METHOD FOR THE PRODUCTION OF ENZYMES BY A STRAIN BELONGING TO A FILAMENTOUS FUNGUS

Abstract

The present invention concerns a process for producing enzymes by a strain belonging to a filamentous fungus, said process comprising two steps: (a) a first step of growing the fungi, in the presence of at least one carbon-based growth substrate, in a stirred and aerated bioreactor in batch phase, at a pH of not more than 4.6; (b) a second step of producing enzymes, starting from the culture medium obtained in the first step (a), in the presence of at least one inductive carbon-based substrate, at a pH of not more than 4.6.

Claims

1. A process for producing enzymes by a strain belonging to a filamentous fungus, characterized in that said process comprises two steps: (a) a first step of growing the fungi, in the presence of at least one carbon-based growth substrate, in a stirred and aerated bioreactor in batch phase, at a pH of not more than 4.6; (b) a second step of producing enzymes, starting from the culture medium obtained in the first step (a), in the presence of at least one inductive carbon-based substrate, at a pH of not more than 4.6.

2. The process as claimed in claim 1, characterized in that the pH in the growth step (a) and/or in the production step (b) is not more than 4.4, in particular between 3.5 and 4.4.

3. The process as claimed in claim 1, characterized in that the pH in the growth step (a) is substantially identical to the pH in the production step (b).

4. The process as claimed in claim 1, characterized in that the pH in the production step (b) is more acidic than the pH in the growth step (a).

5. The process as claimed in claim 1, characterized in that the pH is regulated during the growth step (a) by controlled addition of a nitrogen compound, especially aqueous ammonia.

6. The process as claimed in claim 1, characterized in that the production step (b) operates in batch, fed-batch or continuous mode, or in two or more of these modes successively.

7. The process as claimed in claim 1, characterized in that it comprises an intermediate step (c) between step (a) and step (b), this intermediate step (c) being a step of diluting the culture medium obtained in the growth step (a).

8. The process as claimed in claim 1, characterized in that the concentration of carbon-based growth substrate during the first, growth step (a) is between 15 and 60 g/L.

9. The process as claimed in claim 1, characterized in that the second, production step (b) is operated with a limiting stream of inductive carbon-based substrate, notably of between 30 and 140 mg.Math.g.sup.−1.Math.h.sup.−1 and preferably between 35 and 45 mg.Math.g.sup.−1.Math.h.sup.−1, and preferably with an aqueous solution of inductive carbon-based substrate at a concentration of between 200 and 600 g/L.

10. The process as claimed in claim 1, characterized in that the strain used is a strain of Trichoderma reesei or of Trichoderma reesei modified by selective mutation or genetic recombination.

11. The process as claimed in claim 1, characterized in that the enzymes are cellulolytic and/or hemicellulolytic enzymes.

12. The process as claimed in claim 1, characterized in that it is operated in the absence of antifoams, in particular during the production step (a).

13. A method for the enzymatic hydrolysis of terrestrial or marine cellulosic/hemicellulosic biomass, comprising hydrolysing said biomass by the enzymes obtained by the process of claim 1.

Description

LIST OF FIGURES

[0045] FIG. 1 shows photos of the bioreactors used in examples compliant and not compliant with the process of the invention with one strain, the photos taken during the growth step of the process.

[0046] FIG. 2 shows photos of the bioreactors used in examples compliant and not compliant with the process of the invention with a different strain, the photos taken during the growth step of the process.

[0047] FIG. 3 is a graph showing the concentration in grams per liter of biomass and proteins produced in a comparative example.

[0048] FIG. 4 is a graph showing the concentration in grams per liter of biomass and proteins produced in a working example of the invention.

[0049] FIG. 5 is a graph showing the concentration in grams per liter of biomass and proteins produced in a working example of the invention.

DESCRIPTION OF THE EMBODIMENTS

[0050] The present invention has developed a process for producing enzymes, especially cellulases, in which the incidence of foam is prevented. It has surprisingly emerged that operating at low pH levels during growth (of lower than 4.6, in particular not more than 4.4) does not slow growth of the microorganism and prevents the incidence of foam.

