading of 12 mg/g (7.3 FPU/g), resulted with an 84.4% glucose yield at 96 h. Our results demonstrated that combination of both optimized pretreatment method and fed-batch mode were a favored process model for high-solids hydrolysis of lignocellulose, boosting cellulose hydrolysis efficiency and sugar yields on an industrial scale.

Effect of periodic high-frequency vibration with rigid spheres added on high solids enzymatic hydrolysis of steam-exploded corn straw

The periodic low-frequency force can improve mixing efficiency and reduce water constraint, thus intensifying the high solids enzymatic hydrolysis. However, the availability of intensification methods for the periodic force with different frequencies needs to be studied. The periodic high-frequency vibration with rigid spheres added as another intensification method was proposed to shorten the time of water constraint and improve efficiency. Within this study, glucan and xylan conversions in periodic high-frequency vibration with rigid spheres added enzymatic hydrolysis (PVEH) increased by 8.3%-35% and 5.0%-33.5%, respectively, with solid loading increasing from 15% to 25%, compared with those in static state enzymatic hydrolysis (SEH). The physical properties variations of substrate indirectly proved the higher efficiency of PVEH. Based on the change regularity of constrained water release, PVEH was divided into three stages. The effects of micromixing and macromixing in three stages were analyzed. Additionally, the efficiency of high solids enzymatic hydrolysis can be enhanced by using proper intensification methods in different stages.

High-solids enzymatic hydrolysis of ball-milled corn stover with reduced slurry viscosity and improved sugar yields

BackgroundHigh-solids enzymatic hydrolysis has attracted increasing attentions for the production of bioethanol from lignocellulosic biomass with its advantages of high product concentration, water saving, and low energy and capital costs. However, the increase of solids content would worsen the rheological properties, resulting in heat/mass transfer limitation and higher mixing energy. To address these issues, ball milling was applied to corn stover prior to enzymatic hydrolysis, and the rheological behaviors and digestibility of ball-milled corn stover under high-solids loading were investigated.ResultsBall milling significantly modified the physicochemical properties of corn stover. The apparent viscosity of slurries at 30% solid loading decreased by a factor of 500 after milling for 60 min, and the yield stress was less than 10 Pa. The dramatic decrease of viscosity and yield stress enabled the hydrolysis process to be conducted in shake flask, and remained good mixing. Meanwhile, the estimated energy consumption for mixing during saccharification decreased by 400-fold compared to the untreated one. The resultant hydrolysate using 10 FPU g−1 solids was determined to contain 130.5 g L−1 fermentable sugar, and no fermentation inhibitors were detected.ConclusionsThe proposed ball milling pretreatment improved rheological behavior and sugar yield of high-solids corn stover slurry. Ball milling enables high-solids slurry to maintain low viscosity and yield stress while obtaining a non-toxic high-concentration fermentable syrup, which is undoubtedly of great significance for inter-unit processing, mixing and downstream process. In addition, the energy input for ball milling could be balanced by the reduced mixing energy. Our study indicates ball milling a promising pretreatment process for industrial bioethanol production.

Fed-batch enzymatic hydrolysis of plantain pseudostem to fermentable sugars production and the impact of particle size at high solids loadings

In this work, the performance of a fed-batch high solids enzymatic hydrolysis of plantain pseudostem was studied at different particle sizes. The plantain pseudostem was pretreated with an alkaline hydrogen peroxide medium. Two set of experiments were carried out in a stirred tank bioreactor at substrate concentrations of 10% and 15% w/v, combined each one with particle sizes #20–40, #40–60, and # > 100, in 1 L of reaction volume. Particle sizes of #20–40 and #40–60 allowed around 99% of holocellulose conversion with a substrate concentration of 10% w/v, and 91% with 15% w/v; meanwhile enzymatic hydrolysis at particle size of mesh # > 100 yielded 70% and 73%, with a substrate concentration of 10% and 15% w/v, respectively. The conditions recommended for enzymatic hydrolysis at high solids of 15% w/v through fed-batch operation are particle size #20–40 and 6 h of reaction, achieving 91% of holocellulose conversion and 82 g/L of reducing sugars.

