JP2009535067A - Process for hydrolysis of cellulose mediated by cellulose, Clostridium thermocellum cells and cellulase ternary complexes expressed by these cells - Google Patents

Abstract

A method is disclosed that utilizes reduced cellulase loading to hydrolyze cellulosic substrates. The method determines the amount of purified cellulase required to substantially hydrolyze an amount of cellulosic substrate within a period of time, reducing and reducing the amount of purified cellulase by a factor of 2-5. (1) cells at a concentration equivalent to the reduced cellulase addition amount for a period of time sufficient to determine the amount of cellulase obtained and to allow substantial hydrolysis of the cellulosic substrate under suitable conditions. Including introducing into the cellulosic substrate either a microorganism that expresses the binding cellulase, or (2) a fermentation factor that is engineered to express cell-binding cellulase at a concentration equivalent to the reduced cellulase loading.

Description

(Related application)
This application claims the benefit of priority to US Provisional Patent Application No. 60 / 796,635, filed May 1, 2006, which is incorporated herein by reference.

(Government rights)
Because the research related to development was supported by the National Institute of Standards and Technology (NIST) under contract number 60NANB1 D0064 and by the US Department of Energy under contract number DE-FG02-02ER15350, the US government The invention may have certain rights.

Background Cellulosic biomass represents an inexpensive and readily available resource that may be fermented to produce ethanol or other products. Among biotransformed products, ethanol may be produced as a renewable household fuel that may provide benefits in terms of sustainability, safety and local economic development. Interest is high.

  Bioconversion processes are facing economic challenges with petroleum fuel technology and thus face the challenges associated with the hydrolysis of cellulose to monosaccharides. Processing steps associated with overcoming the difficulty of processing cellulosic biomass are generally the most costly and have the greatest potential for R & D-led improvements. The difficulty of treating cellulose is usually overcome by enzymatic degradation of the pretreated cellulose with a cellulase enzyme following acid pretreatment. Cost estimates for cellulase enzymes currently range from $ 0.30 to $ 0.50 per gallon of ethanol. Along with the cost of enzymes that act as a limiting factor in producing ethanol from cellulosic biomass, the means of reducing the amount of cellulase required for ethanol production remains an important commercial goal. Thus, past efforts have been devoted to understanding the mode of action of the multi-component cellulase enzyme system and improving its effectiveness.

  Enzymatic hydrolysis is mediated by cellulase enzymes acting in the absence of cells and cellulases acting in the presence of cells, but there is no cell-enzyme binding or cellulase bound to the cells. It is. In the latter case, hydrolysis is mediated by a ternary cellulase / enzyme / microorganism (CEM) complex rather than a binary cellulase / enzyme (CE) complex.

  It is known that aerobic microbial cells do not adhere (or only weakly adhere) to cellulose. Thus, the main agent of cellulose hydrolysis in aerobic systems is the cellulase enzyme that binds to cellulose to form a binary CE complex. These CE complexes are characterized by functionally distinct proteins that act separately. In contrast, anaerobic microorganisms mostly adhere to cellulose, and the main agent for cellulose hydrolysis is the ternary CEM complex, often involving all but not all cellulosomes, At that time, a plurality of functionally different proteins work together.

  The synergistic phenomenon between the components of the CE complex, where the ratio realized by two or more components in the combination is greater than the sum of the proportions observed when the components act separately, has been recognized and evaluated in the literature. It has been. However, the synergistic effect of the “enzyme / microorganism” of the CEM complex has not previously been evaluated or quantified.

SUMMARY By providing a method for reducing the amount of enzyme required to achieve hydrolysis of a given amount of biomass, or increasing the amount of biomass that may be hydrolyzed by a given amount of enzyme, The means reported herein advance the art.

