Method involving the production of aminobenzoic acid

Abstract

The invention relates to a method involving the production of aminobenzoic acid, said method having the steps of: (A) fermenting a raw material containing a reducing sugar and a nitrogen-containing compound in the presence of microorganisms in a fermentation reactor in order to form an aminobenzoate anion-containing fermentation product, wherein the pH value is kept in the range of 6.0 to 8.5 during the fermentation process by adding an inorganic base, (B) separating the microorganisms from the fermentation product, thereby obtaining a clarified fermentation product, and (C) precipitating aminobenzoic acid from the clarified fermentation product by setting a pH value of 3.0 to 4.7 by adding a protonic acid and separating the precipitated aminobenzoic acid, thereby leaving a mother liquor. The mother liquor obtained in step (C) is supplied to step (A).

Description

[0001] PROCESS COMPRISING THE PREPARATION OF AMINOBENZOIC ACID

[0002] The work leading to the present invention was financially supported by the German Federal Ministry of Food and Agriculture under grant number 2221NR073X.

[0003] The present invention relates to a process comprising the production of aminobenzoic acid, the process comprising the steps of: (A) fermentation of a raw material containing a reducing sugar and a nitrogen-containing compound in the presence of microorganisms in a fermentation reactor to form a fermentation product containing aminobenzoate anions, wherein the pH during the fermentation is maintained in the range of 6.0 to 8.5 by adding an inorganic base, (B) separating the microorganisms from the fermentation product to obtain a clarified fermentation product and (C) precipitating aminobenzoic acid from the clarified fermentation product by adjusting a pH of 3.0 to 4.7 by adding a protonic acid and separating the precipitated aminobenzoic acid to leave a mother liquor, wherein the mother liquor obtained in step (C) is fed to step (A).

[0004] The production of organic acids through fermentation processes has received particular attention in recent times. Among the organic acids accessible by fermentation, aminobenzoic acid is an economically important product. Aminobenzoic acid is used, for example, in the production of dyes, fragrances, pesticides, and pharmaceuticals. Another example of an application of aminobenzoic acid is its use in the production of aniline by decarboxylation. Aniline, in turn, is particularly important as an intermediate in the production of isocyanates. The ortho isomer of aminobenzoic acid, anthranilic acid, can also serve as a starting material for the production of poly(anthranilamines) and polyamines and the corresponding polyisocyanates.

[0005] The fermentative production of aminobenzoic acid is generally known in the art; see, for example, international patent application WO 2015 / 124687 A1 (which describes the two-step production of aniline via ortho-aminobenzoic acid as an intermediate) and the literature cited therein. Fermentation processes take place in an aqueous environment and, in the case of the production of aminobenzoic acid, generally yield aqueous product mixtures (fermentation broths) with a mass content of aminobenzoic acid, particularly in the range of 10.0 g / L to 100 g / L.

[0006] The ortho isomer of aminobenzoic acid (anthranilic acid) is of particular importance. In the metabolism of bacteria and yeasts, anthranilic acid is formed in the shikimic acid pathway as a natural intermediate in the synthesis of tryptophan. In the biotechnological production of anthranilic acid, its conversion in the pathway is reduced or prevented to achieve accumulation in the fermentation medium. Such a concept for the biotechnological production of anthranilic acid and its subsequent catalytic conversion to aniline is described in the aforementioned international patent applications WO 2015 / 124686 A1 and WO 2015 / 124687 A1. The use of bacteria from the Corynebacteria or Pseudomonads families is described as a possible recombinant microorganism. A more recent application (WO 2017 / 102853 A1) describes the use of yeast.

[0007] Para-aminobenzoic acid is also of interest. The synthesis of para-aminobenzoic acid can occur in the metabolism of bacteria and yeasts via the intermediate chorismate, which is formed as an intermediate in the shikimic acid pathway. Chorismate is first enzymatically converted to 4-amino-4-deoxychorismate and then, through a second enzymatic reaction, to para-aminobenzoic acid. A concept for the biotechnological production of aniline via the intermediate para-aminobenzoic acid is described in the international application WO 2014 / 171205 A1. The use of bacteria from the Corynebacteria family is also described here as a possible recombinant microorganism. T. Kubota et al. also describe the use of Corynebacteria in Metabolie Engineering 2016, 38, 322 - 330 ("Production of para-aminobenzoate bygenetically engineered Corynebacterium glutamicum and non-biological formationofan N-glucosylbyproduct") [1].

