Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2405—Glucanases
  • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
  • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
• • 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
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/06—Ethanol, i.e. non-beverage
  • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
  • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
  • Y02E50/00—Technologies for the production of fuel of non-fossil origin
  • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

• the invention relates to a process for the production of enzymes by a filamentous fungus, in particular enzymes of the cellulase type (cellulolytics and / or hemicellulolytics), which are necessary for the enzymatic hydrolysis of lignocellulosic biomass.
• enzymes of the cellulase type cellulolytics and / or hemicellulolytics
• This enzymatic hydrolysis is implemented for example in the so-called second generation (2G) production processes of biofuels such as bioethanol, or to produce sweet juices which can be used to produce other products chemically or biochemically.
• fermentation for example, alcohols such as ethanol, butanol, propanol, isopropanol or other molecules, for example solvents such as acetone and other bio-based molecules
• solvents such as acetone and other bio-based molecules
• Trichoderma reesei is the most widely used microorganism for the production of cellulases on an industrial scale. Wild strains have the ability to excrete, in the presence of an inducing substrate, cellulose for example, an enzyme complex considered to be well suited to the hydrolysis of lignocellulosic biomass.
• the enzymes in the enzyme complex contain three main types depending on their activity: endoglucanases, exoglucanases and cellobiases.
• Other proteins having properties essential for the hydrolysis of lignocellulosic materials are also produced by Trichoderma reesei, xylanases for example.
• an inducing substrate is essential for the expression of cellulolytic and / or hemicellulolytic enzymes.
• the nature of the carbon substrate has a strong influence on the composition of the enzyme complex. This is the case with xylose, which, associated with a carbon-inducing substrate such as cellulose or lactose, makes it possible to significantly improve the so-called xylanase activity.
• xylose which, associated with a carbon-inducing substrate such as cellulose or lactose, makes it possible to significantly improve the so-called xylanase activity.
• the regulation of cellulase genes on different carbon sources has been studied in detail. They are induced in the presence of cellulose, its hydrolysis products (example: cellobiose) or certain oligosaccharides such as lactose or sophorose (llmen et al., 1997; Appl. Environ. Microbiol. 63: 1298-1306).
• soluble carbon sources such as glucose, xylose or lactose
• lactose also playing the role of an inducing substrate.
• Other soluble sugars such as cellobiose and sophorose have been described as inducers, but they may be considered too expensive to be used on an industrial scale.
• a growth stage in "batch" mode where it is necessary to provide a source of carbon that can be quickly assimilated for the growth of Trichoderma reesei.
• This phase is characterized by an increase in the viscosity of the medium, as well as by a high demand for oxygen.
• a production step in "fed-batch” mode using an inducing substrate for example lactose which allows the expression of cellulases and secretion in the culture centre.
• the optimal flow applied is between 35 and 45 mg (of sugar) .g (of dry cell weight) 1 .h 1 .
• This phase is characterized by a drop in the viscosity of the medium and a lower oxygen demand.
• Protein productivity is equal to the product between the concentration of living biomass and the specific rate of protein production. This productivity can be increased by increasing the production performance of the strain (increase in specific speed) and / or by increasing the concentration of cellular biomass. But the methods to improve the performance of the strains, by genetically modifying them as mentioned above, may end up reaching their limits, and increasing the biomass concentration results in a sharp increase in viscosity, which limits oxygen transfer, especially at the end of the growth stage.
• the production stages proposed in the prior art are carried out in agitated tanks, the growth and production stages being carried out in the same bioreactor. It is an interesting solution because compact in terms of industrial installation, but not necessarily the best solution, because the two stages, growth and production, do not require the same needs, for example in terms of means of agitation or in terms of oxygen transfer: the bioreactor must therefore have the volume and all the equipment and operating means enabling it to carry out all of these two stages, however taking place under very different conditions.
• the object of the invention is therefore to propose an improved process for the production of enzymes of the cellulase type from filamentous fungus strains, in particular a process which makes it possible to obtain increased productivity of enzymes, and, incidentally, which can be implemented on an industrial scale in installations allowing more flexibility.
• the present invention relates to a process for the production of enzymes by a strain belonging to a filamentous fungus, said process comprising three stages:
• step (c) a third step of producing enzymes from the diluted culture medium obtained in the second step (b), in the presence of at least one inducing carbon substrate, in the fed-batch phase.
• the invention includes such a method, where step (b) can start (a little) before the end of step (a) or end (a little) after the start of step (c).
• the invention therefore includes such a method, with an implementation in which steps (a) and (b) on the one hand, and steps (b) and (c) on the other hand can partially overlap.