[0051] The process regime comprises 2 phases: [0052] a batch mode phase lasting preferably between 30 and 50 hours, with a pH advantageously of not more than 4.4 and with a concentration of carbon-based substrate of 15 to 60 g/L preferably [0053] a fed-batch mode phase lasting preferably between 100 and 200 hours, in particular between 100 and 150 hours, advantageously at a pH likewise of not more than 4.4, and with a limiting stream of carbon source of from 35 to 140 mg.Math.g.sup.−1.Math.h.sup.−1 and preferably between 35 and 45 mg.Math.g.sup.−1.Math.h.sup.−1.

[0054] The industrial strains used belong to the species Trichoderma reesei, and are modified to enhance the cellulolytic and/or hemicellulolytic enzymes by mutation-selection methods, an example being the strain CL847 (one such method is described in particular in U.S. Pat. No. 4,762,788). Strains enhanced by genetic recombination techniques may also be used. These strains are cultured in stirred and aerated fermenters under conditions compatible with their growth and the production of the enzymes.

[0055] The main carbon sources may be soluble sugars such as lactose, glucose or xylose:

[0056] The carbon-based growth substrate is preferably selected from lactose, glucose, xylose, residues obtained after ethanolic fermentation of monomeric sugars from the enzymatic hydrolysates of cellulosic biomass, and/or a crude extract of water-soluble pentoses from the pretreatment of a cellulosic biomass.

[0057] The inductive carbon-based substrate is preferably selected from lactose, cellobiose, sophorose, residues obtained after ethanolic fermentation of monomeric sugars from the enzymatic hydrolysates of cellulosic biomass, and/or a crude extract of water-soluble pentoses from the pretreatment of a cellulosic biomass.

[0058] This type of residue/extract may thus also be used as a total carbon source, i.e. both for the growth of the microorganism and for the induction of the expression system. This carbon source can be utilized more particularly by genetically enhanced strains and, especially, recombinant strains.

[0059] The operating conditions of pH and temperature, for the growth step and the production step, are as follows: [0060] pH between 3.5 and 4.4; [0061] temperature between 20 and 35° C. Preference is given to selecting a pH of 4.4 and a temperature of 27° C. during the growth phase, and a pH of 4 or of likewise 4.4 and a temperature of 25° C. during the fed-batch mode production phase.

[0062] The vvm (degree of aeration expressed as volume of air per unit volume of reaction medium per minute) applied during the process is between 0.3 and 1.5 min.sup.−1 and the rpm (stirring speed) must allow the O.sub.2 pressure to be regulated to between 20% and 60%. An aeration of 0.3 to 0.5 vvm and stirring which allows the O.sub.2 pressure to be regulated to 30% or 40% are preferably selected.

[0063] Depending on its nature, the carbon-based substrate selected for producing 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 600 g/L depending on the degree of solubility of the carbon-based substrates used (notably as regards the inductive substrate).

EXAMPLES

[0064] The strains are precultured in 2 Fernbach flasks with a useful capacity of 500 mL, which are seeded with one tube each of T. reesei TR3002 and CL847 spores. They are placed in an INFORS HT Multitron incubator at 30° C., with orbital stirring at 150 rpm for 72 hours. They are then consigned to 8 vacuum-sterilized flasks (80 mL per flask) which will be used to seed each fermenter/bioreactors.

[0065] The operating conditions for producing cellulases from the strains obtained after preculturing are as follows:

[0066] The experiments comprise two phases: [0067] a growth phase on glucose at a temperature of 27° C. and a pH (regulated using 5.5 M aqueous ammonia) ranging from 4.0 to 5.5 according to experiment. Aeration is set at 0.2 L/min (0.3 vvm) and the setpoint pO.sub.2 at 40%. Kept constant by virtue of the stirring is [0068] an enzyme production phase induced by a limiting stream of the fed-batch solution after 30 hours' culturing. The solution is injected with a constant rate of 0.5 g of carbon-based substrate per hour. The setpoint temperature is modified to 25° C. This phase lasts between 160 and 220 hours according to substrate availability and experimental progression.

[0069] Sampling takes place each day, with monitoring of the dry weight and the concentrations of glucose, lactose, galactose, and xylose. 5 mL culture supernatants are stored at 4° C. for protein and enzyme assays conducted at the end of culturing.

