Continuous process of microorganism growth

Abstract

Title: Continuous Process for Growing a Microorganism The present invention relates to a process comprising a step (a) of continuously growing a microorganism capable of transforming a substrate into a compound of formula (I), preferably vanillin. No figure for the abstract

Description

Title of the invention: Continuous method for growing a microorganism Scope of the invention

[0001] The present invention relates to a process comprising a step (a) of continuous growth of a microorganism capable of transforming a substrate into a compound of formula (I), preferably into vanillin. Prior art

[0002] Vanillin can be obtained by various methods known to those skilled in the art, and in particular by the following two methods: • A so-called natural route based on a biotechnological process including the cultivation of a microorganism capable of biotransforming a fermentation substrate into vanillin. One such process, in which the fermentation substrate is ferulic acid, is known from patent application EP0885968. US patent 5017388 describes a process in which the fermentation substrate is eugenol and / or isoeugenol. These processes result in the preparation of vanillin, referred to as natural vanillin. • A so-called synthetic route involving classic chemical reactions from guaiacol without the use of microorganisms. This process results in the preparation of a vanillin known as synthetic vanillin.

[0003] Finally, vanillin can also be prepared via a method described as "bio-based" in which 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.

[0004] As mentioned above, the processes of the so-called natural route include the culture of a microorganism capable of transforming a substrate into vanillin. This step is a growth phase of the microorganism to obtain biomass that will be used as a biocatalyst for the bioconversion phase of a substrate into vanillin.

[0005] 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 to a first fermenter of slightly larger volume to allow the microorganism to grow. This step of biomass growth and transfer to a larger reactor can be repeated as many times as necessary. The total duration of this growth stage can be significant, for example, on the order of 72 hours. Furthermore, this growth stage must be carried out in a sterile environment. An initial sterilization step of the fermenter(s) as well as all inputs and outputs to and from the fermenter(s) is required and must be performed under specific conditions. Consequently, 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.

[0006] The present invention relates to an efficient and industrial process for growing a microorganism in which the risk of contamination is reduced and whose productivity is improved.

[0007] Brief description

[0008] A first object of the present invention relates to a process comprising a step (a) of continuous growth of a microorganism capable of transforming a substrate into a compound of formula (I), preferably into vanillin.

[0009] The present invention also relates to a method 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. Description of the figures

[0010] [Fig-1]: Continuous growth process according to the invention

[0011] [Fig.2]: Method for producing a compound of formula (I) according to the invention

[0012] [Fig.3]: Method for producing a compound of formula (I) according to the invention

[0013] [Fig.4]: Method for producing a compound of formula (I) according to the invention Detailed description

[0014] In the context of the present invention, and unless otherwise indicated, the expression "between .. and ..." includes the limits.

[0015] In the context of the present invention, and unless otherwise specified, 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 as the increase in the concentration of biomass.

[0016] In the context of the present invention, and unless otherwise indicated, the term "bioconversion" refers to a biotechnological process in which a microorganism enables the transformation of a substrate into a bioconversion product. The microorganism may be a wild-type strain, a genetically modified strain obtained by molecular biology, or a mutated strain, in particular by random or targeted mutagenesis.

[0017] 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 germs and / or microorganisms.

[0018] 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 that could be detrimental to the microorganism capable of transforming a substrate into a compound of formula (I), preferably vanillin, used in the process of the present invention.

[0019] In the context of the present invention, and unless otherwise specified, asepsis or sterilization can be achieved by any method, in particular thermal, chemical, mechanical, or radiation-based, enabling the elimination of germs and / or microorganisms. Asepsis or sterilization can be performed on the fluids, raw materials, and / or devices used in the process according to the present invention. By way of illustration, the following methods may be cited in particular: - Sterilization by dry or moist heat, carried out by raising the temperature to a sufficient level, followed by maintaining this temperature for a sufficient duration to eliminate germs and / or microorganisms; for example, the temperature could be 121°C, then maintained at 121°C for a sufficient duration to eliminate germs. - Membrane filtration or several successive membrane filtrations, the smallest cut diameter of which allows the elimination of any germ and / or microorganism.

[0020] In the context of the present invention, and unless otherwise specified, the term "reactor" refers to a container suitable for carrying out chemical reactions. In the context of the present invention, the reactor may be used after having been aseptically or sterilized, or without aseptically or sterilized.

