EP2013355A2

EP2013355A2 - Process for the hydrolysis of cellulose mediated by ternary complexes of cellulose, clostridium thermocellum cells, and cellulase expressed by these cells

Info

Publication number: EP2013355A2
Application number: EP07811862A
Authority: EP
Inventors: Lee R. Lynd; Yi-Heng Percival Zhang; Yanpin Lu
Original assignee: Dartmouth College
Current assignee: Dartmouth College
Priority date: 2006-05-01
Filing date: 2007-05-01
Publication date: 2009-01-14
Also published as: AU2007253987A1; CN101466843A; WO2007136971A2; JP2009535067A; ZA200809448B; BRPI0711163A2; CN101466843B; CA2651753A1; WO2007136971A3

Abstract

Methods of utilizing reduced cellulase loads to hydrolyze cellulosic substrates are disclosed. The methods include determining an amount of purified cellulase necessary to substantially hydrolyze a quantity of cellulosic substrate in 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 to the cellulosic substrate either (1 ) a microorganism expressing cell-bound cellulase in a concentration equal to the reduced cellulase load or (2) a fermentation agent that has been engineered to express cell-bound cellulase in a concentration equal to the reduced cellulase load under suitable conditions and for said period of time sufficient to allow substantial hydrolysis of the cellulosic substrate.

Description

ENZYME-MICROBE SYNERGY

RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. provisional patent application serial no. 60/796,635, filed May 1 , 2006, which is incorporated by reference herein.

GOVERNMENT RIGHTS

[0002] The United States Government may have certain rights in the present invention as research relevant to its development was funded by National Institute of Standards and Technology (NIST) contract number 60NANB1 D0064 and by Department of Energy contract number DE-FG02- 02ER15350.

BACKGROUND

[0003] Cellulosic biomass represents an inexpensive and readily available resource which may be fermented to produce ethanol or other products. Among bioconversion products, interest in ethanol is high because it may be produced as a renewable domestic fuel that could offer benefits in terms of sustainability, security and rural economic development.

[0004] As bioconversion processes strive to become economically competitive with petroleum fuel technologies, they face challenges associated with the hydrolysis of cellulose into simple sugars. Process steps associated with overcoming the recalcitrance of cellulosic biomass are generally the most costly and have the greatest potential for R&D-driven improvement. Cellulose recalcitrance is typically overcome by acid pretreatment followed by enzymatic breakdown of the pretreated cellulose by cellulase enzymes. Cost estimates for cellulase enzymes currently range from $0.30 - 0.50/gallon of ethanol. With the cost of enzymes acting as a limiting factor in the production of ethanol from cellulosic biomass, any means of reducing the amount of cellulase necessary for ethanol production will remain an important commercial goal. Accordingly, efforts have in the past been devoted to understanding the mode of action of multi-component cellulase enzyme systems, and to improving their effectiveness.

[0005] Enzymatic hydrolysis can be mediated by cellulase enzymes acting in the absence of cells, by cellulases acting in the presence of cells but with no cell-enzyme attachment, or by cellulases attached to cells. In the latter case, hydrolysis is mediated by ternary cellulose-enzyme-microbe (CEM) complexes rather than binary cellulose-enzyme (CE) complexes.

[0006] It is known that cells of aerobic microorganisms do not adhere (or only weakly adhere) to cellulose. Thus, the main agent of cellulose hydrolysis in aerobic systems is cellulase enzyme bound to cellulose to form a binary CE complex. These CE complexes feature discretely-acting functionally-distinct proteins. By contrast, most anaerobic microorganisms adhere to cellulose, and the main agent of cellulose hydrolysis is a ternary CEM complex, in many but not all cases involving "cellulosome", wherein multiple functionally-distinct proteins act in concert.

[0007] The phenomenon of synergy between components of CE complexes, whereby the rate realized by two or more components in combination is greater than the sum of the rates observed when the components act separately, has been observed and evaluated in the literature. However, "enzyme-microbe" synergy of CEM complexes has not been previously evaluated and quantified.

