Continuous method for growing a microorganism

Abstract

The present invention relates to a method comprising a step (a) of continuously growing a microorganism capable of transforming a substrate into a compound of formula (I), preferably vanillin.

Description

[0001] CONTINUOUS PROCESS OF GROWTH OF A MICROORGANISM

[0002] Field of invention

[0003] The present invention refers to a method comprising a step (a) of continuous growth of a microorganism capable of transforming a substrate into a compound of formula (I), preferably into vanillin.

[0004] Prior art

[0005] Vanillin can be obtained by various methods known to those skilled in the art, and in particular by the following two routes:

[0006] - A so-called natural route based on a biotechnological process comprising in particular the culture of a microorganism capable of allowing the biotransformation of a fermentation substrate into vanillin. Such a process is known in particular from application EP0885968 in which the fermentation substrate is ferulic acid. US patent 5017388 describes a process in which the fermentation substrate is eugenol and / or isoeugenol. These processes result in the preparation of a vanillin known as natural vanillin.

[0007] - A so-called synthetic route comprising classic chemical reactions from guaiacol not involving microorganisms. This process results in the preparation of a vanillin called synthetic vanillin.

[0008] Finally, vanillin can also be prepared according to a route described as "bio-based" in which the vanillin is derived from lignin; we can cite in particular documents US 2745796, DE1132113 and the article entitled "Preparation of lignin from wood dust as vanillin source and comparison of different extraction method" by Azadbakht et al in International Journal of Biology and Biotechnology, 2004, vol 1, No 4, pp 535-537, or prepared from materials of natural origin; we can cite in particular document WO 2019 / 020773.

[0009] As mentioned above, the processes of the so-called natural route include the cultivation of a microorganism capable of transforming a substrate into vanillin. This step is a growth phase of the microorganism to obtain a biomass that will be used as a biocatalyst for the bioconversion phase of a substrate into vanillin.

[0010] Industrially, this growth phase is time-consuming and delicate. Indeed, it is generally necessary to inoculate a small quantity of microorganism contained in a cryotube, and transfer it into a first fermenter of a slightly larger volume to grow the microorganism. This step of biomass growth and transfer into a larger reactor can be repeated as many times as necessary. The total duration of this growth step can be significant, for example, of the order of 72 hours. Furthermore, this growth step must be carried out in a sterile environment. An initial sterilization step of the fermenter(s) as well as all the inlets and outlets of the fermenter(s) is required and must be carried out under specific conditions. As a result, on the one hand, transfers from one fermenter to another present risks of contamination and, on the other hand, the implementation of this type of process is complex.

[0011] The present invention aims at an efficient and industrial method for growing a microorganism in which the risk of contamination is reduced and whose productivity is improved.

[0012] Brief description

[0013] A first subject of the present invention relates to a method comprising a step (a) of continuous growth of a microorganism capable of transforming a substrate into a compound of formula (I), preferably into vanillin.

[0014] The present invention also relates to a process for producing a compound of formula (I), preferably vanillin, characterized in that it comprises a step (a) of continuous growth of a microorganism capable of transforming a substrate into a compound of formula (I), preferably into vanillin.

[0015] Description of figures

[0016] Eig 1: Continuous growth process according to the invention

[0017] Lig 2: Process for producing a compound of formula (I) according to the invention Lig 3: Process for producing a compound of formula (I) according to the invention Lig. 4: Process for producing a compound of formula (I) according to the invention Detailed description

[0018] For the purposes of the present invention, and unless otherwise indicated, the expression "between ... and..." includes the limits.

[0019] For the purposes of the present invention, and unless otherwise indicated, the growth of a microorganism refers to a process in which said microorganism multiplies. Growth results in the production of biomass. Growth can be defined by the increase in the concentration of biomass.

[0020] In the context of the present invention, and unless otherwise indicated, the term "bioconversion" refers to a biotechnological process in which a microorganism allows the transformation of a substrate into a bioconversion product. The microorganism may be a wild strain, genetically modified obtained by molecular biology or mutated, in particular by random or directed mutagenesis.

[0021] In the context of the present invention, and unless otherwise indicated, the term “process carried out under aseptic conditions” refers to a process carried out under conditions free from any germ and / or microorganism.

[0022] In the context of the present invention, and unless otherwise indicated, the term "process carried out under sterile conditions" refers to a process carried out under conditions free from any germ and / or microorganism likely to be harmful to the microorganism capable of transforming a substrate into a compound of formula (I), preferably into vanillin, used in the context of the process of the present invention.