Improved co-production of ethanol and xylitol from low-temperature aqueous ammonia pretreated sugarcane bagasse using two-stage high solids enzymatic hydrolysis and Candida tropicalis

Process economics of cellulosic ethanol production can be improved by co-production of high value products. Xylitol is a high value nutraceutical and attracted attention as a co-product in cellulosic ethanol process. Here, the production of ethanol and xylitol from sugarcane bagasse pretreated by low-temperature aqueous ammonia soaking was improved by two-stage high solids enzymatic hydrolysis and separate fermentation of glucose and xylose using Candida tropicalis. First-stage high solids fed-batch enzymatic hydrolysis of pretreated bagasse in 3 L bioreactor resulted in 42.6 g/l xylose. The residual solids rich in cellulose were efficiently hydrolyzed by cellulase in the second-stage to glucose. The second stage hydrolysis at 20% solids loading in bioreactor showed 81% efficiency with a glucose concentration of 115.8 g/l. The separate fermentation of the xylose with C. tropicalis in two-stage aeration resulted in 34.5 g/l of xylitol. Fermentation of the glucose by C. tropicalis produced 55.64 g/l of ethanol. Simultaneous saccharification and fermentation of cellulose rich solids from first stage hydrolysis produced 57.2 g/l of ethanol. These results showed that two-stage enzymatic hydrolysis of low-temperature aqueous ammonia pretreated biomass facilitated higher yields and efficiency of production of ethanol and xylitol.

Optimization of an artificial cellulase cocktail for high-solids enzymatic hydrolysis of cellulosic materials with different pretreatment methods

Optimization of the composition of cellulase mixtures is an effective strategy to improve their hydrolytic efficiency and reduce protein demand during enzymatic degradation of lignocelluloses. In this study, the mixture design method was used to optimize the ratios of endoglucanase II (EG II), cellobiohydrolase I (CBH I) and β-glucosidase I (BG I) from Penicillium oxalicum in an artificial cellulase mixture for the hydrolysis of six different cellulosic materials. The optimal composition of enzyme mixture was distinctly different among not only cellulosic materials with different pretreatment methods but hydrolyses at different solids concentrations. CBH I was most critical for the hydrolysis of two acid-pretreated materials, probably due to its strong adsorption on lignin. A higher proportion of EG II was needed for the hydrolysis of ammonium sulfite pretreated wheat straw. The requirements of specific cellulase components were more pronounced at high solids concentrations, highlighting the importance of considering solids loading when optimizing cellulase cocktails.

Enhancement of high-solids enzymatic hydrolysis efficiency of alkali pretreated sugarcane bagasse at low cellulase dosage by fed-batch strategy based on optimized accessory enzymes and additives

Obtaining higher amount of final sugars with low cellulase dosage has great economic benefits for the industrial biorefinery of lignocellulosic biomass. The optimization of accessory enzymes and additives were performed using single factor and orthogonal experiment firstly, after that, fed-batch strategy was applied to enhance the high-solids enzymatic hydrolysis efficiency of alkali pretreated sugarcane bagasse (SCB). A novel enzymatic hydrolysis procedure with 22% (w/v) substrate content and cellulase dosage of only 4 FPU/g dry biomass (DM) was developed, after digested for 48 h, the achieved glucose titer, yield and productivity were 122 g/L, 80% and 2.54 g L−1 h−1, respectively. Results obtained in this study indicated a potential finding for the industrial application of lignocellulosic biomass.

Cellulase recycling in high-solids enzymatic hydrolysis of pretreated empty fruit bunches