  In one embodiment, a method that utilizes a reduced cellulase load to hydrolyze the cellulosic substrate is necessary to substantially hydrolyze the amount of cellulosic substrate within a period of time. Determining the amount of purified cellulase; reducing the amount of purified cellulase by a factor between 2 and 5 and determining the amount of cellulase added reduced; and substantial hydrolysis of the cellulosic substrate under suitable conditions Introducing a microorganism expressing cell-bound cellulase at a concentration equivalent to the amount of cellulase added to the cellulosic substrate for a sufficient time to allow

  In one embodiment, a method that utilizes reduced cellulase loading to hydrolyze cellulosic substrates requires the purification necessary to substantially hydrolyze the amount of cellulosic substrates within a period of time. Determining the amount of cellulase; reducing the amount of purified cellulase by a factor between 2 and 5 and determining the amount of cellulase added reduced; and substantial hydrolysis and fermentation of the cellulosic substrate under suitable conditions Introducing a fermentation factor engineered to express cell-bound cellulase into the cellulosic substrate for a time sufficient to permit

(Detailed explanation)
Described herein are synergistic effects including increased cellulase efficacy by expression on the surface of microbial cells as compared to when the cellulase is not cell-binding. Such “enzyme / microorganism” synergistic effects were quantitatively evaluated and shown to produce substantial increases in the effectiveness of cellulase enzymes.

The study was conducted in Clostridium thermocellum, an anaerobic thermophilic bacterium with the highest cellulose utilization (hydrolysis and fermentation) rate among the described microorganisms. C. Thermocellum produces cellulase complexes or “cellulosomes”, which have a substantial fraction of cellulosomes that bind to the surface of cells under most culture conditions. Hydrolysis of microcrystalline cellulose (Avicel) was analyzed in batch and continuous cultures for the following two systems.
(A) in the absence of added cellulase, C.I. Microbial hydrolysis involving the growth of thermocellum in culture, where the hydrolysis is mediated by both the CEM complex and the CE complex.
(B) purified C. with fermentation of the hydrolysis product by non-cellulolytic thermophilic and anaerobic Thermoanaerobacterium thermosaccharolyticum. Enzymatic hydrolysis by thermocellum cellulosome. In this SSF treatment, hydrolysis is mediated only by the CE complex.

  The specific ratio of cellulose hydrolysis was found to be 2-5 times higher in Clostridium thermocellum growth cultures compared to purified cellulase preparations from the same organism. Thus, the accelerated reaction is C.I. Compared to the case where the cellulase of thermocellum acts without binding to cells, C.I. Occurs when the cellulase of thermocellum is presented on the surface of cellulose adherent cells.

  From an applied scientific point of view, this 2-5 fold synergy is significant in the context of strategies that reduce the cost of enzymatic hydrolysis. In particular, a process in which cellulose is hydrolyzed by an adhesive cellulolytic microorganism as compared to the case where hydrolysis is carried out by a cellulase enzyme in the absence of cellulolytic microorganisms by quantifying the enzyme / microorganism synergistic effect An indication is provided that a high hydrolysis rate may be achieved in the configuration.

  The presence of cellulolytic cellulolytic microorganisms may increase the rate of hydrolysis by reducing the local concentration of inhibitory hydrolysis products via fermentation. For example, cellobiose, and to a lesser extent, glucose is C.I. It is known to inhibit thermocellum cellulosomes. In addition, organisms with improved substrate access (high substrate concentration at the cell surface and / or less loss of substrate to the bulk medium) will likely grow faster and thus have a selective advantage, so that adherent microorganisms You may be rewarded from an evolutionary perspective.

  A list of cellulolytic anaerobic microorganisms that mediate cellulose hydrolysis primarily through CEM complexes is shown in Table 1.

  (Table 1 Cellulolytic anaerobic microorganisms)

酵素／微生物の相乗効果の利益は、セルロース性基質の加水分解に表１で示された嫌気性宿主のいずれかを利用することによって活用されてもよい。さらに、表１の宿主を変えて（たとえば、遺伝子工学又は進化的挑戦に続く選抜を介して）、酵素／微生物の相乗効果を保持する一方で、改善された生成物産出特性（たとえば、力価、収率）を有することが有利であってもよい。

The benefits of enzyme / microorganism synergies may be exploited by utilizing any of the anaerobic hosts shown in Table 1 for hydrolysis of cellulosic substrates. In addition, the hosts in Table 1 can be changed (eg, through genetic engineering or selection following evolutionary challenge) while maintaining the synergistic effect of the enzyme / microbe while improving product production characteristics (eg, titer). It may be advantageous to have a yield).