[0023] In the context of the present invention, and unless otherwise indicated, asepsis or sterilization may be obtained by any method, in particular thermal, chemical, mechanical or by radiation, allowing the elimination of germs and / or microorganisms. Asepsis or sterilization may be carried out on the fluids, raw materials, and / or devices used in the context of the process according to the present invention. By way of illustration, the following methods may be mentioned in particular:

[0024] Sterilization by dry or moist heat, carried out by raising the temperature to a sufficient temperature, followed by maintaining this temperature for a sufficient period of time to eliminate germs and / or microorganisms, for example the temperature may be 121°C, then maintaining this temperature of 121°C for a sufficient period of time to allow the elimination of germs, Membrane filtration or several successive membrane filtrations, the smallest cut-off diameter of which allows the elimination of any germ and / or microorganism.

[0025] For the purposes of the present invention, and unless otherwise indicated, the term "reactor" refers to a container suitable for carrying out chemical reactions. For the purposes of the present invention, the reactor may be used after being sanitized or sterilized or without sanitization or sterilization.

[0026] For the purposes of the present invention, and unless otherwise indicated, the term "fermentation" refers to any biological reaction involving a microorganism such as bacterial growth, bioconversion, biosynthesis or biocatalysis.

[0027] In the context of the present invention, and unless otherwise indicated, the term "fermenter" refers to a reactor capable of carrying out fermentations, for example a bioreactor. In the context of the present invention, the fermenter can be used after having been sanitized or sterilized or without sanitization or sterilization.

[0028] In the context of the present invention, a compound of formula (I) has the following formula:

[0029] Formula (I) in which n is between 0 and 5, preferably between 1 and 2,

[0030] R, identical or different, is chosen from the group consisting of OH, ORi, formyl, CO2H, linear or branched alkyl chains, optionally substituted, comprising between 1 and 6 carbon atoms, alkenyls, linear or branched, optionally substituted, comprising between 1 and 6 carbon atoms.

[0031] According to a particular embodiment, R is chosen from the group consisting of formyl -CHO, OH, OMe, OEt, OPr, OBu, methyl, ethyl, -(CH2)2-C(O)-CH3, -CH=CH-C(O)-CH3, -CH=CH- CO2H, -CO2H, -CH2-OH.

[0032] According to a particular embodiment, RI is chosen from the group consisting of methyl, ethyl, propyl and butyl. According to a particular embodiment, the compound of formula (I) is chosen from vanillin and frambinone.

[0033] A first aspect of the present invention refers to a method comprising a step (a) of continuous growth of a microorganism capable of transforming a substrate into a compound of formula (I), preferably into vanillin.

[0034] In the context of the present invention, the method comprising a step (a) of continuous growth or "continuous growth method" refers to a step or method in which the volume of the reactor is constant. During the continuous growth method, the microorganism multiplies. During the continuous growth method, a stream (F1) comprising at least the biomass is continuously transferred. According to one embodiment, a stream (F1) comprising the entirety of the biomass is continuously transferred. According to another embodiment, a stream (F1) comprising a portion of the biomass is continuously transferred.

[0035] In the context of the present invention, the pH of the growth medium is controlled and optionally regulated by adding acid or base. A person skilled in the art will be able to adapt the pH of the growth medium to the needs of the microorganism capable of transforming a substrate into a compound of formula (I), preferably into vanillin, used in the context of the process of the present invention.

[0036] According to a particular embodiment, the pH of the growth medium is between 3 and 9, in particular between 4 and 8, and very preferably between 6 and 8.

[0037] In the context of the present invention, the continuous growth process is carried out under controlled temperature conditions; the temperature may optionally be regulated. A person skilled in the art will be able to adapt the temperature of the process to the needs of the microorganism capable of transforming a substrate into a compound of formula (I), preferably into vanillin used in the context of the process of the present invention. According to a particular embodiment, the temperature may be between 10 and 55°C, particularly preferably in the range of 30 to 50°C, and very preferably between 35°C and 45°C.

[0038] In the context of the present invention, the pCL of the growth medium is controlled and optionally regulated. A person skilled in the art will be able to adapt the pCL of the growth medium to the needs of the microorganism capable of transforming a substrate into a compound of formula (I), preferably into vanillin used in the context of the method of the present invention.

