Fed-batch Enzymatic Hydrolysis Research Articles

High-solids enzymatic saccharification of starch-rich raw herbal biomass residues for producing high titers of glucose.

The bioresource utilization of herbal biomass residues (HBRs) has been receiving more attention. Herein, three different HBRs from Isatidis Radix (IR) and Sophorae Flavescentis Radix (SFR) and Ginseng Radix (GR) were subjected to batch and fed-batch enzymatic hydrolysis to produce high-concentration glucose. Compositional analysis showed the three HBRs had substantial starch content (26.36-63.29%) and relatively low cellulose contents (7.85-21.02%). Due to their high starch content, the combined action of cellulolytic and amylolytic enzymes resulted in greater release of glucose from the raw HBRs compared to using the individual enzyme alone. Batch enzymatic hydrolysis of 10% (w/v) raw HBRs with low loadings of cellulase (≤ 10 FPU/g substrate) and amylolytic enzymes (≤ 5.0mg/g substrate) led to a high glucan conversion of ≥ 70%. The addition of PEG 6000 and Tween 20 did not contribute to glucose production. Furthermore, to achieve higher glucose concentrations, fed-batch enzymatic hydrolysis was conducted using a total solid loading of 30% (w/v). After 48-h of hydrolysis, glucose concentrations of 125g/L and 92g/L were obtained for IR and SFR residues, respectively. GR residue yielded an 83g/L glucose concentration after 96h of digestion. The high glucose concentrations produced from these raw HBRs indicate their potential as ideal substrate for a profitable biorefinery. Notably, the obvious advantage of using these HBRs is the elimination of the pretreatment step, which is typically required for agricultural and woody biomass in similar studies.

Fed-batch enzymatic hydrolysis of steam-exploded sugarcane bagasse

Statistical design and mathematical modeling were used to investigate the fed-batch enzymatic hydrolysis of steam-exploded sugarcane bagasse (195 °C, 7.5 min). First, a Box-Behnken experimental design was used to evaluate the effect of enzyme loading (8, 24, and 40 FPU g-1 glucans of Cellic CTec3®), stirring speed (100, 150, and 200 rpm), and substrate total solids (5, 12.5, and 20 wt%) on the release of glucose equivalents (GlcEq, mostly glucose) after hydrolysis for 48 h in batch mode. A simplified kinetic model was used to fit the experimental data, in which specific activities in Cellic CTec3 were not differentiated, enzyme adsorption was ignored, and end-product inhibition was only attributed to glucose accumulation. The adjusted kinetic model was used to predict the effects of substrate and enzyme intermittent feedings in fed-batch hydrolysis experiments. Compared with batch experiments at 20 wt%, the proposed fed-batch procedure was able to increase GlcEq productivity by nearly 68% using the same enzyme loading, producing substrate hydrolysates containing 91.8 g L-1 GlcEq.

Effects of combined enzymatic hydrolysis and fed-batch operation on efficient improvement of ferulic acid and p-coumaric acid production from pretreated corn straws

In the present work, the effects of combined enzymatic hydrolysis by cellulase and xylanase (CXEH), fed-batch enzymatic hydrolysis (FBEH) operation and kinetics on production of ferulic acid (FA) and p-coumaric acid (pCA) from pretreated corn straws were investigated. The results showed that CXEH could efficiently increase production of FA and pCA. When performed the FBEH operation by feeding 150 mL enzymatic hydrolysis solution (1.5 % enzyme concentration, 5:4 (v/v) ratio of cellulase to xylanase and 2.0 % substrate loading) to 250 mL batch enzymatic hydrolysis solution at 36 h, the maximum production (2178.58 and 2710.17 mg/L) and production rate (590.95 and 727.89 mg/L.h) of FA and pCA were respectively obtained. Moreover, the disruption of fiber tissues, enhancement of crystallinity and accelerated degradation of hemicelluloses and lignocelluloses caused by CXEH contributed to effectively improving production of FA and pCA in corn straws.

High Concentration of Fermentable Sugars Prepared from Steam Exploded Lignocellulose in Periodic Peristalsis Integrated Fed-Batch Enzymatic Hydrolysis.

High concentrations of fermentable sugars are a demand for economical bioethanol production. A single process strategy cannot comprehensively solve the limiting factors in high-solid enzymatic hydrolysis. The multiple intensification strategies in this study achieved the goal of preparing high-concentration fermentable sugars of corn stalk with high solid loading and low enzyme loading. First, steam explosion pretreatment enhanced the hydrophilicity of substrates and enzymatic accessibility. Second, periodic peristalsis was used to improve the mass transfer efficiency and short the liquefaction time. Additionally, fed-batch feeding and enzyme reduced the enzyme loading. Ultimately, the intensification strategies above showed that the highest fermentable sugar content was 313.8g/L with a solids loading as much as 50% (w/w) and enzyme loading as low as 12.5 FPU/g DM. Thus, these multiple intensification strategies were promising in the high-solid enzymatic hydrolysis of steam-exploded lignocellulose.

