Simultaneous saccharification and fermentation of cellulose: effect of ethanol on enzymatic saccharification of cellulose

It was confirmed that simultaneous saccharification and fermentation are effective for accelerating enzymatic saccharification of cellulose. In this work, the effects of ethanol on the saccharification of tissue paper by Trichoderma cellulase (Meicelase CEPB) have been investigated. The following results were obtained. (1) Saccharification was inhibited by at least 0.2M ethanol. (2) Less than 4M ethanol did not affect the enzymatic activities of beta-glucosidase and endoglucanase (C(x)) at all. The thermal stability of endoglucanase was not also varied by ethanol. (3) It is suggested that ethanol depresses the adsorption of exoglucanase on cellulose. (4) The rate expression of saccharification of cellulose in the presense of ethanol is proposed. (5) The inhibititory effect of ethanol was found to become more significant in the later stages of the reaction than just the initial stage.     

Enhancement of enzymatic saccharification of cellulose by cellulose dissolution pretreatments

Attempts were made to enhance cellulose saccharification by cellulase using cellulose dissolution as a pretreatment step. Four cellulose dissolution agents, NaOH/Urea solution, N-methylmorpholine-N-oxide (NMMO), ionic liquid (1-butyl-3-methylimidazolium chloride; [BMIM]Cl) and 85% phosphoric acid were employed to dissolve cotton cellulose. In comparison with conventional cellulose pretreatment processes, the dissolution pretreatments were operated under a milder condition with temperature <130 °C and ambient pressure. The dissolved cellulose was easily regenerated in water. The regenerated celluloses exhibited a significant improvement (about 2.7- to 4.6-fold enhancement) on saccharification rate during 1st h reaction. After 72 h, the saccharification yield ranged from 87% to 96% for the regenerated celluloses while only around 23% could be achieved for the untreated cellulose. Even with high crystallinity, cellulose regenerated from phosphoric acid dissolution achieved the highest saccharification rates and yield probably due to its highest specific surface area and lowest degree of polymerization (DP).     

Cellulase adsorption-desorption and cellulose saccharification during enzymatic hydrolysis of cellulose materials

Treatment of different cellulose materials with cellulase from Penicillium funiculosum showed a cellulase adsorption-desorption pattern on all materials. The relative rate of adsorption and saccharification (enzyme activity) increases with increasing temperature. At 60° cellulase adsorption increased while the enzyme activity decreased.     

Impact of lignins isolated from pretreated lignocelluloses on enzymatic cellulose saccharification

Lignins were enzymatically isolated from corn stover and wheat straw samples and subjected to hydrothermal or wet oxidation pretreatments for enzyme adsorption experimentations. Lignin contents of the isolates ranged from 26 to 71 % (w/w); cellulose ranged from 3 to 22 % (w/w); xylan from 0.7 to 6 % (w/w) and ash was from 5.8 to 30 % (w/w). ATR-IR analyses indicated significant and similar levels of calcium in all lignin isolates. Commercial cellulase adsorption studies showed that the presence of these lignins had no significant impact on the total amount of adsorbed enzyme in cellulose and cellulose–lignin systems. Consequently, the presence of the lignins had minimal effect, if any, on enzymatic cellulose conversion. Furthermore, this result, coupled with significant calcium levels in the isolated lignins, supports previous work suggesting lignin–calcium complexes reduce enzyme–lignin interactions.     

Effect of crystallinity and degree of polymerization of cellulose on enzymatic saccharification

Lignocellulose materials were pretreated by methods known to improve enzymatic saccharification, and the percentage crystallinity (x-ray diffraction) and degree of polymerization were measured. It was observed that although the percentage crystallinity of cellulose was not altered by alkaline explosion (AE), carbon dioxide explosion (CE), ozone, and sodium chlorite treatment, very great increases were obtained in the extent of enzymatic saccharification. All the pretreatments studied except sodium chlorite caused significant reduction in degree of polymerization. It appears likely that the rate and extent of saccharification is governed by particle size, surface area, and degree of polymerization, since crystallinity effects alone do not explain the observed trends in the hydrolysis data.     

