Enzyme inactivation by ethanol and development of a kinetic model for thermophilic simultaneous saccharification and fermentation at 50 °C with Thermoanaerobacterium saccharolyticum ALK2

Studies were undertaken to understand phenomena operative during simultaneous saccharification and fermentation (SSF) of a model cellulosic substrate (Avicel) at 50°C with enzymatic hydrolysis mediated by a commercial cellulase preparation (Spezyme CP) and fermentation by a thermophilic bacterium engineered to produce ethanol at high yield, Thermoanaerobacterium saccharolyticum ALK2. Thermal inactivation at 50 °C, as shown by the loss of 50% of enzyme activity over 4 days in the absence of ethanol, was more severe than at 37 °C, where only 25% of enzyme activity was lost. In addition, at 50 °C ethanol more strongly influenced enzyme stability. Enzyme activity was moderately stabilized between ethanol concentrations of 0 and 40 g/L, but ethanol concentrations above 40 g/L accelerated enzyme inactivation, leading to 75% loss of enzymatic activity in 80 g/L ethanol after 4 days. At 37 °C, ethanol did not show a strong effect on the rate of enzyme inactivation. Inhibition of cellulase activity by ethanol, measured at both temperatures, was relatively similar, with the relative rate of hydrolysis inhibited 50% at ethanol concentrations of 56.4 and 58.7 g/L at 50 and 37 °C, respectively. A mathematical model was developed to test whether the measured phenomena were sufficient to quantitatively describe system behavior and was found to have good predictive capability at initial Avicel concentrations of 20 and 50 g/L.     

Simultaneous improvement of saccharification and ethanol production from crystalline cellulose by alleviation of irreversible adsorption of cellulase with a cell surface-engineered yeast strain

Enzymatic hydrolysis of cellulosic material is an essential step in the bioethanol production process. However, complete cellulose hydrolysis by cellulase is difficult due to the irreversible adsorption of cellulase onto cellulose. Thus, part of the cellulose remains in crystalline form after hydrolysis. In this study, after 96-h hydrolysis of Avicel crystalline cellulose, 47.1 % of the cellulase was adsorbed on the cellulose surface with 10.8 % crystalline cellulose remaining. In simultaneous saccharification and fermentation of 100 g/L Avicel with 1.0 filter paper unit/mL cellulase, a wild-type yeast strain produced 44.7 g/L ethanol after 96 h. The yield of ethanol was 79.7 % of the theoretical yield. On the other hand, a recombinant yeast strain displaying various cellulases, such as β-glucosidase, cellobiohydrolase, and endoglucanase, produced 48.9 g/L ethanol, which corresponds to 87.3 % of the theoretical yield. Higher ethanol production appears to be attributable to higher efficiency of cellulase displayed on the cell surface. These results suggest that cellulases displayed on the yeast cell surface improve hydrolysis of Avicel crystalline cellulose. Indeed, after the 96-h simultaneous saccharification and fermentation using the cellulase-displaying yeast, the amount of residual cellulose was 1.5 g/L, one quarter of the cellulose remaining using the wild-type strain, a result of the alleviation of irreversible adsorption of cellulases on the crystalline cellulose.     

Hydrolysis of dilute acid pretreated mixed hardwood and purified microcrystalline cellulose by cell-free broth from Clostridium thermocellum

