BRPI0711163A2 - method of using a reduced cellulase charge to hydrolyze a cellulosic substrate - Google Patents

Abstract

METHOD OF USING A REDUCED CELLULASE LOAD TO HYDROLYZE A CELLULOSIC SUBSTRATE. The present invention relates to methods of using reduced cellulase fillers to hydrolyze cellulosic substrates. The methods include determining the amount of purified cellulase needed to substantially hydrolyze an amount of cellulosic substrate over a period of time; reducing the amount of purified cellulase by a factor of between 2 and 5 to determine a reduced cellulase load; and introducing, in the cellulosic substrate, (1) a microorganism expressing cell-bound cellulose at a concentration equal to the reduced cellulase load or (2) a fermentation agent that has been manipulated to express cell-bound cellulase at a concentration equal to the reduced cellulase load under suitable conditions and for a period of time sufficient to allow substantial hydrolysis of the cellulosic substrate.

Description

Patent Descriptive Report for "SYNERGIAENZYME-MICROBIO".

Related Requests

The present application claims the priority benefit of U.S. Provisional Patent Application no. 60 / 796,635, filed May 1, 2005, which is incorporated by reference herein.

Government Rights

The United States Government may have certain rights in this invention as research relevant to its development has been funded by the National Institute of Standards and Technology (NIST) 1 contract number 60NANB1D0064 and the Department of Energy, contract number DE- FG02-02ER15350.

Background

Cellulosic biomass is a cheap and readily available resource that can be fermented to produce ethanol or other products. Among bioconversion products, the interest in ethanol is high because it can be produced as a renewable domestic fuel that could offer benefits in terms of sustainability, safety and rural economic development.

Since bioconversion processes aspire to become economically competitive with petroleum fuel technologies, they face challenges associated with simple sugar cellulose hydrolysis. Process steps associated with overcoming resistance of cellulosic biomass are generally the most expensive and have the greatest potential for improved R&D. Cellulose resistance is typically overcome by acid pretreatment, followed by enzymatic disruption of cellulase enzyme pretreated cellulose. Cost estimates for cellulase enzymes currently range from $ 0.30-0.50 / gallon of ethanol. As the cost of enzymes acting as a limiting factor in the production of ethanola from cellulosic biomass, any means of reducing the amount of cellulase required for ethanol production would remain an important commercial objective. Consequently, efforts have been devoted to understanding the mode of action of multi-component enzyme systems and improving their effectiveness.

Enzymatic hydrolysis may be mediated by cellulase enzymes that quench in the absence of cells, by cellulase acting in the presence of cells but without any enzyme-cell attachment, or by cellulase attached to cells. In the latter case, hydrolysis is mediated by ternary cellulose-enzyme-microbe (CEM) complexes rather than binary cellulose-enzyme (EC) complexes.

Aerobic microorganism cells are known not to adhere (or only poorly) to cellulose. Thus, the main agent of cellulose hydrolysis in aerobic systems is the cellulose-linked cellulase enzyme to form a binary CE complex. These EC complexes are characterized by functionally distinct proteins that act distinctly. In contrast, most anaerobic microorganisms adhere to cellulose and the main cellulose hydrolysis agent is a ternary EMC complex in many, but not all cases involving "cellulosomes" in which multiple functionally distinct proteins act together.

The synergy phenomenon between components of EC complexes, so the rate obtained by two or more components in combination is greater than the sum of the rates observed when the components act distinctly, has been observed and evaluated in the literature. However, the "enzyme-microbe" synergy of EMF complexes has not been previously evaluated and quantified.

summary

The media reported herein refine the technique by providing a method for reducing the amount of enzyme required to hydrolyze a given amount of biomass or, alternatively, increasing the amount of biomass that can be hydrolyzed by a given amount of enzyme. .