  Alternatively, a cellulase derived from one of the organisms in Table 1 may be expressed by manipulating a fermentation factor that is not naturally cellulolytic. Such recombinant organisms may express cellulosomes on the cell surface, and the resulting organisms can form CEM complexes and achieve high hydrolysis rates of cellulose as a result of enzyme / microorganism synergy. Also good. Recombinant organisms that express tethered cellulase enzymes and methods for producing such organisms are described, for example, in US Patent Application No. 60 / 867,018, specifically incorporated herein by reference. Illustrative fermentation factors that can be manipulated to express cellulase are listed in Table 2.

  Table 2 Fermentation factors that can be engineered to express cellulase

本手段に従って加水分解されてもよいセルロース性基質には、たとえば、スイッチグラス、スパルティナ、ホソムギ、クサヨシ、ススキ又はこれらの組み合わせのような草類、たとえば、サトウキビの搾りカスのような、しかし、これに限定されない製糖残留物、たとえば、稲わら、籾殻、オオムギわら、トウモロコシの穂軸、コムギわら、カノーラわら、カラスムギわら、カラスムギ籾殻、及びトウモロコシ繊維、まぐさ、たとえば、大豆まぐさ、トウモロコシ、まぐさのような、しかし、これらに限定されない農業廃棄物、再生木材パルプ繊維、木屑、堅木、軟木、又はこれらの組み合わせのような、しかし、これらに限定されない林業廃棄物、反芻動物消化産物、都市ゴミ、製紙工場廃液、新聞紙、厚紙並びに上述の基質の組み合わせが挙げられるが、これらに限定されない。エタノール製造のために考慮される堅木の例には、ヤナギ、カエデ、カシ、クルミ、ユーカリ、ニレ、カバノキ、トチノキ、ブナ及びセイヨウトネリコが挙げられる。エタノール製造のために考慮される軟木の例には、サザンイエローパイン、モミ、スギ、イトスギ、アメリカツガ、カラマツ、マツ及びトウヒ又はこれらの組み合わせが挙げられる。

Cellulosic substrates that may be hydrolyzed according to the present means include grasses such as switchgrass, spartina, barley, scallops, susuki, or combinations thereof, such as sugarcane squeeze, but this Sugar residues, such as rice straw, rice husk, barley straw, corn cobs, wheat straw, canola straw, oat straw, oat straw, and corn fiber, lintels such as soy lintel, corn, tuna Forest waste, ruminant digestion products such as, but not limited to, agricultural waste, recycled wood pulp fiber, wood chips, hardwood, softwood, or combinations thereof, such as but not limited to Municipal waste, paper mill effluent, newspaper, cardboard, and combinations of the above substrates Including but not limited to. Examples of hardwoods considered for ethanol production include willow, maple, oak, walnut, eucalyptus, elm, birch, cypress, beech and ash. Examples of softwood considered for ethanol production include Southern yellow pine, fir, cedar, cypress, hemlock, larch, pine and spruce or combinations thereof.

Example 1
Isolated Enzyme-Mediated Cellulose Hydrolysis vs. Microbial-Mediated Cellulose Hydrolysis Cellulose Aerobic Thermophilic Thermosaccharolyticum in the Presence and Absence of Soluble Cellulose Hydrolysis Below, for enzyme-mediated hydrolysis performed by purified Clostridium thermocellum cellulosome, A systematic comparison of microorganism-mediated cellulose hydrolysis by thermocellum. In the case of microorganism-mediated hydrolysis, there is a ternary CEM complex, whereas in the case of enzyme-mediated hydrolysis, hydrolysis of cellulose exclusively occurs due to the action of the binary CE complex.

C. Thermocellum and related control batch cultures ₂ g / L AvicelPH105 (FMC Corp., Philadelphia, PA) in triplicate 200 mL closed serum vials (Bellco Biotechnology, Vineland, NJ) under N ₂ atmosphere And 5 mL of Clostridium thermocellum (ATCC 27405) stock culture was inoculated by syringe into 100 mL of MTC medium containing 10 g / L MOPS buffer (initially pH 7.6). The culture was incubated at 60 ° C. in a temperature-controlled water bath with shaking at 200 rpm. Once 2 g / L Avicel has been consumed as measured by visual inspection, supplemental Avicel is added as a 40 g / L sterile suspension to a concentration of 2 g / L and the pH is adjusted by addition of 4 M NaOH. The gas phase was replaced by flushing with N ₂ , adjusted to 7.6 and filter sterilized.