[0039] For illustration purposes, the pO2 can be between 1% and 99%, preferably in the order of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%. The pCh corresponds to the dissolved oxygen concentration in the growth medium relative to the dissolved oxygen concentration at saturation, without biomass. The pCL can in particular be measured with an O2 Sensor Hamilton OXYFERM FDA VP 225.

[0040] In the present invention, the continuous growth process is carried out under stirring. Stirring can be characterized by the blade tip speed or peripheral speed. The blade tip speed can be calculated using the following formula:

[0041] Vp = 2*Pi*D / 2*N / 60, in which Vp is the speed at the blade tip (m / s), D is the diameter of the reactor or fermenter (in m) and N is the rotation speed of the stirrer (rpm). A person skilled in the art will be able to adapt the stirring to the needs of the microorganism capable of transforming a substrate into a compound of formula (I), preferably into vanillin used in the context of the process of the present invention. According to a particular embodiment, the speed at the blade tip is between 1 and 10 m / s, preferably between 2 and 5 m / s.

[0042] According to the present invention, growth is carried out in a medium or "growth medium" suitable for the microorganism whose growth is desired. In general, the growth medium refers to a medium comprising nutrients essential and / or beneficial to the survival and / or growth of the microorganisms.

[0043] In the context of the present invention, the composition of the growth medium is controlled. The composition of the growth medium is adapted continuously. Thus, in the context of the present invention, growth medium is added continuously.

[0044] In the context of the present invention, the growth medium addition rate is equal to the flow removal rate (F1). In the context of the present invention, it is preferable that the growth medium addition or flow removal rate be adapted so as to allow the microorganism sufficient time to multiply and avoid a decrease in its concentration. It is preferable that the growth medium addition or flow removal rate be adapted so as to avoid accumulation or reduction of the amount of glucose in the medium. According to one embodiment, the growth medium addition and flow removal rates (F1) may be constant. According to another embodiment, the growth medium addition and flow removal rates (F1) may be carried out occasionally.

[0045] In general, the continuous growth process according to the present invention is carried out in a fermenter. In general, the continuous growth process is carried out under aseptic conditions, preferably under sterile conditions.

[0046] In the context of the present invention, the continuous growth process is carried out under aseptic, preferably sterile, conditions, indicates that the reactor or fermenter in which the process is carried out, and the various feed or outlet pipes of the reactor or fermenter have been asepticized, preferably sterilized, prior to their use in the process. In the context of the present invention, the continuous growth process is carried out under aseptic, preferably sterile, conditions, indicates that the solvent and growth medium used during the process have been asepticized, preferably sterilized, prior to their use in the process. In the context of the present invention, the continuous growth process is carried out under aseptic, preferably sterile, conditions, indicates that the air or oxygen used during the process is asepticized, preferably sterilized, prior to its use in the process.

[0047] In general, the growth medium comprises a carbon source, an organic or inorganic nitrogen source, inorganic salts and, optionally, growth factors. A person skilled in the art will be able to adapt the composition of the growth medium to the needs of the microorganism capable of transforming a substrate into a compound of formula (I), preferably into vanillin used in the context of the process of the present invention.

[0048] Generally, the term "carbon source" refers to a carbon source that could be used to support the growth of microorganisms. Cells generally use sugars as their primary carbon source and energy source, however other carbon sources may also be used. In a particular aspect, the carbon source may be selected from the group consisting of glucose, fructose, sucrose, mannose, xylose, arabinose, galactose, lactose, ethanol, cellobiose, glycerol and polysaccharides such as cellulose, and mixtures thereof. In a preferred embodiment, the carbon source comprises glucose. In the context of the present invention, the concentration of carbon source is controlled.In the context of the present invention, the concentration of carbon source is adapted to the needs of the microorganism capable of transforming a substrate into a compound of formula (I), preferably into vanillin, used in the context of the process of the present invention.

[0049] In the context of the present invention, the carbon source concentration is maintained at a concentration of between 0 and 20 gL' 1 , preferably between 0.1 and 10 gL' 1 and very preferably between 0.5 and 5 gL' 1 The addition of a carbon source, particularly glucose, is generally carried out continuously.

[0050] Generally speaking, the term "nitrogen source" refers to a source of nitrogen that could be used to support the growth of microorganisms. Examples of suitable nitrogen sources are inorganic nitrogen sources such as nitrates and ammonium salts and organic nitrogen sources such as yeast extract, urea, peptone.