BackgroundThe lignocellulosic biomass feedstocks such as empty fruit bunches (EFBs) prove to be potential renewable resources owing to their abundance, low prices, and high carbohydrate contents. Generally, the conversion of lignocellulosic biomass into chemicals, fuels, and materials mainly includes pretreatment, enzymatic hydrolysis, fermentation, and recovery of final products. To increase the economic viability of such processes, the cost of cellulase production and enzymatic hydrolysis should be reduced. For this, recycling cellulase can be considered for reducing the saccharification cost of lignocellulose. In this study, cellulase recycling for high-solids enzymatic hydrolysis (i.e., 20%) was evaluated in saccharification of hydrothermally-pretreated EFBs.ResultsHigh-solids (20%) enzymatic hydrolysis of hydrothermally-pretreated empty fruit bunches with 40 FPU of Cellic CTec3/g glucan was carried out for cellulase recycling. In the second round of hydrolysis using a recycled enzyme, only 19.3% of glucose yield was obtained. The most important limiting factors for cellulase recycling of this study were identified as the enzyme inhibition by glucose, the loss of enzyme activities, and the non-productive binding of enzymes to insoluble biomass solids. To overcome these limitations, PEG was added prior to the first-round hydrolysis to reduce non-productive enzyme binding, glucose was removed from the enzyme fraction to be reused in the second-round hydrolysis, and EFB solids from the first-round hydrolysis were used in the second-round hydrolysis. These three additional measures with cellulase recycling resulted in a 3.5 times higher glucose yield (i.e., 68.0%) at the second round than that of the control, the second-round hydrolysis with cellulase recycling but without these measures.ConclusionsBecause of the high obstacles found in this study in achieving high saccharification yields in the high-solids saccharification of high-lignin lignocellulose with cellulase recycling, effective measures for improving enzymatic saccharification yields need to be accompanied with cellulase recycling.

Enhanced High-Solids Fed-Batch Enzymatic Hydrolysis of Sugar Cane Bagasse with Accessory Enzymes and Additives at Low Cellulase Loading

High cellulase loading is still a major impediment in the production of fermentative sugars from high-solids enzymatic hydrolysis of lignocellulosic substrates in the enzyme-based “biorefinery” industry. This study attempted a high-solids (20%) enzymatic hydrolysis of lignocellulosic substrate at a very low cellulase loading with mixed use of additives and accessory enzymes by fed-batch mode. To avoid the high initial biomass viscosity, the high-solids enzymatic hydrolysis of lignocellulosic substrates was initiated with a solids content of 8%. Thereafter, 4% of the additional substrates were consecutively fed into the hydrolysis system after 6, 12, and 18 h to reach a final solids content of 20%. Some additive mixtures (40 mg/g substrateTween 80 + 10 mg/g substrate tea saponin +20 mg/g substrate BSA) were observed to enable this fed-batch hydrolysis to increase 30% of the glucose yield after the 48 h. The combination of these additives and accessory enzymes (2.4 mg/g substrate xylanase and 1 mg/g substra...

Pre-senescence Harvest of Switchgrass Inhibits Xylose Utilization by Engineered Yeast

Proper timing of switchgrass harvest for bioenergy is important to maximize yield and optimize end use conversion. Proposed windows range from peak biomass to the following spring after overwintering in the field. There are various pros and cons associated with harvest timing: earlier harvests maximize yield but can remove nutrients from the field that may require replacement, while later harvests have reduced biomass yields due to weathering but maximize nutrient resorption in belowground tissues. Switchgrass composition changes during the harvest period, with losses of potential fermentation nutrients (amino acids and minerals), and sources of pretreatment-derived inhibitors (soluble sugars), which could affect downstream conversion by microorganisms. For this work we investigated whether switchgrass harvest could be timed to maximize beneficial impacts on fermentation. Switchgrass samples were harvested from five replicate field plots in Wisconsin, roughly every 2-3 weeks from peak biomass (Aug. 20) until after the killing frost (Nov. 7). Cell wall composition showed little consistent variation with harvest date while bulk biomass analysis showed a relative increase in cell wall content (lignin and structural sugars) and loss of extractives (minerals, protein, soluble sugars, and others). Following high or low severity AFEX pretreatment and high solids enzymatic hydrolysis (6% glucan loading), two field replicates were fermented using Saccharomyces cerevisiae 424A, a strain engineered to utilize xylose in addition to glucose. For both pretreatment severities, S. cerevisiae 424A grown in hydrolysates from the three earlier harvests utilized only a small fraction of available xylose, while almost complete utilization occurred within 96 hr for the last three harvest dates. Detailed analysis of the hydrolysate low molecular weight aromatics did not indicate any compounds potentially responsible for the inhibition, with most of the observed variation in their concentration due to pretreatment severity. Amino acid composition also did not appear to be limiting. Current indications point to a plant-generated compound that degrades during senescence, which future work will attempt to identify. Ultimately this work demonstrates that, although an attractive option to maximize yield, harvesting switchgrass before it begins senescing could have a negative effect on downstream conversion processes.

