Column cellulose hydrolysis reactor: the effect of retention time,temperature, cellulase concentration and exogenously added cellobiase on the overall process

Summary A novel column cellulose hydrolysis reactor with constant enzyme recycling was operated under various conditions to determine the effects of retention time, temperature, cellulase concentration and exogenously added cellobiase on the concentration of the product stream and the productivities of the reactor. Short term (7 days) hydrolysis was best at 42°C while longer term (14 days) hydrolysis was better at 37°C. A retention time of 11 h and reactor cellulase concentration of 30 filter paper units per gram of cellulose gave the best compromise for efficient operation by minimizing product inhibition, maximizing product concentration and minimizing enzyme consumption. The addition of cellobiase to the reactor increased cellulose hydrolysis and raised the proportion of monomeric sugars in the hydrolysate. Continuous cellulose hydrolyses were maintained for 7 and 14 days at 42°C and 37°C, respectively, resulting in volumetric productivities of 6.82 and 4.84 g/l/h and average sugar concentrations of 7.3% and 6.0% (w/v), respectively. Greater than 95% (w/w) of the sugars produced were in the monomeric state. Average cellulase used for the two runs were 8.4 and 5.3 filter paper units per gram of sugar produced, respectively.     

Continuous hydrolysis of carboxymethyl cellulose with cellulase aggregates trapped inside membranes

Enzymatic hydrolysis of cellulose is often conducted in batch processes in which hydrolytic products tend to inhibit enzyme activity. In this study, we report a method for continuous hydrolysis of carboxymethyl cellulose (CMC) by using cross-linked cellulase aggregate (XCA) trapped inside a membrane. XCA particles prepared by using a millifluidic reactor have a uniform size distribution around 350 nm. Because of their large size, XCA particles in solutions can be filtered through a polyethersulfone membrane to collect 87.1 ± 0.9% of XCA particles. The membrane with impregnated XCA can be used as a catalyst for hydrolysis of CMC in a continuous mode. When the CMC concentration is 1.0 g/l and the flow rate is 2 μl/min, 53.9% of CMC is hydrolyzed to reducing sugars. The membrane with XCA is very stable under continuously flowing solutions. After 72 h of reaction, 97.5% of XCA remains inside the membrane.     

A hyperefficient process for enzymatic cellulose hydrolysis in the intensive mass transfer reactor

Summary A new type of bioreactor, the intensive mass transfer reactor (IMTR), has been developed for enzymatic hydrolysis of cellulose. Using T. reesei cellulases (2 FPU/ml), 3.5–6% of sugars were obtained in the IMTR after 0.5–2 h of cellulose hydrolysis with a productivity of 30–73 g/l.h.     

Column cellulose hydrolysis reactor: Cellulase adsorption profile

Summary A column cellulose hydrolysis reactor was set up using a single passage of cellulase enzyme which was followed with a continuous percolation of buffer. Hydrolysis rates were found to decline precipitously upon the removal of the non-adsorbed cellulase components. By comparing specific activities of the cellulase before and after adsorption on the cellulose column, it was concluded that the adsorption efficiencies for the cellulase components decreased from exoglucanase (1,4--d-glucan cellobiohydrolase EC 3.2.1.91) to endoglucanase [1,4-(1,3;1,4)--d-glucan 4-glucanohydrolase, EC 3.2.1.4] to -glucosidase (-d-glucoside glucohydrolase, EC 3.2.1.21). Of the adsorbed cellulase components, the rate of endoglucanase leaching from the cellulose column was 20 times that for the exoglucanase despite the greater adsorption efficiency of the latter. By analysing the cellulase components which were bound and not bound by the cellulose column and comparing them with a purified exoglucanase enzyme on sodium dodecyl sulfate polyacrylamide gels, it was confirmed that the major cellulase component adsorbed to the cellulose column was an exoglucanase component. The resultant loss of other cellulase components from the reactor was probably the cause for the much reduced rate of cellulose hydrolysis when these components were flushed out of the column.     