[0051] Examples of suitable inorganic salts include, among others, sulfates, nitrates, chlorides, carbonates, hydrogen phosphates and phosphates of sodium, potassium, magnesium, calcium, zinc and iron.

[0052] In the context of the present invention, growth factors which can be used are, for example, D-biotin, nicotinic acid, thiamine (HCl), pyridoxine (HCl), para-aminobenzoic acid, myo-inositol, calcium pantothenate, Na2-EDTA, ZnSO4.7H2O, MnC12.4H2O, COCI2.6H2O, CUSO4.5H2O, Na2MoO4.2H2O, CaCl2.2H2O, FeSO4.7H2O, H3BO3, Kl.

[0053] In the context of the present invention, the concentration of nitrogen source and / or growth factors can be controlled. In the context of the present invention, the concentration of nitrogen source and / or growth factors can be between 0 and 5 gL' 1 , preferably between 0.1 and 2.5 gL' 1 , and very preferably between 0.2 and 1.5 gL' 1 .

[0054] Additionally, magnesium ions, such as magnesium sulfate, can be added at a concentration between 0 and 0.5 gL' 1 , preferably between 0.01 and 0.25 gL' 1 , and very preferably between 0.02 and 0.15 gL' 1 The addition of magnesium ions can be continuous.

[0055] In the context of the present invention, the biomass concentration can be controlled. The biomass concentration, expressed as dry mass, can be between 2 and 50 g / L, preferably between 5 and 25 g / L, very preferably between 8 and 15 g / L. In the context of the present invention, the biomass concentration can be controlled by measuring the optical density. In general, the optical density, measured at 880 nm, is between 5 and 400, preferably between 20 and 200, very preferably between 40 and 100 and even more preferably between 60 and 80. The measurement of the optical density at 880 nm can be carried out continuously, online. The optical density at 880 nm can be measured using a Hamilton Dencytee Unit probe.

[0056] In the context of the present invention, the biomass concentration can be controlled by measuring the optical density at 600 nm. In general, the optical density, measured at 600 nm, is between 2 and 100, preferably between 5 and 80, very preferably between 10 and 60 and even more preferably between 25 and 45. The measurement of the optical density at 600 nm can be measured by taking a sample of growth medium. The sample is then diluted 50 times in water. The optical density at 600 nm can be measured using a spectrophotometer set at 600 nm.

[0057] In the context of the present invention, the microorganism may be any microorganism capable of forming a compound of formula (I), preferably vanillin by bioconversion of a suitable substrate. The microorganism, in particular a bacterium or fimgus, may be a wild microorganism, genetically modified by molecular biology, mutated, by random or site-directed mutagenesis. Preferably, the microorganism is chosen from bacteria belonging to the order AesActinomycetales, Caulobacterales or P seudomonadales, preferably belonging to the family Streptomycetacae, Pseudonocardiacae, Micrococcaceae, Caulobacteraceae, or Pseudomonadaceae, very preferably Streptomyces setonii, Amycolatopsis sp. Streptomyces psammoticus, Amycolatopsis thermoflava, Micrococcus sp, Caulobacter segnis, Pseudomonasfluorescens, or their mutants, even more preferentially Streptomyces setonii, Amycolatopsis sp., Streptomyces psammoticus.Preferably, the bioconversion reaction is carried out in the presence of a strain available under the number ATCC39116, DSMZ 9991, DSMZ 9992, CCTCC 2015329, CCTCC 2011265, IMI 390106 or Zyl 926 or their mutants, preferentially ATCC39116, DSMZ 9991, DSMZ 9992, CCTCC 2015329, IMI 390106 or Zyl 926 or their mutants and very preferentially ATCC39116, DSMZ 9991, DSMZ 9992, CCTCC 2015329, IMI 390106 or Zyl 926. According to another embodiment, the microorganism may be chosen from genetically modified microorganisms belonging to the Saccharomyces family. cerevisiae, Schizosaccharomyces pombe, E. coli, Corynebacterium glutamicul. The microorganism used in the context of the present invention may also be a strain as described in WO 2014 / 102368, WO 2016 / 001203 or EP 2721148.

[0058] According to another embodiment, the microorganism is chosen from fungi belonging to the genus Aspergillus, Pycnoporus, Penicillium, preferably belonging to the family Aspergillus luchuensis, Aspergillus niger, Picnoporus cinnabarinus, Penicillium camamberti. In the context of the present invention, the substrate can be transformed by bioconversion into a compound of formula (I), preferably into vanillin. The substrate is generally chosen from the group consisting of glucose, ferulic acid, eugenol, isoeugenol, coumaric acid, caffeic acid, L-tyrosine, vanillic acid, 4-vinyl guaiacol, preferably chosen from glucose, ferulic acid and eugenol.

