Process for producing enzymes with a strain belonging to a filamentous fungus

Abstract

A process for producing cellulolytic and/or hemicellulolytic enzymes with a strain of microorganism belonging to the family of filamentous fungi. The process includes growing the fungi in an aqueous culture medium, in the presence of at least one carbon-based growth substrate, in a stirred and aerated bioreactor. It also includes the production of enzymes, starting with the aqueous culture medium in the presence of at least one inductive carbon-based substrate and also inducing the production of hydrophobins. Further, at least a portion of the hydrophobins produced in step (b) are reintroduced into the growth step (a).

Claims

1. A process for producing cellulolytic and/or hemicellulolytic enzymes with a strain of Trichoderma reesei or of Trichoderma reesei modified by selective mutation or genetic recombination, said process comprising: a step (a) of growing the fungi in an aqueous culture medium, in the presence of at least one carbon-based growth substrate in a stirred and aerated bioreactor, a step (b) of producing enzymes from the aqueous culture medium obtained in the first step (a), in the presence of at least one inductive carbon-based substrate and also producing hydrophobins, wherein step (b) results in the aqueous culture medium comprising enzymes, fungi and hydrophobins; at least one separation step (c) performed on the aqueous culture medium from the production step (b) comprising separating out the hydrophobins in a liquid phase by filtration, and a step (d), wherein the hydrophobins produced in step (b) and separated in step (c) are reintroduced into the growth step (a), and wherein the process during step a), has a volumetric oxygen transfer coefficient K.sub.La of 185 to 480.

2. Process according to claim 1, wherein step (c) is performed by direct separation of at least a portion of the hydrophobins in liquid phase from the culture medium of the production step (b), wherein direct separation comprises a single step where hydrophobins are separated from the fungi and enzymes of the aqueous culture medium.

3. Process according to claim 1, wherein the, a separation step (c) comprises a first substep (C1) of separation between the fungi and the rest of the culture medium, and then a second substep (c2) of separation of the remainder of culture medium between the hydrophobins in liquid phase, and the enzymes.

4. Process according to claim 1, wherein the separation step (c) comprises at least one ultrafiltration of the hydrophobins from the liquid medium in which they are present so as to isolate the hydrophobins in the filtrate in the liquid phase.

5. Process according to claim 3, wherein the substep (c1) of separation between the fungi and the rest of the reaction medium is performed by filtration.

6. Process according to claim 1, wherein the hydrophobins isolated after separation step (c) are stored and diluted or concentrated before reintroduction step (d).

7. Process according to claim 1, wherein the hydrophobins produced in the production step (b) that are reintroduced into the growth step (a) are at least 50% type II hydrophobins.

8. Process according to claim 1, wherein the growth step (a) is performed in batch mode, fed-batch mode, continuous mode, or in several of these modes successively.

9. Process according to claim 1, wherein in step (d), the hydrophobins are reintroduced into the culture medium of the growth step (a) continuously, throughout the duration of said growth step (a).

10. Process according to claim 1, wherein the hydrophobins produced in the production step (b) are reintroduced into the growth step (a) in the form of a filtrate in aqueous phase obtained from the culture medium of step (b), the water of the culture medium of the production step (a) coming totally or partly from said filtrate.

11. Process according to claim 1, wherein the hydrophobins are reintroduced into the growth step (a) in solution in an aqueous medium at a concentration of between 10 and 400 mg/l.

12. Process according to claim 1, wherein during the growth step (a), the concentration of carbon-based growth substrate is between 15 and 100 g/l, and wherein the production step (b) is performed with a limiting stream of inductive carbon-based substrate, of between 30 and 140 mg.Math.g.sup.−1.Math.h.sup.−1.

13. Process according to claim 1, wherein the production step (b) is performed in batch mode, fed-batch mode, continuous mode, or in several of these modes successively.

14. The process of claim 1 wherein the growth step (a) is performed in batch mode and the production step (b) is performed in fed-batch mode.