Cellulose hydrolysis by a methanogenic culture enriched from landfill waste in a semi-continuous reactor

This study investigates the hydrolysis of cellulose by a mixed culture enriched from landfill waste in a continuous reactor operated under prolonged residence times to accommodate methanogenic conditions. Chemostat studies of hydrolysis under balance methanogenic conditions are rarely reported, despite the importance of hydrolysis under these conditions in waste management and renewable energy industries. Continuous digestion was studied in a 1.25l digester, fed with a 1% (w/v) slurry of 50mum cellulose in sterilized leachate drawn from a 220l digester operated on a feedstock of mixed municipal solid waste. Unsterilized leachate was used as the inoculum. Stable and rapid hydrolytic conditions were established at residence time of 2.5, 3.5 and 5d with a 1st order hydrolysis rate 0.45+/-0.07d(-1) and high methane yields ranging from 57% to 62% of solubilised cellulose on a COD basis. Biomass yields were between 32% and 35% of solubilised cellulose on a COD basis, over three times that observed with fermentative cultures. This is attributed to the diversity of the microbial population which fully converted solubilised COD to methane, as evident by VFA yields of less than 8% on a COD basis.     

Enzymatic hydrolysis of sodium-hydroxide-pretreated sallow in an ultrafiltration membrane reactor

An ultrafiltration membrane reactor was used to investigate the recovery of biocatalysts during enzymatic hydrolysis of pretreated sallow. Product inhibition could be eliminated by continuous removal of products through the ultrafiltration membrane, thus retaining the macromolecular substrate and enzymes. In this way, the degree of conversion was improved from 40% in a batch hydrolysis to 95% (within 20 h), and the initial hydrolysis rate was increased up to seven times. The recovery studies were focused on mechanical deactivation and irreversible adsorption on to the nonconvertible fraction of the substrate. Cellulase deactivation during mechanical agitation was not significant, and the loss of activity was attributed mainly to strong adsorption of the enzymes onto undigested material. This process was studied in semicontinuous hydrolyses, where fresh substrate was added intermittently. The amount of reducing sugars produced in this experiment was 25.7 g/g enzyme, compared to 4.7 g/g enzyme in a batch hydrolysis.     

Supercritical CO2 pretreatment of lignocellulose enhances enzymatic cellulose hydrolysis

The supercritical carbon dioxide (SC-CO2) pretreatment of lignocellulose for enzymatic hydrolysis of cellulose was investigated. Aspen (hardwood) and southern yellow pine (softwood) with moisture contents in the range of 0-73% (w/w) were pretreated with SC-CO2 at 3100 and 4000 psi and at 112-165 degrees C for 10-60 min. Each pretreated lignocellulose was hydrolyzed with commercial cellulase to assess its enzymatic digestibility. Untreated aspen and southern yellow pine (SYP) gave final reducing sugar yields of 14.5 +/- 2.3 and 12.8 +/- 2.7% of theoretical maximum, respectively. When no moisture was present in lignocellulose to be pretreated, the final reducing sugar yield from hydrolysis of SC-CO2-pretreated lignocellulose was similar to that of untreated aspen. When the moisture content of lignocellulose was increased, particularly in aspen, significantly increased final sugar yields were obtained from enzymatic hydrolysis of SC-CO2-pretreated lignocellulose. When the moisture content of lignocellulose was 73% (w/w) before pretreatment, the sugar yields from the enzymatic hydrolysis of aspen and southern yellow pine pretreated with SC-CO2 at 3100 psi and 165 degrees C for 30 min were 84.7 +/- 2.6 and 27.3 +/- 3.8% of theoretical maximum, respectively. The SC-CO2 pretreatments of both aspen and SYP with moisture contents of 40, 57, and 73% (w/w) showed significantly higher final sugar yields compared to the thermal pretreatments without SC-CO2.     