The cellulase activity in cell-free broths from the thermophilic, ethanol-producing anaerobic bacterium Clostridium thermocellum is examined on both dilute-acid-pretreated mixed hardwood (90% maple, 10% birch) and Avicel. Experiments were conducted in vitro in order to distinguish properties of the cellulase from properties of the organism and to evaluate the effectiveness of C. thermocellum cellulase in the hydrolysis of a naturally occurring, lignin-containing substrate. The results obtained establish that essentially quantitative hydrolysis of cellulose from pretreated mixed hardwood is possible using this enzyme system. Pretreatment with 1% H(2)SO(4) and a 9-s residence time at 220, 210, 200, and 180 degrees C allowed yields after enzymatic hydrolysis (percentage of glucan solubilized/ glucan potentially solubilized) of 97.8, 86.1, 82.0, and 34.6%, respectively. Enzymatic hydrolysis of mixed hardwood with no pretreatment resulted in a yield of 10.1%. Hydrolysis yields of >95% were obtained from approximately 0.6 g/L mixed hardwood pretreated at 220 degrees C in 7 h at broth strengths of 60 and 80% (v/v) and in approximately 48 h with 33% broth. Hydrolysis of pretreated mixed hardwood is compared to hydrolysis of Avicel, a pure microcrystalline cellulose studied previously. The initial rate of Avicel hydrolysis saturates with respect to enzyme, whereas the initial rate of hydrolysis of pretreated wood is proportional to the amount of enzyme present. Initial hydrolysis rates for pretreated wood and Avicel at 0.6 g/L are greater for wood at low broth dilutions (1.25: 1 to 5 :1) by up to 2.7-fold and greater for Avicel at high broth dilutions (5 : 1 to 50 : 1) by up to 4.3-fold. Maximum rates of hydrolysis are achieved at <2 g substrate/L for both pretreated wood and Avicel. The substrate concentration at one-half the maximum observed rate for C. thermocellum broths is smaller for pretreated mixed hardwood than for Avicel and decreases with increasing broth dilution for both substrates. An initial activity per volume broth of approximately 11 mumol soluble glucose equivalent produced/L broth/min is observed for mixed hardwood pretreated at 220 degrees C and for Avicel at high broth dilutions; the initial activity per volume broth for Avicel is lower at low broth dilutions. The results indicate that pretreated wood is hydrolyzed at rates comparable to Avicel under many conditions and at rates significantly faster than Avicel under several conditions.     

BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates

Cellulase and bovine serum albumin (BSA) were added to Avicel cellulose and solids containing 56% cellulose and 28% lignin from dilute sulfuric acid pretreatment of corn stover. Little BSA was adsorbed on Avicel cellulose, while pretreated corn stover solids adsorbed considerable amounts of this protein. On the other hand, cellulase was highly adsorbed on both substrates. Adding a 1% concentration of BSA to dilute acid pretreated corn stover prior to enzyme addition at 15 FPU/g cellulose enhanced filter paper activity in solution by about a factor of 2 and beta-glucosidase activity in solution by about a factor of 14. Overall, these results suggested that BSA treatment reduced adsorption of cellulase and particularly beta-glucosidase on lignin. Of particular note, BSA treatment of pretreated corn stover solids prior to enzymatic hydrolysis increased 72 h glucose yields from about 82% to about 92% at a cellulase loading of 15 FPU/g cellulose or achieved about the same yield at a loading of 7.5 FPU/g cellulose. Similar improvements were also observed for enzymatic hydrolysis of ammonia fiber explosion (AFEX) pretreated corn stover and Douglas fir treated by SO(2) steam explosion and for simultaneous saccharification and fermentation (SSF) of BSA pretreated corn stover. In addition, BSA treatment prior to hydrolysis reduced the need for beta-glucosidase supplementation of SSF. The results are consistent with non-specific competitive, irreversible adsorption of BSA on lignin and identify promising strategies to reduce enzyme requirements for cellulose hydrolysis.     

Access of cellulase to cellulose and lignin for poplar solids produced by leading pretreatment technologies

Adsorption of cellulase on solids resulting from pretreatment of poplar wood by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid (DA), flowthrough (FT), lime, and sulfur dioxide (SO₂) and pure Avicel glucan was measured at 4°C, as were adsorption and desorption of cellulase and adsorption of β‐glucosidase for lignin left after enzymatic digestion of the solids from these pretreatments. From this, Langmuir adsorption parameters, cellulose accessibility to cellulase, and the effectiveness of cellulase adsorbed on poplar solids were estimated, and the effect of delignification on cellulase effectiveness was determined. Furthermore, Avicel hydrolysis inhibition by enzymatic and acid lignin of poplar solids was studied. Flowthrough pretreated solids showed the highest maximum cellulase adsorption capacity (σ_solids = 195 mg/g solid) followed by dilute acid (σ_solids = 170.0 mg/g solid) and lime pretreated solids (σ_solids = 150.8 mg/g solid), whereas controlled pH pretreated solids had the lowest (σ_solids = 56 mg/g solid). Lime pretreated solids also had the highest cellulose accessibility (σ_cellulose = 241 mg/g cellulose) followed by FT and DA. AFEX lignin had the lowest cellulase adsorption capacity (σ_lignin = 57 mg/g lignin) followed by dilute acid lignin (σ_lignin = 74 mg/g lignin). AFEX lignin also had the lowest β‐glucosidase capacity (σ_lignin = 66.6 mg/g lignin), while lignin from SO₂ (σ_lignin = 320 mg/g lignin) followed by dilute acid had the highest (301 mg/g lignin). Furthermore, SO₂ followed by dilute acid pretreated solids gave the highest cellulase effectiveness, but delignification enhanced cellulase effectiveness more for high pH than low pH pretreatments, suggesting that lignin impedes access of enzymes to xylan more than to glucan, which in turn affects glucan accessibility. In addition, lignin from enzymatic digestion of AFEX and dilute acid pretreated solids inhibited Avicel hydrolysis less than ARP and flowthrough lignin, whereas acid lignin from unpretreated poplar inhibited enzymes the most. Irreversible binding of cellulase to lignin varied with pretreatment type and desorption method. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Cellulase adsorption and relationship to features of corn stover solids produced by leading pretreatments