[0008] The fermentative production of aminobenzoic acid produces an aqueous product stream. When fermentation is carried out at pH values ​​significantly above the isoelectric point (which is common when bacteria are used as microorganisms), the valuable product, aminobenzoic acid, is present predominantly to fully charged as an anion (aminobenzoate anion). To neutralize the aminobenzoic acid formed during fermentation, a base, particularly sodium hydroxide, is typically added as a pH adjuster. After performing the workup steps typical for fermentation, the valuable product, aminobenzoic acid, can be precipitated in an electroneutral form as a solid (crystallization) by adjusting the pH to a value near or at the isoelectric point. The aminobenzoic acid can then be separated, for example, by filtration. The filtered product initially occurs in a highly watery state ("slurry").This product is then, for example, washed and, if necessary (depending on the intended use), dried, or dissolved in a solvent such as aniline or 1-dodecanol (see WO 2015 / 124687 A1). Mineral acids, especially hydrochloric acid, are typically used to adjust the pH during crystallization. This is described, for example, in the international application WO 2017 / 085170 A1, which deals with a process for producing aminobenzoic acid or an aminobenzoic acid derivative by fermenting a suitable raw material under the influence of suitable microorganisms to obtain a fermentation broth comprising aminobenzoate and / or aminobenzoic acid, with the focus being in particular on obtaining the aminobenzoic acid from the fermentation broth by crystallization using a one-step acid treatment in the presence of seed crystals.

[0009] As an alternative to the use of mineral acids, in a specific embodiment of the fermentative production of aminobenzoic acid, carbon dioxide can also be introduced under pressure to crystallize the aminobenzoic acid (crystallization with "carbonic acid"). Such a process is described in WO 2019 / 234092 A1 (also published as US 2021 / 0222215 A1). In this process, the fermentation takes place in the presence of a calcium salt, wherein (I) the aminobenzoic acid present in the fermentation broth is partially bound, optionally as far as possible due to the solubility equilibrium, as insoluble calcium aminobenzoate,This insoluble calcium aminobenzoate is then (II) isolated either as such or in admixture with the microorganisms used in the fermentation and converted into a water-soluble form by separating an insoluble calcium salt different from calcium aminobenzoate [ion exchange], and then (III) aminobenzoic acid is precipitated by introducing carbon dioxide under pressure into the aqueous solution freed from the precipitated calcium salt [crystallization]. The advantages of this process are a reduction in the concentration of dissolved aminobenzoic acid in the fermentation reactor, the possibility of crystallizing the majority of the aminobenzoic acid with carbon dioxide, and a saving of base in the fermentation.

[0010] When anthranilic acid is separated by filtration, a mother liquor remains that still contains a significant residual concentration of aminobenzoic acid (corresponding to the solubility of aminobenzoic acid under the respective conditions). Therefore, this dissolved aminobenzoic acid should be removed as quantitatively as possible before being disposed of as wastewater. In addition, the mother liquor usually contains dissolved residual components of usable media components, such as carbon or nitrogen sources, which were not fully converted to aminobenzoic acid or biomass during fermentation. The effort required to remove these usable media components from the mother liquor usually exceeds the economic benefit, which is why disposal with wastewater is preferred in the current state of the art.