15. A process for producing cellulolytic and/or hemicellulolytic enzymes with a strain of Trichoderma reesei or of Trichoderma reesei modified by selective mutation or genetic recombination, said process comprising: a step (a) of growing the fungi in an aqueous culture medium, in the presence of at least one carbon-based growth substrate in a stirred and aerated bioreactor, a step (b) of producing enzymes from the aqueous culture medium obtained in the first step (a), in the presence of at least one inductive carbon-based substrate and also producing hydrophobins, wherein step (b) results in the aqueous culture medium comprising enzymes, fungi and hydrophobins; at least one separation step (c) performed on the aqueous culture medium from the production step (b) comprising separating out the hydrophobins in a liquid phase by filtration, and a step (d), wherein at least a portion of the hydrophobins produced in step (b) and separated in step (c) are reintroduced into the growth step (a), and wherein the reintroduced hydrophobin concentration is in the range of 10-400 mg/l and wherein the process during step (a), has a volumetric oxygen transfer coefficient K.sub.La of 185 to 480.

16. The process of claim 15, wherein the reintroduced hydrophobin concentration is in the range of 50-200 mg/l.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a block diagram of an enzyme production process according to a first variant of the invention.

(2) FIG. 2 is a block diagram of an enzyme production process according to a second variant of the invention.

(3) FIG. 3 is a graph representing, on the x-axis, the concentration of fungi (cellular biomass) in g/l, and, on the y-axis, the corresponding kLa in h.sup.−1 for water (curve CO), and for Example 1 and Example 2 described below (curves C1 and C2, respectively).

DESCRIPTION OF THE EMBODIMENTS

(4) FIG. 1 corresponds to the first variant of the enzyme production process according to the invention. The coalescence-limiting molecules are hydrophobins produced during the carbon-based substrate limitation phase. The process involves: a step (a) of growing the fungi and then a growth step (b) according to the invention, a step (c) of separation by ultrafiltration is added, making it possible to obtain, on the one hand, a retentate F+E rich in fungi and in enzymes which they have produced, and, on the other hand, a filtrate containing water (the culture medium of steps (a) and (b) is aqueous) enriched in hydrophobins. This filtrate is then reinjected into the growth step (a). Before reinjection, it may be diluted or, more frequently, concentrated, and it may also be temporarily stored.

(5) In this variant, the hydrophobins are thus filtered directly from the fermentation medium, and the fungi and enzymes are left together. This variant is particularly advantageous when the downstream process using the enzymes can exploit these enzymes without prior separation from the fungi: only one separation is necessary to perform the invention.

(6) FIG. 2 corresponds to a second variant of the process according to the invention. The growth step (a) and then the production step (b) are found. Two successive separations according to the invention are envisaged here: first a separation (c1), for example by filtration with a filter press, making it possible to separate, on the one hand, a retentate F rich in fungi, and, on the other hand, a filtrate enriched in enzymes and hydrophobins. Next, this filtrate undergoes a separation (c2) by ultrafiltration making it possible to recover, on the one hand, a retentate enriched in enzymes, and, on the other hand, a filtrate comprising water and enriched in hydrophobins, this filtrate then being reinjected, as in the preceding variant, into the growth step (a).

(7) The enzymes have thus been separated here from the rest of the culture medium, which is advantageous when the downstream process uses these enzymes in pure form or to market them.

(8) In these two variants notably, but for any other variant of the invention, there is a separation in the production step (b). It should be noted that this separation preferentially takes place at the end of step (b), and that this reinjection preferentially takes place at the start of step (a). However, it is also possible to perform the separation via methods other than filtration, and it is also possible for the separation to be performed throughout step (b) or over only a portion of its duration. Similarly, the reinjection of the hydrophobins into step (a) may also take place gradually over all or part of the duration of step (a).

(9) The hydrophobins are preferably reintroduced in aqueous liquid form (they are obtained directly in this form by performing the separations by filtration). After adjusting the concentration in the liquid phase, this liquid phase may even entirely replace the water used for the culture medium of the growth step (a).

Implementation Examples

(10) Two comparative growths of Trichoderma reesei in a 30 L bioreactor were performed: The first is performed with a conventional culture medium, and the second is performed by replacing the water with a hydrophobin-rich filtrate obtained from a previous production. This filtrate was obtained by filtering the culture medium by ultrafiltration with membranes having a 10 kDa cut-off threshold; it was then concentrated.

(11) Rheological measurements and kLa measurements are performed in both cases. In the first case, it is seen that the viscosity greatly affects the oxygen transfer (as demonstrated in the abovementioned 2012 article by Gabelle J.-C.). In the second case, despite the high viscosity, supplying hydrophobins has a positive impact on the kLa of the medium, which becomes equivalent to, or even higher than, that of water, obtained under the same stirring and aeration conditions.