Enhancement of cellulose accessibility and enzymatic hydrolysis by simultaneous wet milling

It has been shown that the rate of enzymatic saccharification of cellulosic materials including “pure” cellulose (Whatman CF?11 cellulose), newsprint, lignocellulose (prehydrolyzed to remove hemicelluloses), and wood can be substantially increased by simultaneous wet milling. An enhanced hydrolysis rate was sustained above that observed for ball milling: providing a more extensive saccharification. The cellulosic substrates were wet milled with a variety of grinding elements, such as sand, glass beads, and stainless-steel beads, agitated in a shaker bath. Simultaneous hydrolysis was achieved with a 2% substrate slurry in a 0.1M acetate buffer at 45°C and pH 5. The effectiveness of this process was dependent upon the lignified matrix of the cellulose microfibrils, the grinding elements, and the oscillation frequency of the shaker bath. Wet milling “pure” cellulose for 48 hr, with 3.5 mm glass beads and 200 oscillations/min (opm), yielded 1031 mg reducing sugar/g substrates (93% saccharification) as compared to 483 mg (44%) for the ball-milled sample and 253 mg (23%) for the unmilled material. With the lignified substrates stainless-steel beads (3.5 mm) were more effective than glass. For lignocellulose 529 mg sugar/g substrate (93% saccharification) could be obtained by wet milling with cellulase for 24 hr. This was about three times greater than that of the ball milled (169 mg, 30%) and 10 times greater than that of the unmilled (52 mg, 9%) substrates. The method was also effective for wood particles (60 mesh) giving 143 mg sugar/g wood (approximately 38% saccharification) in 48 hr, whereas the ball-milled sample gave only 79 mg (21%) and the unmlilled substrate 38 mg (10%). These observations can be explained on the basis of the current crystalline theory for the morphology of the cellulosic microfibrils. The advantage of wet milling and simultaneous hydrolysis apparently depends on a continuous generation of accessible sites and sustained rapid hydrolysis rate as the saccharification proceeds, where in the pretreated substrates the hydrolysis rate slow down as the active sites are reduced.     

玉米秸秆分批补料获得高还原糖浓度酶解液的条件优化

木质纤维素高浓度还原糖水解液的获得是纤维乙醇产业化发展的方向。在发酵工业领域,分批补料法是实现这一目标的重要研究途径。本研究采用分批补料法对获得高浓度玉米秸秆酶解还原糖的条件进行了优化。以稀硫酸预处理的玉米秸秆为原料,考察了液固比、补加量与补加时间对分批补料糖化的影响。结果表明,秸秆高浓度酶解液条件的初始物料为20％ (重量/体积),木聚糖酶220 U/g (底物),纤维素酶6 FPU/g (底物),果胶酶50 U/g (底物),在24 h、48 h后分批补加8％预处理后的物料,同时添加与补料量相应的木聚糖酶20 U/g (底物),纤维素酶2 FPU/g (底物),72 h后,最终糖化结果与非补料法相比,还原糖浓度从48.5 g/L提高到138.5 g/L,原料的酶解率最终达到理论值的62.5％。试验结果表明补料法可以显著提高秸秆水解液还原糖浓度。     

Kinetics of the enzymatic hydrolysis of cellulose: 2. A mathematical model for the process in a plug-flow column reactor

The kinetics of enzymatic cellulose hydrolysis in a plug-flow column reactor catalysed by cellulases [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] from Trichoderma longibrachiatum adsorbed on cellulose surface have been studied. The maximum substrate conversion achieved was 90–94%. The possibility of enzyme recovery for a reactor of this type is discussed. A mathematical model for enzymatic cellulose hydrolysis in a plug-flow column reactor has been developed. The model allows for the component composition of the cellulase complex, adsorption of cellulases on the substrate surface, inhibition by reaction products, changes in cellulose reactivity and the inactivation of enzymes in the course of hydrolysis. The model affords a reliable prediction of the kinetics of d-glucose and cellobiose formation from cellulose in a column reactor as well as the degree of substrate conversion and reactor productivity with various amounts of adsorbed enzymes and at various flow rates.     