Example 1

[0070] Example 1 is carried out starting from the strain TR3002. This strain is described in the following publications: Ben Chaabane F, Jourdier E, Licht R, Cohen C and Monot F (2012) “Kinetic characterization of Trichoderma reesei CL847 TR3002: an engineered strain producing highly improved cellulolytic cocktail”, Journal of Chemistry and Chemical Engineering 6 (2), 109-117, and Ayrinhac C, Margeot A, Ferreira N L, Ben Chaabane F, Monot F, Ravot G, Sonet J.-M and Fourage L (2011) “Improved saccharification of wheat straw for biofuel production using an engineered secretome of Trichoderma reesei”, Organic Process Research and Development 15 (1), 275-278. [0071] The growth phase is conducted at pH 4, at 27° C. and with a glucose concentration of 15 g/L [0072] The production phase is conducted at pH 4, at 25° C. and with a lactose concentration of 220 g/L, corresponding to a specific fed-batch lactose flow rate of 45 mg per gram of biomass per hour.

Example 2: (Comparative)

[0073] Example 2 is carried out starting from the strain TR3002. [0074] The growth phase is conducted at pH 4.8, at 27° C. and with a glucose concentration of 15 g/L [0075] The production phase is conducted at pH 4.8, at 25° C. and with a lactose concentration of 220 g/L, corresponding to a specific fed-batch lactose flow rate of 45 mg per gram of biomass per hour.

Example 3: (Comparative)

[0076] Example 3 is carried out starting from the strain TR3002. [0077] The growth phase is conducted at pH 5.5, at 27° C. and with a glucose concentration of 15 g/L [0078] The production phase is conducted at pH 5.5, at 25° C. and with a lactose concentration of 220 g/L, corresponding to a specific fed-batch lactose flow rate of 45 mg per gram of biomass per hour.

Example 4

[0079] Example 4 is carried out starting from the strain TR3002. [0080] The growth phase is conducted at pH 4.4, at 27° C. and with a glucose concentration of 15 g/L [0081] The production phase is conducted at pH 4.4, at 25° C. and with a lactose concentration of 220 g/L, corresponding to a specific fed-batch lactose flow rate of 45 mg per gram of biomass per hour.

Example 5

[0082] Example 5 is carried out starting from the strain CL847. This strain is described in the following publications:—Jourdier E, Poughon L, Larroche C, Monot F and Ben Chaabane F (2012) “A new stoichiometric miniaturization strategy for screening of industrial microbial strains: application to cellulase hyper-producing Trichoderma reesei strains”, Microbial Cell Factories 11, 70 (Impact Factor: 3,60), and Jourdier E, Ben Chaabane F, Poughon L, Larroche C and Monot F (2012) “Simple Kinetic Model of Cellulase Production by Trichoderma Reesei for Productivity or Yield Maximization”, Chemical Engineering Science 27, 313-318. [0083] The growth phase is conducted at pH 4, at 27° C. and with a glucose concentration of 15 g/L [0084] The production phase is conducted at pH 4, at 25° C. and with a lactose concentration of 220 g/L, corresponding to a specific fed-batch lactose flow rate of 45 mg per gram of biomass per hour.

Example 6

[0085] Example 6 is carried out starting from the strain CL847. [0086] The growth phase is conducted at pH 4.4, at 27° C. and with a glucose concentration of 15 g/L [0087] The production phase is conducted at pH 4.4, at 25° C. and with a lactose concentration of 220 g/L, corresponding to a specific fed-batch lactose flow rate of 45 mg per gram of biomass per hour.

Example 7: (Comparative)

[0088] Example 7 is carried out starting from the strain CL847. [0089] The growth phase is conducted at pH 4.8, at 27° C. and with a glucose concentration of 15 g/L [0090] The production phase is conducted at pH 4.8, at 25° C. and with a lactose concentration of 220 g/L, corresponding to a specific fed-batch lactose flow rate of 45 mg per gram of biomass per hour.

Example 8: (Comparative)

[0091] Example 8 is carried out starting from the strain CL847. [0092] The growth phase is conducted at pH 5.5, at 27° C. and with a glucose concentration of 15 g/L [0093] The production phase is conducted at pH 5.5, at 25° C. and with a lactose concentration of 220 g/L, corresponding to a specific fed-batch lactose flow rate of 45 mg per gram of biomass per hour.