[0021] In the context of the present invention, and unless otherwise indicated, the term "fermenter" refers to a reactor suitable for carrying out fermentations. In the context of the present invention, the fermenter may be used after having been aseptically or sterilized, or without aseptically or sterilized.

[0022] In the context of the present invention, a compound of formula (I) has the following formula:

[0023] [Chem.l] Formula (1)

[0024] in which n is between 0 and 5, preferably between 1 and 2,

[0025] 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.

[0026] According to a particular embodiment, R is chosen from the group consisting of formyl -CHO, OH, OMEt, OEt, OPr, OBu, methyl, ethyl, -(CH2)2-C(O)-CH3, -CH=CH-C(O)-CH3, -ch=ch-co2h, -co2h, -ch2-oh.

[0027] According to a particular embodiment, the compound of formula (I) is chosen from vanillin and frambinone.

[0028] A first aspect of the present invention relates to a process comprising a step (a) of continuous growth of a microorganism capable of transforming a substrate into a compound of formula (I), preferably into vanillin.

[0029] In the context of the present invention, the process comprising a step (a) of continuous growth or "continuous growth process" refers to a step or process in which the reactor volume is constant. During the continuous growth process, the microorganism multiplies. During the continuous growth process, a stream (Fl) comprising at least the biomass is continuously transferred. In one embodiment, a stream (Fl) comprising all of the biomass is continuously transferred. In another embodiment, a stream (Fl) comprising a portion of the biomass is continuously transferred.

[0030] In the context of the present invention, the pH of the growth medium is controlled and optionally regulated by the addition of acid or base. Those skilled in the art can 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 vanillin, used in the process of the present invention.

[0031] 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.

[0032] In the context of the present invention, the continuous growth process is carried out under controlled temperature conditions, the temperature of which may optionally be regulated. Those skilled in the art can adapt the process temperature to the needs of the microorganism capable of transforming a substrate into a compound of formula (I), preferably vanillin, used in 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 most preferably between 35°C and 45°C.

[0033] In the context of the present invention, the pO2 of the growth medium is controlled and optionally regulated. Those skilled in the art can adapt the pO2 of the growth medium to the needs of the microorganism capable of transforming a substrate into a compound of formula (I), preferably vanillin, used in the process of the present invention.

[0034] By way of illustration, the pO2 can be between 1% and 99%, preferably in the range of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. The pO2 corresponds to the concentration of dissolved oxygen in the growth medium relative to the dissolved oxygen concentration at saturation, without biomass. The pO2 can notably be measured with a Hamilton OXYFERM FDA VP 225 O2 Sensor.

[0035] In the context of the present invention, the continuous growth process is carried out under agitation. Agitation can be characterized by the blade tip velocity or peripheral velocity. The blade tip velocity can be calculated using the following formula:

[0036] Vp = 2*Pi*D / 2*N / 60, where Vp is the blade tip velocity (m / s), D is the diameter of the reactor or fermenter (in m), and N is the rotational speed of the agitator (rpm). Those skilled in the art can adapt the agitation to the needs of the microorganism capable of transforming a substrate into a compound of formula (I), preferably vanillin, used in the process of the present invention. According to a particular embodiment, the blade tip velocity is between 1 and 10 m / s, preferably between 2 and 5 m / s.

[0037] According to the present invention, growth is carried out in a medium or "growth medium" adapted to 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 microorganisms.

[0038] In the context of the present invention, the composition of the growth medium is controlled. The composition of the growth medium is continuously adjusted. Thus, in the context of the present invention, growth medium is added continuously.

[0039] In the context of the present invention, the rate of addition of the growth medium is equal to the flow withdrawal rate (Fl). In the context of the present invention, it is preferable that the flow rate for adding the growth medium or removing the flow be adjusted to allow the microorganism sufficient time to multiply and prevent a decrease in its concentration. It is also preferable that the flow rate for adding the growth medium or removing the flow be adjusted to prevent the accumulation or reduction of glucose in the medium.

[0040] According to one embodiment, the rates of addition of the growth medium and removal of the flux (Fl) may be constant. According to another embodiment, the rates of addition of the growth medium and removal of the flux (Fl) may be carried out occasionally.

[0041] In general, the continuous growth process according to the present invention is carried out in a fermenter. Generally, the continuous growth process is carried out under aseptic conditions, preferably under sterile conditions.