The effect of crystallinity of cellulose on the rate of reducing sugars production by heterogeneous enzymatic hydrolysis

A kinetic model is devised, from the reaction mechanism steps, to predict the rate of reducing sugar production by hydrolysis of two types of cellulose, namely, amorphous carboxymethylcellulose (CMC) and highly crystalline wood shavings, using Aspergillus niger cellulase. Experimental results in a stirred batch reactor at 40 degrees C show that the production of reducing sugar reduced at much shorter times for wood shavings in comparison to CMC at the same initial substrate concentration. The experimental results are used to determine the kinetic parameters of the model equations. The significance of crystallinity was determined using inert fraction coefficient, which is assumed to be constant and equals 0.05 and 0.98 for CMC and wood shavings, respectively. It is shown there is a good agreement between the experimental results and proposed kinetic model predictions. The effect of the inert fraction coefficient on the production of reducing sugar by the enzymatic hydrolysis of cellulose is also determined. It is found that the cellulase used extracted from A. niger is much more sensitive towards the substrate structure in comparison to that extracted from Trichoderma reesei.     

Continuous enzymatic saccharification of cellulose with culture filtrates of trichoderma viride QM 6a.

Continuous saccharification of Solka Floc (cellulose pulp) in single and four-vessel stirred-tank reactor systems has been possible employing enzymes obtained directly from submerged fermentation of Trichoderma viride QM 6a. Studies on the effect of modification of the solid substrate, enzyme stability, substrate concentration, and the influence of reducing sugar concentration on the rate of hydrolysis are reported. While susceptibility of substrate to digestion is not affected by heating alone, it is strikingly increased by heating plus grinding, or by grinding following heating. Batch and steady state continuous saccharification experiments have yielded more than 5% reducing sugar in the effluent with a dilution rate of 0.025 hr^?1 at 50°C, at a substrate level of 10%. An average glucose concentration of 3.4% has been obtained in the effluent of a continuous saccharification using 5% substrate at the same dilution rate and temperature.     

Tryptic hydrolysis of caseinomacropeptide in membrane reactor: Preparation of bioactive peptides

Summary Tryptic hydrolysis of caseinomacropeptide (CMP: C-terminal part of k-Casein) was used as a model in membrane reactor to study continuous production and isolation of bioactive peptides from milk proteins. Compared to the batch reactor, productivity of the proposed continuous process was 3 times higher after 3.5 hours of hydrolysis, but only 50% of the substrate is converted at steady state of the system. As shown by reverse-phase HPLC analysis, a good selectivity of the ultrafiltration membrane to various products was also obtained.     

Ethanol fermentation in an immobilized cell reactor using Saccharomyces cerevisiae