  Immediately after the supplemental Avicel is added, microbial cellulose utilization data is taken at the first (time zero) data point. Microbial control 1 was performed as described above except that 1M sodium azide solution sterilized at a final concentration of 38.5 mM was added in conjunction with supplemental Avicel addition. Addition of azide as specified above results in a fermentation stop as indicated by a constant concentration of fermentation product over time as measured by HPLC.

Cellulosome preparation and purification Cellulosomes used in batch SSF experiments were grown in MTC medium in 200 mL flasks with Avicel as growth substrate at an initial concentration of 4 g / L. Obtained from a batch culture of thermocellum. Cellulosomes for continuous SSF experiments were grown in MTC medium at a dilution rate of 0.052 hr ⁻¹ (flow rate / fermentation working volume) and a feed cellulose concentration of 4 g / L. Obtained from a continuous culture of thermocellum. Cellulosomes were purified from the culture broth supernatant by affinity digestion. The purified cellulosome preparation used for batch and continuous SSF experiments was about 1.2 g / L with a specific activity of 2.8 IU / mg cellulase in Tris buffer (50 mM, 10 mM CaCl ₂ , pH 6.8). Contains cellulase. The concentration of soluble hydrolysis products in the purified cellulosome preparation was confirmed by HPLC to be low enough (<0.002 g / L) so as not to complicate interpretation of the SSF experiment.

Batch SSF and associated enzyme control In N ₂ atmosphere, in triplicate 200 mL serum vials, 5 mL Thermoanaerobacter thermoaccharolyticum (ATCC 31960) in 100 mL MTC medium containing 2 g / L AvicelPH105 and 2 g / L cellobiose. ) Stock culture. The culture was incubated at 60 ° C. in a temperature-controlled water bath with shaking at 200 rpm. Once cellobiose has been consumed as measured by HPLC, the pH is adjusted to 7.6 and the purified cellulosome preparation (above) is filter sterilized (Millex-GV, 0.22um pore size, Millipore, Billerica) , MA), added to the culture via syringe at a final concentration of 100 mg / L. SSF data were taken at the first (time zero) data point immediately after adding cellulase. Control 1 without cells was performed as described above in the presence of 2 g / L Avicel and 100 mg / L purified cellulosome, except that no fermenting organisms were present. Control 2 without cells was performed like Control 1 without cells, except that 1M sterile sodium azide was added at a final concentration of 38.5M.

Continuous culture A modified 1 L fermentor (Applikon, Dependable Instruments, Foster City, CA, modified by NDS) with an overflow sidearm (id 0.38 ") and a working volume of 0.5 L by C. thermocellum Used for both microbial fermentation and SSF performed in continuous mode, pH controlled at 6.8 by Delta V process control system (New England Controls Inc., Mansfield, Mass.) By addition of 4M NaOH, fermentor Was stirred at 250 rpm and the temperature was controlled by circulating water through the fermenter jacket at 60 ° C. MTC medium containing 4 g / L AvicelPH105 was fed with a peristaltic pump, The desired residence time was achieved.For SSF experiments, an additional peristaltic pump was used to deliver purified cellulosomes stored in 50 mM Tris buffer (pH 6.8) at 4 ° C. Used for SSF experiments. The composition of the MTC medium and the concentration of Avicel were adjusted to provide the same concentration used in the C. thermocellum fermentation experiments (eg, 4 g / LAvicel final concentration), at 2 g / L of cellobiose. The SSF experiment was started by inoculating 50 mL of late log phase culture of T. thermosaccharolyticum in MTC medium containing supplemented 4 g / L Avicel Once cell growth was apparent, cellulase addition was started. Used to calculate steady state values for continuous fermentation Samples were taken at intervals of at least one residence.