SUMMARY

[0008] The instrumentalities reported herein advance the art by providing a method for reducing the amount of enzyme necessary to achieve hydrolysis of a given amount of biomass, or alternatively increasing the amount of biomass that may be hydrolyzed by a given amount of enzyme.

[0009] In one embodiment, a method of utilizing a reduced cellulase load to hydrolyze a cellulosic substrate, includes: determining an amount of purified cellulase necessary to substantially hydrolyze a quantity of cellulosic substrate in 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 to the cellulosic substrate a microorganism expressing cell-bound cellulase in a concentration equal to the reduced cellulase load under suitable conditions and for said period of time sufficient to allow substantial hydrolysis of the cellulosic substrate. [0010] In one embodiment, a method of utilizing a reduced cellulase load to hydrolyze a cellulosic substrate, includes: determining an amount of purified cellulase necessary to substantially hydrolyze a quantity of cellulosic substrate in 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 a fermentation agent that has been engineered to express cell- bound cellulase to the cellulosic substrate under suitable conditions and for said period of time sufficient to allow substantial hydrolysis and fermentation of the cellulosic substrate, wherein the cell-bound cellulase is present in a concentration equal to the reduced cellulase load.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 shows cellulose utilization by a microbe, where hydrolysis is mediated by both CEM and CE complexes.

[0012] FIG. 2 shows enzymatic hydrolysis of cellulose during a Simultaneous Saccharification and Fermentation (SSF) reaction involving a CE complex.

[0013] FIG. 3 illustrates changes in pH and product concentration over time for the experiment depicted in FIG. 1.

[0014] FIG. 4 illustrates changes in pH and product concentration over time for the experiment depicted in FIG. 2. [0015] FIG. 5 shows cellulose concentration curves for the microbial and SSF experiments depicted in FIGS. 1 and 2, as well as control experiments.

[0016] FIG. 6 shows cellulose hydrolysis and product accumulation for cell-free control 1. [0017] FIG. 7 shows cellulose hydrolysis and product accumulation for continuous SSF of Avicel by T. thermosaccharolyticum in the presence of 0.064 g/L purified C. thermocellum cellulosome.

[0018] FIG. 8 shows cellulose hydrolysis and product accumulation for continuous SSF of Avicel by T. thermosaccharolyticum in the presence of 0.052 g/L purified C. thermocellum cellulosome.

DETAILED DESCRIPTION

[0019] Synergy involving enhancement of the effectiveness of cellulase by virtue of expression on the surface of a microbial cell as compared to when the cellulase is not cell-bound is disclosed herein. Such

"enzyme-microbe" synergy was quantitatively evaluated, and was shown to give rise to a substantial increase in the effectiveness of cellulase enzymes.

[0020] The studies were carried out on Clostridium thermocellum, which is an anaerobic, thermophilic bacterium that exhibits one of the highest rates of cellulose utilization (hydrolysis and fermentation) among described microorganisms. C. thermocellum produces a cellulase complex, or "cellulosome", with a substantial fraction of the cellulosome bound to the cell surface under most culture conditions. Hydrolysis of microcrystalline cellulose (Avicel) was analyzed in batch and continuous cultures for the following two systems:

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

(b) Enzymatic hydrolysis by purified C. thermocellum cellulosome with fermentation of hydrolysis products by the non-cellulolytic thermophilic anaerobe Thermoanaerobacterium thermosaccharolyticum. In this SSF process, hydrolysis is mediated by CE complexes only. [0021] Specific rates of cellulose hydrolysis were found to be about 2 to 5-fold higher for growing cultures of Clostridium thermocellum as compared with purified cellulase preparations from the same organism. Thus, accelerated reactions occur when C. thermocellum cellulase is presented on the surface of a cellulose-adhered cell as compared to when the C. thermocellum cellulase acts without cell attachment.