Effects of drying-induced fiber hornification on enzymatic saccharification of lignocelluloses

This study investigated the effect of fiber hornification during drying on lignocellulosic substrate enzymatic saccharification. Two chemically pretreated wood substrates and one commercial bleached kraft hardwood pulp were used. Heat drying at 105 and 150°C and air drying at 50% RH and 23.8°C for different durations were applied to produce substrate with various degrees of hornification. It was found that substrate enzymatic digestibilities (SEDs) of hornified substrates made from the same never-dried sample correlate very well to an easily measurable parameter, water retention value (WRV), and can be fitted by a Boltzmann function. The hornification-produced SED reduction at a given degree of hornification as the percentage of the total SED reduction when the substrate is completely hornified depends on two parameters. The first is WRVˉ, which is primarily a function of the effective enzyme molecule size, and Δ, which is related to the substrate pore size distribution shape. The low values of SED(CH), SED of a completely hornified substrate, obtained from curve fittings for the three sets of samples studied, suggest that enzyme accessibility to cellulose is mainly through the pores in the cell wall rather than substrate external surface. The SEDs of hornified substrates were found to correlate to Simons' staining measurements well. A new parameter was proposed to better correlate enzyme accessibility to cellulose using the two-color Simons' staining technique.     

Ultrasound effect on enzymatic saccharification

Summary An ultrasound field during enzymatic saccharification ehanced the rate and extent of reaction of sugarcane bagasse that had been previously pretreated inducing a matrix swelling and partial delignification.     

A model for continuous enzymatic saccharification of cellulose with simultaneous removal of glucose syrup

Cellulase of Trichoderma viride was concentrated in various molecular cutoff membranes, and flux rates and retention of activity were studied under ultra-filtration conditions. Little or no Cellulase was discharged through the membranes tested. The concentrated (5–8-fold) enzymes were used to saccharify finely ground substrate (Solka Floe) in stirred tank (STR) and membrane reactors (MR). A pressure filtration vessel provided with a membrane for simultaneous removal of low molecular weight products (glucose) from the reacting system (Cellulose-Cellulase) is designated as a membrane reactor. Continuous digestion of dense cellulose suspension in the membrane reactor was achieved. Using PM-30 (Amicon) membrane reasonably high mass flux values (9.7–23.3 gals/ft²—day) were obtained in separating glucose from a digest of 30% cellulose suspension. Abcor membrane (HFA 300) was equally effective and necessitated less care in handling. Nearly 14% glucose concentration has been achieved in less than 50 hrs in STR by digesting a 30% cellulose suspension. Based on experimental data a model system is proposed for the continuous steady state Saccharification of ground substrate in which there is continuous removal of concentrated glucose syrup, and a feedback of enzyme.     

Mild pretreatment and enzymatic saccharification of cellulose with recycled ionic liquids towards one-batch process

The development of second-generation bioethanol involves minimizing the energy input throughout the processing steps. We report here that efficient ionic liquid pretreatments of cellulose can be achieved with short duration times (20min) at mild temperature (45°C) with [Emim](+)[MeO(H)PO(2)](-) and at room temperature (25°C) with [Emim](+)[CH(3)COO](-). In these conditions, yields of glucose were increased by a factor of 3. In addition, the recycling of these two imidazolium-based ILs can be performed in maintaining their efficiency to pretreat cellulose. The short time and mild temperature of cellulose solubilization allowed a one-batch processing of [Emim](+)[MeO(H)PO(2)](-) IL-pretreatment and saccharification. In the range from 0 to 100% IL in an aqueous enzymatic medium, the glucose yields were improved at IL proportions between 10 and 40%. The maximum yield at 10% IL is very promising to consider one batch process as efficient as two-step process.     