[0042] In the context of the present invention, the continuous growth process is carried out under aseptic, preferably sterile, conditions. This 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 aseptic, 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. This indicates that the solvent and growth medium used in the process have been aseptic, 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. This indicates that the air or oxygen used in the process is aseptic, preferably sterilized, prior to its use in the process.

[0043] In general, the growth medium comprises a carbon source, an organic or inorganic nitrogen source, inorganic salts, and optionally growth factors. Those skilled in the art can 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 vanillin, used in the process of the present invention.

[0044] Generally speaking, the term "carbon source" refers to a source of carbon 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. Depending on a particular aspect, the carbon source may be chosen from the group consisting of glucose, fructose, sucrose, mannose, xylose, arabinose, galactose, lactose, ethanol, cellobiose, glycerol, and polysaccharides such as cellulose, and mixtures thereof. According to a preferred embodiment, the carbon source includes glucose.

[0045] In the context of the present invention, the carbon source concentration is controlled. In the context of the present invention, the carbon source concentration is adapted to the needs of the microorganism capable of transforming a substrate into a compound of formula (I), preferably vanillin, used in the process of the present invention.

[0046] In the context of the present invention, the carbon source concentration is maintained at a concentration between 0 and 20 gL*, preferably between 0.1 and 10 gL1 and very preferably between 0.5 and 5 gL1. The addition of a carbon source, in particular glucose, is generally carried out continuously.

[0047] Generally speaking, the expression "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, and peptone.

[0048] Usable inorganic salts include, for example, among others, sulfates, nitrates, chlorides, carbonates, hydrogen phosphates and phosphates of sodium, potassium, magnesium, calcium, zinc and iron.

[0049] Within the scope of the present invention, usable growth factors include, for example, D-biotin, nicotinic acid, thiamine (HCl), pyridoxine (HCl), para-aminobenzoic acid, myo-inositol, calcium pantothenate, Na2-EDTA, ZnSO4.7H2O, MnCl2.4H2O, CoC12.6H2O, CuSO4.5H2O, Na2MoO4.2H2O, CaCl2.2H2O, FeSO4.7H2O, H3BO3, KL

[0050] Within the framework of the present invention, the concentration of the nitrogen source and / or growth factors can be controlled. Within the framework of the present invention, the concentration of the nitrogen source and / or growth factors can be between 0 and 5 gL*, preferably between 0.1 and 2.5 gL*, and most preferably between 0.2 and 1.5 g.L1.

[0051] In addition, magnesium ions, such as magnesium sulfate, may be added at a concentration of between 0 and 0.5 gL*, preferably between 0.01 and 0.25 gL*, and most preferably between 0.02 and 0.15 gL*. The addition of magnesium ions may be continuous.

[0052] Within the framework 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, and most preferably between 8 and 15 g / L.

[0053] 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 The optical density at 880 nm is between 5 and 400, preferably between 20 and 200, most preferably between 40 and 100, and even more preferably between 60 and 80. Optical density measurement at 880 nm can be performed continuously online. Optical density at 880 nm can be measured using a Hamilton Dencytee Unit probe.

[0054] In the context of the present invention, the biomass concentration can be controlled by measuring the optical density at 600 nm. Generally, the optical density, measured at 600 nm, is between 2 and 100, preferably between 5 and 80, most preferably between 10 and 60, and even more preferably between 25 and 45. The optical density at 600 nm can be measured by taking a sample of the growth medium. The sample is then diluted 50 times in water. The optical density at 600 nm can be measured using a spectrophotometer set to 600 nm.

[0055] In the context of the present invention, the microorganism can 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 fungus, can be a wild-type microorganism, genetically modified by molecular biology, mutated, or mutagenetic, whether random or site-directed. Preferably, the microorganism is chosen from bacteria belonging to the order Actinomycetales, Caulobacterales, or Pseudomonadales, preferably belonging to the family Streptomycetaceae, Pseudonocardiacae, Micrococcaceae, Caulobacteraceae, or Pseudomonadaceae, most preferably Streptomyces setonii or 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 conducted in the presence of a strain available under the numbers ATCC39116, DSMZ 9991, DSMZ 9992, CCTCC 2015329, CCTCC 2011265, IMI 390106, or Zyl 926, or their mutants, preferably ATCC39116, DSMZ 9991, DSMZ 9992, CCTCC 2015329, IMI 390106, or Zyl 926, or their mutants, and most preferably ATCC39116, DSMZ 9991, DSMZ 9992, CCTCC 2015329, IMI 390106, or Zyl 926. In another embodiment, the microorganism may be selected from genetically modified microorganisms belonging to the Saccharomyces family. cerevisiae, Schizosaccharomyces pombe, E.coli, Corynebacterium glutamicul. .