Fermentation of sugar by Saccharomyces cerevisiae, for production of ethanol in an immobilized cell reactor (ICR) was successfully carried out to improve the performance of the fermentation process. The fermentation set-up was comprised of a column packed with beads of immobilized cells. The immobilization of S. cerevisiae was simply performed by the enriched cells cultured media harvested at exponential growth phase. The fixed cell loaded ICR was carried out at initial stage of operation and the cell was entrapped by calcium alginate. The production of ethanol was steady after 24 h of operation. The concentration of ethanol was affected by the media flow rates and residence time distribution from 2 to 7 h. In addition, batch fermentation was carried out with 50 g/l glucose concentration. Subsequently, the ethanol productions and the reactor productivities of batch fermentation and immobilized cells were compared. In batch fermentation, sugar consumption and ethanol production obtained were 99.6% and 12.5% v/v after 27 h while in the ICR, 88.2% and 16.7% v/v were obtained with 6 h retention time. Nearly 5% ethanol production was achieved with high glucose concentration (150 g/l) at 6 h retention time. A yield of 38% was obtained with 150 g/l glucose. The yield was improved approximately 27% on ICR and a 24 h fermentation time was reduced to 7 h. The cell growth rate was based on the Monod rate equation. The kinetic constants (K(s) and mu(m)) of batch fermentation were 2.3 g/l and 0.35 g/lh, respectively. The maximum yield of biomass on substrate (Y(X-S)) and the maximum yield of product on substrate (Y(P-S)) in batch fermentations were 50.8% and 31.2% respectively. Productivity of the ICR were 1.3, 2.3, and 2.8 g/lh for 25, 35, 50 g/l of glucose concentration, respectively. The productivity of ethanol in batch fermentation with 50 g/l glucose was calculated as 0.29 g/lh. Maximum production of ethanol in ICR when compared to batch reactor has shown to increase approximately 10-fold. The performance of the two reactors was compared and a respective rate model was proposed. The present research has shown that high sugar concentration (150 g/l) in the ICR column was successfully converted to ethanol. The achieved results in ICR with high substrate concentration are promising for scale up operation. The proposed model can be used to design a lager scale ICR column for production of high ethanol concentration.     

Hydrothermal treatment of wheat straw at pilot plant scale using a three-step reactor system aiming at high hemicellulose recovery, high cellulose digestibility and low lignin hydrolysis

A pilot plant (IBUS) consisting of three reactors was used for hydrothermal treatment of wheat straw (120-150 kg/h) aiming at co-production of bioethanol (from sugars) and electricity (from lignin). The first reactor step was pre-soaking at 80 degrees C, the second extraction of hemicellulose at 170-180 degrees C and the third improvement of the enzymatic cellulose convertibility at 195 degrees C. Water added to the third reactor passed countercurrent to straw. The highest water addition (600 kg/h) gave the highest hemicellulose recovery (83%). With no water addition xylose degradation occurred resulting in low hemicellulose recovery (33%) but also in high glucose yield in the enzymatic hydrolysis (72 g/100g glucose in straw). Under these conditions most of the lignin was retained in the fibre fraction, which resulted in a lignin rich residue with high combustion energy (up to 31 MJ/kg) after enzymatic hydrolysis of cellulose and hemicellulose.     

Effect of surfactants on cellulose hydrolysis

The effect of surfactants on the heterogeneous enzymatic hydrolysis of Sigmacell 100 cellulose and of steam-exploded wood was studied. Certain biosurfactants (sophorolipid, rhamnolipid, bacitracin) and Tween 80 increased the rate of hydrolysis of Sigmacell 100, as measured by the amount of reducing sugar produced, by as much as seven times. The hydrolysis of steam-exploded wood was increased by 67% in the presence of sophorolipid. At the same time, sophorolipid was found to decrease the amount of enzyme adsorbed onto the cellulose at equilibrium. Sophorolipid had the greatest effect on cellulose hydrolysis when it was present from the beginning of the experiment and when the enzyme/cellulose ratio was low. (c) 1993 John Wiley & Sons, Inc.     

