Use of CO2 to deactivating a cellulolytic microorganism used in the biochemical conversion of lignocellulosic materials

Abstract

The invention concerns a process for deactivating a cellulolytic microorganism enabling the production of an enzymatic cocktail, said cocktail being used without separating the cellulolytic microorganism during the biochemical conversion of lignocellulosic materials, comprising at least one step for bringing a gaseous stream into contact with a medium containing said microorganism, said gaseous stream comprising more than 25% by weight of CO.sub.2 and comprising less than 0.5 molar % of O.sub.2.

Claims

1. A process for the production of an enzymatic cocktail and for the biochemical conversion of lignocellulosic materials with the enzymatic cocktail, said process comprising: producing the enzymatic cocktail with a cellulolytic microorganism to obtain a medium comprising the enzymatic cocktail and the cellulolytic microorganism; deactivating the cellulolytic microorganism in the medium by bringing a gaseous stream into contact with the medium obtained from producing the enzymatic cocktail which contains said microorganism, wherein the gaseous stream originates from a simultaneous saccharification and fermentation (SSF) step and wherein said gaseous stream comprises more than 25% by weight of CO.sub.2 and comprising less than 0.5 molar % of O.sub.2, and whereby the gaseous stream deactivates the cellulolytic microorganism in the medium; and without separating the cellulolytic microorganism from the enzymatic cocktail in the medium, contacting the medium with lignocellulosic materials to effect biochemical conversion of the lignocellulosic materials.

2. The process according to claim 1, wherein the gaseous stream has undergone a treatment for reducing its ethanol and volatile organic compounds content by at least 25% before the contact step.

3. The process according to claim 2, wherein the treatment is washing said gaseous stream with water.

4. The process according to claim 1, wherein the cellulolytic microorganism is selected from strains of fungi belonging to the genera Trichoderma, Aspergillus, Penicillium or Schizophyllum.

5. The process according to claim 4, wherein the cellulolytic microorganism belongs to the species Trichoderma reesei.

6. The process according to claim 1, wherein the gaseous stream comprises less than 0.25 molar % of O.sub.2.

7. The process according to claim 1, wherein the contacting of the medium with lignocellulosic materials for biochemically converting the lignocellulosic materials is an SSF step comprising saccharification and fermentation of the lignocellulosic materials.

8. The process according to claim 7, wherein the SSF comprises saccharification of the lignocellulosic materials to products comprising glucose and fermentation of the glucose to ethanol.

9. The process according to claim 1, wherein the step of bringing the gaseous stream into contact with the medium is carried out during the step for producing the enzymatic cocktail.

10. The process according to claim 1, wherein the step of bringing the gaseous stream into contact with the medium is carried out during the step for biochemical conversion of the lignocellulosic materials.

11. The process according to claim 1, wherein the step of bringing the gaseous stream into contact with the medium is carried out between the step for producing the enzymatic cocktail and the step for biochemical conversion of the lignocellulosic materials.

12. The process according to claim 7, wherein the step of bringing the gaseous stream into contact with the medium is carried out in the SSF step for biochemical conversion of the lignocellulosic materials.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) The invention concerns a process for deactivating a cellulolytic microorganism enabling the production of an enzymatic cocktail, said cocktail being used without separating the cellulolytic microorganism during the biochemical conversion of lignocellulosic materials, comprising at least one step for bringing a gaseous stream into contact with a medium containing said microorganism, said gaseous stream comprising more than 25% by weight of CO.sub.2 and comprising less than 0.5 molar % of O.sub.2.

(2) Preferably, said gaseous stream originates from a SSF step.

(3) Preferably, said gaseous stream has undergone a treatment for reducing its ethanol and volatile organic compounds content by at least 25% before the contact step.

(4) Preferably, said treatment is washing said gaseous stream with water.

(5) Preferably, said contact step is carried out in a SSF step.

(6) Preferably, said contact step is carried out in the enzymatic cocktail production step at the end of the phase for production of said cocktail.

(7) Preferably, said contact step is carried out between the enzymatic cocktail production step and a SSF step.

(8) Preferably, the cellulolytic microorganism is selected from strains of fungi belonging to the genera Trichoderma, Aspergillus, Penicillium or Schizophyllum.

(9) Preferably, the cellulolytic microorganism belongs to the species Trichoderma reesei.

(10) The enzymatic cocktail production step employs a cellulolytic microorganism. Said production process is carried out in submerged culture. The term submerged culture means culture in a liquid medium.

(11) The cellulolytic microorganisms used in the process for producing an enzymatic cocktail are strains of fungi belonging to the genera Trichoderma, Aspergillus, Penicillium or Schizophyllum, preferably belonging to the species Trichoderma reesei. The best performing industrial strains are strains belonging to the species Trichoderma reesei, modified to improve the enzymatic cocktail by mutation-selection processes such as, for example, the strain IFP CL847 (French patent FR-B-2 555 803). Strains improved by genetic recombination techniques may also be used. These strains are cultivated in stirred, aerated reactors under conditions compatible with their growth and production of enzymes.