[0056] 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.

[0057] According to another embodiment, the microorganism is chosen from among the fungi ap belonging to the genera Aspergillus, Pycnoporus, Penicillium, preferably belonging to the family Aspergillus luchuensis, Aspergillus niger, Pycnoporus cinnabarinus, Penicillium camamberti

[0058] In the context of the present invention, the substrate can be transformed by bioconversion into a compound of formula (I), preferably 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 to obtain a sufficient quantity of biomass. This sufficient quantity of biomass can then be used in a continuous growth process according to the present invention.

[0060] The bacteria are generally cultured in an aqueous medium, in the presence of nutrients. Generally, the culture medium comprises a carbon source, an organic or inorganic nitrogen source, inorganic salts, and growth factors. Those skilled in the art can 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 vanillin, used in the process of the present invention. The carbon source concentration is generally between 5 and 50 g L*, preferably between 20 and 34 g L*. The nitrogen source, such as a yeast extract, and the growth factors are generally added at a concentration between 2 and 20 g L*, preferably between 5 and 10 g L*. In addition, magnesium ions, such as magnesium sulfate, may be added at a concentration between 0.1 and 5 g.L*, preferably between 0.5 and 1 gL*. In general, the pH of the culture is between 3 and 9, particularly 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, 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 most 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 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 entirely consumed, preferably such that the carbon source concentration is less than or equal to 15 gL*, preferably less than or equal to 5 gL*, most preferably less than or equal to 3 gL*. 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 beginning of the 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 vanillin.

[0063] In this embodiment, step (a) is carried out under the conditions described above for the continuous process of 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 include a step (aO) prior to step (a) as described above.

[0065] According to one embodiment, the process 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, with step (a) being carried out before step (b). According to another embodiment, steps (a) and (b) can be carried out simultaneously.

[0067] The bioconversion step (b) can in particular be carried out under the conditions described in EP0885968, EP0761817, or WO2017 / 025339.

[0068] According to an alternative, step (b) can be carried out in batch or fed-batch mode. The bioconversion substrate can be added all at once or in several stages. According to another embodiment, step (b) can be carried out continuously. In particular, the bioconversion substrate can be fed continuously. Optionally, a stream (F2) comprising the compound of formula (I), preferably vanillin, can be withdrawn continuously. The stream (F2) can also optionally include fermentation substrate, impurities, and biomass.

[0069] Step (b) can be carried out under aseptic, preferably sterile, conditions. According to another embodiment, step (b) can be carried out without aseptic or sterile conditions.

[0070] In particular the bioconversion substrate can be selected from glucose, ferulic acid, eugenol, isoeugenol, coumaric acid, caffeic acid, L-tyrosine, vanillic acid, 4-vinyl guaiacol, preferably selected from glucose, ferulic acid and eugenol.

[0071] In the context of the present invention, the microorganism can 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 fungus, can be a wild-type microorganism, genetically modified by Molecular biology, mutated, by random or directed mutagenesis. Preferably the microorganism is chosen from bacteria belonging to the order Actinomycetales, Caulobacterales or Pseudomonadales, preferably belonging to the family Streptomycetacae, Pseudonocardiacae, Micrococcaceae, Caulobacteraceae, or PseudomonadaceaeM^ preferably Streptomyces setonii, Amycolatopsis sp. Streptomyces psammoticus, Amycolatopsis thermoflava, Micrococcus sp., Caulobacter segnis, Pseudomonas fluorescens. or their mutants, even more preferably Streptomyces setonii, Amycolatopsis sp. Streptomyces psammoticus.Preferably the bioconversion reaction is conducted 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, preferably ATCC39116, DSMZ 9991, DSMZ 9992, CCTCC 2015329, IMI 390106 or Zyl 926 or their mutants and very preferably ATCC39116, DSMZ 9991, DSMZ 9992, CCTCC 2015329, IMI 390106 or Zyl 926. .