Kinetic modeling for enzymatic hydrolysis of pretreated creeping wild ryegrass

A semimechanistic multi‐reaction kinetic model was developed to describe the enzymatic hydrolysis of a lignocellulosic biomass, creeping wild ryegrass (CWR; Leymus triticoides). This model incorporated one homogeneous reaction of cellobiose‐to‐glucose and two heterogeneous reactions of cellulose‐to‐cellobiose and cellulose‐to‐glucose. Adsorption of cellulase onto pretreated CWR during enzymatic hydrolysis was modeled via a Langmuir adsorption isotherm. This is the first kinetic model which incorporated the negative role of lignin (nonproductive adsorption) using a Langmuir‐type isotherm adsorption of cellulase onto lignin. The model also reflected the competitive inhibitions of cellulase by glucose and cellobiose. The Matlab optimization function of “lsqnonlin” was used to fit the model and estimate kinetic parameters based on experimental data generated under typical conditions (8% solid loading and 15 FPU/g‐cellulose enzyme concentration without the addition of background sugars). The model showed high fidelity for predicting cellulose hydrolysis behavior over a broad range of solid loading (4–12%, w/w, dry basis), enzyme concentration (15–150 FPU/ g‐cellulose), sugar inhibition (glucose of 30 and 60 mg/mL and cellobiose of 10 mg/mL). In addition, sensitivity analysis showed that the incorporation of the nonproductive adsorption of cellulase onto lignin significantly improved the predictability of the kinetic model. Our model can serve as a robust tool for developing kinetic models for system optimization of enzymatic hydrolysis, hydrolysis reactor design, and/or other hydrolysis systems with different type of enzymes and substrates. Biotechnol. Bioeng. 2009;102: 1558–1569. © 2008 Wiley Periodicals, Inc.     

The effect of bovine serum albumin on batch and continuous enzymatic cellulose hydrolysis mixed by stirring or shaking

Bovine serum albumin (BSA) was applied as a model non-catalytic protein to enzymatic hydrolysis of Avicel and dilute acid pretreated corn stover at different reaction conditions to improve the understanding of its ability to enhance cellulose hydrolysis. Addition of BSA improved the 72 h hydrolysis yields in shake flasks by up to 26% for both substrates by reducing de-activation of the exoglucanases and by facilitating reductions in particle size and crystallinity during a magnetically stirred pre-incubation step. The enzyme stabilizing effect of BSA addition was most striking for batch hydrolysis in a stirred tank reactor, with glucose yields increasing by 76% after 72 h for Avicel and by 40% after 145 h for corn stover. Application of BSA to continuous hydrolysis for a mean residence time of 24h gave 33% and 40% higher glucose yields for corn stover and Avicel compared to the controls.     

Development of a stable continuous flow immobilized enzyme reactor for the hydrolysis of inulin

A 23.5-fold purified exoinulinase with a specific activity of 413 IU/mg and covalently immobilized on Duolite A568 has been used for the development of a continuous flow immobilized enzyme reactor for the hydrolysis of inulin. In a packed bed reactor containing 72 IU of exoinulinase from Kluyveromyces marxianus YS-1, inulin solution (5%, pH 5.5) with a flow rate of 4 mL/h was completely hydrolyzed at 55 degrees C. The reactor was run continuously for 75 days and its experimental half-life was 72 days under the optimized operational conditions. The volumetric productivity and fructose yield of the reactor were 44.5 g reducing sugars/L/h and 53.3 g/L, respectively. The hydrolyzed product was a mixture of fructose (95.8%) and glucose (4.2%) having an average fructose/glucose ratio of 24. An attempt has also been made to substitute pure inulin with raw Asparagus racemosus inulin to determine the operational stability of the developed reactor. The system remained operational only for 11 days, where 85.9% hydrolysis of raw inulin was achieved.     

Application of immobilized invertase to continuous hydrolysis of concentrated sucrose solutions

Invertase immobilized onto corn grits was utilized in the hydrolysis of highly concentrated sucrose solutions producting liquid sugar solutions containing glucose and fructose. Comparisons of conversion efficiencies of this immobilized invertase in a continuous stirredtank reactor and a plug-flow reactor indicated that the plug-flow reactor has an higher efficiency. Continuous sucrose hydrolysis was then performed in 0.1- and 1-L tubular reactors. This tenforld scaling-up was achieved without any noticeable loss in efficiency. This process thus was scaled-up to a 17.6-L pilot reactor set in a cane sugar refinery. This reactor was fed with highly concentrated sucrose solutions [71% (w/w)] to produce invert sugar syrup with the desired inversion degree. It allows a productivity equal to 9.1 kg sucrose hydrolyzed/h in the case of a 69% (w/w) sucrose initial concentration with a 72% conversion rate.