In one embodiment, a method of using a cellulase charge to hydrolyze a cellulosic substrate includes: determining an amount of purified cellulase required to substantially hydrolyze an amount of cellulosic substrate over a period of time; reducing the amount of purified cellulase by a factor of between 2 and 5 to determine a reduced cellulose load; and introducing into the cellulosic substrate a cell-bound cellulase expressing microorganism at a concentration equal to the reduced cellulase charge under suitable conditions for a period of time to permit substantial hydrolysis of the cellulosic substrate.

In one embodiment, a method of using a cellulase charge to hydrolyze a cellulosic substrate includes: determining an amount of purified cellulase required to substantially hydrolyze an amount of cellulosic substrate over a period of time; reducing the amount of purified cellulase by a factor of between 2 and 5 to determine a reduced cellulase charge; and introducing a fermentation agent that has been engineered to express cell-cell-bound to cellulosic substrate under conditions and for a suitable time period to permit substantial hydrolysis and fermentation of cellulosic substrate, wherein cell-bound cellulase is present at a concentration equal to the reduced charge. of cellulase.

Brief Description of the Drawings

Figure 1 shows the use of cellulose by a microbe, where hydrolysis is mediated by CEM and CE complexes.

Figure 2 shows the enzymatic hydrolysis of cellulose during a Concurrent Saccharification and Fermentation (SSF) reaction involving a CÉ complex.

Figure 3 illustrates changes in pH and yield concentration as time for the experiment shown in Figure 1.

Figure 4 illustrates changes in pH and yield concentration as time for the experiment shown in Figure 2.

Figure 5 shows concentration curves for the microbial and SSF experiments depicted in FIGS. 1 and 2, as well as control experiments.

Figure 6 shows cellulose hydrolysis and product accumulation for cell free control 1.

Figure 7 shows cellulose hydrolysis and product accumulation for Avicel SSF by T. thermosaccharolyticum in the presence of 0.064 g / L purified C. thermocellum cellulosome.

Figure 8 shows hydrolysis and product accumulation for continuous Avicel SSF by T. thermosaccharolyticum in the presence of 0.052 g / L of purified C. thermocellum cellulosome.

Detailed Description

The synergy involving enhancement of cellulase efficacy in surface expression of a microbial cell when compared to when the cellulase is not cell-bound is disclosed herein. "Enzyme-microbe" talsinergia has been quantitatively evaluated and has been shown to give rise to a substantial increase in the effectiveness of cellulase enzymes.

Studies have been performed on Clostridium thermocellum, which is a thermophilic anaerobic bacterium that exhibits one of the highest rates of cellulose utilization (hydrolysis and fermentation) among the described microorganisms. C. thermocellum produces a cellulase complex or "cellulosome-ma" with a substantial fraction of the cell surface bound cellulosome under most culture conditions. Microcrystalline cellulose hydrolysis (AviceI) was analyzed in batch and continuous cultures for the following two systems:

(a) Microbial hydrolysis involving growth of deC cultures. thermocellum in the absence of added cellulase, in which hydrolysis is mediated by CEM and CEM complexes

(b) Enzymatic hydrolysis by C. thermocellumpurified cellulosome with fermentation of hydrolysis products by non-cellulolytic thermophilic anaerobic Thermoanaerobacterium thermosaccharolyticum. In this SSF process, hydrolysis is mediated by EC complexes only.

Specific cellulose hydrolysis rates have been found to be about 2 to 5 times higher for growing Clostridiumthermocellum cultures as compared to organism purified cellulase preparations. Thus, accelerated reactions occur when deC cellulase. thermocellum is presented on the surface of a cellulose-adhered cell when compared to when C. thermocellum cellulase acts in cell attachment.

From an applied scientific perspective, this 2- to 5-fold synergistic effect is significant in the context of strategies that lower the cost of enzymatic hydrolysis. In particular, quantification of synergiaenzyme-microbe provides an indication that higher hydrolysis rates can be obtained in process configurations in which cellulose is hydrolyzed by adherent cellulolytic microbes when compared to processes in which hydrolysis is performed by cellulase enzymes in the presence of cellulolytic microbes. .