[0072] According to another embodiment the microorganism can be chosen from among the genetically modified microorganisms belonging to the family Saccharomyces ce-revisiae, Schizosaccharomyces pombe, E.coli, Corynebacterium glutamicul.

[0073] 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.

[0074] According to another embodiment, the microorganism is chosen from among the fungi belonging to the genera Aspergillus, Pycnoporus, Penicillium, preferably belonging to the family Aspergillus luchuensis, Aspergillus niger, Pycnoporus cinnabarinus, Penicillium camamberti

[0075] According to a first aspect of the present invention, step (b) is carried out in a batch process as shown in [Fig. 2]. In this embodiment, step (a) is carried out in a fermenter. A stream (Fl) is withdrawn from this fermenter to feed a reactor or fermenter. The bioconversion is carried out under batch conditions as described in EP0885968, with or without sterilization of the reactor or fermenter used in step (b) of bioconversion. In these embodiments, the stream (Fl) can feed several reactors or fermenters operated in batch mode in parallel.

[0076] According to another aspect of the present invention, step (b) is carried out in a continuous process following step (a), as shown in [Fig. 3]. In this embodiment, step (a) is carried out in a fermenter. A stream (Fl) is withdrawn from this fermenter to feed a reactor, fermenter, or cascade of reactors or fermenters in which the bioconversion step (b) is performed. Ferulic acid is continuously fed into the reactor or fermenter in which the bioconversion is carried out. In this embodiment, the stream (Fl) can feed several reactors or fermenters. liars operated on continuously in parallel.

[0077] According to another aspect of the present invention, step (b) is carried out in a continuous process simultaneously with step (a), as shown in [Fig. 4]. In this embodiment, the fermenter is continuously fed with growth medium and fermentation substrate. A stream (F3) can be withdrawn comprising the compound of formula (I), preferably vanillin, and optionally fermentation substrate, impurities, and biomass.

[0078] The continuous processes for growing and producing a compound of formula (I), preferably vanillin, according to the present invention are particularly advantageous in that they allow for gains in time and productivity. These processes also allow for a reduction in the frequency of maintenance operations. The risk of contamination in the reactor or fermenter, which is operated continuously, is also reduced, since the biomass concentration is high relative to the concentration of any potential contaminant, thus reducing the risk of colonization by a contaminating agent. Finally, the biomass composition 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 composition of crude vanillin. This, in particular, facilitates the purification processes.

[0079] In the context of the present invention, step (b) may be followed by a purification step (c) of the compound of formula (I), preferably vanillin, obtained. Generally, the purification includes a first step in which the fermentation mash 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, distillation, crystallization, evaporation in a scraped-film evaporator, a falling-film evaporator, a partition evaporator, and / or by stripping. Nanofiltration and ultrafiltration steps may also be carried out as part of the purification step (c). For illustrative purposes, the purification process can be carried out according to the processes described in WO2013 / 087795, WO 2014 / 114590, WO2018 / 146210, WO2021 / 019005.

[0080] According to another embodiment, vanillin can be continuously extracted from the fermenter using a vanillin separation device. By way of illustration, the purification process can be carried out according to the process described in WO2017 / 025339. There are no particular limitations regarding the choice of the vanillin separation device. Preferably, the vanillin separation device is a membrane filtration device. This type of device allows for the recovery of the fermentation wort, a retentate containing the microorganism, and a filtrate free of microorganisms. The vanillin separation device can also allow selective extraction of vanillin from extracted fermentation must.

[0081] The present invention also relates to a compound of formula (I), preferably vanillin, which can be obtained according to the process of the present invention. Finally, the present invention relates to the use of the compound of formula (I), preferably vanillin obtained according to the present invention, as a flavoring in the fields of human and animal food, pharmaceuticals, or as a fragrance in the cosmetics, perfumery, and detergent industries. Examples

[0082] Example 1: Cultivation

[0083] The Streptomyces setonii ATCC 39116 strain is cultured under the conditions described in document WO2017 / 025339.

[0084] Example 2: Continuous process according to the invention

[0085] The microorganism Streptomyces setonii is cultured according to example 1.

[0086] When the glucose concentration in the medium falls below 3 g / L, a A new growth medium containing 30 g / L glucose and 0.8 g / L MgSO4·7H2O is continuously introduced into the fermenter. This medium has been previously sterilized. A drain line removes the growth medium at the same flow rate as the incoming flow rate to maintain a constant fermenter volume. The culture conditions are identical to those in Example 1. Under these conditions, the biomass concentration remains constant, and the culture is maintained for 13 days.