Although essential to enzymatic hydrolysis of cellulosic biomass to sugars for fermentation to ethanol or other products, enzyme adsorption and its relationship to substrate features has received limited attention, and little data and insight have been developed on cellulase adsorption for promising pretreatment options, with almost no data available to facilitate comparisons. Therefore, adsorption of cellulase on Avicel, and of cellulase and xylanase on corn stover solids resulting from ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, lime, and sulfur dioxide (SO₂) pretreatments were measured at 4°C. Langmuir adsorption parameters were then estimated by non‐linear regression using Polymath software, and cellulase accessibility to cellulose was estimated based on adsorption data for pretreated solids and lignin left after carbohydrate digestion. To determine the impact of delignification and deacetylation on cellulose accessibility, purified CBHI (Cel7A) adsorption at 4°C and hydrolysis with whole cellulase were followed for untreated (UT) corn stover. In all cases, cellulase attained equilibrium in less than 2 h, and upon dilution, solids pretreated by controlled pH technology showed the greatest desorption followed by solids from dilute acid and SO₂ pretreatments. Surprisingly, the lowest desorption was measured for Avicel glucan followed by solids from AFEX pretreatment. The higher cellulose accessibility for AFEX and lime pretreated solids could account for the good digestion reported in the literature for these approaches. Lime pretreated solids had the greatest xylanase capacity and AFEX solids the least, showing pretreatment pH did not seem to be controlling. The 24 h glucan hydrolysis rate data had a strong relationship to cellulase adsorption capacities, while 24 h xylan hydrolysis rate data showed no relationship to xylanase adsorption capacities. Furthermore, delignification greatly enhanced enzyme effectiveness but had a limited effect on cellulose accessibility. And because delignification enhanced release of xylose more than glucose, it appears that lignin did not directly control cellulose accessibility but restricted xylan accessibility which in turn controlled access to cellulose. Reducing the acetyl content in corn stover solids significantly improved both cellulose accessibility and enzyme effectiveness. Biotechnol. Bioeng. 2009;103: 252–267. © 2009 Wiley Periodicals, Inc.     

Cellulase kinetics as a function of cellulose pretreatment

Microcrystalline cellulose (Avicel) was subjected to three different pretreatments (acid, alkaline, and organosolv) before exposure to a mixture of cellulases (Celluclast). Addition of beta-glucosidase, to avoid the well-known inhibition of cellulase by cellobiose, markedly accelerated cellulose hydrolysis up to a ratio of activity units (beta-glucosidase/cellulase) of 20. All pretreatment protocols of Avicel were found to slightly increase its degree of crystallinity in comparison with the untreated control. Adsorption of both cellulase and beta-glucosidase on cellulose is significant and also strongly depends on the wall material of the reactor. The conversion-time behavior of all four states of Avicel was found to be very similar. Jamming of adjacent cellulase enzymes when adsorbed on microcrystalline cellulose surface is evident at higher concentrations of enzyme, beyond 400 U/L cellulase/8 kU/L beta-glucosidase. Jamming explains the observed and well-known dramatically slowing rate of cellulose hydrolysis at high degrees of conversion. In contrast to the enzyme concentration, neither the method of pretreatment nor the presence or absence of presumed fractal kinetics has an effect on the calculated jamming parameter for cellulose hydrolysis.     

Fermentation of lactose by yeast cells secreting recombinant fungal lactase.