[0022] From an applied science perspective, this 2 to 5-fold synergistic effect is significant in the context of strategies that decrease the cost of enzymatic hydrolysis. In particular, quantification of enzyme-microbe synergy provides an indication that higher hydrolysis rates may be achieved in process configurations in which cellulose is hydrolyzed by adherent cellulolytic microbes as compared to processes in which hydrolysis is carried out by cellulase enzymes in the absence of cellulolytic microbes. [0023] The presence of a cellulose-adherent cellulolytic microbe may increase hydrolysis rates by lowering the local concentration of inhibitory hydrolysis products through fermentation. For example, cellobiose, and to a lesser extent glucose, are known to inhibit the C. thermocellum cellulosome. Additionally, adherent microorganisms may be rewarded from an evolutionary perspective, since organisms with improved substrate access (higher concentration of substrate at the cell surface and/or less loss of substrate to the bulk medium) would presumably grow faster and thus have a selective advantage.

[0024] A list of cellulolytic anaerobes that mediate cellulose hydrolysis primarily via CEM complexes is presented in Table 1.

Table 1. Cellulolvtic Anaerobes

Clostridium straminosolvens

Acetivibrio cellulolyticus

Clostridium cellulolyticum Clostridium stercorarium subs, stercorarium

Clostridium stercorarium subs, thermolacticum

Clostridium stercorarium subs, leptospartum

Clostridium hungatei

Caldicellulosiruptor kristjanssonii Clostridium phytofermentans

Clostridium thermocellum

Ruminococcus albus Ruminococcus flavefaciens Butyrivibrio fibrisolvens Caldicellulosiruptor saccharolyticum Eubacterium cellulosolvens Fervidobacterium cellulosolvens Fibrobacter succinogenes Spirochaeta thermophila Thermotoga neapolitana Neocallimastix sp. (anaerobic cellulolytic fungi) Pyromyces sp. (anaerobic cellulolytic fungi)

[0025] The benefits of enzyme-microbe synergy may be exploited by utilizing any of the anaerobic hosts shown in Table 1 for hydrolysis of a cellulosic substrate. Further, it may be advantageous to alter the hosts in Table 1 (e.g., via genetic engineering or selection following an evolutionary challenge) to have improved product producing properties (e.g., titer, yield) while retaining enzyme-microbe synergy.

[0026] Alternatively, a fermentation agent that is not naturally cellulolytic 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 CEM complexes and achieve higher rates of cellulose hydrolysis as a result of enzyme-microbe synergy. Recombinant organisms that express tethered 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 may be engineered to express cellulase are listed in Table 2.

Table 2. Fermentation agents that may be engineered to express cellulase Saccharomyces cerevisiea Zymomonas mobilis Escherichia coli Klebsiella oxytoca Clostridium acetobutylicum Schizosaccharomyces pombe Candida albicans Kluyveromyces lactis Pichia pastoris Pichia stipitis Yarrowia lipolytica Hansenula polymorpha Phaffia rhodozyma Candida utilis

Arxula adeninivorans Debaryomyces hansenii Debaryomyces polymorphus Schwanniomyces occidentalis [0027] Cellulosic substrates that may be hydrolyzed according to the present instrumentalities include, but are not limited to, grasses, such as switch grass, cord grass, rye grass, reed canary grass, miscanthus, or a combination thereof; sugar-processing residues, such as but not limited to sugar cane bagasse; agricultural wastes, such as but not limited to rice straw, rice hulls, barley straw, corn cobs, wheat straw, canola straw, oat straw, oat hulls, and corn fiber; stover, such as but not limited to soybean stover, corn stover; and forestry wastes, such as but not limited to recycled wood pulp fiber, sawdust, hardwood, softwood, or any combination thereof; ruminant digestion products; municipal wastes; paper mill effluent; newspaper; cardboard; and combinations of any of the above mentioned substrates. Examples of hardwoods considered for ethanol production may include willow, maple, oak, walnut, eucalyptus, elm, birch, buckeye, beech, and ash. Examples of softwoods considered for ethanol production may include southern yellow pine, fir, cedar, cypress, hemlock, larch, pine, and spruce, or combinations thereof. EXAMPLE 1 MICROBIALLY-MEDIATED VERSUS ISOLATED-ENZYME-MEDIATED

CELLULOSE HYDROLYSIS

[0028] Microbially-mediated cellulose hydrolysis by Clostridium thermocellum was systematically compared to enzymatically-mediated hydrolysis carried out by purified C. thermocellum cellulosomes in the presence and absence of Thermoanaerobacterium thermosaccharolyticum, a non-cellulolytic thermophile capable of utilizing soluble products of cellulose hydrolysis. Ternary CEM complexes are present in the case of microbially- mediated hydrolysis whereas cellulose hydrolysis occurs exclusively due to the action of binary CE complexes in the case of enzymatically-mediated hydrolysis.