The presence of a cellulose-adherent cellulolytic microbe may increase hydrolysis rates by decreasing the local concentration of inhibitory hydrolysis products by fermentation. For example, cellobiose and, to a lesser extent, glucose, are known to inhibit C. thermocellum cellulosum. In addition, adherent microorganisms may be rewarded from a historical perspective, as organisms with improved substrate access (higher cell surface undubstrate concentration and / or less substrate loss to the general medium) would presumably grow faster and thus , would have a selective advantage.

A list of cellulolytic anaerobes that mediate cellulose hydrolysis primarily via EMF complexes is presented in Table 1.

Table 1. Cellulolytic Anaerobes

Clostridiumstraminosolvens

Acetivibrio cellulolyticus

Clóstridium cellulolyticum

Clostridium stercorarium subs. stercorarium

Clóstridium stercorarium subs, thermolacticum

Clóstridium stercorarium subs, Ieptospartum

Clóstridium hungatei

Caldicellulosiruptor kristjanssoniiClostridium phytofermentansClostridium thermocellumRuminococcus albusRuminococcus flavefaeiensButyrivibrio fibrisolvensEubaeterium eellulosolvensFervidobaeterium eellulosolvensFibrobaeter sueeinogenesSpiroehaitta nataTepaehaeta thermocellum (anaerobic cellulolytic fungus) Pyromyees sp. (anaerobic cellulolytic fungus)

The benefits of enzyme-microbe synergy can be exploited using any of the anaerobic hosts shown in Table 1 for hydrolysis of a cellulosic substrate. In addition, it may be advantageous to change the hosts in Table 1 (e.g. via genetic manipulation or selection after an evolutionary stimulus) to have improved product production properties (eg titration, yield), while retaining enzyme synergy. -microbe.

Alternatively, a non-naturally cellulolytic fermenting agent may be engineered to express cellulase from one of the organisms in Table 1. Such a recombinant organism may express a cellulosome on the cell surface and the resulting organism may form EMF complexes and obtain higher cellulose hydrolysis rates as a result of enzyme-microbe synergy. Recombinant organisms expressing trapped cellulase enzymes and methods of producing such organisms are disclosed, for example, in U.S. Patent Application No. 60 / 867,018, which is expressly incorporated herein by reference. Exemplary fermentation agents that can be manipulated to express cellulase are listed in Table 2.

Table 2. Fermentation Agents That Can Be Handled to Express Cellulase

Saccharomyces cerevisiaeZymomonas mobilis Escherichia coliKlebsieiia oxytoeaCiostridiumacetobutyiicumSchizosaceharomyees pombeCandida aibieansKluyveromyces IaetisPiehia pastorisPiehia stipitisYarrowia IipoiyticaHansenula polymorphaPhaffia rhodozymaCandidautilisArxuia adeninivoransDebaryomyees hanseniiDebaryomyees polymorphusSchwanniomyees oeeidentalis

Cellulosic substrates which may be hydrolyzed according to the present means include, but are not limited to, grams such as perennial grass, common grass, rye grass, yellow grass, mis-canthus or a combination thereof; sugar processing wastes such as, but not limited to, sugarcane bagasse; agricultural waste such as, but not limited to, rice straw, rice husks, barley straw, corn cobs, wheat straw, canola straw, oat straw, oat husks and corn fiber; silage such as, but not limited to, soybean silage, corn silage and forest waste such as, but not limited to, recycled wood pulp fiber, sawdust, hardwood, softwood or any combination thereof; ruminant management products; municipal waste; effluent from shredding paper, newspaper; cardboard; and combinations of any of the above substrates. Examples of hardwoods considered for ethanol production may include willow, maple, oak, imbui, eucalyptus, elm, birch, chestnut, beech and ash. Examples of softwoods considered for ethanol production may include southern yellow pine, spruce, cypress, cypress, tsuga, larch, pine and pine or combinations thereof.

