EP2013355A2 - Verfahren zur durch dreifach-zellulose-komplexe, clostridium-thermocellum-zellen und von diesen zellen exprimierte zellulase vermittelten zellulose-hydrolyse - Google Patents

Verfahren zur durch dreifach-zellulose-komplexe, clostridium-thermocellum-zellen und von diesen zellen exprimierte zellulase vermittelten zellulose-hydrolyse

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Publication number
EP2013355A2
EP2013355A2 EP07811862A EP07811862A EP2013355A2 EP 2013355 A2 EP2013355 A2 EP 2013355A2 EP 07811862 A EP07811862 A EP 07811862A EP 07811862 A EP07811862 A EP 07811862A EP 2013355 A2 EP2013355 A2 EP 2013355A2
Authority
EP
European Patent Office
Prior art keywords
cellulase
clostridium
hydrolysis
cellulosic substrate
cellulose
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP07811862A
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English (en)
French (fr)
Inventor
Lee R. Lynd
Yi-Heng Percival Zhang
Yanpin Lu
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Dartmouth College
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Dartmouth College
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dartmouth College filed Critical Dartmouth College
Publication of EP2013355A2 publication Critical patent/EP2013355A2/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase

Definitions

  • Cellulosic biomass represents an inexpensive and readily available resource which may be fermented to produce ethanol or other products.
  • 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.
  • 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.
  • CEM ternary cellulose-enzyme-microbe
  • CE binary cellulose-enzyme
  • 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.
  • 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.
  • FIG. 1 shows cellulose utilization by a microbe, where hydrolysis is mediated by both CEM and CE complexes.
  • FIG. 2 shows enzymatic hydrolysis of cellulose during a Simultaneous Saccharification and Fermentation (SSF) reaction involving a CE complex.
  • SSF Simultaneous Saccharification and Fermentation
  • FIG. 3 illustrates changes in pH and product concentration over time for the experiment depicted in FIG. 1.
  • FIG. 4 illustrates changes in pH and product concentration over time for the experiment depicted in FIG. 2.
  • FIG. 5 shows cellulose concentration curves for the microbial and SSF experiments depicted in FIGS. 1 and 2, as well as control experiments.
  • FIG. 6 shows cellulose hydrolysis and product accumulation for cell-free control 1.
  • 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.
  • 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.
  • enzyme-microbe synergy was quantitatively evaluated, and was shown to give rise to a substantial increase in the effectiveness of cellulase enzymes.
  • thermocellum 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:
  • thermocellum cellulosome 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.
  • thermolacticum Clostridium stercorarium subs, thermolacticum
  • 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.
  • 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.
  • 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.
  • grasses such as switch grass, cord grass, rye grass, reed canary grass, miscanthus, or a combination thereof
  • 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.
  • thermocellum 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.
  • PH105 FMC Corp., Philadelphia, PA
  • 10 g/L MOPs buffer initial pH 7.6
  • 10 g/L MOPs buffer initial pH 7.6
  • Cultures were incubated at 60 0 C in a temperature controlled water bath with rotary shaking at 200 rpm.
  • 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 N 2 .
  • 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.
  • Cellulosome preparation and purification were 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.
  • 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.
  • 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.
  • 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) H 2 SO 4 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).
  • FIG. 5 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.
  • 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.
  • 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.
  • 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.
  • thermosaccharolyticum was carried out at conditions chosen to achieve similar conversion and total cellulosome concentrations to those observed for microbial cellulose utilization.
  • 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).
  • 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.
  • the cellulosome-specific hydrolysis rate on a cellulosome basis ( r c £ , g cellulose * g cellulosome "1 • hr "1 ) may be calculated using:
  • C 0 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
  • ⁇ is the elapsed time (batch) or the residence time (continuous),
  • E is the average cellulosome concentration in g/L over the elapsed time (batch) or for multiple steady-state points (continuous). The degree of
  • DS EM enzyme-microbe synergy
  • the degree of synergy on a total cellulosome basis, DS E E M ' is found by using the total cellulosome concentration, E 7 -, in equation (1 ).
  • 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.
  • Ep pellet cellulase concentration
  • 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.
  • 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.

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EP07811862A 2006-05-01 2007-05-01 Verfahren zur durch dreifach-zellulose-komplexe, clostridium-thermocellum-zellen und von diesen zellen exprimierte zellulase vermittelten zellulose-hydrolyse Withdrawn EP2013355A2 (de)

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US79663506P 2006-05-01 2006-05-01
PCT/US2007/067954 WO2007136971A2 (en) 2006-05-01 2007-05-01 Process for the hydrolysis of cellulose mediated by ternary complexes of cellulose, clostridium thermocellum cells, and cellulase expressed by these cells

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JP2009033993A (ja) * 2007-07-31 2009-02-19 Toyota Central R&D Labs Inc セルラーゼ担持材料及びその利用
CA2724036C (en) * 2008-05-11 2018-09-25 Mascoma Corporation Construction of protrophic/celluloytic yeast strains expressing tethered and secreted cellulases
JP5352882B2 (ja) * 2009-12-04 2013-11-27 独立行政法人国際農林水産業研究センター クロストリジウム属微生物を利用したセルロース分解酵素の生産方法及びクロストリジウム属微生物の培養増殖方法。
CN103131733A (zh) * 2011-11-26 2013-06-05 嘉兴慧升生物技术咨询有限公司 一种利用生物废弃物组合生产生物质能的新方法
CN102643748A (zh) * 2012-05-09 2012-08-22 刘建伦 污水处理中污泥减量的微生物组合优势菌群
CN103688869B (zh) * 2013-01-22 2015-10-28 厦门嘉烨兴农业科技有限公司 一种用于养猪的发酵床
CN103664357B (zh) * 2013-11-26 2015-04-22 新疆晨源生物科技有限公司 一种有机发酵复合肥及其制备方法
CN104450829B (zh) * 2014-12-11 2018-06-12 中国科学院过程工程研究所 高温木质纤维素酶协同酶解制备可发酵糖的方法
CN108866025B (zh) * 2017-05-10 2021-04-13 中国科学院青岛生物能源与过程研究所 一种纤维素酶制剂及其应用
CN108866112B (zh) * 2018-08-17 2021-04-27 青岛中科潮生生物技术有限公司 采用木质纤维素制备角鲨烯的方法
CN109022506B (zh) * 2018-08-17 2021-04-27 青岛中科潮生生物技术有限公司 采用木质纤维素制备葡萄糖酸钠的方法
CN108977421B (zh) * 2018-08-17 2021-04-13 中国科学院青岛生物能源与过程研究所 用于催化木质纤维素糖化的全菌酶制剂
CN109019874B (zh) * 2018-09-08 2022-12-30 佛山市森昂生物科技有限公司 一种用于造纸废水的生物促生剂及其制备方法
CN110540982B (zh) * 2019-09-30 2021-11-02 江南大学 一种提高梭热杆菌纤维素酶酶活的发酵方法

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CN101466843B (zh) 2013-05-08
WO2007136971A2 (en) 2007-11-29
AU2007253987A1 (en) 2007-11-29
BRPI0711163A2 (pt) 2011-08-23
JP2009535067A (ja) 2009-10-01
WO2007136971A3 (en) 2008-04-10
ZA200809448B (en) 2010-08-25
CA2651753A1 (en) 2007-11-29

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