[0059] In general, the continuous growth process may be preceded by a step (aO) during which a microorganism is cultured so as to obtain a sufficient quantity of biomass. This sufficient quantity of biomass may then be used in a continuous growth process according to the present invention.

[0060] The culture of the bacteria is generally carried out in an aqueous medium, in the presence of nutrients. In general, the culture medium comprises a carbon source, an organic or inorganic nitrogen source, inorganic salts and growth factors. A person skilled in the art will be able to adapt the composition of the culture medium to the needs of the microorganism capable of transforming a substrate into a compound of formula (I), preferably into vanillin used in the context of the process of the present invention. The concentration of carbon source is generally between 5 and 50 gL 4 , preferably between 20 and 34 gL4 The nitrogen source, such as yeast extract, and growth factors are generally added at a concentration between 2 and 20 gL' 1 , preferably between 5 and 10 gL . In addition, magnesium ions, such as magnesium sulfate, may be added at a concentration between 0.1 and 5 gL 4 , preferably between 0.5 and 1 gL 4 . In general, the pH of the culture is between 3 and 9, in particular between 4 and 8, and very preferably between 6 and 8. In general, the temperature of the culture is between 10 and 55°C, particularly preferably in the range of 30 to 50°C, preferably between 30 and 42°C and very preferably between 35°C and 45°C.

[0061] The culture period generally lasts between 15 minutes and 80 hours, preferably between 1 hour and 50 hours and very preferably between 5 hours and 40 hours. The duration of the culture period is variable and can be adapted according to the microorganism capable of transforming a substrate into a compound of formula (I), preferably into vanillin used in the context of the present invention. By way of illustration, the culture period can last until the carbon source, generally glucose, is almost completely consumed, preferably such that the concentration of carbon source is less than or equal to 15 gL' 1 , preferably less than or equal to 5 gL' 1 , very preferably less than or equal to 3 gL' 1 . According to another embodiment, the culture period can last until 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% of the carbon source introduced at the start of step (aO) has been consumed.

[0062] According to another aspect, the present invention relates to a process for producing a compound of formula (I), preferably vanillin, characterized in that it comprises a step (a) of continuous growth of a microorganism capable of transforming a substrate into a compound of formula (I), preferably into vanillin.

[0063] In the context of this embodiment, step (a) is carried out under the conditions described previously for the continuous process for growing a microorganism capable of transforming a substrate into a compound of formula (I), preferably into vanillin.

[0064] The process for producing a compound of formula (I), preferably vanillin, may further comprise a step (aO) prior to step (a) as described above.

[0065] According to one embodiment, the method for producing a compound of formula (I), preferably vanillin, further comprises a bioconversion step (b) in which the microorganism transforms a substrate into a compound of formula (I), preferably vanillin.

[0066] Steps (a) and (b) can be carried out successively, step (a) is carried out before step (b). According to another embodiment, steps (a) and (b) can be carried out simultaneously. The bioconversion step (b) can in particular be carried out under the conditions described in EP0885968, EP0761817, or WO2017 / 025339.

[0067] Alternatively, step (b) may be carried out in batch or fed-batch. The bioconversion substrate may be added all at once or in several times. According to another embodiment, step (b) may be carried out continuously. In particular, bioconversion substrate may be fed continuously. Optionally, a stream (F2) comprising the compound of formula (I), preferably vanillin, may be removed continuously. Stream (F2) may also optionally comprise fermentation substrate, impurities and biomass. Step (b) may be carried out under aseptic, preferably sterile, conditions. According to another embodiment, step (b) may be carried out without aseptic or sterility conditions.

[0068] In particular, the bioconversion substrate may be chosen from glucose, ferulic acid, eugenol, isoeugenol, coumaric acid, caffeic acid, L-tyrosine, vanillic acid, 4-vinyl guaiacol, preferably chosen from glucose, ferulic acid and eugenol.