Example 1

1 Microbially Mediated Cellulose Hydrolysis Versus Isolated-Mediated Enzyme

Microbially mediated cellulose hydrolysis by Clostridi-um thermocellum was systematically compared with enzymatically mediated hydrolysis by purified C. thermocellum cellulosomes in the presence and absence of Thermoanaerobacterium thermosaccharoly-ticum, a non-cellulolytic thermophile capable of using soluble products. cellulose dahydrolysis. Ternary CEM complexes are present in the case of microbially mediated hydrolysis, while cellulose hydrolysis occurs exclusively due to the action of binary CE complexes in the case of enzymatically mediated hydrolysis.

Batch Culture and C. Thermocellum and Relevant Controls

Five ml of Clostridium thermocellum stock culture (ATCC 27405) was inoculated via a syringe into 100 ml MTC medium containing 2 g / l de-Avicel PH105 (FMC Corp., Philadelphia, PA) and 10 g / l MOP buffer (pH 7.6 ml) in sealed 200 ml triplicate serum vials (Bélico Biotechnology, Vineland, NJ) under an N 2 atmosphere. Ascultures were incubated at 60 ° C in a temperature-controlled water bath with rotary shaking at 200 rpm. Since 2 g / l of Avicel was consumed as determined by visual inspection, supplemental Avicels was added via a syringe as 40 g / l of a sterile suspension to a concentration of 2 g / l, the pH was adjusted to 7.6 through 4M NaOH was added and the gas phase was replaced by filter sterile N 2 flow.

Microbial cellulose utilization data are taken as the initial data point (time zero) exactly after addition of supplemental Avicel. Microbial control 1 was performed as above except that a sterile 1M sodium azide solution was added to a final concentration of 38.5 mM together with the addition of additional Avicel. Addition of azide as specified above resulted in termination of defermentation as indicated by constant concentrations of fermentation products over time as determined by HPLC.

Cellulosome Preparation and Purification

Cellulosome used for SSF experiments on C. thermocellum batch cultures grown in MTC medium in a 200 ml flask with Avicel as the growth substrate at an initial concentration of 4 g / L. Cellulosome for continuous SSF experiments was obtained from uniformly continuous C. thermo-cellum cultures grown in MTC medium at a dilution rate (flow rate / fermenter working volume) of 0.052 h'1 and a cellulose concentration 4 g / L. Cellulosome was purified from the broth culture supernatant by affinity digestion. Purified decellulosome preparations used for batch continuous SSF experiments contained approximately 1.2 g / l cellulase with a specific activity of 2.81 U / mg cellulase in Tris buffer (50 mM, with 10 mM CaC2, pH of 6.8). The concentration of soluble hydrolysis products in the purified cellulosome preparation was verified by HPLC as sufficiently small (<0.002 g / L) so as not to complicate interpretation of batch SSF.SFF experiments and relevant enzyme controls.

Five ml of a stock culture of Thermoanaerobacterthermosaccharolyticum (ATCC 31960) was inoculated into 100 ml TCM medium containing 2 g / l Avicel PH105 and 2 g / l cellobiose in 200 ml desiccant vials in triplicate under an N2 atmosphere. The cultures were incubated at 60 ° C in a temperature controlled water bath with rotary comagitation at 200 rpm. Once cellobiose was consumed as determined by HPLC, the pH was adjusted to 7.6 and a purified cellulosome preparation (above) was filter sterilized (Millex-GV, pore size 0, 22 μm, Millipore, Billerica, MA) and a syringe is added to the culture to a final concentration of 100 mg / L. SSF data was taken with the initial data point (time zero) just after the addition of cellulase. Cell free control 1 was performed in the presence of 2 g / L Avicei and 100 mg / L purified Ceylonome as above, except that no fermentation organism was present. Cell-free control 21 was performed as per cell-free control 1, except that a sterile 1M sodium azide solution was added to a final concentration of 38.5 mM.