Measurement of residual cellulose and fermentation product The residual cellulose by quantitative saccharification was measured. Sugar (cellbioose, glucose) by HPLC using Bio-Rad HPX-87H column operated at 55 ° C. with 0.01% (v / v) H ₂ SO ₄ as eluent and refractive index detector. ) And fermentation products (lactic acid, acetic acid and ethanol) were analyzed. Ehrman, C.I. I. , M.M. E. Himmel. The oligomeric sugars were analyzed according to the modified NREL hydrolysis procedure reported by Biotechnology Technologies, 8 (2): 99 (1994).

Determination of protein, cellulosome and cellulase activity The protein content in the supernatant samples was determined according to the Bradford protein assay (Bradford, MM Anal. Biochem. 72, 24 (1976)) with bovine serum albumin as a standard. Previously, Zhang et al. The protein content in the pellets was measured using the pellet protein assay described by (Zhang, Y.-H.P, LR Lynd. J. Bacteriol. 187, 99 (2005)). Cellulosome concentrations in the supernatant and pellet were determined by Dubois, M. et al. , K .; Gilles, J.M. K. Hamilton, P.A. A. Rebers, F.A. Smith. Nature. 168, 167 (1951). The avicelase activity in supernatant and pellet samples was measured at 60 ° C. using the method of Zhang (2005) based on the soluble sugar product as measured by the Dubois (1951) phenol sulfate method. The results are expressed in terms of international units (IU) = 1 micromole glucose equivalent / L / min.

Batch results In batch cultures under controlled conditions, microbial hydrolysis requires 16 hours to fully hydrolyze 2 g / L of cellobiose, during which time the cellulosome and cellular protein concentrations are respectively Rough doubling from 48 mg / L to 98 mg / L and from 125 mg / L to 264 mg / L (FIG. 1). As shown in FIG. SSF (enzymatic hydrolysis) using purified cellulosomes in the presence of thermosaccharolyticum requires 32 hours for complete hydrolysis, during which the cellulosome concentration is 100 mg / L and the cellular protein concentration is from 160 mg / L Increased to 260 mg / L.

  The conditions under which microbial hydrolysis and SSF occur were similar. The product concentration 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 equivalent (Glu Eqv). The hydrolysis products in the growth medium (total soluble glucan or total glucose equivalent) is always ≦ 0.02 g / L for both microbial hydrolysis and SSF, with 50% inhibition generally observed The value was two orders of magnitude lower than the concentration. As shown in Table 3, the specific activity of cellulosome was quite similar for both microbial and enzymatic hydrolysis and remained nearly constant throughout the experiment.

  Table 3. Comparison of batch cultures of C. thermocellum with enzyme controls associated with SSF

＊ Ｃ．ｔｈｅｒｍｏｃｅｌｌｕｍのバッチ培養及びＳＳＦの相２（図１及び２の０時間〜４８時間）の開始０時間を参照のこと。
＃ Ｃ．ｔｈｅｒｍｏｃｅｌｌｕｍのバッチ培養では１６時間を参照し、ＳＳＦ及び酵素対照では３２時間を参照する相２（図１及び２の０時間〜４８時間）の終了
図５にて、時間に対してセルロース濃度を、微生物による加水分解（図１から）、ＳＳＦ（図２から）、及び以下の対照について；微生物対照１：３８．５ｍＭのアジ化ナトリウムと共にＣ．ｔｈｅｒｍｏｃｅｌｌｕｍの培養（１００ｍｇ／Ｌのセルロソーム、２６４ｍｇ／Ｌの細胞タンパク質）、細胞を含まない対照１：発酵生物を伴わずに１００ｍｇ／Ｌの精製セルロソーム、細胞を含まない対照２：３８．５ｍＭのアジ化ナトリウムを伴って細胞を含まない対照１と同様、についてプロットした。セルロソームの濃度が微生物対照１（１００ｍｇ／ｍＬ、セルロソーム）よりも微生物による加水分解に関するほとんどの実験（図１を参照）で低いという事実にもかかわらず、加水分解率は、代謝的に不活性の細胞（微生物対照１）よりも増殖している細胞（微生物による加水分解）の方が実質的に高い。代謝的に不活性の細胞による加水分解の比率の低さは、アジド存在下又は非存在下で観察される細胞を含まない加水分解率が類似しているので、一義的にはセルロソームに対するアジドの効果によるものではない。