Fed-batch strategies for saccharification of pilot-scale mild-acid and alkali pretreated sugarcane bagasse: Effects of solid loading and surfactant addition

High-solid enzymatic hydrolysis of lignocellulosic biomass is a key approach for high titer and reduced downstream costs in second generation ethanol production. Combining a pilot-scale (80 L reactor) mild acid pretreatment and an alkali pretreatment of sugarcane bagasse (SCB) with fed-batch strategies for high-solid enzymatic hydrolysis, more than 150 g/L of glucose was found in the hydrolysate. The best result of the fed-batch study (2.03 g/L/h of glucose productivity) was obtained with a total solids content of 27%w/w (being 20%w/w of solids added at 0 h), and the addition of the total amount of the non-ionic surfactant PEG 4000 and the enzymes solely at the beginning of the reaction. A semi-simultaneous saccharification and fermentation strategy of the pretreated SCB (with the yeast inoculum occurring after 12 h of a single hydrolysis stage at most proper conditions for the cellulases) resulted in 62 g/L of ethanol after 48 h, and a productivity of up to 6.6 g/L/h. The strategy here reported indicates that it is possible to achieve ethanol titers suitable for industrial distilleries avoiding mass transfer limitations that incur in operational problems during high-solid enzymatic hydrolysis.

Comparison of One-Stage Batch and Fed-Batch Enzymatic Hydrolysis of Pretreated Hardwood for the Production of Biosugar.

Fed-batch method has shown a great promise in debottlenecking the high-solid enzymatic hydrolysis for the commercialization of cellulosic biosugar conversion for biofuel/biochemical production. To further improve enzymatic hydrolysis efficiency at high solid loading, fed-batch methods of green liquor-pretreated hardwood were performed to evaluate their effects on sugar recovery by comparing with one-stage batch method in this study. Among all the explored conditions, the fed-batch at 15% consistency gave higher sugar recovery on green liquor-pretreated hardwood compared to that of one-stage batch. By using general linear model analysis, the percentage of enzymatic sugar recovery in fed-batch consistency method (increasing consistency from the initial 10.7 to 15% at intervals of 24 and 48h) was higher than that of batch hydrolysis at higher density of 15% consistency. Under that best fed-batch condition, the total sugar recovery of pretreated hardwood in enzymatic hydrolysate reached approximately 48.41% at Cellic® enzyme loading of 5filter-paper unit (FPU)/g and 58.83% at Cellic® enzyme loading of 10FPU/g with a hydrolysis time of 96h.

Investigation of different pretreatment methods of Mediterranean-type ecosystem agricultural residues: characterisation of pretreatment products, high-solids enzymatic hydrolysis and bioethanol production

ABSTRACTAgricultural and agro-industrial lignocellulosic residues represent an important renewable resource for the production of fuels and chemicals towards a bio-based economy. Olive pruning, vineyard pruning and almond shells are important residues from agricultural activities in Mediterranean-type ecosystems. In the current work, bioethanol production from the above three types of agro-residues was studied, focusing on the effect of different pretreatment methods on enzymatic saccharrification efficiency of cellulose and production of second-generation bioethanol. Dilute acid, hydrothermal and steam explosion pretreatments were compared in order to remove hemicellulose and facilitate the subsequent enzymatic hydrolysis of the hemicellulose-deficient biomass to glucose. Enzymatic hydrolysis was performed in a free-fall mixing reactor enabling high solids loading of 23% w/w. This allowed hydrolysis of up to 67% of available cellulose in almond shells and close to 50% in olive pruning samples, and facilitated high ethanol production in the subsequent fermentation step; the highest ethanol concentrations achieved were 47.8 g/L for almond shells after steam explosion and 42 g/L for hydrothermally pretreated olive pruning residue.