Revalorization of sunflower stalk pith as feedstock for the coproduction of pectin and glucose using a two-step dilute acid pretreatment process

BackgroundSunflower stalk pith, residue from the processing of sunflower, is rich in pectin and cellulose, thereby acting as an economic raw material for the acquisition of these compounds. In order to increase the commercial value of sunflower processing industry, a two-step dilute sulfuric acid treatment process was conducted on spent sunflower stalk pith to obtain the value-added products, pectin and glucose.ResultsIn this study, pectin was firstly extracted under mild acid condition to avoid pectin degradation, which was conducted at 90 °C with a pH of 2.0 for 2 h, and ~0.14 g/g of pectin could be recovered. Then the remaining solids after pectin extraction were subjected to the reinforced treatment process with 0.75% H2SO4 at 150 °C for 30 min to further improve enzymatic hydrolysis efficiency. Moreover, by combining a fed-batch enzymatic hydrolysis strategy, a solid loading content of 16% was successfully achieved and the glucose titer reached 103.1 g/L with a yield of 83.6%.ConclusionFinally, ~140 g pectin and 260 g glucose were produced from 1 kg of raw sunflower stalk pith using the integrated biorefinery process. This work puts forward a two-step dilute acid pretreatment combined with enzymatic hydrolysis method to produce pectin and glucose from sunflower spent waste.

Production of high-concentration fermentable sugars from lignocellulosic biomass by using high solids fed-batch enzymatic hydrolysis

The synergy of multiple lignocellulolytic enzymes for lignocellulosic biomass saccharification at high substrate loading to obtain high-concentration fermentable sugar is of great economic interest for industrial biorefineries. In this study, the mixture of cellulase, β-glucosidase, hemicellulose, and pectinase was optimized for hydrolysis of different cellulosic feedstocks at different substrate concentrations. It was found that the enzyme combination of 43.8% cellulase, 41.8% hemicellulase, 14.4% pectinase was more suitable for high-concentration alkaline hydrogen peroxide pretreated corncob hydrolysis. The optimized enzyme mixture was then applied to fed-batch enzymatic hydrolysis. The cumulative solid loading was 40% (w / w). After 48 h of hydrolysis, the glucose and xylose concentration reached 110.47 g/L and 61.88 g/L, respectively. The fermentable sugar productivity was up to 3.6 g/L/h. In addition, the enzyme components for high-concentration substrate hydrolysis were optimized. The substrate concentration was increased by fed-batch hydrolysis, and the high-concentration fermentable sugar was obtained in a shorter time.

Conversion of High-Solids Hydrothermally Pretreated Bioenergy Sorghum to Lipids and Ethanol Using Yeast Cultures

Glucose and xylose are the major sugars present in cellulosic hydrolysates. The cellulosic sugars can be used for the production of platform chemicals. In this study, productions of lipid and ethanol by yeasts were compared for concentrated bioenergy sorghum syrup. Bioenergy sorghum was hydrothermally pretreated at 50% w/w solids in a continuous industrial reactor and sequentially mechanically refined using a burr mill to improve biomass accessibility for hydrolysis. Fed-batch enzymatic hydrolysis was conducted with 50% w/v solids loading and cellulase cocktail (50 FPU/g biomass) to achieve 230 g/L sugar concentration. Various strains of Rhodosporidium toruloides were evaluated for converting sugars into lipids, and strain Y-6987 had the highest lipid titer (9.2 g/L). The lipid titer was improved to 19.0 g/L by implementing a two-stage culture scheme, where the first stage was optimized for yeast growth and the second for lipid production. For ethanol production, the engineered Saccharomyces cerevisiae SR8ΔADH6 was utilized to coferment glucose and xylose. Ethanol fermentation was optimized for media nutrients (YP, YNB/urea, and urea), cellulosic sugar concentration, and sulfite conditioning to maximize the ethanol concentration from sorghum syrups. Fermentation of 70% v/v concentrated hydrolysate conditioned with sulfite produces 50.1 g/L ethanol from 141 g/L of sugars.