[0087] No degradation of biomass is observed during the 13-day culture period.

[0088] Example 3: Continuous process according to the invention

[0089] The microorganism Streptomyces setonii is cultured according to example 1.

[0090] When the glucose concentration in the medium falls below 3 g / L, a A new growth medium containing 30 g / L glucose and 0.8 g / L MgSO4·7H2O is continuously fed into the fermenter. This medium has been previously sterilized. A drain line removes the growth medium at the same flow rate as the incoming flow rate to maintain a constant fermenter volume. The culture conditions are identical to those in 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 30 g / L glucose and 0.8 g / L MgSO4·7H2O is immediately fed into the fermenter. Feeding and draining are then stopped. Aeration, temperature, and agitation conditions remain unchanged. After 8.5 h, the glucose concentration is less than 4 g / L. Feeding with growth medium containing glucose at 30 g / L and MgSO4, 7H2O at 0.8 g / L is restarted, as well as the withdrawal through the drain line.

[0091] No degradation of biomass is observed during a sudden change in the composition of the growth medium. It is also noted that when feeding into the growth medium and withdrawal are restarted continuously, the growth of the microorganism continues without change.

[0092] Example 4: Continuous process according to the invention

[0093] The microorganism Streptomyces setonii is cultured according to example 1.

[0094] When the glucose concentration in the medium falls below 3 g / L, a A new growth medium containing 30 g / L glucose and 0.8 g / L MgSO4·7H2O is continuously introduced into the fermenter. This medium has been previously sterilized. A drain line removes the growth medium at the same flow rate as the incoming flow rate to maintain a constant fermenter volume. The culture conditions are identical to those in Example 1.

[0095] 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 MgSO4·7H2O at 0.8 g / L is immediately introduced into the fermenter. Feeding and emptying are then stopped. The aeration, temperature, and agitation conditions remain unchanged.

[0096] After a further 146-hour growth period, a volume of growth medium containing the biomass is withdrawn from the fermenter. The same volume of growth medium containing 30 g / L glucose and 0.8 g / L MgSO4·7H2O is immediately introduced into the fermenter. Feeding and emptying are then stopped. The aeration, temperature, and agitation conditions remain unchanged.

[0097] 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.

[0098] Example 5: Vanillin preparation

[0099] 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 molar sodium hydroxide and maintained constant under stirring.

[0100] A solution containing ferulic acid (78 g), 30% molar sodium hydroxide (60 g), and water (522 g) is prepared. This solution is then added to the fermenter containing the biomass.

[0101] Regardless of the biomass preparation method, a vanillin concentration of approximately 15 g / L and a ferulic acid concentration of approximately 0.3 g / L are measured by HPLC after 24 hours. The ferulic acid conversion rate is approximately 99%.

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

[0103] However, continuous growth processes as described in Examples 2 to 4 allow for time and productivity gains of approximately 20%. These processes also reduce the frequency of maintenance and cleaning operations. The equipment required for the annual production of vanillin is also reduced.

Claims

1. Claims Continuous process comprising a step (a) of growing a microorganism capable of transforming a substrate into a compound of formula (I), preferably into vanillin in which the compound of formula (I) is [Chem. 2] OH

2.

3.

4.

5.

6. Formula H) in which n is between 0 and 5, preferably between 1 and 2, R, identical or different, is selected 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, in which growth medium is added continuously and in which a stream (Fl) comprising at least the biomass is transferred continuously characterized in that the rate of addition of the growth medium is equal to the rate of removal of the stream (Fl). Continuous process according to claim 1 characterized in that the growth process is carried out in a growth medium. Continuous process according to claim 2 characterized in that the growth medium is added continuously. Continuous process according to any one of claims 1 to 3, characterized in that step (a) is preceded by a step (aO) of culturing the microorganism. Continuous process according to any one of claims 1 to 4, characterized in that the substrate is 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. Process for the production of 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 according to any one of the re-

7.

8. claims 1 to 5 and comprising a bioconversion step (b). Process for producing a compound of formula (I), preferably vanillin according to claim 6, characterized in that steps (a) and (b) are successive. Process for the production of a compound of formula (I), preferably vanillin according to any one of claims 6 to 7, characterized in that steps (a) and (b) are simultaneous.

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