Strains of Saccharomyces cerevisiae transformed with a yeast multicopy expression vector carrying the cDNA for Aspergillus niger secretory beta-galactosidase under the control of ADH1 promoter and terminator were studied for their fermentation properties on lactose (V. Kumar, S. Ramakrishnan, T. T. Teeri, J. K. C. Knowles, and B. S. Hartley, Biotechnology 10:82-85, 1992). Lactose was hydrolyzed extracellularly into glucose and galactose, and both sugars were utilized simultaneously. Diauxic growth patterns were not observed. However, a typical biphasic growth was observed on a mixture of glucose and galactose under aerobic and anaerobic conditions with transformants of a haploid S. cerevisiae strain, GRF167. Polyploid distiller's yeast (Mauri) transformants were selected simply on the basis of the cloned gene expression on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) plates. Rapid and complete lactose hydrolysis and higher ethanol (0.31 g/g of sugar) and biomass (0.24 g/g of sugar) production were observed with distiller's yeast grown under aerobic conditions. A constant proportion (10%) of the population retained the plasmid throughout the fermentation period (48 h). Nearly theoretical yields of ethanol were obtained under anaerobic conditions on lactose, glucose, galactose, and whey permeate media. However, the rate and the amount of lactose hydrolysis were lower under anaerobic than aerobic conditions. All lactose-grown cells expressed partial galactokinase activity.     

Saccharification and fermentation of Sugar Cane bagasse by Klebsiella oxytoca P2 containing chromosomally integrated genes encoding the Zymomonas mobilis ethanol pathway

Pretreatment of sugar cane bagasse is essential for a simultaneous saccharification and fermentation (SSF) process which uses recombinant Klebsiella oxytoca strain P2 and Genencor Spezyme CE. Strain P2 has been genetically engineered to express Zymomonas mobilis genes encoding the ethanol pathway and retains the native ability to transport and metabolize cellobiose (minimizing the need for extracellular cellobiase). In SSF studies with this organism, both the rate of ethanol production and ethanol yield were limited by saccharification at 10 and 20 filter papaer units (FPU) g(-1) acid-treated bagasse. Dilute slurries of biomass were converted to ethanol more efficiently (over 72% of theoretical yield) in simple batch fermentations than slurries containing high solids albeit with the production of lower levels of ethanol. With high solids (i.e., 160 g acid-treated bagasse L(-1)), a combination of 20 FPU cellulase g(-1) bagasse, preincubation under saccharification conditions, and additional grinding (to reduce particle size) were required to produce ca. 40 g ethanol L(-1). Alternatively, almost 40 g ethanol L(-1) was produced with 10 FPU cellulase g(-1) bagasse by incorporating a second saccharification step (no further enzyme addition) followed by a second inoculation and short fermentation. In this way, a theoretical ethanol yield of over 70% was achieved with the production of 20 g ethanol 800 FPU(-1) of commercial cellulase. (c) 1994 John Wiley & Sons, Inc.     

Evaluating the distribution of cellulases and the recycling of free cellulases during the hydrolysis of lignocellulosic substrates

The recycling of cellulase enzymes is one potential strategy for reducing the cost of the enzymatic hydrolysis step during the bioconversion of lignocellulosics to ethanol. To determine the influence of lignin on the post-hydrolysis distribution of cellulase enzymes between the liquid and solid phases, the hydrolysis of Avicel was compared to an organosolv-pretreated Douglas fir substrate with a lignin content of 3.0%. After a 12 h hydrolysis reaction on Avicel, 90% of the added cellulases (including beta-glucosidases) remained "free" in the liquid phase compared to only 65% in the case of the hydrolysis of the organosolv-pretreated Douglas fir substrate. The readsorption of free cellulases by supplementing the hydrolysis reaction with fresh substrate was explored as a potential means of recovering the free cellulases that remain in the liquid phase after hydrolysis. The Langmuir adsorption isotherm was used to develop a model predicting that 82% of the free cellulases could be recovered via readsorption onto fresh substrates during the hydrolysis of an ethanol-pretreated mixed softwood substrate with a lignin content of 6%. Recoverable free cellulase values of 85% and 88% based on cellulase activity and protein content, respectively, were obtained after experimental verification of the model. The readsorption of free cellulases onto fresh lignocellulosic substrates was shown to be an effective method for free enzyme recovery.     