(12) The rheology measurement is used according to the method described in the following article:

(13) Nicolas Hardy, Frederic Augier, Alvin W. Nienow, Catherine Béal, Fadhel Ben Chaabane. Scale-up agitation criteria for Trichoderma reesei fermentation: Chemical Engineering Science, Elsevier, 2017, 172, pages 158-168(10.1016/j.ces.2017.06.034).

(14) Calculation of the kLa is performed via the known gas balance method described in the abovementioned article: Gabelle J. C., Jourdier E., Licht R. B., Ben Chaabane F., Henaut I., Morchain J., and Augier F. (2012) Impact of rheology on the mass transfer coefficient during the growth phase of Trichoderma reesei in stirred bioreactors. Chemical Engineering Science 75, 408-417. The reactor used is a 30 L bioreactor. Its configuration is described in the same article.

(15) Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The preceding preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

(16) In the foregoing and in the examples, all temperatures are set forth uncorrected in degrees Celsius and, all parts and percentages are by weight, unless otherwise indicated.

(17) The entire disclosures of all applications, patents and publications, cited herein and of corresponding French application No. 19/12.856, filed Nov. 18, 2019, are incorporated by reference herein.

Example 1 (Comparative Example—Invention Preliminary)

(18) The growth of Trichoderma reesei is performed in the abovementioned mechanically stirred 30 L fermenter with a working volume of 20 L. The mineral medium has the following composition: KOH 1.66 g/L, 85% H.sub.3PO.sub.4 2 mL/L, (NH.sub.4).sub.2SO.sub.4 2.8 g/L, MgSO.sub.4.7H.sub.2O 0.6 g/L, CaCl.sub.2) 0.6 g/L, MnSO.sub.4 3.2 mg/L, ZnSO.sub.4.7H.sub.2O 2.8 mg/L, CoCl.sub.2 104.0 mg/L, FeSO.sub.4.7H.sub.2O 10 mg/L, Corn Steep 1.2 g/L, antifoam 0.5 mL/L (which will be at least partly consumed by the microorganism). Mains water is used to dilute the various components of the medium and to fill the reactor so as to obtain a final volume of 20 L.

(19) The fermenter containing the mineral medium is sterilized at 120° C. for 20 minutes, the carbon-based glucose source is sterilized separately at 120° C. for 20 minutes and then added under sterile conditions to the bioreactor so as to obtain a final concentration of 50 g/L. The fermenter is seeded with a liquid preculture of 1 L of the strain of Trichoderma reesei CL847. The mineral medium of the preculture is identical to that of the fermenter, except for the addition of potassium phthalate at 5 g.Math.L.sup.−1 to buffer the pH of the medium. The growth of the fungus in preculture is performed using glucose as carbon-based substrate, at a concentration of 30 g.Math.L.sup.−1. The growth of the inoculum lasts 2 to 3 days, and is performed at 28° C. in a shaking incubator. Transfer to the fermenter is performed when the residual glucose concentration is less than 15 g/L.

(20) The growth step is performed for 50 hours in the stirred 30 L bioreactor at a temperature of 27° C. and a pH of 4.8 (adjusted with 5.5 M aqueous ammonia). The aeration is 0.5 vvm (volume/volume/minute) and the concentration percentage of dissolved oxygen relative to saturation in the liquid medium is adjusted to 40%. The fermenter is equipped with a stirrer containing two impellers with inclined straight paddles, rotating at a speed of 1200 rpm.

(21) Samples are taken regularly to monitor the rheology of the medium and the concentration of cellular biomass. Since there are no insoluble components in the culture medium, the dry weight, determined by filtration and drying to constant weight, represents the mass of fungi, also known as the cellular biomass. An analyser at the bioreactor outlet makes it possible to monitor the O.sub.2 and CO.sub.2 composition of the gas.