Cellulosic Bioethanol from Industrial Eucalyptus globulus Bark Residues Using Kraft Pulping as a Pretreatment

The pulp and paper industry faces an emerging challenge for valorising wastes and side-streams generated according to the biorefinery concept. Eucalyptus globulus bark, an abundant industrial residue in the Portuguese pulp and paper sector, has a high potential to be converted into biobased products instead of being burned. This work aimed to evaluate the ethanol production from E. globulus bark previously submitted to kraft pulping through separate hydrolysis and fermentation (SHF) configuration. Fed-batch enzymatic hydrolysis provided a concentrated hydrolysate with 161.6 g·L−1 of cellulosic sugars. S. cerevisiae and Ethanol Red® strains demonstrated a very good fermentation performance, despite a negligible xylose consumption. S. passalidarum, a yeast known for its capability to consume pentoses, was studied in a simultaneous co-culture with Ethanol Red®. However, bioethanol production was not improved. The best fermentation performance was achieved by Ethanol Red®, which provided a maximum ethanol concentration near 50 g·L−1 and fermentation efficiency of 80%. Concluding, kraft pulp from E. globulus bark showed a high potential to be converted into cellulosic bioethanol, being susceptible to implementing an integrated biorefinery on the pulp and paper industrial plants.

Co-production of polysaccharides, ginsenosides and succinic acid from Panax ginseng residue: A typical industrial herbal waste

Co-production of polysaccharides, ginsenosides and succinic acid was achieved from Panax ginseng residue (PGR) in this study. Physico-chemical separation was first applied to recover the released polysaccharides and ginsenoside. Enzymatic hydrolysis was then conducted to covert the left PGR into mono-sugars which was following transformed into succinic acid by constructing a succinic acid-producing strain of Escherichia coli—ZW333. Results indicated that the yields of polysaccharides and ginsenosides increased according to the increase of deconstruction content of PGR. A total sugar yield reached 52 g/L at 10% PGR loading and increased to 94.33 g/L following fed-batch enzymatic hydrolysis. Finally, 56.28 g/L succinic acid was produced. In total, 18 g ginseng polysaccharides, 230 mg ginsenosides and 39 g succinic acid were produced from 100 g PGR. Accordingly, the total economic output could reach RMB 80,149 from 1 t PGR, illustrating the great value increasement of PGR by this industrially possible process.

Recycling of Black Liquor for Treating Sugarcane Bagasse at Low Temperature to Attain High Ethanol Production without Washing Step

Alkaline pretreatment has been commonly used for bioconversion of lignocellulose into fuel ethanol due to its gentle condition. However, the large quantity of water consumption and wastewater generation from the pretreatment and washing steps have been two bottleneck problems for the practical application of alkaline pretreatment. In order to save water consumption and reduce wastewater generation, black liquor (BL) generated in the sodium hydroxide (NaOH) pretreatment process was recycled for pretreating sugarcane bagasse (SCB), and the BL-NaOH-treated SCB without being washed was directly enzymatically hydrolyzed and fermented. After 120-h fed-batch enzymatic hydrolysis with the addition of 20 FPU cellulase/g cellulose and 2.51 μL Tween80/mL hydrolytic slurry, the maximum glucose concentration and enzymatic hydrolysis efficiency achieved 99.95 g/L and 71.02%, respectively. After 36-h fermentation with the domesticated Saccharomyces cerevisiae, the ethanol production reached 44.53 g/L, which amounted to 87.35% of theoretical ethanol yield. Compared with other water-saving and wastewater-reducing alkaline pretreatments, this technology saved more than 20% water consumption and reduced over 20% wastewater discharge. It provides a promising applied technology for cellulosic ethanol production.

Conversion of Exhausted Sugar Beet Pulp into Fermentable Sugars from a Biorefinery Approach

In this study, the production of a hydrolysate rich in fermentable sugars, which could be used as a generic microbial culture medium, was carried out by using exhausted sugar beet pulp pellets (ESBPPs) as raw material. For this purpose, the hydrolysis was performed through the direct addition of the fermented ESBPPs obtained by fungal solid-state fermentation (SSF) as an enzyme source. By directly using this fermented solid, the stages for enzyme extraction and purification were avoided. The effects of temperature, fermented to fresh solid ratio, supplementation of fermented ESBPP with commercial cellulase, and the use of high-solid fed-batch enzymatic hydrolysis were studied to obtain the maximum reducing sugar (RS) concentration and productivity. The highest RS concentration and productivity, 127.3 g·L−1 and 24.3 g·L−1·h−1 respectively, were obtained at 50 °C and with an initial supplementation of 2.17 U of Celluclast® per gram of dried solid in fed-batch mode. This process was carried out with a liquid to solid ratio of 4.3 mL·g−1 solid, by adding 15 g of fermented solid and 13.75 g of fresh solid at the beginning of the hydrolysis, and then the same amount of fresh solid 3 times every 2.5 h. By this procedure, ESBPP can be used to produce a generic microbial feedstock, which contains a high concentration of monosaccharides.