A modified ILfermentor (Applikon, Dependable Instruments, Foster City, CA, NDS-modified) with an extra-leak lateral arm (0.38 "di) and a working volume of 0.5 L was used for fermentation. C. thermocellum and SSF performed at continuous pH The pH was controlled at 6.8 by a Delta V process control system (New England Controls Inc., Mansfield, MA) with the addition of 4M NaOH. , the fermenter was stirred at 250 rpm and the temperature controlled at 60 ° C by circulating hot water through the fermenter jacket MTC medium containing 4 g / l Avicel PH105 was fed through a peristaltic pump to obtain the desired residence times. For SSF experiments, an additional peristaltic pump was used to dispense purified cellulose, stored at 4 ° C, in 50mM Tris buffer (pH 6.8) .MTC medium compositions and Avicelus concentration for experiments of SSF were adjusted for to provide the same concentrations as those used in C. thermocellum fermentation experiments (eg final concentration of 4 g / L Avi-cel). SSF experiments were initiated by inoculation of 50 ml of a late exponential phase culture of T. thermosaccharolyticum in MTC medium containing 4 g / l Avicel supplemented with 2 g / l cellobiose. Once growth was evident, the addition of Samples used to calculate uniform state values for continuous fermentations were taken at intervals of at least one residence. Measurement of Residual Cellulose and Fermentation Products.

Residual cellulose was determined by quantitative saccharification. The concentrations of sugars (cellobiose, glucose) and fermentation products (lactic acid, acetic acid and ethanol) were analyzed by HPLC using a Bio-Rad HPX-87H column operated at 55 ° C with 0.01% (v / v ) of H2SO4 as effluent and a refractive index detector. Oligomeric sugars were analyzed according to the modified NREL posthydrolysis procedure reported by Ehrman1 C.I., M.E. Himmel. Biotechnology Techniques, 8 (2): 99 (1994).

Protein measurement. Cellulosome and Cellulase Activity

Protein content in supernatant samples was determined with bovine serum albumin as the standard according to the Bradford Protein Assay (Bradford, M.M. Anal. Biochem. 72, 24 (1976)). Protein content in the pellet was measured using the pellet protein assay previously described by Zhang and colleagues (Zhang, Y.-H. P, L.R. Lynd. J. BacterioL 187, 99 (2005)). Cellulosome concentrations in the supernatant and pellet were determined by an ELISA method reported by Dubois, M., K. Gilles, J.K. Hamilton, P.A. Rebers, F. Smith. Natu-re. 168, 167 (1951). Avicelase activity in pellet supernatant samples was measured at 60 ° C using the Zhang (2005) method based on soluble sugar production as determined using the Dubois comphenol-sulfuric acid method (1951). Results are expressed in terms of International Units (IU) = 1 μιποΙ glycoside equivalent / L / min.

Batch Results

In batch culture under controlled conditions, hydrolysemyrobial required 16 hours for complete hydrolysis of 2 g cellulose / L, during which cellulosome and cell protein concentrations almost doubled from 48 to 98 mg / L and 125 to 264 mg / L respectively (Figure 1). As shown in Figure 2, SSF using purified cellulosome in the presence of T. thermosaccharolyticum (enzymatic hydrolysis) required 32 hours for complete hydrolysis, during which the concentration of cellosomes was 100 mg / L and the concentration of cellular protein increased from 160 to 260 mg / L.

The conditions under which microbial hydrolysis and SSF occurred were similar. Product concentrations and pH versus time to reactions of FIGS. 1 and 2 are shown in FIGS. 3 and 4, respectively. The monitored products were ethanol (Et) 1 acetate (Act), cellobiose (CB) and glucose equivalents (Eq. Gli). Hydrolysis products (soluble distal glycans or glucose equivalents) in the growth medium were <0.02 g / Lem all times for microbial hydrolysis and SSF, two orders of magnitude less than the concentrations at which 50% inhibition is generally observed. As shown in Table 3, specific cell-soma activity was very similar for microbial and enzymatic hydrolysis and remained approximately constant throughout the experiment.