(12) In accordance with the invention, a gaseous stream comprising less than 0.5 molar % of oxygen (O.sub.2), preferably less than 0.25 molar %, and more preferably free of oxygen, is then brought into contact with the medium containing said cellulolytic microorganism.

(13) Alcoholic fermentation is a biochemical reaction during which organisms, for example the yeast Saccharomyces cerevisiae, transform sugars into ethanol and carbon dioxide (CO.sub.2).

(14) A stream of gas comprising the CO.sub.2 produced during the alcoholic fermentation step and/or during the SSF step is separated during fermentation by continuously degassing the fermenter. A gas stream comprising the fraction of CO.sub.2 dissolved in the fermentation medium is produced during the ethanol purification step.

(15) The gaseous stream used in the present invention is advantageously constituted by a mixture of the gaseous stream separated by continuously degassing the fermenter and a stream produced during the ethanol purification step.

(16) The gas stream produced during the alcoholic fermentation step comprises at least 25% by weight of CO.sub.2, preferably at least 50% by weight, and more preferably at least 75% by weight. Said gaseous stream also comprises in the range 0 to 10% by weight of ethanol, preferably in the range 0 to 5% by weight. Said gaseous stream also comprises water and volatile organic compounds (VOC), these latter being defined in accordance with Article 2 of Council Directive 1999/13/CE of 11 Mar. 1999.

(17) During said ethanol purification step, said dissolved fraction of CO.sub.2 is separated from the medium containing the ethanol by any means known to the skilled person, for example by flashing, which consists of reducing the pressure of the medium to vaporize the dissolved CO.sub.2, by distillation, by membrane separation or by a combination of these means or other means known to the skilled person.

(18) Preferably, the gaseous stream containing the CO.sub.2 separated by continuous degassing and/or the gaseous stream containing CO.sub.2 separated in the ethanol purification step is treated in a scrubbing step so as to reduce its ethanol and VOC content as well as its oxygen content, if necessary. Said scrubbing step may be carried out using any method known to the skilled person. Preferably, said scrubbing step is washing with water or membrane separation.

(19) Said scrubbing step is intended to reduce the ethanol and VOC content in the gaseous stream by 25% to 100%, preferably by 50% to 100% and more preferably by 75% to 100%.

(20) In a preferred arrangement, the gaseous stream is brought into contact with the medium containing the cellulolytic microorganism without prior treatment in said scrubbing step.

(21) During said contact, the ethanol which might be contained in said gaseous stream is absorbed into said medium containing the cellulolytic microorganism. By recycling, then, the concentration of ethanol at the end of the alcoholic fermentation step is increased. This increase in the concentration of ethanol has the effect of reducing the energy consumption of the ethanol purification step.

(22) During said contact step, the CO.sub.2 will acidify the medium. Contact of the gaseous stream and the medium containing the cellulolytic microorganism is always carried out with a check and possible adjustment of the pH.

(23) Contact of the gaseous stream and the medium containing the cellulolytic microorganism, said medium behaving in a similar manner to a liquid, may be carried out by any means for bringing a gas and a liquid into intimate contact which is known to the skilled person. Means of this type are described, for example, but not in an exhaustive manner, in Adsorption en traitement d'air [Adsorption in air treatment], Michel Roustan, Techniques d'ing?nieur, G1750.

(24) Preferably, the step for bringing the gaseous stream into contact with the medium containing the cellulolytic microorganism is carried out in a SSF step. In order to obtain this contact, the equipment in which a SSF step is carried out is provided with a system for dispersing gas in the liquid medium. Another method consists of extracting a fraction of the reaction medium contained in said equipment, intimately mixing said fraction with said gaseous stream and reintroducing said fraction, which may or may not have been freed from the gaseous portion, into said equipment. Another method consists of injecting said gaseous stream into the gas overhead of said equipment.

(25) Preferably, the step for bringing the gaseous stream into contact with the medium containing the cellulolytic microorganism is carried out during the step for producing the enzymatic cocktail, at the end of phase (b) for producing said cocktail. Since the equipment for producing the enzymatic cocktail is aerated and stirred, as is known to the skilled person and described, for example, in patent EP 1 690 944, air injection is cut off at the end of said production phase (b) and instead, said gaseous stream is injected until the partial pressure of oxygen is less than 0.5 molar %, preferably less than 0.25 molar % and more preferably 0%. Another advantage of adding CO.sub.2 is to allow the pH to be reduced, preferably to between 3.5 and 3.7. These low pHs mean that the risk of contamination with respect to the culture pH, which is in the range 4.8 to 4, can be limited. Care should be taken in this step that the pH does not fall below 3.3, which would have a negative effect on the activity of certain enzymes of the cocktail.