[0029] Batch culture of C. thermocellum and relevant controls. Five ml of a Clostridium thermocellum (ATCC 27405) stock culture was inoculated via syringe into 100 ml MTC medium containing 2 g/L Avicel

PH105 (FMC Corp., Philadelphia, PA) and 10 g/L MOPs buffer (initial pH 7.6) in triplicate 200 ml sealed serum vials (Bellco Biotechnology, Vineland, NJ) under a N₂ atmosphere. Cultures were incubated at 60⁰C in a temperature controlled water bath with rotary shaking at 200 rpm. Once 2 g/L Avicel was consumed, as determined by visual inspection, supplemental Avicel was added via syringe as a 40 g/L sterile suspension to a concentration of 2 g/L, pH was adjusted to 7.6 by addition of 4M NaOH, and the gas phase was replaced by flushing with filter-sterilized N₂.

[0030] Microbial cellulose utilization data are taken with the initial (time zero) data point just after supplemental Avicel addition. Microbial control 1 was carried out as above except that a sterilized 1 M sodium azide solution was added to a final concentration of 38.5 mM in conjunction with supplemental Avicel addition. Addition of azide as specified above resulted in cessation of fermentation as indicated by constant concentrations of fermentation products over time as determined by HPLC.

[0031] Cellulosome preparation and purification. Cellulosome used for batch SSF experiments was obtained from batch cultures of C. thermocellum 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 steady-state continuous cultures of C. thermocellum grown in MTC medium at a dilution rate (flow rate/fermentor working volume) of 0.052 hr^"1 and feed cellulose concentration of 4 g/L. Cellulosome was purified from the supernatant of the culture broth by affinity digestion. Purified cellulosome preparations used for batch and continuous SSF experiments contained approximately 1.2 g/L cellulase with a specific activity of 2.8 IU/mg cellulase in Tris buffer (5OmM, with 1OmM CaCb, pH 6.8). The concentration of soluble hydrolysis products in the purified cellulosome preparation was verified by HPLC to be sufficiently small (< 0.002 g/L) so as not to complicate the interpretation of SSF experiments.

[0032] Batch SSF and relevant enzyme controls. Five ml of a Thermoanaerobacter thermosaccharolyticum (ATCC 31960) stock culture was inoculated into 100 ml MTC medium containing 2 g/L Avicel PH105 and 2 g/L cellobiose in triplicate 200 ml serum vials under a N2 atmosphere. Cultures were incubated at 6O⁰C in a temperature controlled water bath with rotary shaking at 200 rpm. Once cellobiose was consumed, as determined by HPLC, pH was adjusted to 7.6, and a purified cellulosome preparation (above) was filter sterilized (Millex-GV, 0.22um pore size, Millipore, Billerica, MA) and added to the culture via syringe to a final concentration of 100 mg/L. SSF data were taken with the initial (time zero) data point just after cellulase addition. Cell-free control 1 was carried out in the presence of 2 g/L Avicel and 100 mg/L purified cellulosome as above except that no fermenting organism was present. Cell-free control 2 was carried out as for cell-free control 1 except that a sterilized 1 M sodium azide solution was added to a final concentration of 38.5 mM.