• the dilution stage (b) can last several hours, and it may therefore be advantageous to start it between 0 and 4 hours before the end of stage (a), and / or to finish it between 0 and 4 hours after going to step (c).
• the invention has thus developed a new fermentation line, with an intermediate dilution stage between the growth stage and the production stage. Surprisingly, it turned out that the specific productivities obtained after diluting the culture medium at the end of the growth phase are significantly higher than those obtained with the culture medium without dilution (l 'increase of up to 30% and more), and this for the same specific flux of carbonaceous substrate (calculated relative to the mass of biomass in the reactors).
• the third production stage (c) is carried out in a bubble column:
• the invention also judiciously exploits the idea that the growth and production stages do not have the same needs , do not have the same operating mode, in particular in terms of agitation and oxygen transfer, and that it is therefore more relevant to carry out these stages in two different fermenters:
• the production of biomass can take place in good conditions in a stirred tank, preferably targeting a high biomass concentration (for example greater than or equal to 20 g / L).
• agitated bioreactor has the same meaning as “agitated tank”.
• the second stage (b) of dilution is carried out in the bioreactor at the end of the stage (a) of mushroom growth and / or in the bubble column before or at the start of the third stage (c) of production. enzymes.
• the dilution step which therefore leads to lowering the viscosity of the culture medium, can thus be carried out in one or the other of the reactors, or even online during the transfer. from the first reactor to the second reactor. It may be preferable to carry out the dilution in the agitated tank, so as to facilitate the transfer of the medium, already diluted, from the agitated tank to the bubble column.
• the dilution factor of the culture medium in stage (b) of dilution is at least 1, 1 or 1, 2, in particular at least 1, 5, preferably about 2, and in particular at most 6.
• the dilution must be adjusted so that the culture medium has a biomass concentration making it possible to limit the viscosity of the medium, that is to say between 5 and 20 g / L.
• biomass corresponds to fungi.
• the dilution factor is understood as a volume dilution factor, and corresponds to the ratio between the volume after dilution and before dilution.
• the culture medium being an aqueous medium
• the dilution liquid is water or an aqueous medium, such as a culture medium also.
• means are provided for fluid connection between the bioreactor and the bubble column to ensure the transfer of the culture medium obtained in the growth step in the bioreactor to the bubble column, means in particular in the form pipes fitted with manual or piloted valves and in particular pump (s).
• the second stage (b) of dilution in whole or in part, can be carried out in the fluid connection means during the transfer of the culture medium by these fluid connection means from the bioreactor to the bubble column.
• the concentration of carbonaceous growth substrate in the bioreactor is greater than 20 g / L, it is in particular between 30 and 100 g / L, and preferably between 50 and 80 g / L.
• the inducing carbon substrate feeds the bubble column at a specific speed of between 30 and 140 mg per gram of cellular biomass per hour, preferably between 35 and 45 mg per gram of cellular biomass per hour.
• the biomass growth step is continued until a biomass (mushroom) concentration of at least 20 g / L.
• a concentrated culture medium is thus obtained, which will then be diluted to proceed with production.
• the method of the invention can be implemented with a number n of agitated and aerated bioreactors and a number m of bubble columns, with n less than or equal to m, a bubble column being able to be connected fluidly to many bioreactors or vice versa. It is possible to have bubble columns of much larger capacity than agitated bioreactors, which allows several agitated bioreactors to feed a single bubble column for example, and to take into account that the step of growth is faster than the production stage.
• the carbonaceous growth substrate is chosen from at least one of the following compounds: lactose, glucose, xylose, the residues obtained after ethanolic fermentation of the monomeric sugars of the enzymatic hydrolysates of cellulosic biomass, a crude extract of water-soluble pentoses from pretreatment of cellulosic biomass.
• the inducing carbon substrate is chosen from at least one of the following compounds: lactose, cellobiose, sophorose, the residues obtained after ethanolic fermentation of the monomeric sugars of the enzymatic hydrolysates of cellulosic biomass, a crude extract of water-soluble pentoses from pretreatment of cellulosic biomass.
• the growth substrates and inducers mentioned above can be used alone or as a mixture.
• the carbonaceous growth substrate chosen for obtaining the biomass is introduced into the bioreactor before sterilization, or is sterilized separately and introduced into the bioreactor after sterilization.
• the inducing carbon substrate introduced during the fed-batch production step is preferably sterilized independently, before being introduced into the bubble column. If the inducing carbon substrate is pretreated biomass or hemicellulosic hydrolysates, it can be used without sterilizing it.