(22) The gas balances make it possible to continuously calculate the rate of O.sub.2 consumption, rO.sub.2 and the kLa:

(23) This gives, at the pseudo-stationary state
rO.sub.2=Qin*% O.sub.2in−Qout*% O.sub.2out
rCO.sub.2=Qout*% CO.sub.2out−Qin*% CO.sub.2in
with:
Qin: air flow rate at the inlet in mol/h
Qout: air flow rate at the outlet in mol/h
% O.sub.2in: mol % of O.sub.2 at the inlet
% O.sub.2out: mol % of O.sub.2 at the outlet
% CO.sub.2in: mol % of CO.sub.2 at the inlet
% CO.sub.2out: mol % of CO.sub.2 at the outlet

(24) The rO.sub.2 is used to calculate the culture kLa by means of the combination of the two O.sub.2 material balances on the liquid phase and the gas phase at the pseudo-stationary state:
kLa(t)=rO.sub.2/(O.sub.2*−O.sub.2L)
with:
O.sub.2*: concentration of O.sub.2 at saturation
O.sub.2L: concentration of oxygen in the liquid

(25) According to Henrys law, the maximum concentration of a gas in solution, at equilibrium with an atmosphere containing this gas, is proportional to the partial pressure of this gas at this point.

(26) This thus gives, for the case of O.sub.2:
O.sub.2*(mol/m.sup.3)=1.25pO (bar)
with:
pO: partial pressure of O.sub.2

(27) It should be noted that the partial pressure of O.sub.2 is equal to the product of the mole fraction of O.sub.2 in the gas and of the pressure. The O.sub.2* in an industrial fermenter is thus maximal at the bottom of the reactor (maximum pressure and percentage of O.sub.2 at the inlet of 21%) and minimal at the top (headspace pressure and percentage of O.sub.2 in the gas at the outlet). It is calculated at each moment in the experiment, since the O.sub.2 composition of the exiting gas decreases due to the consumption of O.sub.2 by the microorganism. In the case of a laboratory reactor, the pressure difference between the top and the bottom of the reactor is negligible.

(28) The oxygen concentration in the liquid is calculated by means of the pO.sub.2 probe measurement, which gives a percentage of O.sub.2 relative to saturation.

(29) Thus, during the growth of the fungus, it was possible to measure at different moments the concentration of cellular biomass (X), the viscosity (pa) of the fermentation medium at a shear of 10 s.sup.−1, and a coefficient of gas-liquid transfer kLa in h.sup.−1.

(30) The kLa measurements are compared with a measurement taken in water, at the same air flow rate and the same stirring speed. Table 1 below presents the results for the transfer coefficients obtained during the fermentation according to the prior art.

(31) TABLE-US-00001 TABLE 1 kLa (h.sup.−1) kLa water (h.sup.−1) 2.5 0.03 180 217 3.8 0.055 150 217 9.2 0.21 82 217 16 0.48 45 217

(32) The production phase was then performed at pH 4 at 25° C., with a lactose concentration of 220 g/L, corresponding to a specific fed-batch lactose flow rate of 45 mg per gram of biomass and per hour.

(33) Example 2 (according to the invention) An experiment is performed under the same conditions, but replacing the water with a solution obtained from a previous cellulase production experiment on which a concentration of 100 mg/L of HFBII hydrophobins (only the HFBIIs were assayed, it is possible that the hydrophobins used also comprise other types of hydrophobins, in minor amount) was determined by HPLC (high-performance liquid chromatography) with a Wide Pore C5 column (150×2.1 mm; 5 μm) and UV detection. Filtration was performed at the end of this experiment to separate the fungus from the cellulases, and ultrafiltration with membranes having a porosity of 10 kDa (the recommended membranes are UFX 10 pHt membranes sold by the company Alfa Laval. The supplier's information should be adhered to for their use (pressure, temperature, etc. conditions). For the product, it is preferable not to exceed 30° C. during this ultrafiltration, 20 to 25° C. being a preferred temperature range. After ultrafiltration, the permeates contain the hydrophobins. Analysis of the mean permeate sample makes it possible to assay the sample. The flow rates obtained are 20 l/h/m.sup.2 of membrane. Ultrafiltration makes it possible to concentrate the cellulases and to recover in the filtrate a medium containing hydrophobins. The hydrophobins concentration was then measured again by HPLC and is close to 100 mg/L. It is this solution which was used as dilution water in the 30 L bioreactor. It should be noted that additional water was added, thus diluting the hydrophobin concentration at the start (time T0) of the experiment by 25%. The operating conditions are the same as for Example 1.

(34) The production phase is also conducted, after the growth phase, under the same operating conditions as for Example 1.