[0069] In the context of the present invention, the microorganism may be any microorganism capable of forming the compound of formula (I), preferably vanillin by bioconversion of a suitable substrate. The microorganism, in particular a bacterium or fimgus, may be a wild microorganism, genetically modified by molecular biology, mutated, by random or directed mutagenesis. Preferably, the microorganism is chosen from bacteria belonging to the order AesActinomycetales, Caulobacterales or Pseudomonadales, preferably belonging to the family Streptomycetacae, Pseudonocardiacae, Micrococcaceae, Caulobacteraceae, or Pseudomonadaceae, preferably Streptomyces setonii, Amycolatopsis sp. Streptomyces psammoticus, Amycolatopsis thermoflava, Micrococcus sp, Caulobacter segnis, Pseudomonasfluorescens. or their mutants, even more preferentially Streptomyces setonii, Amycolatopsis sp. Streptomyces psammoticus.Preferably, the bioconversion reaction is carried out in the presence of a strain available under the number ATCC39116, DSMZ 9991, DSMZ 9992, CCTCC 2015329, CCTCC 2011265, IMI 390106 or Zyl 926 or their mutants, preferentially ATCC39116, DSMZ 9991, DSMZ 9992, CCTCC 2015329, IMI 390106 or Zyl 926 or their mutants and very preferentially ATCC39116, DSMZ 9991, DSMZ 9992, CCTCC 2015329, IMI 390106 or Zyl 926.

[0070] According to another embodiment, the microorganism may be chosen from genetically modified microorganisms belonging to the family Saccharomyces cerevisiae, Schizosaccharomyces pombe, E.coli, Corynebacterium glutamicul.

[0071] The microorganism used in the context of the present invention may also be a strain as described in WO 2014 / 102368, WO 2016 / 001203 or EP 2721148.

[0072] According to another embodiment, the microorganism is chosen from fungi belonging to the genus Aspergillus, Pycnoporus, Penicillium, preferably belonging to the family Aspergillus luchuensis, Aspergillus niger, Picnoporus cinnabarinus, Penicillium camamberti According to a first aspect of the present invention, step (b) is carried out in a batch process according to figure 2. According to this embodiment, step (a) is carried out in a fermenter. A stream (E1) is withdrawn from this fermenter to feed a reactor or a fermenter. The bioconversion is carried out under the batch conditions as described in EP0885968, with or without sterilization of the reactor or fermenter used in the context of the bioconversion step (b). According to these embodiments, the stream (F1) can feed several reactors or fermenters operated in batch in parallel.

[0073] According to another aspect of the present invention, step (b) is carried out in a continuous process at the end of step (a), according to Figure 3. According to this embodiment, step (a) is carried out in a fermenter. A stream (F1) is withdrawn from this fermenter to feed a reactor, a fermenter or a cascade of reactors or fermenters in which the bioconversion step (b) is carried out. The ferulic acid is continuously fed into the reactor or fermenter in which the bioconversion is carried out. According to this embodiment, the stream (F1) can feed several reactors or fermenters operated continuously in parallel.

[0074] According to another aspect of the present invention, step (b) is carried out in a continuous process simultaneously with step (a), according to Figure 4. According to this embodiment, the fermenter is continuously supplied with growth medium and fermentation substrate. A stream (F3) may be withdrawn comprising compound of formula (I), preferably vanillin, and optionally fermentation substrate, impurities and biomass.

[0075] The continuous processes for growing and producing the compound of formula (I), preferably vanillin, according to the present invention are particularly advantageous in that they allow time and productivity savings. These processes also allow a gain in terms of reducing the frequency of maintenance operations. The risk of contamination in the reactor or fermenter, operated continuously, is also reduced, given that the biomass concentration is high compared to the concentration of possible contaminant, the risk of colonization by a contaminating agent is thus reduced. Finally, the composition of the biomass is more homogeneous, unlike batch processes in which the biomass composition could vary from one growth batch to another. This homogeneity makes it possible to obtain a more homogeneous crude vanillin composition. This makes it possible, in particular, to facilitate the purification processes.

[0076] In the context of the present invention, step (b) may be followed by a step (c) of purification of the compound of formula (I), preferably vanillin, obtained. In general, the purification comprises a first step in which the fermentation must is separated from the biomass. The purification of the compound of formula (I), preferably vanillin, obtained at the end of step (b) may in particular be carried out by liquid / liquid extraction, by distillation, by crystallization, by evaporation in a scraped film evaporator, a falling film evaporator, in a partition evaporator, and / or by stripping. Nanofiltration and ultrafiltration steps may also be carried out within the framework of the purification step (c). By way of illustration, the purification process can be carried out according to the processes described in WO2013 / 087795, WO 2014 / 114590, WO2018 / 146210, WO2021 / 019005.