[0094] Visual observation of the incidence or nonincidence of foam leads to the results collated in table 1 below:

TABLE-US-00001 TABLE 1 Example Strain Growth pH Visual observation Example 1 TR3002 4 No foam Example 2 TR3002 4.8 Foam from 24 h then continuously Example 3 TR3002 5.5 Foam from 24 h then continuously Example 4 TR3002 4.4 No foam Example 5 CL847 4 No foam Example 6 CL847 4.4 No foam Example 7 CL847 4.8 Foam from 24 h then continuously until sporulation Example 8 CL847 5.5 Foam from 24 h then continuously until sporulation

[0095] FIGS. 1 and 2 show photographs of the bioreactors of examples 1, 2, 3, 5, 6, 7 and 8 after 24 hours: (reference 1 corresponds to example 1 and so on).

[0096] From table 1 and FIGS. 1 and 2 it is noted that whichever strain is used (CL847 or TR3002), a growth-phase pH greater than or equal to 4.8 results in production of foam from 24 hours onward. After that, this foam cannot be controlled, and so the fermentations stop at a fairly high pH owing to overflow or sporulation. Conversely there was no foaming with the cultures at a pH of not more than 4.4.

[0097] To ascertain whether the selection of the growth pH at values of not more than 4.4 in accordance with the invention had any effect otherwise on the performance of the strain, a calculation was made of the specific protein production rate qp. This specific rate qp is equal to rp/X, where rp is the protein productivity in g/L/h, and X is the concentration of biomass in g/L. The values obtained for qp are as follows:

[0098] Example 1 (pH 4 growth, strain TR3002): qp=28.4 mgP/gX/h

[0099] Example 2 (pH 4.8 growth, strain TR3002): qp=27.7 mgP/gX/h

[0100] Example 4 (pH 4.4 growth, strain TR3002): qp=25.4 mgP/gX/h

[0101] Example 5 (pH 4 growth, strain CL847): qp=8.5 mgP/gX/h

[0102] Example 6 (pH 4.4 growth, strain CL847): qp=9.4 mgP/gX/h

[0103] For examples 7 and 8, the values of qp are not significantly different from those of examples 5 and 6 while no foaming has started, but foaming thereafter interrupted the experiments for these two examples.

[0104] It is therefore found that, for a given strain, lowering the growth pH had no significant effect on either the specific rate qp or the overall productivity of the process. Indeed, the final concentration of proteins, for a given production time, is no longer affected by the lowering of the growth pH: amounts of around 40 g/L are obtained for the examples with strain TR3002 irrespective of growth pH, and amounts of around 25 g/L for the examples with strain CL847 irrespective of growth pH.

[0105] FIG. 3 indicates the production in g/L of biomass (line with dots) and of proteins (line with triangles) as a function of time expressed in hours for comparative example 2, with FIG. 4 showing the same type of graph for inventive example 1, and FIG. 5 for inventive example 4: comparison of these graphs confirms that protein production remains at the same level whether or not the growth pH is lowered. It is also apparent (example 4, FIG. 5) that adopting the same pH for growth and production no longer has any adverse effect on protein production. Additionally the enzymatic activity levels of the enzymes produced were evaluated, by measurement of what is called the Filter Paper Activity (“FPase”). This method allows assaying of the overall activity of the enzymic pool (endoglucanases and exoglucanases). The FPase activity is measured on Whatman #1 paper (procedure recommended by the IUPAC biotechnology commission): A determination is made of the sample of the enzymatic solution that produces a 4% advancement of the enzymatic reaction in 60 minutes. The principle of the filter paper activity is to use a DNS (dinitrosalicylic acid) assay to determine the amount of reduced sugars obtained from a Whatman #1 paper.

[0106] The FPase values obtained for the 8 examples, which thus convey the overall activity of the enzyme cocktail, and hence its quality, exhibit values which are conventional for the strains used, of between 0.8 and 1 IU/mg.

[0107] The conclusion drawn from this is that although the invention enables particularly simple and effective prevention of foaming, it has no negative impact on either the enzyme production yield or the quality of the enzymes produced.