* C. See thermocellum batch culture and 0 hour start of SSF phase 2 (0 to 48 hours in FIGS. 1 and 2).
#C. Termination of Phase 2 (0 to 48 hours in FIGS. 1 and 2) with reference to 16 hours for thermocellum batch cultures and 32 hours for SSF and enzyme controls. In FIG. For microbial hydrolysis (from FIG. 1), SSF (from FIG. 2), and the following controls; microbial control 1: C. with 38.5 mM sodium azide. Thermocellum culture (100 mg / L cellulosome, 264 mg / L cell protein), cell-free control 1: 100 mg / L purified cellulosome without fermenting organisms, cell-free control 2: 38.5 mM azimuth Plotted as for Control 1 with no cells with sodium chloride. Despite the fact that the concentration of cellulosome is lower in most experiments on microbial hydrolysis (see Figure 1) than microbial control 1 (100 mg / mL, cellulosome), the rate of hydrolysis is metabolically inactive. Proliferating cells (microorganism hydrolysis) are substantially higher than cells (microbe control 1). The low rate of hydrolysis by metabolically inactive cells is similar to the rate of cell-free hydrolysis observed in the presence or absence of azide, and is therefore primarily azide to cellulosome. It is not due to effects.

  FIG. 6 shows cellulose hydrolysis and product accumulation for Control 1 without cells. It can be observed that the hydrolysis product concentration is similar to Control 1 without cells than SSF by an order of magnitude higher (Fig. 6 compared to Fig. 2) than SSF. Thus, the accumulation of hydrolyzable organisms in the bulk fermentation broth is not a plausible explanation for the significant difference between microbial hydrolysis and SSF.

  The results of the batch support the degree of enzyme / microbe synergy and the cellulosome normalized hydrolysis rate seen in the microbial system between 2.8 and 4.7 (Table 4). Equal to the ratio divided by that of the system. See Example 2 for further description of quantification of enzyme / microorganism synergy.

Results of continuous culture Hydrolysis by microorganisms and SSF were also compared in steady-state continuous culture. The average of four or more steady state data points is reported in Table 4, along with the specific activity of cellulase in Table 5. Steady state 1 and 2 of the microorganisms were obtained with residence times (τ = fermentor capacity / feed flow rate) of 6.8 hours and 9.8 hours, respectively. For microbial steady state 1, 65.3% of the supplied cellulose was hydrolyzed in the presence of a total concentration of 39 mg / L cellulosome, whereas in microbial steady state 2 it was 76.8 at 63 mg / L cellulosome. % Hydrolysis was achieved. T.A. In addition to fermentation with thermosaccharolyticum, purified C.I. Thermocellum cellulosome mediated steady state continuous SSF was performed under selected conditions to achieve conversion and total cellulosome concentrations similar to those observed for cellulose utilization by microorganisms. In the steady state 1 of SSF, 67% hydrolysis of cellulose was observed at τ = 24.4 hours compared to the steady state 1 of the microorganism, and the added cellulosome was 52 mg / L. For SSF steady state 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 the detection limit (2.5 mg / L) for both the microbial steady state and the SSF steady state. The time-dependent change of the SSF data is shown in FIG. 7 (SSF steady state 1) and FIG. 8 (SSF steady state 2). The experiment converted from continuous mode to batch mode in 168 hours to prevent further accumulation of cellobiose; continuous feeding resumed at 184 hours. The specific activity of cellulase was similar between the microbial steady state and the SSF steady state (Table 5).

  (Table 4 Result of continuous culture and degree of calculation of synergy)

（表５ Ｃ．ｔｈｅｒｍｏｃｅｌｌｕｍとＳＳＦの連続培養）

(Table 5 Continuous culture of C. thermocellum and SSF)

連続培養のデータは、酵素／微生物の相乗効果の程度を支持し、２．８〜４．８（表４）の間の、微生物の系で見られたセルロソームで正規化された加水分解率を酵素の系のそれで割った比率に等しい。酵素／微生物の相乗効果の定量のさらなる記載については実施例２を参照のこと。

Continuous culture data support the extent of enzyme / microorganism synergy, and the cellulosome normalized hydrolysis rate seen in the microbial system between 2.8 and 4.8 (Table 4). Equal to the ratio divided by that of the enzyme system. See Example 2 for further description of quantification of enzyme / microorganism synergy.