[0077] According to another embodiment, vanillin can be continuously extracted from the fermenter using a vanillin separation device. For illustration purposes, the purification method can be carried out according to the method described in WO2017 / 025339. There is no particular limitation as to the choice of the vanillin separation device. Preferably, the vanillin separation device is a membrane filtration device. This type of device makes it possible to recover from the fermentation must a retentate containing the microorganism and a microorganism-free filtrate. The vanillin separation device can also allow the selective extraction of vanillin from the extracted fermentation must.

[0078] The present invention also relates to a compound of formula (I), preferably a vanillin, capable of being obtained according to the process of the present invention. Finally, the present invention refers to the use of the compound of formula (I), preferably vanillin, obtained according to the present invention, as a flavoring in the field of human and animal food, pharmacy, or as a perfume in the cosmetics, perfumery and detergent industry.

[0079] Examples

[0080] Example 1: cultivation

[0081] The strain Streptomyces setonii ATCC 39116 is cultivated under the conditions described in document WO2017 / 025339.

[0082] Example 2: Continuous process according to the invention

[0083] The microorganism Streptomyces setonii is cultured according to Example 1. When the glucose concentration in the medium falls below 3 g / L, a new growth medium containing glucose at 30 g / L and MgSCL, 7H2O at 0.8 g / L is continuously introduced into the fermenter. This medium has been previously sterilized. A drain line withdraws the growth medium at the same flow rate as the growth medium inlet flow rate to keep the fermenter volume constant. The culture conditions are identical to those of Example 1. Under these conditions, the biomass concentration remains constant and the culture is maintained for 13 days.

[0084] No biomass degradation was observed during the 13-day culture period.

[0085] Example 3: Continuous process according to the invention

[0086] The microorganism Streptomyces setonii is cultured according to example 1.

[0087] When the glucose concentration in the medium drops below 3 g / L, a new growth medium containing glucose at 30 g / L and MgSO4, 7H2O at 0.8 g / L is continuously introduced into the fermenter. This medium has been previously sterilized. A drain line withdraws the growth medium at the same flow rate as the growth medium inlet flow rate to keep the fermenter volume constant. The culture conditions are identical to those of Example 1. After 146 h of growth (approximately 6 days), a volume of growth medium containing the biomass is withdrawn from the fermenter. The same volume of growth medium containing glucose at 30 g / L and MgSCl, 7H2O at 0.8 g / L is introduced instantly into the fermenter. Feeding and draining are then stopped. The aeration, temperature and agitation conditions do not change. After 8.5 hours, the glucose concentration is less than 4 g / L.Feeding with growth medium containing glucose at 30 g / L and MgSCL, 7H2O at 0.8 g / L is restarted, as well as withdrawal through the drain line.

[0088] No biomass degradation is observed during a sudden change in the composition of the growth medium. It is also noted that when the growth medium supply and withdrawal are restarted continuously, the growth of the microorganism continues unchanged.

[0089] Example 4: Continuous process according to the invention

[0090] The microorganism Streptomyces setonii is cultured according to Example 1. When the glucose concentration in the medium falls below 3 g / L, a new growth medium containing glucose at 30 g / L and MgSCl, 7H2O at 0.8 g / L is continuously introduced into the fermenter. This medium has been previously sterilized. A drain line withdraws the growth medium at the same flow rate as the growth medium inlet flow rate to keep the fermenter volume constant. The culture conditions are identical to those of Example 1.

[0091] After 146 hours of growth (approximately 6 days), a volume of growth medium containing the biomass is withdrawn from the fermenter. The same volume of growth medium containing glucose at 30 g / L and MgSCL, 7H2O at 0.8 g / L is introduced instantly into the fermenter. Feeding and emptying are then stopped. The aeration, temperature and agitation conditions do not change.

[0092] After a further 146h of growth, a volume of growth medium containing the biomass is withdrawn from the fermenter. The same volume of growth medium containing glucose at 30 g / L and MgSO4, 7H2O at 0.8 g / L is introduced instantly into the fermenter. Feeding and emptying are then stopped. The aeration, temperature and agitation conditions do not change.

[0093] No degradation of biomass is observed during sudden variations in the composition of the growth medium. It is also noted that with each of these variations, the microorganism is able to continue its growth.