• the aqueous solution is prepared at the concentration of 200-250 gL 1 .
• the inducing carbon substrate is the pretreated biomass, it is preferably aimed at an average sugar intake of between 35 and 140 mg per gram of cell and per hour.
• the strains used belong to the species Trichoderma reesei, possibly modified to improve the cellulolytic enzymes and / or hemicellulolytics by mutation-selection processes; strains improved by genetic recombination techniques can also be used. These strains are cultivated under conditions compatible with their growth and the production of enzymes. Other strains of microorganisms producing enzymes according to processes similar to those used for Trichoderma can be used. *
• the enzymes are cellulolytic or hemicellulolytic enzymes.
• the filamentous fungus strain used is therefore a Trichoderma reesei or Trichoderma reesei strain modified by genetic mutation, selection or recombination.
• the strain is a CL847, RutC30, MCG77, or MCG80 strain.
• the invention also relates to an installation for the production of enzymes by a strain belonging to a filamentous fungus, such that said installation comprises: - a stirred and aerated bioreactor operating in batch phase to operate a stage of growth of the fungi, in the presence at least one carbonaceous growth substrate; - a bubble column operating in fed-batch phase to operate a step of producing enzymes from the culture medium originating from the bioreactor; - Fluid connection means connecting the bioreactor to the bubble column to effect the transfer of the culture medium from the bioreactor to the bubble column; - means for injecting dilution liquid into the bioreactor and / or into the bubble column and / or into the fluid connection means.
• the bioreactor and / or the bubble column contain a culture medium with a strain belonging to a filamentous fungus.
• This installation can advantageously implement the process described above, with the same advantages which result therefrom: better specific productivity than fermentation without intermediate dilution, in a single agitated tank, easier operability and maintenance of the installation, adaptation to productions of variable quantities ...
• the bubble column has an interior volume at least twice greater than the interior volume of the stirred bioreactor, which allows the bubble column to "absorb" the increased volume of the diluted culture medium and / or to be supplied by several agitated bioreactors.
• the installation described above can thus include n agitated and aerated bioreactors and m bubble columns, with n less than or equal to m (and with n greater than or equal to 1). DESCRIPTION OF THE FIGURES
• Figure 1 is a very schematic representation of the installation implementing the method of the invention.
• Figures 2a, 2b and 2c are graphs showing the evolution of the concentration of the mass of the proteins produced and of the mass of the cellular biomass according to three different examples.
• the present invention relates to a process for the production of cellulases by a strain belonging to the species Trichoderma reesei, in a stirred and aerated bioreactor comprising three stages:
• the first growth step in the presence of at least one carbonaceous growth substrate in the batch phase with a concentration of carbonaceous growth substrate of between 30 and 100 g / L, preferably from 50 to 80 g / L. This step is done in a stirred tank
• a second stage of dilution of the fermentation must by a factor greater than 1, 1 and preferably by 2.
• This stage of dilution can be carried out in the growth tank.
• Another solution is to carry out an online dilution during the transfer from the agitated tank to the bubble column.
• a third stage of production in a bubble column in the presence of at least one inducing carbon substrate in the fed-batch phase, this substrate being fed at a specific speed of between 35 and 140 mg per gram of cells and per hour, and preferably between 35 and 45 mg per gram of cells per hour.
• the production step is carried out under conditions of limitation of the inducing carbon substrate with a flow lower than the maximum consumption capacity of the strain.
• the inducing carbon source is lactose
• the aqueous solution is prepared at the concentration of 200-250 gL 1 .
• the inducing carbon substrate is pretreated biomass, we aim for an average sugar intake of between 35 and 140 mg per gram of cell and per hour.
• the process according to the present invention makes it possible to obtain a higher productivity in cellulases by better using agitated bioreactors: dedicated to the growth of biomass, they can be arranged, best suited to this stage only.
• the biomass is then transferred to bubble columns, which are large volume fermenters and which will be dedicated to the production of enzymes.
• the whole process is more economical in utilities (energy consumption %) and more flexible.
• control mode was thus adapted compared to conventional methods, on the one hand by diluting the concentration of mushrooms at the end of growth, and on the other hand by carrying out the production in bubble column.
• FIG. 1 An exemplary embodiment of the method according to the invention is understood from FIG. 1, representing an example of an installation implementing it.
• the installation thus comprises, first of all, a stirred tank 1, in which the growth stage will be carried out.
• This tank 1 has a volume of between 20 and 500 m 3 , preferably between 40 and 400 m 3 .