[0011] For the recovery of aminobenzoic acid from dilute aqueous solutions, various concepts have been developed in the prior art, which are described in WO 2023 / 117756 A1 on pages 3 to 5. As explained therein, none of these concepts is without disadvantages. WO 2023 / 117756 A1 itself proposes extracting the aqueous mother liquor containing dissolved aminobenzoic acid obtained in the process described therein with an alkanol having 8 to 12 carbon atoms. Then, the aminobenzoic acid dissolved in the alkanol phase is recovered by basic or acidic back-extraction in the form of an aqueous solution of an aminobenzoic acid salt, and finally, precipitating it from the salt solution by adjusting the pH to 3.0 to 4.7.Although the use of Cs-C alkanols as extraction agents in conjunction with basic or acidic back-extraction represents a good compromise between the requirement for the most complete extraction of the aminobenzoic acid from the mother liquor (to achieve the highest possible yield) on the one hand and the requirement to obtain the purest possible extracted mother liquor (to simplify wastewater treatment or disposal) on the other, this process is not without disadvantages. For example, the necessity of extracting the entire mother liquor is associated with considerable effort. The previously mentioned application WO 2019 / 234092 A1 (also published as US 2021 / 0222215 A1) proposes recycling the mother liquor remaining after separation of the aminobenzoic acid precipitated in the carbon dioxide crystallization to the ion exchange step.It is further proposed to crystallize further aminobenzoic acid from the aqueous fermentation solution remaining after separation of the calcium aminobenzoate obtained in the fermentation by adding a mineral acid. The additional mother liquor remaining after separation of the precipitated aminobenzoic acid can be subjected to an adsorption step to recover any residual aminobenzoic acid dissolved therein, and adsorbed aminobenzoic acid can be recovered by a subsequent desorption step. Depending on the pH at which the desorption is carried out, the resulting desorbate can be recycled to the ion exchange step (desorption at pH values ​​of 6.0 to 11) or subjected to post-crystallization by addition of base (desorption at pH values ​​of less than 3.0). The recycling of mother liquor (regardless of its origin) to the fermentation is not disclosed in WO 2019 / 234092 A1.These process steps for recovering the anthranilic acid remaining in the mother liquor also involve considerable effort. To achieve high purity, the process steps for recovering anthranilic acid from the mother liquor are deliberately designed so that media components added as nutrients during fermentation but not fully converted are not isolated from the mother liquor along with the anthranilic acid. As a result, nutrients remain in the aqueous wastewater after the recovery of anthranilic acid from the mother liquor. Selective recovery of these nutrients for reuse in the fermentation has not yet been considered in the prior art.

[0012] There was therefore a need for further improvements in the field of fermentative production of aminobenzoic acid. In particular, it would be desirable to reduce the losses of usable media components as well as the effort required to extract aminobenzoic acid from the diluted aqueous mother liquor.

[0013] In response to this need, the present invention provides a process comprising the preparation of aminobenzoic acid, the process comprising the following steps:

[0014] (A) [Fermentation] Fermentation of a raw material (1) containing a reducing sugar and a nitrogen-containing compound in the presence of microorganisms in a fermentation reactor (100, 100-X) to a fermentation product (2) containing aminobenzoate anions, wherein the pH during the fermentation is maintained in the range of 6.0 to 8.5 by adding an inorganic base (3), preferably with the addition of an oxygen-containing gas (9; in particular air),

[0015] (B) [Clarification] Separating the microorganisms (biomass, 4) from the fermentation product (2) to obtain a clarified fermentation product (5) (by means of a suitable separation device (200, 200-X), which can also be integrated into the fermentation reactor (100, 100-X)), and

[0016] (C) [Crystallization] Precipitation (in particular in a crystallizer (300, 300-X)) of aminobenzoic acid from the clarified fermentation product (5) by adjusting a pH of 3.0 to 4.7, preferably 3.2 to 3.7, particularly preferably 3.4 to 3.6, very particularly preferably 3.5, by adding a protic acid (6) and separating the precipitated aminobenzoic acid (7) (by means of a suitable separation device (400, 400-X), which can also be integrated into the crystallizer (300, 300-X)) while leaving a mother liquor (8), wherein a partial separation of water can take place before or after step (C), wherein the mother liquor (8) obtained in step (C), optionally after treatment (such as in particular sterile filtration and / or thermal inactivation), is fed to step (A).

[0017] Completely surprisingly, it was found that the mother liquor obtained during crystallization can be successfully recycled to the fermentation, which, in conjunction with the use of reducing sugars, enables an increase in the space-time yield. Despite the acidic nature of the mother liquor, a suitable pH can be maintained during the fermentation by adding increasing amounts of inorganic base to the fermentation reactor or, preferably, by adjusting the mother liquor obtained in step (C) to a pH in the range of 6.0 to 8.5 by adding an inorganic base before feeding it to the fermentation, and in particular by adjusting it to a pH that deviates by a maximum of 0.2, preferably a maximum of 0.1, from the pH maintained during the fermentation and, in particular, corresponds to this pH.Without wishing to be bound by any theory, it is assumed that during fermentation, a reaction occurs between the aldehyde group of the reducing sugar and the amino group of the aminobenzoate anions, forming a glycosylamine. This glycosylamine is broken down into the parent sugar and aminobenzoic acid at the low pH values ​​of crystallization. After separation of the precipitated aminobenzoic acid, a mother liquor remains, the sugar components of which, after the mother liquor is returned to the fermentation, can serve as an additional food source for the microorganisms. Furthermore, by returning the mother liquor, the portion of aminobenzoic acid present in dissolved form at the pH value of crystallization is returned to the fermentation reactor.As a result, the effort required to recover aminobenzoic acid dissolved in the mother liquor as well as the effort required to recover the sugars released during crystallization is considerably reduced by the process according to the invention.