[0094] Example 5: Preparation of vanillin

[0095] Growth medium containing biomass, prepared according to Examples 2, 3 or 4, is used. The pH of the growth medium is adjusted to 8.4 by adding 30 mol% sodium hydroxide and kept constant, with stirring.

[0096] A solution containing ferulic acid (78 g), 30% mol sodium hydroxide (60 g), and water (522 g) is prepared. This solution is then added to the fermenter containing the biomass. Regardless of the biomass preparation method, a vanillin concentration of around 15 g / L, 0.3 g / L in ferulic acid, is measured after 24 hours by HPLC. The ferulic acid conversion rate is around 99%. The vanillin selectivity of the process is greater than 80%. Example 6: Preparation of vanillin according to Figure 3

[0097] The microorganism Streptomyces setonii is cultured in a fermenter 1 according to example 1.

[0098] When the glucose concentration in the medium falls below 3 g / L, a new growth medium containing glucose at 30 g / L and MgSCL, 7H2O at 0.8 g / L is continuously introduced into the fermenter 1 at a flow rate PL This medium has been previously sterilized. A drain line withdraws the growth medium at the same flow rate Fl as the inlet flow rate of the growth medium to keep the volume of the fermenter 1 constant. The culture conditions are identical to those of example 1.

[0099] This flow Fl feeds a fermenter 2 also fed by a flow of a 20 g / L ferulic acid solution at a flow rate F2. A drain line withdraws the bioconversion medium at a flow rate F3 which is the sum of the flow rate Fl and the flow rate F2 to keep the volume of the fermenter 2 constant. The bioconversion conditions are identical to those of example 5.

[0100] The withdrawal flow at a flow rate F3 from fermenter 2 is analyzed at regular intervals. A vanillin concentration of around 5 g / L, 0.5 g / L in ferulic acid is measured by HPLC.

[0101] The results obtained show a ferulic acid conversion rate of around 95%. The vanillin selectivity of the process is greater than 80%.

[0102] Thus, Examples 5 and 6 show that the continuous process of growth of the microorganism has no deleterious effect on the microorganism. The microorganism allows the bioconversion of a substrate into vanillin as efficiently as the processes of the prior art in which the growth is carried out in batch.

[0103] However, continuous growth processes as described in examples 2 to 4 allow a saving in time and productivity of the order of 20%. These processes also allow a gain in terms of reduction in the frequency of maintenance and cleaning operations. The sizing of the equipment required for the production of an annual volume of vanillin is reduced.

Claims

CLAIMS A continuous process comprising a step (a) of growing a microorganism capable of transforming a substrate into a compound of formula (I), preferably vanillin, wherein the compound of formula (I) is Formula (I) in which n is between 0 and 5, preferably between 1 and 2, R, identical or different, is selected from the group consisting of OH, ORi, formyl, -CHO, -CO2H, linear or branched alkyl chains, optionally substituted, comprising between 1 and 6 carbon atoms, alkenyl chains, linear or branched, optionally substituted, comprising between 1 and 6 carbon atoms. RI is selected from the group consisting of methyl, ethyl, propyl, and butyl. A continuous process according to claim 1 characterized in that the growth process is carried out in a growth medium. A continuous process according to claim 2 characterized in that the growth medium is added continuously. A continuous process according to any one of claims 1 to 3 characterized in that the concentration of the carbon source in the growth medium is controlled and adapted to the needs of the microorganism capable of transforming a substrate into a compound of formula (I), preferably vanillin.A continuous process according to any one of claims 1 to 4, characterized in that step (a) is preceded by a step (a0) of culturing the microorganism. A continuous process according to any one of claims 1 to 5, characterized in that the substrate is selected from the group consisting of glucose, ferulic acid, eugenol, etc. Isoeugenol, coumaric acid, caffeic acid, L-tyrosine, vanillic acid, 4-vinyl guaiacol, preferably selected from glucose, ferulic acid, and eugenol. A process for producing a compound of formula (I), preferably vanillin, characterized in that it comprises a step (a) of continuous growth of a microorganism capable of transforming a substrate into a compound of formula (I), preferably vanillin, according to any one of claims 1 to 6. A process for producing a compound of formula (I), preferably vanillin, according to claim 7, characterized in that it comprises a step (b) of bioconversion. A process for producing a compound of formula (I), preferably vanillin, according to any one of claims 7 to 8, characterized in that steps (a) and (b) are successive.A process for producing a compound of formula (I), preferably vanillin according to any one of claims 7 to 8, characterized in that steps (a) and (b) are simultaneous.

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