High gravity enzymatic hydrolysis of hydrothermal and ultrasonic pretreated big bluestem with recycling prehydrolysate water

Enhancing sugar concentration and minimizing water consumption are key objectives for future cellulosic biofuel economics. To achieve those objectives, high-solids loading pretreatment and enzymatic hydrolysis (up to 20%, w/v) were studied. Big bluestem was selected and combined hydrothermal and ultrasonic treatment without chemicals addition was carried out in this study. Optimal high-solids loading pretreatment (16%, w/v) was identified in the ultrasonic reactor at 200 °C for 30 min. High-solids enzymatic hydrolysis (12%, w/v) was inefficient in the laboratory rotary shaker. However, using a horizontal reactor with good mixing is effective for high solids loading (20%, w/v), yielding 75 g/L of glucose. Minimum water to detoxify pretreated biomass while maintaining high sugar yields was 10 mL/g, which reduced by 50% as compared to the conventional washing process for steam-treated wheat straw. Recycling pretreatment liquor to treat the next batch of biomass was proved to be feasible without affecting the sugar yields.

Evaluation of the solvent water effect on high solids saccharification of alkali-pretreated sugarcane bagasse

Solvent water is an essential factor for high solids enzymatic hydrolysis. To investigate its effect on substrate conversion efficiency in high solids hydrolysis of sugarcane bagasse (SCB), oleyl alcohol was used to partially substitute the solvent water. The results in batch hydrolysis tests in which diverse ratio of solvent water was replaced found that the majority of the substrate was insoluble. Then high solids fed-batch hydrolysis with the reaction solution mixed with solvent water and oleyl alcohol in the ratio of 3:1 (solids concentration correspond to 24% (w/v)) was carried out at the final real solids loading of 18% (w/v). The produced sugars were found to be less than pure water system, which indicated that water played a significant role in high solids hydrolysis process, and solids effect was related to the solvent water content.

Toward Biochemical Conversion of Lignocellulose On-Farm: Pretreatment and Hydrolysis of Corn Stover in Situ

Abstract. High-solids lignocellulosic pretreatment using NaOH followed by high-solids enzymatic hydrolysis was evaluated for an on-farm biochemical conversion process. Increasing the solids loadings for these processes has the potential for increasing glucose concentrations and downstream ethanol production; however, sequential processing at high-solids loading similar to an on-farm cellulose conversion system has not been studied. This research quantified the effects of high-solids pretreatment with NaOH and subsequent high-solids enzymatic hydrolysis on cellulose conversion. As expected, conversion efficiency was reduced; however, the highest glucose concentration (40.2 g L-1), and therefore the highest potential ethanol concentration, resulted from the high-solids combined pretreatment and hydrolysis. Increasing the enzyme dosage improved cellulose conversion from 9.6% to 36.8% when high-solids loadings were used in both unit operations; however, increasing NaOH loading and pretreatment time did not increase the conversion efficiency. The enzyme-to-substrate ratio had a larger impact on cellulose conversion than the NaOH pretreatment conditions studied, resulting in recommendations for an on-farm bioconversion system. Keywords: Corn stover, Enzymatic hydrolysis, Enzyme loading, High solids, Low solids, Sodium hydroxide.

Response Surface Optimization of Enzymatic Hydrolysis of Sugar Beet Leaves into Fermentable Sugars for Bioethanol Production

Sugar beet leaves are the major crop waste from sugar beet production, while the unused leaves contain a high number of sugars and polysaccharides. The effects of different enzyme products (cellulase, Cellic CTec2; xylanase, Cellic HTec2; and pectinase, Pectinex Ultra SPL) were determined during high-solids enzymatic hydrolysis of sugar beet leaves at 10% total solids (TS) content. Response surface methodology was used to study the effects of enzyme loadings during the hydrolysis of sugar beet leaves for producing fermentable sugars. It was found that both cellulases and pectinases are important enzymes for the hydrolysis of sugar beet leaves. Enzyme loading and reaction time were important factors. Based on the amount of sugars released, a maximum sugar conversion of 82% was achieved after 72 h of hydrolysis using 30 filter paper unit (FPU) g-1 glucan for cellulase and 150 polygalacturonase unit (PGU) g-1 polygalacturonic acid for pectinase, or 37 FPU g-1 glucan for cellulase and 100 PGU g-1 polygalacturonic acid for pectinase. The corresponding sugar yield and sugar concentration were 0.35 g·g-1 TS, and 35 g·l-1, respectively. Sugar conversion ranged from 59% - 70%, 68% - 80%, and 74% - 82% after 24 h, 48 h, and 72 h of hydrolysis depending on the design conditions.