(35) The viscosity and the transfer coefficient are measured during the growth of the fungus. The results obtained are presented in Table 2, which indicates the concentration of cellular biomass (X), the viscosity of the fermentation medium at a shear of 10 s.sup.−1 (μa), and a coefficient of gas-liquid transfer kLa in h.sup.−1 as in the preceding Table 1.

(36) TABLE-US-00002 TABLE 2 kLa (h.sup.−1) kLa water (h.sup.−1) 2 0.021 480 217 4 0.06 370 217 10 0.24 185 217 15 0.44 120 217

(37) A comparison of the performance obtained according to the two Examples 1 and 2 is shown on the graph of FIG. 3: It is noted that the transfer coefficients kLa obtained according to the invention (curve C1 for Comparative Example 1, curve C2 for Example 2 according to the invention) are at least two times higher than according to the prior art. Depending on the biomass concentration, the transfer coefficient is sometimes lower and sometimes higher than that measured in water (curve CO) under the same aeration and stirring conditions.

(38) These examples show that the oxygen transfer is greatly facilitated by the invention. This advantage may be exploited in various ways, and notably: to lower the stirring speed in the bioreactor, to reach the target transfer coefficient while consuming less energy, to lower the air flow rate in the bioreactor, for the same reasons, to perform culturing with more cellular biomass, while at the same time remaining above a minimum target concentration of dissolved oxygen, which makes it possible to increase the productivity of the bioreactors (also known as fermenters).

(39) It was also checked that the enzyme production performance of the two examples, evaluated at the end of the production phase, are the same or virtually the same for the two examples:

(40) these advantages are thus not obtained at the expense of the performance or the production yield of the process.

(41) As the transfer coefficient kLa is proportional to the power dissipated per unit volume (P/V, expressed in W/m.sup.3), within a power range per unit volume P/V of between 0.4 and 0.5 kW/m.sup.3 (according to the abovementioned 2012 article by Gabelle et al.), increasing the kLa by a factor of at least 2 makes it possible to save between 75% and 85% of the power dissipated per unit volume, which is an enormous saving at the industrial scale.

(42) It should moreover be noted that while Examples 1 and 2 use specific carbon sources for the growth and for the production, the invention naturally applies with other carbon sources, such as soluble sugars, for instance lactose, glucose or xylose. The carbon-based growth substrate may be chosen from lactose, glucose, xylose, residues obtained after ethanolic fermentation of monomer sugars from the enzymatic hydrolysates of cellulose-based biomass, and/or a crude extract of water-soluble pentoses obtained from the pretreatment of a cellulose-based biomass. The inductive carbon-based substrate is preferably chosen from lactose, cellobiose, sophorose, residues obtained after ethanolic fermentation of monomer sugars from the enzymatic hydrolysates of cellulose-based biomass, and/or a crude extract of water-soluble pentoses obtained from the pretreatment of a cellulose-based biomass. This type of residue/extract may thus also be used as a source of total carbon, i.e. both for the growth of the microorganism and for the induction of the expression system. This carbon source may be used more particularly by genetically enhanced strains and notably recombinant strains.

(43) Similarly, the invention also applies under operating conditions different from those expressly envisaged in the examples. Thus, the pH and the temperature, for the growth step and the production step, may be as follows: pH between 3.5 and 4.4; temperature between 20 and 35° C.

(44) The vvm (degree of aeration expressed as volume of air per volume of reaction medium and per minute) applied during the process is between 0.3 and 1.5 min.sup.−1 and the rpm (stirring speed) must make it possible to regulate the concentration percentage of dissolved oxygen relative to saturation in the liquid medium to between 20% and 60% of O.sub.2. An aeration of 0.3 to 0.5 min.sup.−1 and stirring which makes it possible to regulate the concentration percentage of dissolved oxygen to between 30% and 40% of O.sub.2 are preferably chosen.

(45) Depending on its nature, the carbon-based substrate chosen for the production of the biomass is introduced into the fermenter before sterilization, or is sterilized separately and introduced into the fermenter after sterilization. The concentration of carbon-based substrate is between 200 and 700 g/L depending on the degree of solubility of the carbon-based substrates used (notably as regards the inductive substrate which forms part of these carbon-based substrates).

(46) The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples.

(47) From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention and, without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.