High titer ethanol production from simultaneous enzymatic saccharification and fermentation of aspen at high solids: a comparison between SPORL and dilute acid pretreatments

Native aspen (Populus tremuloides) was pretreated using sulfuric acid and sodium bisulfite (SPORL) and dilute sulfuric acid alone (DA). Simultaneous enzymatic saccharification and fermentation (SSF) was conducted at 18% solids using commercial enzymes with cellulase loadings ranging from 6 to 15 FPU/g glucan and Saccharomyces cerevisiae Y5. Compared with DA pretreatment, the SPORL pretreatment reduced the energy required for wood chip size-reduction, and reduced mixing energy of the resultant substrate for solid liquefaction. Approximately 60% more ethanol was produced from the solid SPORL substrate (211 L/ton wood at 59 g/L with SSF efficiency of 76%) than from the solid DA substrate (133 L/ton wood at 35 g/L with SSF efficiency 47%) at a cellulase loading of 10 FPU/g glucan after 120 h. When the cellulase loading was increased to 15 FPU/g glucan on the DA substrate, the ethanol yield still remained lower than the SPORL substrate at 10 FPU/g glucan.     

Effect of glucose supplements on the fermentation of xylose by Pachysolen tannophilus

Hemicellulosic sugars, predominantly D-xylose, comprise about one-half the total carbohydrate that can be obtained from hardwoods and agricultural residues through dilute acid hydrolysis. Because rates and yields in the xylose fermentation are low, economic utilization of these materials as fermentation feedstocks is difficult. Pachysolen tannophilus formed 5.5% ethanol from 12% glucose but only 2% ethanol from 12% xylcose. Aeration doubled the specific rate of D-glucose fermentation by P. tannophilus, as compared to anaerobic fermentation, but the specific rate of the xylose fermentation remained unchanged. Periodic additions of 0.5% D-glucose to aerobic fermentations of 3% xylose increased the yield of ethanol from 0.28 g/g xylose to greater than 0.41 g/g xylose utilized. The rate of xylose utilization remained unchanged, and radiotracer studies showed that addition of 0.5% glucose did not inhibit xylose utilization under aerobic or anaerobic conditions. No enhancement was observed anaerobically, nor was enhancement observed with acid hydrolysates, apparently because of the presence of acetic acid which inhibited growth and fermentation.     

Aerobic and sequential anaerobic fermentation to produce xylitol and ethanol using non-detoxified acid pretreated corncob

Background

For economical bioethanol production from lignocellulosic materials, the major technical challenges to lower the production cost are as follows: (1) The microorganism should use efficiently all glucose and xylose in the lignocellulose hydrolysate. (2) The microorganism should have high tolerance to the inhibitors present in the lignocellulose hydrolysate. The aim of the present work was to combine inhibitor degradation, xylitol fermentation, and ethanol production using a single yeast strain.

Results

A new process of integrated aerobic xylitol production and anaerobic ethanol fermentation using non-detoxified acid pretreated corncob by Candida tropicalis W103 was proposed. C. tropicalis W103 is able to degrade acetate, furfural, and 5-hydromethylfurfural and metabolite xylose to xylitol under aerobic conditions, and the aerobic fermentation residue was used as the substrate for ethanol production by anaerobic simultaneous saccharification and fermentation. With 20% substrate loading, furfural and 5-hydroxymethylfurfural were degraded totally after 60 h aerobic incubation. A maximal xylitol concentration of 17.1 g l^-1 was obtained with a yield of 0.32 g g^-1 xylose. Then under anaerobic conditions with the addition of cellulase, 25.3 g l^-1 ethanol was produced after 72 h anaerobic fermentation, corresponding to 82% of the theoretical yield.

Conclusions

Xylitol and ethanol were produced in Candida tropicalis W103 using dual-phase fermentations, which comprise a changing from aerobic conditions (inhibitor degradation and xylitol production) to anaerobic simultaneous saccharification and ethanol fermentation. This is the first report of integrated xylitol and ethanol production from non-detoxified acid pretreated corncob using a single microorganism.