  For both batch and continuous culture experiments, under the conditions studied, during microbial hydrolysis compared to SSF, C.I. The thermocellum cellulase complex is substantially more effective. Such enzyme / microorganism synergies require the presence of metabolically active cellulolytic microorganisms and are not explained by the removal of hydrolysis products from the bulk fermentation broth. The key obvious difference between microbial hydrolysis and SSF is that CEM complexes are present during microbial hydrolysis, but this is not the case for SSF.

Example 2 Quantification of enzyme / microorganism synergy Cellulosome-specific hydrolysis rate (r ^E _C , cellulose g / cellulosome g / time) based on cellulosome may be calculated using the following equation.

式中、Ｃ_０は、当初の（バッチ反応の）又は供給における（定常状態の連続反応の）いずれかのｇ／Ｌでのセルロース濃度であり、Ｃは時間ｔ後（バッチ）又は定常状態での（連続）発酵槽のセルロース濃度であり、τは、経過時間（バッチ）又は滞留時間（連続）であり、Ｅは、経過時間（バッチ）にわたる又は複数の滞留時間ポイント（連続）の間のｇ／Ｌでの平均セルロソーム濃度である。酵素／微生物の相乗効果、ＤＳ_ＥＭは、以下の式を用いて微生物による加水分解及びＳＳＦで認められるセルロソーム特異的な加水分解率から算出されてもよい。

Where C ₀ is the cellulose concentration in g / L either at the beginning (in batch reaction) or at the feed (in steady state continuous reaction) and C is after time t (batch) or at steady state. (Continuous) fermenter cellulose concentration, τ is the elapsed time (batch) or residence time (continuous), E is the elapsed time (batch) or between multiple residence time points (continuous) Average cellulosome concentration in g / L. Synergy enzyme / microorganism, DS _EM may be calculated from the hydrolysis and cellulosome specific hydrolysis rate observed in SSF by microorganisms using the following equation.

総セルロソームを基にした相乗効果の程度、ＤＳ^ＥＲ _ＥＭは、総セルロソーム、方程式（１）におけるＥ_τを用いて見い出される。或いは、ペレットのセルロソームを基にした相乗効果の程度、ＤＳ^ＥＲ _ＥＭは、ペレットのセルラーゼ濃度（Ｅ_ｐ、ＣＥ及びＣＥＭ双方の複合体を潜在的に含む）を使用するならば見い出される。

The degree of synergy based on total cellulosome, DS ^ER _EM, is found using total cellulosome, E _τ in equation (1). Alternatively, a degree of synergy based on pellet cellulosomes, DS ^ER _EM is found if pellet cellulase concentrations (potentially containing complexes of both E _p , CE and CEM) are used.

The DS ^ER _EM values calculated from the batch data and the continuous data are presented in Table 4. The DS ^ER _EM based on the batch data after 8 hours was 4.69. Once the complete cellulose hydrolysis is achieved, a DS ^ER _EM is calculated of 2.78. Continuous culture gives a DS ^ER _EM value of 2.80 based on the steady state 2 of the microorganism and SSF, about which about 75% of the cellulose is hydrolyzed. For microbial and SSF steady state 1, DS ^ER _EM equals 4.81 when about 66% hydrolysis is achieved. The pellet cellulase-based enzyme / microbe synergy value, DS ^Ep _EM, was 2.84 at steady state 2 for continuous culture, microbe and SSF, and 4.77 at steady state 1 for microbe and SSF. Similar. A decrease in synergistic effect is seen with increasing degree of cellulose hydrolysis and decreasing substrate to enzyme ratio in both batch and continuous cultures.

  Changes may be made in the above methods and systems without departing from the scope of the specification. Accordingly, it should be noted that what is contained in the above description or shown in the accompanying drawings is to be interpreted as illustrative and not limiting. The following claims are intended to cover all the general and specific features described herein, and all statements within the scope of the method that may be said to fall between them as a language. Is intended to cover.