[0018] In the attached figures, FIG. 1 and FIG. 2 show various embodiments of the method according to the invention:

[0019] FIG. 1 shows an embodiment of the process according to the invention with recycling of the mother liquor to the same fermentation reactor from which the fermentation product originates, by the treatment of which said mother liquor was obtained in steps (B) and (C).

[0020] FIG. 2 shows an embodiment of the process according to the invention with several fermentation reactors connected in series, wherein the mother liquor is fed to the subsequent fermentation reactor.

[0021] FIG. 3 to FIG. 8 illustrate the results of the experiments described in the examples section in graphic form. The following is plotted on the ordinate axis, for three experiments each, and as a function of the fermentation time in hours plotted on the abscissa axis:

[0022] FIG. 3: the dry microbial biomass as a concentration value in g / L in step (A),

[0023] FIG. 4: the concentration of aminobenzoic acid produced in g / L in step (A),

[0024] FIG. 5: the concentration of free, unbound aminobenzoic acid in g / L in step (A),

[0025] FIG. 6: the concentration of bound glucose in g / L in step (A),

[0026] FIG. 7: the absolute values ​​of aminobenzoic acid produced in g in step (A) and

[0027] FIG. 8: the microbial biomass dry mass as absolute amount in g in step (A).

[0028] In the context of the present invention, all pH values ​​refer to the temperature at which the corresponding step (e.g. step (A)) is carried out and can be easily measured using a glass electrode.

[0029] Carrying out the fermentation according to step (A) in a fermentation reactor means, in the simplest case, that the mother liquor obtained in step (C) is returned to the same fermentation reactor from which the fermentation product was taken, the treatment of which in steps (B) and (C) resulted in the mother liquor being originally obtained (see also FIG. 1). Of course, several such fermentation reactors can also be operated in parallel (not shown in FIG. 1). This embodiment is suitable for both batch and continuous operation. However, carrying out the fermentation according to step (A) in a fermentation reactor also encompasses the case where several (m, where m is a natural number from, in particular, 2 to 6) fermentation reactors are operated in series (see also FIG. 2), and is therefore to be read as "in at least one fermentation reactor."This embodiment is particularly suitable for continuous operation of the process. In this case, the mother liquor obtained in step (C) is then fed to the respective subsequent fermentation reactor. The left side (1000) of FIG. 2 shows the first execution of steps (A) to (C) in the series. The right side (2000) represents each subsequent ("X-th") execution of steps (A) to (C). For clarity, the apparatuses on the left side are provided with the suffix "-1" and those on the right side with the suffix "-X". The mother liquor obtained by treating the fermentation product obtained in the last fermentation reactor in the series according to steps (B) and (C) can be disposed of, preferably after recovery of dissolved portions of aminobenzoic acid using conventional methods (not shown in FIG. 2).If the salt load of the mother liquor is not too high (see below for details), it can also be recycled to the first fermentation reactor in the series (not shown in FIG. 2). It is also possible to use several parallel lines of series-connected fermentation reactors (not shown in FIG. 2).

[0030] First, a brief summary of various possible embodiments of the invention follows:

[0031] In a first embodiment of the process according to the invention, which can be combined with all other embodiments except those which are limited to a continuously carried out procedure, steps (A) to (C) are carried out discontinuously, wherein a further fermentation is started by feeding the mother liquor obtained in step (C) into step (A), which is followed by a further implementation of steps (B) and (C), wherein steps (A) to (C) are carried out a total of n times, wherein n is a natural number in the range from 2 to 6.