Evaluation of an efficient fed-batch enzymatic hydrolysis strategy to improve production of functional xylooligosaccharides from maize straws

The present study investigated the production of xylooligosaccharides (XOS) from the xylans extracted from maize straws (XMSs) using the fed-batch enzymatic hydrolysis strategy (FBEH). When fed 300 mL of the fresh XMSs with 2 % of the substrate loading and 12 U/g of the enzyme activity at the enzymatic hydrolysis time of 7 h, the yield of XOS was obtained to be 0.67 g/g of XMSs. Characterization analysis indicated that XOS contained typical molecular groups and side chains of oligosaccharides and was mainly composed of xylobiose, xylotriose and xylose. Furthermore, XOS exhibited strong antioxidant capacity in vitro and had a significant inhibition effect on HepG2 cells, indicating that XOS might be potentially used as natural antioxidants and anticancer additives in food or pharmaceutical industry.

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.

Fed-batch enzymatic hydrolysis of alkaline organosolv-pretreated corn stover facilitating high concentrations and yields of fermentable sugars for microbial lipid production

BackgroundLignocellulosic biomass has been commonly regarded as a potential feedstock for the production of biofuels and biochemicals. High sugar yields and the complete bioconversion of all the lignocellulosic sugars into valuable products are attractive for the utilization of lignocelluloses. It is essential to pretreat and hydrolyze lignocelluloses at high solids loadings during industrial processes, which is more economical and environmentally friendly as capital cost, energy consumption, and water usage can be reduced. However, oligosaccharides are inevitably released during the high solids loading enzymatic hydrolysis and they are more recalcitrant than monosaccharides for microorganisms.ResultsA fed-batch enzymatic hydrolysis of corn stover pretreated by the sodium hydroxide–methanol solution (SMs) at high solids loading was demonstrated to reach the high concentrations and yields of fermentable sugars. Glucose, xylose, cello-oligosaccharides, and xylo-oligosaccharides achieved 146.7 g/L, 58.7 g/L, 15.6 g/L, and 24.7 g/L, respectively, when the fed-batch hydrolysis was started at 12% (w/v) solids loading, and 7% fresh substrate and a standardized blend of cellulase, β-glucosidase, and hemicellulase were fed consecutively at 3, 6, 24, and 48 h to achieve a final solids loading of 40% (w/v). The total conversion of glucan and xylan reached 89.5% and 88.5%, respectively, when the oligosaccharides were taken into account. Then, a fed-batch culture on the hydrolysates was investigated for lipid production by Cutaneotrichosporon oleaginosum. Biomass, lipid content, and lipid yield were 50.7 g/L, 61.7%, and 0.18 g/g, respectively. The overall consumptions of cello-oligosaccharides and xylo-oligosaccharides reached 74.1% and 68.2%, respectively.ConclusionsHigh sugars concentrations and yields were achieved when the enzyme blend was supplemented simultaneously with the substrate at each time point of feeding during the fed-batch enzymatic hydrolysis. Oligosaccharides were co-utilized with monosaccharides during the fed-batch culture of C. oleaginosum. These results provide a promising strategy to hydrolyze alkaline organosolv-pretreated corn stover into fermentable sugars with high concentrations and yields for microbial lipid production.

Techno-Economic Implications of Fed-Batch Enzymatic Hydrolysis

Fed-batch enzymatic hydrolysis has the potential to improve the overall process of converting cellulosic biomass into ethanol. This paper utilizes a process simulation approach to identify and quantify techno-economic differences between batch and fed-batch enzymatic hydrolysis in cellulosic ethanol production. The entire process of converting corn stover into ethanol was simulated using SuperPro Designer simulation software. The analysis was conducted for a plant capacity of 2000 metric tons of dry biomass per day. A literature review was used to identify baseline parameters for the process. The sensitivity of the ethanol production cost to changes in sugar conversion efficiency, plant capacity, biomass cost, power cost, labor cost, and enzyme cost was evaluated using the process simulation. For the base scenario, the ethanol unit production cost was approximately $0.10/gallon lower for fed-batch hydrolysis. The greatest differences were seen in facilities costs, labor costs, and capital costs. Using a fed-batch operation decreased facilities costs by 41%, labor costs by 21%, and capital costs by 15%. The sensitivity analysis found that cost of biomass had the greatest effect on ethanol production cost, and in general, the results support the proposition that fed-batch enzymatic hydrolysis does improve the techno-economics of cellulosic ethanol production.