Table 3. Comparison of enzymatic activity in deC batch culture. thermocellum, SSF and relevant enzymatic control

<table> table see original document page 13 </column> </row> <table>

Beginning of phase 2 (FIGS. 1 and 2, 0h - 48h) of batch culture with C. thermocellum and SSF1 for 0h.

# End of phase 2 (FIGS. 1 and 2, 0h - 48h), referring to 6h parabatelated with C. thermocellum and 32h for SSF and enzymatic control.

The cellulose concentration is plotted vs. Time in Figure 5 Microbial parahydrolysis (Figure 1), SSF (Figure 2) and controls as follows: microbial control 1, a C. thermocellum culture (100 mg / L decellulosome, 264 mg / L cellular protein) with azide 38.5 mM sodium, cell-free control 1, 100 mg / L purified cellulosome without fermentation organism; cell free control 2 as per cell free control with 38.5 mM sodium azide. The hydrolysis rate is substantially higher for growing cells (microbial hydrolysis) than for metabolically inactive cells (microbial control 1), despite the fact that the concentration of cellulocyte is lower in most of the hydrolysis experiment. microbial (see figure 1) than for controlemicrobial 1 (100 mg / L of cellulosome). The lower rates of metabolically inactive cell hydrolysis are not primarily due to the affect of azide on the cellulosome, since the cell-free hydrolysis rates observed in the presence and absence of azide are similar.

Figure 6 shows cellulose hydrolysis and product accumulation for cell free control 1. It can be seen that although the concentrations of hydrolysis products are an order of magnitude larger for cell-free control 1 than for SSF (Figure 6 compared to Figure 2), hydrolysis rates are similar. Accumulation of hydrolysis products in crude fermentation broth, therefore, is not a reasonable explanation for the marked difference between microbial hydrolysis and SSF.

The batch results support a degree of enzyme-microbe synergy, equal to the proportion of the cell-normalized hydrolysis rates observed for the microbial system divided by that for the enzyme system, from 2.8 to 4.7 ( Table 4). See Example 2 for additional description of enzyme-microbe synergy quantification.

Continuous Culture Results

Microbial hydrolysis and SSF were also compared in continuous uniform cultures. Mean values for four or more uniform data points are reported in Table 4, with specific cellulase activities in Table 5. Microbial uniform states 1 and 2 were obtained at residence times (τ = fermenter volume / feed flow rate). ) of 6.8 and 9.8 hours, respectively. For microbial state 1, 65.3% of the fed cellulose was hydrolyzed in the presence of a total concentration of 39 mg cellulosome / L, while 76.8% hydrolysis was obtained at 63 mg cellulosome / L for the uniform state. 2. Continuous SSF mediated SSF of C. thermocellum purified in conjunction with fermentation by T. thermosaccharolyticum was performed under conditions chosen to obtain conversion and total cellulosome concentrations similar to those observed for the use of microbial cellulose. For SSF in state 1, comparable to microbial uniform state 1, 67% cellulose hydrolysis was observed at τ = 24.4 hours and cellulosome added at 52 mg / L. For uniform SSF 2, 75.3% cellulose hydrolysis was observed at τ = 19.23 hours and 63 mg / L cellulosome. The concentration of cellulose hydrolysis products was below detection limits (2.5mg / L) for microbial and SSF uniform states. Time course data in SSF are shown in Figure 7 (Uniform State SSF 1) and Figure 8 (Uniform State SSF 2). The experiment was switched from continuous to batch at 168 hours to prevent further accumulation of cellobiosis, and continuous feeding was restarted at 184 hours. The specific activity of cellulase was similar for microbial and SSF uniform states (Table 5). <table> table see original document page 16 </column> </row> <table> <table> table see original document page 17 </ column > </row> <table> Continuous culture data support a degree of enzyme-microbe synergy, equal to the proportion of the cellulosome-normalized hydrolysis rates observed for the microbial system divided by that for the enzyme system, from 2.8 to 4. .8 (Table 4). See Example 2 for additional description of enzyme-microbe synergy quantification.