[0032] In a second embodiment of the process according to the invention, which is a special embodiment of the first embodiment, step (B) is carried out in each case after reaching a time ti, wherein the time ti denotes the time at which a specific minimum amount of reducing sugar ?RZ_MIN was converted for the first time, wherein the specific minimum amount of converted reducing sugar ?RZ_MIN is given as the difference between the total mass m Rz_i(ti) of reducing sugar added up to the time ti and the mass of reducing sugar m Rz z(ti) present in the fermentation product at the time ti (determined by an enzymatic-photometric measuring method as described in the description), based on the total volume Vpp(ti) of the fermentation product (FP) present in the fermentation reactor at the time ti, wherein the specific minimum amount of reducing sugar converted has a predetermined value in the range from 20 g / L to 300 g / L, and wherein the mass of reducing sugar m Rz_z(t) present in the fermentation product is measured continuously or at intervals of 5 min to 240 min. For the purposes of the present invention, the determination of the value m Rz_2(t) (and thus the verification of whether ?RZ_MIN is reached or not) is carried out enzymatically photometrically, as described in more detail below.

[0033] In a third embodiment of the process according to the invention, which is a particular embodiment of the second embodiment, the predetermined value for the specific minimum amount of reducing sugar converted is in the range from 40 g / L to 200 g / L.

[0034] In a fourth embodiment of the method according to the invention, which is a special embodiment of the second and third embodiments, n = 2 or 3.

[0035] In a fifth embodiment of the process according to the invention, which is a particular embodiment of the second, third and fourth embodiments, the aminobenzoic acid obtained in each of the n steps (C) is combined to form an aminobenzoic acid product fraction, wherein the aminobenzoic acid can be purified by washing and / or recrystallization before or after the combination.

[0036] In a sixth embodiment of the process according to the invention, which can be combined with all other embodiments except those limited to a batchwise procedure, steps (A) to (C) are carried out continuously.

[0037] In a seventh embodiment of the process according to the invention, which can be combined with all other embodiments except those which necessarily provide for the serial operation of several fermentation reactors, the mother liquor obtained in step (C) is fed back to the same fermentation reactor from which the fermentation product originates, by the treatment of which in steps (B) and (C) said mother liquor was obtained (it is also possible for several fermentation reactors connected in parallel, in each of which fermentations are carried out).

[0038] In an eighth embodiment of the method according to the invention, which is compatible with all other embodiments except those which involve serial operation of several

[0039] Fermentation reactors can be combined, several, in particular 2 to 6, fermentation reactors are operated in series, the mother liquor obtained in step (C) being fed to a fermentation reactor which is arranged in the flow direction after the one from which the fermentation product originates, by the treatment of which in steps (B) and (C) said mother liquor was obtained (it is also possible for several parallel strands of fermentation reactors connected in series, in each of which fermentations are carried out continuously).

[0040] In a ninth embodiment of the process according to the invention, which can be combined with all other embodiments, the nitrogen-containing compound is selected from ammonia gas, ammonia water, (at least) one ammonium salt, soy protein, urea or a mixture of two or more of the aforementioned nitrogen-containing compounds.

[0041] In a tenth embodiment of the process according to the invention, which can be combined with all other embodiments, the reducing sugar is selected from the group of monosaccharides (aldoses or ketoses), disaccharides, oligosaccharides (oligomers of three to ten linked monosaccharides) or mixtures of two or more of the aforementioned saccharides.

[0042] In an eleventh embodiment of the process according to the invention, which can be combined with all other embodiments, the reducing sugar is selected from glucose, fructose, xylose, arabinose, mannose, galactose, ribose, maltose, lactose or mixtures of two or more of the aforementioned reducing sugars.

[0043] In a twelfth embodiment of the process according to the invention, which can be combined with all other embodiments, the reducing sugar is a component of a fermentable carbon-containing compound selected from starch hydrolysate, sugar cane juice, sugar beet juice, hydrolysates from lignocellulose-containing raw materials or a mixture of two or more of the aforementioned carbon-containing compounds.

[0044] In a thirteenth embodiment of the method according to the invention, which can be combined with all other embodiments, the microorganisms are selected from Escherichia coli, Pseudomonas putida, Corynebacterium glutamicum, Bacillus coagulans or a mixture of two or more of the aforementioned microorganisms.

[0045] In a fourteenth embodiment of the process according to the invention, which can be combined with all other embodiments except those limited to the preparation of meta- or para-aminobenzoic acid, ortho-aminobenzoic acid is prepared.