[0033] Continuous culture. A modified 1 L fermentor (Applikon, Dependable Instruments, Foster City, CA, modified by NDS) with an overflow sidearm (i.d. 0.38") and 0.5 L working volume was used for both microbial fermentation by C. thermocellum and for SSF carried out in continuous mode. pH was controlled at 6.8 by a Delta V process control system (New England Controls Inc., Mansfield, MA) with addition of 4M NaOH, the fermentor was stirred at 250 rpm, and temperature was controlled at 60⁰C by circulating hot water through the fermentor jacket. MTC medium containing 4 g/L Avicel PH105 was fed by a peristaltic pump to achieve the desired residence times. For SSF experiments, an additional peristaltic pump was used to deliver purified cellulosome, stored at 4°C, in 5OmM Tris buffer (pH 6.8). The compositions of MTC medium and the concentration of Avicel used for SSF experiments were adjusted to provide the same concentrations as those used in C. thermocellum fermentation experiments (e.g., final concentration of 4 g/L Avicel). SSF experiments were initiated by inoculating 50 ml of a late- exponential phase culture of T. thermosaccharolyticum into MTC medium containing 4 g/L Avicel supplemented with 2 g/L cellobiose. Once growth was evident, cellulase addition commenced. Samples used to calculate steady- state values for continuous fermentations were taken at intervals of at least one residence.

[0034] Measurement of residual cellulose and fermentation products. Residual cellulose was determined by quantitative saccharification. 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) H₂SO₄ as effluent and a refractive index detector. Oligomer sugars were analyzed according to the modified NREL post-hydrolysis procedure reported by Ehrman, C.I., M. E. Himmel. Biotechnology Techniques, 8(2): 99 (1994).

[0035] Measurement of protein, cellulosome, and cellulase activity. The protein content in supernatant samples was determined with bovine serum albumin as the standard in accordance with the Bradford protein assay (Bradford, M. M. Anal. Biochem. 72, 24 (1976)). Protein content in the pellet was measured using the pellet protein assay described previously by Zhang et al. (Zhang, Y.-H. P, L. R. Lynd. J. Bacteriol. 187, 99 (2005)). Supernatant and pellet cellulosome concentrations were determined by an ELISA method reported by Dubois, M., K. Gilles, J. K. Hamilton, P.A. Rebers, F. Smith. Nature. 168, 167 (1951 ). Avicelase activity in supernatant and pellet samples was measured at 60°C, using the method of Zhang (2005), based on soluble sugar production as determined by the phenol-sulfuric acid method of Dubois (1951 ). Results are expressed in terms of International Units (IU) = 1 μmole glucose equivalent/L/min. [0036] Batch Results. In batch culture under controlled conditions, microbial hydrolysis required 16 hours for complete hydrolysis of 2 g cellulose/L during which the cellulosome and cell protein concentrations roughly doubled from 48 to 98 mg/L and 125 to 264 mg/L respectively (FIG. 1 ). As shown in FIG. 2, SSF using purified cellulosome in the presence of T. thermosaccharolyticum (enzymatic hydrolysis) required 32 hours for complete hydrolysis, during which the cellulosome concentration was 100 mg/L and the cell protein concentration increased from 160 to 260 mg/L.

[0037] The conditions under which microbial hydrolysis and SSF occurred were similar. Product concentrations and pH versus time for the reactions of FIGS. 1 and 2 are shown in FIGS. 3 and 4, respectively. The products monitored were ethanol (Eth), acetate (Act), cellobiose (CB) and glucose equivalents (GIu Eqv). Hydrolysis products (total soluble glucans, or glucose equivalents) in the growth medium were < 0.02 g/L at all times for both microbial hydrolysis and SSF, two orders of magnitude less than concentrations at which 50% inhibition is generally observed. As shown in Table 3, the cellulosome specific activity was quite similar for both microbial and enzymatic hydrolysis and remained nearly constant throughout the experiment.

Table 3. Comparison of enzyme activity in C. thermocellum batch culture, SSF and relevant enzyme control

* Beginning of phase 2 (FIGS. 1 and 2, Ohr- 48hr) of C. thermocellum batch culture and SSF, referring to 0 hr.