• the agitated tank 1 comprises between one and five agitation mobiles 4, with an agitation power of between 0.1 and 7 kW / m 3 , preferably with a maximum power of between 1 and 4 kW / m 3 by the motor 3.
• the tank 1 has a height / diameter ratio of between 1 and 5, preferably between 1, 5 and 3.
• the tank 1 is also provided with a bubbling device 5 in the lower part, fluidly connected to an external air inlet 6, and with one (or more) inlet (s) 2 in carbonaceous growth source and in mushrooms.
• the tank 1 operates between 1 and 5 bar abs. It is supplied with air (5.6) at a flow rate between 0.1 and 2 Volume of air (under normal conditions) per volume of liquid and per hour, preferably between 0.2 and 1 Volume of air per volume of liquid per hour.
• a bubble column is a reaction chamber composed of a cylindrical column, with a height to diameter ratio conventionally between 1 and 10, preferably between 2.5 and 6.
• the column is equipped with an injection device for air, possibly enriched with oxygen.
• This injection device is positioned at the bottom of the column so that the injected air supplies the entire volume with bubbles and therefore with oxygen.
• the injection device can be a perforated tray, a system of perforated tubes or any other system known to those skilled in the art.
• the book “Bubble Column Reactors” by WDDeckwer (J. Wiley & Sons, 1992) provides a set of description of bubble columns, which can be declined in multiple variants according to whether they are empty or equipped with internal type perforated trays, recirculation line or other.
• the simplified representation of the bubble column 7 according to FIG. 1 represents the supply 8 of the bubble column with an inducing carbon substrate, a bubbling device 1 1 at the bottom in fluid connection with an external air inlet 12.
• the bubble column at a volume between 40 and 1000 m 3 , it generally defines an interior volume at least 2 times greater than that of the agitated tank 1, to allow the dilution of the culture medium resulting from the growth in tank 1 .
• the bubble column 7 has a height / diameter ratio of between 2 and 10, preferably between 2.5 and 7.
• the bubble column can be empty, or equipped with an internal cylinder to promote the recirculation of the liquid.
• a person skilled in the art then speaks of an “airlift” reactor, which is a well-known variant of the simple bubble column and which can just as easily be used in the context of the present invention.
• the bubble column 7 operates between 1 and 5 bar abs. It is supplied with air 1 1, 12 at a flow rate of between 0.1 and 2 Volume of air (under normal conditions) per volume of liquid and per hour, preferably between 0.2 and 1 Volume of air per volume of liquid and per hour.
• Transfer of the culture medium from the agitated tank 1 to the ball column 7 is effected by a fluid connection 13 between the two reactors produced by one or more pipes connecting the two reactors.
• these pipes are arranged in the lower parts of the two reactors, both essentially oriented along a vertical axis.
• These lines are provided with a pump 15, and with manual or pilot-operated valves making it possible to control the transfer of the medium from the tank 1 to the bubble column 7.
• the stage of dilution of the culture medium between the stage of growth in tank 1 and the stage of production in the bubble column can be carried out in three different ways: - either in tank 1, at the end of growth, by supplying aqueous liquid to injection point 2, before transfer to column 7 - either at the fluid connection, by supplying aqueous liquid to the culture medium being transferred (injection point 14), - either at the inlet of column 7 by adding aqueous liquid to column 7 at the injection point 9. It is also possible to choose to mix these three dilution modes, that is to say to add a portion of the water required in the tank and another during the transfer and / or in the bubble column for example. The main thing is to reach the required dilution level, for example around 2, depending on the respective volumes of the two reactors, the viscosity of the culture medium at the end of the growth stage, etc.
• the prior dilution step according to the invention therefore makes it possible, in particular, to adjust the final biomass concentration to a target concentration of between 5 and 20 g / L.
• the industrial process for the production of enzymes according to the invention can use a lower number of stirred tanks than the number of bubble columns, which maximizes the use of both types of fermenters.
• step (a) lasts 50 hours and step (c) lasts 200 hours
• the contents of the agitated tank can be transferred after dilution (b) into a first bubble column, then the tank can be reused to perform step (a) again. His new content will then be diluted and transferred to another bubble column.
• the number of bubble columns and agitated reactors is to be adjusted according to the duration of each stage.
• Example 1 is an example of comparison of two productions carried out at concentrations of 12.5 g / L and 25 g / L respectively of biomass stabilized during the fed-batch production step.
• Example 2 according to the invention demonstrates the feasibility and the production performances obtained with a pipe carried out in a stirred tank during the growth step and in a bubble column during the enzyme production step.