     

Allocation of ATP to synthesis of cells and hydrolytic enzymes in cellulolytic fermentative microorganisms: bioenergetics, kinetics, and bioprocessing

Under anaerobic, carbon limited conditions, celluloytic fermentative microorganisms face a metabolic choice with respect to the allocation of relatively scarce ATP: to invest it in cells or in hydrolytic enzymes. A model is proposed that defines an allocation parameter reflecting the fractional expenditure of ATP on cell synthesis relative to the total ATP available (gross ATP synthesized less maintenance). This parameter is then incorporated into an ATP-centered model of anaerobic cellulose fermentation based on the ethanol fermentation of yeast and the cellulase system of Trichoderma reesei. Results indicate that high processing rates are possible via a consolidated bioprocessing strategy, especially at high cellulase specific activities, and that cell/cellulase allocation represents an interesting system in which to study, and perhaps exploit, microbial evolution and metabolic control.     

OPTIMIZATION OF AFFINITY DIGESTION FOR THE ISOLATION OF CELLULOSOMES FROM Clostridium thermocellum

The affinity digestion process for cellulase purification consisting of binding to amorphous cellulose, and amorphous cellulose hydrolysis in the presence of dialysis (Morag et al., 1991), was optimized to obtain high activity recoveries and consistent protein recoveries in the isolation of Clostridium thermocellum cellulase. Experiments were conducted using crude supernatant prepared from C. thermocellum grown on either Avicel or cellobiose. While no difference was observed between Avicel-grown or cellobiose-grown cellulase in the adsorption step, differences were observed during the hydrolysis step. The optimal amorphous cellulose loading was found to be 3 mg amorphous cellulose per milligram supernatant protein. At this loading, 90–100% of activity in the crude supernatant was adsorbed. Twenty-four-hour incubation with the amorphous cellulose during the adsorption stage was found to result in maximal and stable adsorption of activity to the substrate. By fitting the adsorption data to the Langmuir model, an adsorption constant of 410 L/g and a binding capacity of 0.249 g cellulase/g cellulose were obtained. The optimal length of time for hydrolysis was found to be 3 hr for cellulase purified from Avicel cultures and 4 hr for cellulase purified from cellobiose cultures. These loadings and incubation times allowed for more than 85% activity recovery.     

Hydrolysis of lignocellulosic materials for ethanol production: a review

Lignocellulosic biomass can be utilized to produce ethanol, a promising alternative energy source for the limited crude oil. There are mainly two processes involved in the conversion: hydrolysis of cellulose in the lignocellulosic biomass to produce reducing sugars, and fermentation of the sugars to ethanol. The cost of ethanol production from lignocellulosic materials is relatively high based on current technologies, and the main challenges are the low yield and high cost of the hydrolysis process. Considerable research efforts have been made to improve the hydrolysis of lignocellulosic materials. Pretreatment of lignocellulosic materials to remove lignin and hemicellulose can significantly enhance the hydrolysis of cellulose. Optimization of the cellulase enzymes and the enzyme loading can also improve the hydrolysis. Simultaneous saccharification and fermentation effectively removes glucose, which is an inhibitor to cellulase activity, thus increasing the yield and rate of cellulose hydrolysis.     

Adsorption of Clostridium thermocellum cellulases onto pretreated mixed hardwood, avicel, and lignin

Adsorption of Avicel-hydrolyzing activity was examined with respect to: mixed hardwood flour pretreated with 1% sulfuric acid for 9 s at 220 degrees C (PTW220), lignin prepared from PTW220 by either acid or enzymatic hydrolysis, and Avicel. Experiments were conducted at 60 degrees C for all materials, and also at 25 degrees C for PTW220. Based on transient adsorption results and reaction rates, times were selected at which to characterize adsorption at 60 degrees C as follows: PTW220, 1 min; lignin, 30 min; and Avicel, 45 min. Similar results were obtained for adsorption of cellulase activity to PTW220 at 25 and 60 degrees C, and for lignin prepared by enzymatic and acid hydrolysis. For all materials, adsorption was described well by a Langmuir equation, although the reversibility of adsorption was not investigated. Langmuir affinity constants (L/g) were: PTW220, 109; lignin, 17.9; Avicel, 4.3; cellulose from PTW220, >/=187. Langmuir capacity constants were 760 for PTW220 and 42 for Avicel; the cellulase binding capacity of lignin appeared to be very high under the conditions examined, and could not be determined. At low and moderate cellulase loadings at least, the majority of cellulase activity adsorbed to PTW220 is bound to the cellulosic component. The results indicate that PTW220, and its cellulose component in particular, differ radically from Avicel with respect to adsorption. Avicel-hydrolyzing activity and CMC-hydrolyzing activities were found to bind to Avicel with a constant ratio of essentially one, consistent with adsorption of a multi-activity complex. (c) 1993 John Wiley & Sons, Inc.     