(26) Preferably, the step for bringing the gaseous stream into contact with the medium containing the cellulolytic microorganism is carried out between the step for producing the enzymatic cocktail and the SSF step. This contact may be brought about in dedicated equipment using conventional gas/liquid contact technologies which are known to the skilled person.

(27) The gaseous stream containing the CO.sub.2 may also be used for the production of microalgae intended for the production of a biofuel known as third generation fuel after extracting the lipids from said microalgae, thus meaning that a co-product with a high added value can be produced, i.e. directly for carrying out aquaculture.

(28) Other gaseous streams comprising less than 0.5 molar % oxygen may also be used, for example effluents from units for the anaerobic treatment of water, waste combustion fumes, or effluents from organic waste methanation units.

(29) The following examples illustrate the invention without limiting its scope. Hereinbelow, the activity of the enzymatic cocktail was measured in Filter Paper Units denoted FPU. This activity was measured on Whatman No 1 paper (procedure recommended by the IUPAC biotechnology commission) at an initial concentration of 50 g/L; a sample of the enzymatic solution to be analysed which released the equivalent of 2 g/L of glucose (colorimetric assay) in 60 minutes was determined.

EXAMPLE 1

Comparison of Stored Trichoderma reesei Mash

(30) Example 1 presents the change in the activity of the enzymatic cocktail of a medium containing both said enzymatic cocktail and the cellulolytic microorganism T. reesei, termed the mash, stored for 3 weeks and having undergone three different treatments. This example shows that the FPU activity is conserved in the mash which has been placed under an atmosphere of CO.sub.2 or N.sub.2, while it is substantially reduced in that conserved in air.

(31) An enzymatic cocktail was produced in a fermenter using Trichoderma reesei CL847 and by means of a conventional protocol described, for example, in patent application EP 1 690 944. The concentration of enzymatic cocktail obtained was 39.9 g/L and the activity was 32 FPU/mL.

(32) The mash was divided into different pre-sterilized flasks, without separation of the cellulolytic microorganism and the supernatant containing the enzymatic cocktail.

(33) In flask A, a gaseous stream comprising more than 99 molar % of CO.sub.2 and containing less than 0.5 molar % of O.sub.2 was bubbled through until the gas overhead contained less than 0.5 molar % CO.sub.2. Said gaseous stream had been obtained by harvesting the degassing product from an alcoholic fermentation carried out elsewhere.

(34) In flask B, a gaseous stream comprising more than 99 molar % of N.sub.2 and containing less than 0.5 molar % of O.sub.2 was bubbled through until the gas overhead contained less than 0.5 molar % O.sub.2.

(35) In flask C, the mash was stored in air in a sterile flask with a 0.2 ?m filter.

(36) The % O.sub.2 was measured by gas phase chromatography (GPC) using a sample taken from the gas overhead.

(37) The flasks were then stored at 33? C. for 3 weeks.

(38) The activities obtained after three weeks are shown in Table 1. More than 90% of the activity was maintained in a CO.sub.2 atmosphere or nitrogen atmosphere, while 50% of the activity was lost when the fungus was in the presence of air. The cellulolytic microorganism is thus indeed deactivated in the absence of air.

(39) TABLE-US-00001 TABLE 1 Change in enzymatic activity Activity after Initial activity 3 weeks FPU/mL FPU/mL Flask A 32.00 29.08 Flask B 30.44 Flask C 15.15

EXAMPLE 2

Comparison of Two SSFs

(40) Example 2 compares two SSFs carried out at 33? C. using a mash containing both the cellulolytic microorganism and the enzymatic cocktail with or without the addition of CO.sub.2 to the gas overhead.

(41) Two SSFs were carried out using the enzyme production mash directly (enzymatic cocktail+cellulolytic microorganism) in the hydrolysis and fermentation reactor with a 30 mg dose of enzyme per gram of dry matter (DM). The percentage by weight of dry matter is the ratio of the weight of the sample obtained after drying at 105? C. for 24 hours over the initial weight of the sample. The weight of dry matter is the product of the percentage by weight of dry matter and the weight of the sample.

(42) The experiment was carried out at 18% DM (wheat straw steam exploded under acid conditions, washed and dried) in a 2 L reactor. The temperature was adjusted to 33? C. and the pH was adjusted to 5 using 5 N sodium hydroxide (NaOH). The yeast was added 1 hour after starting hydrolysis, at a concentration of 0.5 g of yeast per kg of fermentation medium. The first SSF (SSF1) was carried out with a slight bubbling through of a gaseous stream comprising 99.8 molar % of CO.sub.2 and 0.2 molar % of O.sub.2. The second SSF (SSF2) was carried out without bubbling CO.sub.2 through.

(43) SSF1 finished with a final ethanol concentration of 40.2 g/L, while SSF2 finished with a final concentration of only 34.1 g/L. The SSF1 yield was greater than that of SSF2 by 17.9%. In the case of SSF2, the cellulolytic microorganism had consumed a portion of the sugar liberated by hydrolysis of the cellulose, causing the final yield for ethanol production to be reduced.