# End of phase 2 (FIGS. 1 and 2, Ohr- 48hr), referring to 16 hr for C. thermocellum batch and 32 hr for SSF and enzyme control. [0038] Cellulose concentration is plotted vs time in FIG. 5 for microbial hydrolysis (from FIG. 1 ), SSF (from FIG. 2) and for controls as follows: microbial control 1 , a C. thermocellum culture (100 mg/L cellulosome, 264 mg/L cell protein) with 38.5 mM sodium azide; cell-free control 1 , 100 mg/L purified cellulosome with no fermenting organism; cell-free control 2, as for cell-free control 1 with 38.5 mM sodium azide. The rate of hydrolysis is substantially higher for growing cells (microbial hydrolysis) than for metabolically-inactive cells (microbial control 1 ), in spite of the fact that the cellulosome concentration is lower through most of the experiment for microbial hydrolysis (see FIG. 1 ) than for microbial control 1 (100 mg/L cellulosome). The lower rates of hydrolysis by metabolically-inactive cells are not primarily due to the affect of azide on the cellulosome, since the rates of cell-free hydrolysis observed in the presence and absence of azide are similar.

[0039] FIG. 6 shows cellulose hydrolysis and product accumulation for cell-free control 1. It can be observed that although concentrations of hydrolysis products are an order of magnitude higher for cell-free control 1 than SSF (FIG. 6 compared to FIG. 2), the hydrolysis rates are similar. Accumulation of hydrolysis products in the bulk fermentation broth is therefore not a plausible explanation for the marked difference between microbial hydrolysis and SSF. [0040] The batch results support a degree of enzyme-microbe synergy, equal to the ratio of the cellulosome-normalized hydrolysis rates observed for the microbial system divided by that for the enzymatic system, of between 2.8 and 4.7 (Table 4). See Example 2 for further description of the quantification of enzyme-microbe synergy. [0041] Continuous Culture Results. Microbial hydrolysis and SSF were also compared in steady-state continuous cultures. Mean values for four or more steady-state data points are reported in Table 4, with cellulase specific activities in Table 5. Microbial steady states 1 and 2 were obtained at residence times (τ = fermentor volume/feed flow rate) of 6.8 and 9.8 hours, respectively. For microbial steady state 1 , 65.3% of the feed cellulose was hydrolyzed in the presence of a total cellulosome concentration of 39 mg/L, whereas 76.8% hydrolysis was achieved at 63 mg cellulosome/L for microbial steady state 2. Steady-state continuous SSF mediated by purified C. thermocellum cellulosome in conjunction with fermentation by T. thermosaccharolyticum was carried out at conditions chosen to achieve similar conversion and total cellulosome concentrations to those observed for microbial cellulose utilization. For SSF steady state 1 , comparable to microbial steady state 1 , cellulose hydrolysis of 67% was observed at τ = 24.4 hr and added cellulosome at 52 mg/L. For SSF steady state 2, cellulose hydrolysis of 75.3% was observed at τ = 19.23 hr and 63 mg/L cellulosome. The concentration of cellulose hydrolysis products was below detection limits (2.5 mg/L) for both microbial and SSF steady states. Time course SSF data are presented in FIG. 7 (SSF steady state 1 ) and FIG. 8 (SSF steady state 2). The experiment was switched from continuous to batch at 168 hours to prevent further accumulation of cellobiose; continuous feeding was reinitiated at 184 hours. Cellulase specific activity was similar for microbial and SSF steady states (Table 5).

Table 4. Continuous culture data and degree of synergy calculation.

Cellulose (g/L) Xf Cellulase (g/L) τ Spec Rate (hr ¹) Synergy

C₀(g/L) C(g/L) Ep Et (hr) c ς< r_Et u nb_E ^ε _Mi J pJiύ_E ^E _Mi

Batch

Microbial hydrolysis

After 8 hours 2 1 0 95 0 55 0 057 8 2 52 - 4 69 -

Complete reaction 2 1 0 0 1 0 0 073 16 1 80 - 2 78 -

SSF (enzymatic hydrolysis)

After 8 hours 2 07 1 64 0 21 0 10 8 0 54 -

Complete reaction 2 07 0 0 1 0 0 10 32 0 65 -

Continuous

Microbial hydrolysis

Steady state 1 4 68 1 63 0 653 0 0280 039 6 80 1 1 50 16 01 4 81 4 77

Steady state 2 4 66 1 08 0 768 0 0290 046 9 80 7 94 10 66 2 80 2 84

SSF (enzymatic hydrolysis)