Effect of enzyme supplementation at moderate cellulase loadings on initial glucose and xylose release from corn stover solids pretreated by leading technologies

Moderate loadings of cellulase enzyme supplemented with beta-glucosidase were applied to solids produced by ammonia fiber expansion (AFEX), ammonia recycle (ARP), controlled pH, dilute sulfuric acid, lime, and sulfur dioxide pretreatments to better understand factors that control glucose and xylose release following 24, 48, and 72 h of hydrolysis and define promising routes to reducing enzyme demands. Glucose removal was higher from all pretreatments than from Avicel cellulose at lower enzyme loadings, but sugar release was a bit lower for solids prepared by dilute sulfuric acid in the Sunds system and by controlled pH pretreatment than from Avicel at higher protein loadings. Inhibition by cellobiose was observed to depend on the type of substrate and pretreatment and hydrolysis times, with a corresponding impact of beta-glucosidase supplementation. Furthermore, for the first time, xylobiose and higher xylooligomers were shown to inhibit enzymatic hydrolysis of pure glucan, pure xylan, and pretreated corn stover, and xylose, xylobiose, and xylotriose were shown to have progressively greater effects on hydrolysis rates. Consistent with this, addition of xylanase and beta-xylosidase improved performance significantly. For a combined mass loading of cellulase and beta-glucosidase of 16.1 mg/g original glucan (about 7.5 FPU/g), glucose release from pretreated solids ranged from 50% to75% of the theoretical maximum and was greater for all pretreatments at all protein loadings compared to pure Avicel cellulose except for solids from controlled pH pretreatment and from dilute acid pretreatment by the Sunds pilot unit. The fraction of xylose released from pretreated solids was always less than for glucose, with the upper limit being about 60% of the maximum for ARP and the Sunds dilute acid pretreatments at a very high protein mass loading of 116 mg/g glucan (about 60 FPU).     

Enhanced microbial utilization of recalcitrant cellulose by an ex vivo cellulosome-microbe complex

A cellulosome-microbe complex was assembled ex vivo on the surface of Bacillus subtilis displaying a miniscaffoldin that can bind with three dockerin-containing cellulase components: the endoglucanase Cel5, the processive endoglucanase Cel9, and the cellobiohydrolase Cel48. The hydrolysis performances of the synthetic cellulosome bound to living cells, the synthetic cellulosome, a noncomplexed cellulase mixture with the same catalytic components, and a commercial fungal enzyme mixture were investigated on low-accessibility recalcitrant Avicel and high-accessibility regenerated amorphous cellulose (RAC). The cell-bound cellulosome exhibited 4.5- and 2.3-fold-higher hydrolysis ability than cell-free cellulosome on Avicel and RAC, respectively. The cellulosome-microbe synergy was not completely explained by the removal of hydrolysis products from the bulk fermentation broth by free-living cells and appeared to be due to substrate channeling of long-chain hydrolysis products assimilated by the adjacent cells located in the boundary layer. Our results implied that long-chain hydrolysis products in the boundary layer may inhibit cellulosome activity to a greater extent than the short-chain products in bulk phase. The findings that cell-bound cellulosome expedited the microbial cellulose utilization rate by 2.3- to 4.5-fold would help in the development of better consolidated bioprocessing microorganisms (e.g., B. subtilis) that can hydrolyze recalcitrant cellulose rapidly at low secretory cellulase levels.     

Ethanol from whey: Continuous fermentation with cell recycle

The production of ethanol from cheese whey lactose has been demonstrated using a single-stage continuous culture fermentation with 100% cell recycle. In a two-step process, an aerobic fed batch operation was used initially to allow biomass buildup in the absence of inhibitory ethanol concentrations. In the anaerobic ethanol-producing second step, a strain of Kluyveromyces fragilis selected on the basis of batch fermentation data had a maximum productivity of 7.1 g ethanol/L/h at a dilution rate of 0.15 h(-1), while achieving the goal of zero residual sugar concentration. The fermentation productivity diminished when the feed sugar concentation exceeded 120 g/L despite the inclusion of a lipid mixture previous shown to enhance batch fermentation productivities.