FIG. 1 shows the utilization of cellulose by microorganisms mediated by both CEM and CE complexes in hydrolysis. FIG. 2 shows the enzymatic hydrolysis of cellulose while the CE complex is involved and the saccharification and fermentation reactions are performed simultaneously (SSF). FIG. 3 is a diagram illustrating changes in pH and product concentration over time for the experiment shown in FIG. FIG. 4 is a diagram illustrating changes in pH and product concentration over time for the experiment shown in FIG. FIG. 5 shows the cellulose concentration curve for the microorganism and SSF experiment shown in FIGS. 1 and 2 as in the control experiment. FIG. 6 shows cellulose hydrolysis and product accumulation for Control 1 with no cells. FIG. 7 shows 0.064 g / L of purified C.I. in the presence of thermocellum cellulosome, FIG. 2 shows cellulose hydrolysis and product accumulation for Avicel continuous SSF by thermosaccharolyticum. FIG. 8 shows 0.052 g / L of purified C.I. in the presence of thermocellum cellulosome, FIG. 2 shows cellulose hydrolysis and product accumulation for Avicel continuous SSF by thermosaccharolyticum.

Claims (18)

A method that utilizes reduced cellulase loading to hydrolyze cellulosic substrates,
Determining the amount of purified cellulase required to substantially hydrolyze an amount of cellulosic substrate within a time;
Reducing the amount of purified cellulase by a factor between 2 and 5 to determine the amount of cellulase added reduced;
Under suitable conditions, the microorganism expressing cell-bound cellulase at a concentration equivalent to the reduced amount of cellulase added during the time during which the cellulosic substrate can be substantially hydrolyzed. Introducing into a cellulosic substrate. The method of claim 1, wherein the factor is between 2.8 and 4.8. The microorganism is a member of Clostridium, Acetivibrio, Caldicellosiruptor, Ruminococcus, Butyravibrio, Eubacterium, Ferbacterium, Fibrobacter, Spirochata, Thermopia. The method of claim 1, wherein the microorganism is a member of the genus Clostridium. The microorganism may be Clostridium stranosolvens, Acetibrio cellulolyticus, Clostridium cellulolyticum, Clostridium stercorium subs. stercorarium, Clostridium stercorium subs. thermolyticum, Clostridium stercorium 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 thermophil , Thermotoga neapolitana, Neocallimastix sp. And Pyromyces sp. The method of claim 1, wherein the method is selected from the group consisting of: The method according to claim 1, wherein the microorganism is a strain of Clostridium thermocellum. 2. The cellulosic substrate is selected from grasses, sugar residues, agricultural waste, lintels, forestry waste, ruminant digestion products, municipal waste, paper mill effluent, newspaper, cardboard and combinations thereof. the method of. 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. A method of hydrolyzing a cellulosic substrate using a reduced amount of cellulase added,
Determining the amount of purified cellulase required to substantially hydrolyze an amount of cellulosic substrate within a time;
Reducing the amount of purified cellulase by a factor between 2 and 5 to determine the amount of cellulase added reduced;
Fermentation factors engineered to express cell-bound cellulase for a period of time sufficient to allow substantial hydrolysis and fermentation of the cellulosic substrate under suitable conditions. Introducing into a sex substrate,
The method wherein the cell-bound cellulase is present at a concentration equal to the reduced cellulase loading. 11. The method of claim 10, wherein the factor is between 2.8 and 4.8. Said fermentation factor, Saccharomyces, Zymomonas, Escherichia, Klebsiella, Clostridium, Schizosaccharomyces, Candida, Kluyveromyces, Pichia, Yarrowia, Hansenula, Phaffia, Arxula, Debaryomyces, The method of claim 10 which is a member of the genus selected from Debaryomyces and Schwanniomyces . 11. The method of claim 10, wherein the fermentation factor is a member of the genus Saccharomyces. Said fermentation factor, 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, Debaryomyce polymorphus and is selected from the group consisting of Schwanniomyces occidentalis, The method of claim 10. The method of claim 10, wherein the fermentation factor is Saccharomyces cerevisiae. 11. The cellulosic substrate is selected from grasses, sugar residues, agricultural waste, lintels, forestry waste, ruminant digestion products, municipal waste, paper mill effluent, newspaper, cardboard and combinations thereof. the method of. The method of claim 10, wherein the fermentation factor is an anaerobic fermentation factor. The method of claim 17, wherein the suitable conditions comprise a substantially oxygen free atmosphere.

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