Process for the continuous production of cellulases by a filamentous fungus using a carbon substrate obtained from an acid pretreatment

• US 9,249,402 B2
• Filed: 09/26/2012
• Issued: 02/02/2016
• Est. Priority Date: 10/14/2011
• Status: Active Grant

• Current Assignee: IFP Energies Nouvelles
• Original Assignee: IFP Energies Nouvelles
• Inventors: Ben Chaabane, Fadhel, Chaussepied, Bernard
• Primary Examiner(s): NOAKES, SUZANNE MARIE
• Application Number:
  US14/350,827
• Publication Number:
  US 20140295524A1
• Time in Patent Office: 1,224 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12N 1/14 Fungi culture of mushrooms A01G18/00; as new plants A01H15/00; Culture media therefor
• CPC Class Codes: C12N 1/38 Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound C12N1/34 takes precedence
• CPC Class Codes: C12N 9/18 Carboxylic ester hydrolases (3.1.1)
• CPC Class Codes: C12N 9/2437 Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
• CPC Class Codes: C12P 21/00 Preparation of peptides or proteins single cell protein C12N1/00
• CPC Class Codes: C12Y 301/01073 Feruloyl esterase (3.1.1.73)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

A process for the production of cellulases and hemicellulases using a strain of a filamentous fungus in a stirred and aerated bioreactor, in at least two phases, a phase a) for growth of said strain in the presence of at least one carbonaceous growth substrate in a closed reactor, a phase b) for the continuous production of cellulases, in which at least one carbonaceous inducer substrate is supplied at a supply rate which is constant over a period of at least more than 200 h, said carbonaceous substrate being at least one aqueous hemicellulosic hydrolysate solution obtained from an acid pre-treatment of a lignocellulosic substrate, said aqueous hemicellulosic hydrolysate solution not undergoing prior sterilization and not undergoing pH rectification, being operated at a dilution rate in the range 0.001 to 0.02 h⁻¹.

Claims

1. A process for the production of cellulases and hemicellulases with a strain of a filamentous fungus in a stirred and aerated bioreactor, comprising:

producing cellulases and hemicellulases with a strain of a filamentous fungus in a stirred and aerated bioreactor in at least two phases;

a phase a) for growth of said strain in the presence of at least one carbonaceous growth substrate in a closed reactor, said growth phase being carried out with a concentration of carbonaceous growth substrate of 10 to 90 g/L; and

a phase b) for the continuous production of cellulases, in which at least one carbonaceous inducer substrate is supplied at a supply rate which is constant over a period of at least more than 200 h, said carbonaceous inducer substrate being at least one aqueous hemicellulosic hydrolysate solution obtained from an acid pre-treatment of a lignocellulosic substrate, said aqueous hemicellulosic hydrolysate solution not undergoing prior sterilization and not undergoing pH rectification, said pH of the aqueous solution being 0.5 to 3, the mass of the reaction volume being kept constant by withdrawing a fraction of said reaction volume, said phase b) being operated at a dilution rate of 0.002 to 0.008 h<sup>−

1</sup>.

2. The process according to claim 1, in which said strain is a strain of Trichoderma reesei modified by mutation, selection or genetic recombination.

3. The process according to claim 1, in which the carbonaceous growth substrate in said growth phase is glucose, lactose, xylose, a residue obtained after ethanolic fermentation of monomeric sugars of an enzymatic hydrolysate of a lignocellulosic substrate, an extract of a hemicellulosic fraction in the form of monomers deriving from a pre-treated lignocellulosic substrate, or a combination thereof.

4. The process according to claim 1, in which the acid pre-treatment is an acid hydrolysis, acid cooking or steam explosion with prior impregnation of said lignocellulosic substrate with an aqueous sulphuric acid solution.

5. The process according to claim 1, in which the carbonaceous inducer substrate in phase b) is an aqueous hemicellulosic hydrolysate solution obtained from an acid pre-treatment of a lignocellulosic substrate, as a mixture with at least one other carbonaceous substrate of inducer or non-inducer sugars.

6. The process according to claim 1, in which the duration of phase b) for the continuous production of cellulases is more than 300 h.

7. The process according to claim 6, in which the duration of phase b) for the continuous production of cellulases is more than 400 h.

8. The process according to claim 1, in which the supply flow rate of said carbonaceous inducer substrate is 35 to 140 mg of carbonaceous inducer substrate per gram of the dry weight of the strain per hour.

9. The process according to claim 1, in which the supply flow rate is gradually increased in phase b).

10. The process according to claim 1, in which an optional phase a′

) for growth and production in a reactor with a continuous supply of at least one carbonaceous inducer substrate during which no continuous withdrawal from the fermenter occurs, is carried out between phase a) and phase b).

11. The process according to claim 10, in which said carbonaceous inducer substrate in phase a′

) is identical to the carbonaceous inducer substrate in the production phase b).

12. The process according to claim 10, in which phase a′

) is carried out for a period of 50 to 150 h.

13. The process according to claim 1, wherein the vvm is 0.3 to 1.5 min⁻

1 wherein the vvm is the degree of aeration expressed as the volume of air under normal temperature and pressure conditions per volume of reaction medium per minute.

14. The process according to claim 13, wherein the vvm is 0.3 to 1 min⁻

1.

15. The process according to claim 13, wherein the vvm is 0.5.