Steady state 1 4 53 1 50 0 67 0 0370 052 24 4 2 39 3 36

Steady state 2 4 65 1 16 0 753 0 041 0 064 19 2 2 84 4 43 ^a(C₀ - C)/C_o All cellulose concentrations reported in terms of glucose equivalent Table 5. Continuous culture of C. thermocellum and SSF

X - conversion of cellulose E₁ - total cellulosome E_ads - adsorbed cellulosome

[0042] The continuous culture data support a degree of enzyme- microbe synergy, equal to the ratio of the cellulosome-normalized hydrolysis rates observed for the microbial system divided by that for the enzymatic system, of between 2.8 and 4.8 (Table 4). See Example 2 for further description of the quantification of enzyme-microbe synergy.

[0043] For both batch and continuous culture experiments the C. thermocellum cellulase complex is substantially more effective during microbial hydrolysis as compared to SSF under the conditions examined. Such enzyme-microbe synergy requires the presence of metabolically active cellulolytic microbes, and is not explained by removal of hydrolysis products from the bulk fermentation broth. The key apparent difference between microbial hydrolysis and SSF appears to be that CEM complexes are present during microbial hydrolysis whereas this is not the case for SSF. EXAMPLE 2

QUANTIFICATION OF ENZYME-MICROBE SYNERGY

[0044] The cellulosome-specific hydrolysis rate on a cellulosome basis ( r_c ^£ , g cellulose * g cellulosome^"1 • hr^"1) may be calculated using:

_E _ (C₀ - C)/τ

(1 ) where C₀ is the cellulose concentration in g/L either initially (for batch reactions) or in the feed (for steady-state continuous reactions), C is the fermentor cellulose concentration in g/L after time t (batch) or at steady-state

(continuous), τ is the elapsed time (batch) or the residence time (continuous),

and E is the average cellulosome concentration in g/L over the elapsed time (batch) or for multiple steady-state points (continuous). The degree of

enzyme-microbe synergy, DS_EM , may be calculated from the cellulosome-

specific hydrolysis rates observed for microbial hydrolysis and SSF using:

[0045] The degree of synergy on a total cellulosome basis, DS_E ^E _M' , is found by using the total cellulosome concentration, E₇-, in equation (1 ). Alternatively, the degree of synergy on a pellet cellulosome basis, DS^E _M' , is found if the pellet cellulase concentration (Ep, potentially including both CE and CEM complexes) is used. [0046] Values for DS_E ^C _M' calculated from batch and continuous data are presented in Table 4. DS_E ^E _M' based on batch data after eight hours is 4.69. If DS_E ^E _M' is calculated after complete cellulose hydrolysis is achieved, a value of 2.78 is obtained. In continuous culture, a DS^E _M' value of 2.80 is obtained based on microbial and SSF steady states 2, for which about 75% of the feed cellulose is hydrolyzed. For microbial and SSF steady states 1 , for which about 66% hydrolysis is achieved, DS_E ^E _M' is equal to 4.81. Values for enzyme- microbe synergy on a pellet cellulase basis, DS^E _M' , are quite similar to DS^E _M' values observed in continuous culture: 2.84 for microbial and SSF steady states 2, and 4.77 for microbial and SSF steady states 1. Decreasing synergy is seen with increasing extents of cellulose hydrolysis, and with decreasing substrate to enzyme ratios, for both batch and continuous culture.

[0047] Changes may be made in the above methods and systems without departing from the scope hereof. It should thus be noted that the matter 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 statements of the scope of the present method, which, as a matter of language, might be said to fall there between.

Claims

CLAIMSWe claim:

1. A method of utilizing a reduced cellulase load to hydrolyze a cellulosic substrate, comprising: determining an amount of purified cellulase necessary to substantially hydrolyze a quantity of cellulosic substrate in 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 to the cellulosic substrate a microorganism expressing cell- bound cellulase in a concentration equal to the reduced cellulase load under suitable conditions and for said period of time to allow substantial hydrolysis of the cellulosic substrate.