For batch and continuous culture experiments, the C. thermocellum eelulase complex is substantially more effective during microbial hydrolysis as compared to SSF under the depleted conditions. Such enzyme-microbe synergy requires the presence of metabolically active cellulolytic microbes and is not explained by the removal of hydrolysis products from the crude fermentation broth. The obvious key difference between microbial hydrolysis and SSF appears to be that EMF complexes are present during microbial hydrolysis, while this is not the case for SSF.

EXAMPLE 2

QUANTIFICATION OF ENZYME-MICROBIO SYNERGY

The cellulosome-specific hydrolysis rate on a cellulosome basis (Γγ, g cellulose · g cellulosome-1 h "1) can be calculated using:

<formula> formula see original document page 18 </formula>

where C0 is the cellulose concentration in g / L initially (for blunt reactions) or in the feed (for continuous uniform reactions), C is the fermenter cellulose concentration in g / L after time t (beat-lada) or in a uniform (continuous) state, τ is the elapsed time (batch) or the residence time (continuous) and E is the mean cellulosic concentration in g / L for the elapsed time (batch) or for uniform points (to be continued). The degree of enzyme-microbe synergy, DSEm, can be calculated from the observed cellulosome-specific hydrolysis rates for microbial hydrolysis and SSF using: <formula> formula see original document page 19 </formula>

The degree of synergy over a total cellulosome base, <formula> formula see original document page 19 </formula>, is found using the total cellulosome concentration, ET, in equation (1). Alternatively, the degree of synergy in a cellulosome depelota base, <formula> formula see original document page 19 </formula>, is found if the concentration of cellulase in the pellet (Ep, including potentially CE and CEM complexes) is used.

Values for <formula> formula see original document page 19 </formula> calculated from continuous and batch data are presented in Table 4. <formula> formula see original document page 19 </formula> based on batch data after eight hours is 4.69. If <formula> formula see original document page 19 </formula> is calculated after complete hydrolysis of cellulose is obtained, a value of 2.78 is obtained. In continuous culture, a value of <formula> formula see original document page 19 </formula> 2.80 is obtained based on SSF microbial uniform states, for which about 75% of the fed cellulose is hydrolyzed. For microbial and SSF uniform states for which about 66% hydrolysis is obtained, <formula> formula see original document page 19 </formula> equals 4.81. Values for synergiaenzyme-microbe in a pellet cellulase base, m, are very similar to the values of <formula> formula see original document page 19 </formula> observed in continuous culture: 2.84 for uniform states 2 microbial and SSF and 4.77 for 1 microbe and SSF uniform states. Decreased synergy is observed with increasing cellulose hydrolysis extensions and decreasing undubstrate to enzyme ratios for batch and continuous culture.

Changes can be made to the above methods and systems without deviating from their scope. Thus, it should be noted that the subject contained in the above description or shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense. The following claims are intended to cover all generic and specific features described herein, as well as all scope instructions of this method which, as a matter of language, could be referred to as falling within it.