Effects of growth stage on enzymatic saccharification and simultaneous saccharification and fermentation of bamboo shoots for bioethanol production

Bamboo is a fast-growing renewable biomass that is widely distributed in Asia. Although bamboo is recognised as a useful resource, its utilization is limited and further development is required. Immature bamboo shoots harvested before branch spread were found to be a good biomass resource to achieve a high saccharification yield. The saccharification yield of the shoots increased (up to 98% for immature Phyllostachys bambusoides) when xylanase was used in addition to cellulase. Simultaneous saccharification and fermentation (SSF) processing converted immature shoots of P. bambusoides and Phyllostachys pubescens to ethanol with an ethanol yield of 169 and 139 g kg⁻¹, respectively (98% and 81%, respectively, of the theoretical yields based on hexose conversion) when 12 FPU g⁻¹ enzyme and the yeast Saccharomyces cerevisiae were used.     

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.     

Mechanism of the enzymatic hydrolysis of cellulose: Effects of major structural features of cellulose on enzymatic hydrolysis

The susceptibility of cellulose to enzymatic hydrolysis is affected by the structural features of cellulosic materials. It has been suggested that the crystallinity and surface area of cellulose fibers are the most important structural features in this regard. This study investigated in depth the relative effects of these two structural features upon the enzymatic hydrolysis of cellulose and the change of the structural parameters of cellulose during the course of hydrolysis. It was found that the hydrolysis rate is mainly dependent upon the fine structural order of cellulose which can best be represented by the crystallinity rather than the simple surface area. Monitoring the changes in the structural parameters during the course of reaction showed that surface area is not a major limiting factor that slows hydrolysis in its late stages as has been suggested. This information concerning structural features is used to elucidate the mode of action of cellulase.     

Effects of fungal pretreatment and steam explosion pretreatment on enzymatic saccharification of plant biomass

The effects of consecutive treatments by a lignin-degrading fungus Phanerochaete chrysosporium and by steam explosion for the enzymatic saccharification of plant biomass were studied experimentally, and the optimal operational conditions for obtaining the maximum saccharification were evaluated. Beech wood-meal was treated by the fungus for 98 days and then by high steam temperatures of 170-230 degrees C with steaming times of 0-10 min. The treatment of the wood-meal by fungus prior to steam explosion enhanced the saccharification of wood-meal. The treated wood-meal was separated into holo-cellulose, water soluble material, methanol soluble lignin, and Klason lignin. The saccharification decreased linearly with the increase in the amount of Klason lignin. It was estimated by the equation for the saccharification of exploded wood-meal expressed as a function of steam temperature and steaming time that the maximum saccharification of wood-meal was obtained by consecutive treatments such as fungal treatment for 28 days and then steam explosion at a steam temperature of 215 degrees C and a steaming time of 6.5 min. (c) 1995 John Wiley & Sons, Inc.     

Fractal kinetic analysis of polymers/nonionic surfactants to eliminate lignin inhibition in enzymatic saccharification of cellulose

The profile of enzymatic saccharification of Avicel in the presence and absence of lignin has been described with a fractal kinetic model (Wang and Feng, 2010), in which the retarded hydrolysis rate of enzymatic saccharification of cellulose has been represented with a fractal exponent. The lignin inhibition in the enzymatic saccharification of cellulose is indexed by the increase of fractal exponent, which can not be fully counterbalanced by high cellulase loading due to the high fractal exponent at high cellulase loading. On the contrary, fractal kinetic analysis indicates that an addition of some nonionic surfactant/polymers decrease the fractal exponent to the original values of enzymatic saccharification of Avicel without lignin and the corresponding toxicity of nonionic surfactants/polymers on the consecutive ethanol fermentation strain Saccharomyces cerevisiae is also examined.     

Engineering and economics of cellulose saccharification systems

The design of cellulose saccharification systems will govern the economics of biomass conversion to ethanol and other oxygenated compounds. Solids handling of bulky cellulosic materials, chemical processing of a physically and chemically heterogeneous substrate, cellulose pretreatment and product recovery present formidable engineering challenges. Marketing strategy must also be carefully formulated given the variety of hexoses, pentoses, organic acids, as well as lignin which result from biomass processing. Since the intrinsic cost of the biomass is $0.015 to $0.03/lb, and the processing costs are $0.03 to $0.10/lb, the key is to identify products having a value in excess of $0.10/lb which are uniquely suited for production from biomass-derived sugars. Competitive pressures from other carbohydrate sources such as corn and sugar cane must also be considered in the economic analysis. Process concepts and associated costs are presented in a comparison of corn and biomass saccharification routes.     