2. The method of claim 1 , wherein the factor is between 2.8 and 4.8.

3. The method of claim 1 , wherein the microorganism is a member of a genus selected from Clostridium, Acetivibrio, Caldicellulosiruptor, Ruminococcus, Butyrivibrio, Eubacterium, Fervidobacterium, Fibrobacter, Spirochaeta, Thermotoga, Neocallimastix and Pyromyces.

4. The method of claim 1 , wherein the microorganism is a member of the Clostridium genus.

5. The method of claim 1 , wherein the microorganism is selected from the group consisting of Clostridium straminosolvens, Acetivibrio cellulolyticus, Clostridium cellulolyticum, Clostridium stercorarium subs, stercorarium, Clostridium stercorarium subs, thermolacticum, Clostridium stercorarium subs, leptospartum, Clostridium hungatei, Caldicellulosiruptor kristjanssonii, Clostridium phytofermentans, Clostridium thermocellum, Ruminococcus albus, Ruminococcus flavefaciens, Butyrivibrio fibrisolvens, Caldicellulosiruptor saccharolyticum, Eubacterium cellulosolvens, Fervidobacterium cellulosolvens, Fibrobacter succinogenes, Spirochaeta thermophila, Thermotoga neapolitana, Neocallimastix sp. and Pyromyces sp.

6. The method of claim 1 , wherein the microorganism is a strain of Clostridium thermocellum.

7. The method of claim 1 , wherein the cellulosic substrate is selected from grasses, sugar-processing residues, agricultural wastes, stover, forestry wastes, ruminant digestion products, municipal wastes, paper mill effluent, newspaper, cardboard and combinations thereof.

8. The method of claim 1 , wherein the microorganism is an anaerobic microorganism.

9. The method of claim 8, wherein the suitable conditions comprise a substantially oxygen-free atmosphere.

10. A method of utilizing a reduced cellulase load to hydrolyze a cellulosic substrate, comprising: determining an amount of purified cellulase necessary to substantially hydrolyze a quantity of cellulosic substrate in 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 a fermentation agent that has been engineered to express cell-bound cellulase to the cellulosic substrate under suitable conditions and for said period of time sufficient to allow substantial hydrolysis and fermentation of the cellulosic substrate, wherein the cell-bound cellulase is present in a concentration equal to the reduced cellulase load.

11. The method of claim 10, wherein the factor is between 2.8 and 4.8.

12. The method of claim 10, wherein the fermentation agent is a member of a genus selected from Saccharomyces, Zymomonas, Escherichia, Klebsiella, Clostridium, Schizosaccharomyces, Candida, Kluyveromyces, Pichia, Yarrowia, Hansenula, Phaffia, Arxula, Debaryomyces, Debaryomyces and Schwanniomyces.

13. The method of claim 10, wherein the fermentation agent is a member of the Saccharomyces genus.

14. The method of claim 10, wherein the fermentation agent is selected from the group consisting of Saccharomyces cerevisiea, Zymomonas mobilis, Escherichia coli, Klebsiella oxytoca, Clostridium acetobutylicum, Schizosaccharomyces pombe, Candida albicans, Kluyveromyces lactis, Pichia pastoris, Pichia stipitis, Yarrowia lipolytica, Hansenula polymorpha, Phaffia rhodozyma, Candida utilis, Arxυla adeninivorans, Debaryomyces hansenii, Debaryomyces polymorphus and Schwanniomyces occidentalis.

15. The method of claim 10, wherein the fermentation agent is a

Saccharomyces cerevisiea.

16. The method of claim 10, wherein the cellulosic substrate is selected from grasses, sugar-processing residues, agricultural wastes, stover, forestry wastes, ruminant digestion products, municipal wastes, paper mill effluent, newspaper, cardboard and combinations thereof.

17. The method of claim 10, wherein the fermentation agent is an anaerobic fermentation agent.

18. The method of claim 17, wherein the suitable conditions comprise a substantially oxygen-free atmosphere.