Claims (18)

A method of using a reduced cellulase charge to hydrolyze a cellulosic substrate comprising: determining the amount of purified cellulase required to substantially hydrolyze a quantity of cellulosic substrate over a period of time; reducing the amount of purified cellulase by a factor of between 2 and 5 to determine a reduced cellulase charge; introduction into the cellulosic substrate of a microorganism expressing cell-bound cellulase at a concentration equal to the reduced cellulase charge under conditions and for a suitable period of time to permit substantial hydrolysis of the cellulosic substrate. A method according to claim 1 wherein the factor is between 2.8 and 4.8. The method according to claim 1 wherein the microorganism is a member of a selected genus of Clostridium, Acetivibrio, Caldicellulosiruptor, Ruminococcus, Butyrivibrio, Eubacterium, Fervidobacterium, Fibrobacter, Spirochaeta, Thermotoga, Neomycimastix and Pyromycix. A method according to claim 1 wherein the microorganism is a member of the genus Clostridium. A method according to claim 1 wherein the microorganism is selected from the group consisting of Clostridium straminosolvens, Acetivibrio cellulolyticus, Clostridium cellulolyticum, Clostridium stercorariumsubs. stercorarium, Clostridium stercorarium subs, thermolacticum, Clostridi-a stercorarium subs, leptospartum, Clostridium hungatei, Caldicellulosirup-tor kristjanssonii, Clostridium phytofermentans, Clostridium thermocellum, Ruminococcus albus, Ruminocumulus saccharum, Bacterium carcinoma, Cephalopharyngeal carcinoma cellulosolvens, Fibrobacter succinogenes, Spirochaeta thermophil-la, Thermotoga neapolitana, Neocallimastix sp. and Pyromyces sp. The method of claim 1 wherein the microorganism is a strain of Clostridium thermocellum. A method according to claim 1 wherein the cellulosic substrate is selected from grasses, sugar processing wastes, agricultural wastes, silage, forest wastes, ruin digestion products, municipal wastes, paper shredding effluent, newspaper, cardboard and combinations thereof. The method of claim 1 wherein the microorganism is an anaerobic microorganism. The method of claim 8 wherein the appropriate conditions comprise a substantially oxygen-free atmosphere. A method of using a reduced cellulase charge to hydrolyze a cellulosic substrate comprising: determining the amount of purified cellulase required to substantially hydrolyze a quantity of cellulosic substrate over a period of time, reducing the amount of purified cellulase by a factor of between. 2 and 5 to determine a reduced cellulase charge; introduction of a fermentation agent that has been engineered to express cell-bound cellulase to the cellulosic substrate under conditions and for a suitable period of time to allow substantial hydrolysis and fermentation of the cellulosic substrate, wherein the cell-bound cellulase is present in a concentration. -traction equal to reduced cellulase charge. A method according to claim 10 wherein the factor is between 2.8 and 4.8. A method according to claim 10 wherein the fermentation agent is a member of a selected genus of Saeeharomy-ces, Zymomonas, Escherichia, Klebsiellal Clostridium, Schizosaccharomy-ees, Candida, Kluyveromyees, Piehia, Yarrowia, Hansenula, Phaffia, Arxula. , Debaryomyees, Debaryomyees and Sehwanniomyees. A method according to claim 10 wherein the fermentation agent is a member of the genus Saeeharomyees. A method according to claim 10 wherein the fermentation agent is selected from the group consisting of Saccharomyces ce-revisiae, Zymomonas mobifis, Eseheriehia coli, Klebsiella oxytoea, Clostridi-a aeetobutylicum, Pigeon schizosaceharomyees, Candida albieans, Kluy-veromyces laetis Piehia pastoris, Piehia stipitis, Yarrowia lipolytiea, Uanse-null polymorpha, Phaffia rhodozyma, Candida utilis, Arenula adeninivorans, Debaryomyees hansenii, Debaryomyees polymorphus and Schwanniomyeesoeeidentalis. The method of claim 10 wherein the fermentation agent is a Saccharomyees eerevisiae. A method according to claim 10 wherein the cellulosic substrate is selected from grasses, sugar processing wastes, agricultural wastes, silage, forest wastes, deruminant digestion products, municipal wastes, waste crushing effluent. paper, newspaper, paper, and combinations thereof. The method of claim 10 wherein the fermentation agent is an anaerobic fermentation agent. A method according to claim 17 wherein the suitable conditions comprise a substantially oxygen free atmosphere.