Enzymatic saccharification of cellulose in membrane reactors

The kinetics of enzymatic saccharification of ball-milled sugar-cane bagasse, sorghum stubble and peanut shells was studied and their conversions compared. Particle size analyses were performed on the bagasse sample and pure cellulose (Solka-Floc). It was revealed that most of the size reduction of cellulose particle took place between 0 5% conversion. Means of using commercially available ultrafiltration units as continuous-flow membrane reactors to reduce glucose inhibition were tested and compared using Solka-Floc as substrate. It was pointed out that a low conversion CSTR placed between a ball-mill and a hollow-fibre cartridge could reduce the cost of pretreatment and prevent possible blockage of hollow fibres.     

Continuous enzymatic liquefaction of starch for saccharification

A process was explored for continuous enzymatic liquefaction of corn starch at high concentration and subsequently saccharification to glucose. The process appears to be quite efficient for conversion of starch to glucose and enzymatic liquefaction and should be readily adaptable to industrial fermentation processes. Preliminary work indicated that milled corn or other cereal grains also can be suitably converted by such a process. Essentially, the process involved incorporation of a thermostable, bacterial alpha-amylase for liquefaction and, subsequently, of a glucoamylase into the continuous mixer under conditions conductive to rapid enzymatic hydrolyses. Also studied was the effect on substrate liquefaction of variable such as starch concentration (40-70 degrees ), level of alpha-amylase (0.14-0.4%, dry starch basis), temperature (70-100 degrees C), pH (5.8-7.1), and residence time (6 and 12 min). The degree of liquefaction was assessed by determining (1) the Brookfield viscosity, (2) the amount of reducing groups, and (3) the rate and extent of glucose formed after glucoamylase treatment. Best liquefaction process conditions were achieved by using 50-60% starch concentration, at 95 degrees C, with 0.4% alpha-amylase, and a 6-min residence period in the mixture. Under these conditions, rate and extents of glucose obtained after glucoamylase treatment approached those obtained in longer laboratory batch liquefactions. The amount of glucose formed in 24h with the use of 0.4% glucoamylase was 86% of theory after a 6-min continuous liquefaction, compared to 90% for a 30-min laboratory batch liquefaction (95 degrees C, 0.4% alpha-amylase).     

Effects of preheating on briquetting and subsequent hydrothermal pretreatment for enzymatic saccharification of wheat straw

Briquetting of plant biomass with low bulk density is an advantage for handling, transport, and storage of the material, and heating of the biomass prior to the briquetting facilitates the densification process and improves the physical properties of the briquettes. This study investigates the effects of preheating prior to briquetting of wheat straw (WS) on subsequent hydrothermal pretreatment and enzymatic conversion to fermentable sugars. WS (11% moisture content) was densified to briquettes under different conditions; without preheating or with preheating at 75 or 125°C for either 5 or 10 min. Subsequent hydrothermal pretreatment was done for both un-briquetted WS and for briquettes. Enzymatic saccharification was afterwards performed for all samples. The results showed that as expected, nonpretreated WS briquettes gave very low sugar yields (22–29% of the cellulose content), even though preheating at 125°C prior to briquetting (without pretreatment) improved sugar yields somewhat. When combined with pretreatment, briquetting with preheating showed neutral or negative effects on sugar yield. This result suggests that moderate preheating (75°C for 5 min) before briquetting improved bulk density and compressive resistance of briquettes without impeding subsequent enzymatic conversion. However, excessive preheating (75 or 125°C for 10 min) before briquetting may result in irreversible structural modifications that hinder the interaction between biomass and water during pretreatment, thereby decreasing the accessibility of cellulose to enzymatic saccharification.