• Comparative example 3 demonstrates the difficulty in carrying out the growth step in a bubble column without respecting the conditions of the invention.
• the production of cellulases is carried out in a mechanically stirred fermenter (not shown in Figure 1).
• the mineral medium has the following composition: KOH 1, 66 g./L, H3PO4 85% 2 mL / L, (NH 4 ) 2S0 4 2.8 g / L, MgS04, 7 H 2 0 0.6 g / L , CaCI 2 0.6 g / L, MnS0 4 3.2 mg / L, ZnS0 4 , 7 H2 o 2.8 mg / L, CoCI 2 10 4.0 mg / L, FeS0 4 , 7 H 2 0 10 mg / L, Corn Steep 1, 2 g / L, anti-foam 0.5 ml / L.
• the fermenter containing the mineral medium is sterilized at 120 ° C for 20 minutes, the carbonaceous glucose source is sterilized separately at 120 ° C for 20 minutes and then added sterile to tank 1 so as to have a final concentration of 25 g / L .
• the fermenter is seeded at 10% (v / v) with a liquid preculture of the Trichoderma reesei CL847 strain.
• the mineral medium of the preculture is identical to that of the fermenter, except for the addition of potassium phthalate at 5 gL 1 to buffer the pH of the medium.
• the growth of the mushroom in preculture is done using glucose as carbonaceous substrate, at the concentration of 30 gL 1 .
• the growth of the inoculum lasts 2 to 3 days, and is carried out at 28 ° C in a shaken incubator.
• the transfer to the fermenter is carried out when the residual concentration of glucose is less than 15 g / L.
• the growth stage is carried out for 50 hours in the agitated bioreactor 1 with an initial concentration of 50 g / L of glucose at a temperature of 27 ° C and a pH of 4.8 (regulated by ammonia 5.5 M).
• the aeration is 0.5 vvm (volume / volume / minute), and the partial pressure of dissolved oxygen in the medium is 40% of P 0 2 sat.
• the 50 g / L of glucose makes it possible, after exhaustion of the substrate, to a biomass concentration of approximately 25 g / L.
• Part of the medium is drawn off and diluted by a factor of two in another sterile bioreactor identical to the previous one, so as to have, at the start of the fed-batch phase of production, a bioreactor at 25 g / L of biomass and another at 12.5 g / L of biomass.
• a lactose solution at 250 g / L is injected continuously at a rate of 35 to 45 mg per g of cells and per hour up to 164 hours in the two reactors (approximately 2 ml_ / h for the experiment at 12, 5 g / L of biomass and 4 mL / h for the experiment at 25 g / L of biomass).
• the temperature is lowered to 25 ° C, and the pH is maintained at 4 until the end of the culture.
• the pH is regulated by adding a 5.5 N ammonia solution which provides the nitrogen necessary for the synthesis of the excreted proteins.
• the dissolved oxygen content is maintained at 40% P 0 2 sat.
• Figure 2a shows the evolution of the mass of cellular biomass and proteins for the experiment at 25 g / L of biomass and Figure 2b, the same curves but at a biomass stabilized at 12.5 g / L.
• Figure 2b shows the same curves but at a biomass stabilized at 12.5 g / L.
• 47 g of proteins are obtained with the experiment at 25 g / L of biomass, and only 36 g of proteins with the experiment at 12.5 g / L of biomass.
• the specific speed of protein production noted qp is significantly higher with experience at 12.5 g / L of biomass (about 65% higher). Indeed, it is 12 ⁇ 2 mg / g / h at 25 g / L of biomass and 20 ⁇ 1 mg / g / h with the experiment at 12.5 g / L of biomass.
• Example 2 The experiment is carried out under the same conditions as Example 1, except that after the dilution step (to go from a concentration of from 25 to 12.5 g / L of cellular biomass) in the agitated tank 1 , the production step is carried out in the bubble column 7 after transfer from one reactor to another by the system of pipes 13, the stirring being carried out therefore in production only by injecting air at a vvm of 1 in the bubble column.
• Example 3 is carried out under the same conditions as Example 1, but simulating a growth step in a bubble column, as in production: In this example, the growth is carried out in a tank stirred but without stirring, to mimic operation in a bubble column.
• the invention incorporates an intermediate dilution step with very interesting and unexpected effects on the performance of the process. It has also proved to be very advantageous to use two different types of reactors to operate the growth and production stages, so that these stages take place under industrially optimal conditions: the invention allows a transition from production of proteins to the industrial scale which is robust, efficient, which remains simple and which is very flexible in its implementation.

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