Process optimization for the production of high-concentration ethanol with Scenedesmus raciborskii biomass

Scenedesmus raciborskii WZKMT was subjected to fed-batch enzymatic hydrolysis and fermentation to facilitate the saccharification of high-solid-loading substrate for high-concentration ethanol. In this work, process factors affecting enzymatic hydrolysis, including enzyme loading, temperature, pH, and solid loading, were optimized. Results showed that 58.03 g L−1 glucose, 12.57 g L−1 xylose, and 1.45 g L−1 cellobiose were obtained after the enzymatic hydrolysis of 330 g L−1 substrates under the optimal conditions of 30 FPU g−1 enzyme loading, 50 °C, and pH 5.5. Meanwhile, 89.60% yield and 30.43 g L−1 content of ethanol were obtained after the fermentation of 330 g L−1 hydrolysate. The maximum ethanol concentration of 79.38 g L−1 could be achieved through repeated fed-batch process, indicating that S. raciborskii WZKMT is a promising feedstock for ethanol production.

An eco-friendly biorefinery strategy for xylooligosaccharides production from sugarcane bagasse using cellulosic derived gluconic acid as efficient catalyst

A novel approach was proposed for the production of xylooligosaccharides by direct pre-hydrolysis using gluconic acid as catalyst. Maximum xylooligosaccharides (degree of polymerization 2–6) yield of 53.2% could be obtained in 60 min through 5% gluconic acid hydrolysis of sugarcane bagasse at 150 °C. Furthermore, the yield of glucose from solids following gluconic acid hydrolysis treatment was 86.2% after fed-batch enzymatic hydrolysis with 10% solids loading. Results indicated that gluconic acid pretreatment combined with enzymatic hydrolysis could be successfully applied to sugarcane bagasse substrate. Subsequently, glucose could be efficiently bio-oxidized to gluconic acid by Gluconobacter oxydans ATCC 621H with 93.1% yield, and sugarcane bagasse derived gluconic acid has been proved to be an effective catalyst for xylooligosaccharides production. In this study, xylooligosaccharides production from sugarcane bagasse by gluconic acid hydrolysis demonstrated a great potential with respect to the production of these probiotics around the world.

Batch and fed-batch enzymatic hydrolysis of pretreated sugarcane bagasse – Assays and modeling

Choosing the right pretreatment for a specific biomass in order to improve sugar recovery is a key step towards the commercial feasibility of cellulosic ethanol. This article deals with the enzymatic hydrolysis of sugarcane bagasse subjected to either delignification or hydrothermal pretreatment. Two operational strategies were studied: batch experiments were carried out with different solids loads (5–20% w/v) and fed-batch tests reached cumulative solids content of 24% (w/v). The highest glucose concentration (127 g/L) was achieved when working with the delignified material in fed-batch systems. However, the reactions using delignified bagasse presented sharp decrease in conversion when the solids loads exceeded 15% (w/v). During the modeling phase, the hydrolysis behavior was described according to a semi-mechanistic approach. The goodness of fit evaluation showed that the models performed well when compared to the collected data. Finally, fed-batch simulations suggested new feeding polices to increase the final glucose content.

Alkaline-assisted Microwave Pretreatment of Tetraselmis suecica Biomass for Fed-batch Enzymatic Hydrolysis

A two-part study on pretreatment and fed-batch enzymatic hydrolysis of pretreated Tetraselmis suecica using a high initial biomass concentration was conducted. First, the effect of different pretreatment processes, i.e. microwave (MC), dilute alkaline (AK), and microwave-alkaline assisted (MAK) pretreatment, on enzymatic hydrolysis of T. suecica biomass was evaluated. Furthermore, high initial biomass concentration enzymatic hydrolysis improvement via a fed-batch strategy was performed. Among the pretreatments tested, the MAK pretreatment produced the highest sugar concentration at 9.83 ± 0.24 mg/mL, corresponding to a conversion yield of up to 85.58% of carbohydrate content available in the pretreated biomass. The solid fraction generated after pretreatment was characterized using Fourier transform infrared (FTIR) spectroscopy. The FTIR analysis revealed a significant change in the functional hydroxyl and acetyl groups of the biomass, which is favorable for enzymatic hydrolysis. Introducing an initial microalgal biomass concentration beyond 15% (w/v) exhibited a low enzymatic hydrolysis yield. The fed-batch enzymatic hydrolysis strategy of the MAK pretreated T. suecica was further investigated by adding the substrate at different time intervals. The findings indicate that the fed-batch operation system could enhance sugar production and enzymatic hydrolysis yield one-fold.