[0046] In a fifteenth embodiment of the process according to the invention, which can be combined with all other embodiments except those limited to the preparation of ortho- or meta-aminobenzoic acid, para-aminobenzoic acid is prepared.

[0047] In a sixteenth embodiment of the process according to the invention, which is a particular embodiment of the fourteenth embodiment, the ortho-aminobenzoic acid is converted to a poly(anthranilamide). In a seventeenth embodiment of the process according to the invention, which is a further particular embodiment of the fourteenth embodiment, the ortho-aminobenzoic acid is converted to an anthranilic acid derivative selected from an anthranilic acid halide, isatoic anhydride, or a mixture thereof, and the anthranilic acid derivative is converted to a polyamine with a polyol.

[0048] In an eighteenth embodiment of the process according to the invention, which is a particular embodiment of the seventeenth embodiment, the polyamine is phosgenated to a polyisocyanate.

[0049] In a nineteenth embodiment of the process according to the invention, which is a particular embodiment of the fourteenth and fifteenth embodiments, preferably the fourteenth embodiment, the ortho- or para-aminobenzoic acid is converted to aniline with elimination of carbon dioxide.

[0050] In a twentieth embodiment of the process according to the invention, which is a particular embodiment of the nineteenth embodiment, the aniline is reacted with formaldehyde to form methylenediphenylenediamine and polymethylenepolyphenylenepolyamine.

[0051] In a twenty-first embodiment of the process according to the invention, which is a particular embodiment of the twentieth embodiment, the methylenediphenylenediamine and / or the polymethylenepolyphenylenepolyamine is phosgenated to methylenediphenylene diisocyanate and / or polymethylenepolyphenylene polyisocyanate.

[0052] In a twenty-second embodiment of the process according to the invention, which is a particular embodiment of the twenty-first embodiment, the methylenediphenylene diisocyanate and / or the polymethylenepolyphenylene polyisocyanate is reacted with a polyol to form a polyurethane.

[0053] In a twenty-third embodiment of the process according to the invention, which is a further particular embodiment of the nineteenth embodiment, the aniline is converted to an azo compound.

[0054] In a twenty-fourth embodiment of the process according to the invention, which can be combined with all other embodiments, the inorganic base is selected from sodium hydroxide solution, potassium hydroxide solution, aqueous ammonia, gaseous ammonia or a mixture of two or more of the aforementioned inorganic bases.

[0055] In a twenty-fifth embodiment of the process according to the invention, which can be combined with all other embodiments, the protonic acid is selected from hydrochloric acid, sulfuric acid, phosphoric acid or a mixture of two or more of the aforementioned protonic acids.

[0056] In a twenty-sixth embodiment of the process according to the invention, which can be combined with all other embodiments, the mother liquor obtained in step (C) is fed into step (A) until the fermentation product containing aminobenzoate anions reaches or exceeds a predetermined osmolality limit for the first time, wherein the osmolality limit is in the range from 1.0 osmol / kg to 3.0 osmol / kg, preferably in the range from 1.5 osmol / kg to 2.5 osmol / kg, and most preferably in the range from 2.0 osmol / kg to 2.2 osmol / kg. The measurement of osmolality is described further below.

[0057] In a twenty-seventh embodiment of the process according to the invention, which can be combined with all other embodiments, the mother liquor obtained in step (C) is adjusted to a pH in the range from 6.0 to 8.5 by adding an inorganic base before being fed into step (A), wherein the mother liquor obtained in step (C) is preferably adjusted to a pH which deviates by a maximum of 0.2, preferably by a maximum of 0.1, from the pH maintained in the fermentation and in particular corresponds to the pH maintained in the fermentation.

[0058] The embodiments briefly described above and other possible configurations of the invention are explained in more detail below. All of the embodiments described above and the other configurations of the invention described below can be combined with one another as desired, unless the context clearly indicates otherwise to a person skilled in the art or unless expressly stated otherwise.

[0059] Fermentation to a fermentation product containing aminobenzoate anions and its clarification (steps (A) and (B))

[0060] Step (A) of the process comprises at least the fermentation of a raw material containing a reducing sugar and a nitrogen-containing compound in the presence of microorganisms to form a fermentation product containing aminobenzoate anions, wherein the pH during the fermentation is maintained in the range of 6.0 to 8.5 by adding an inorganic base. The fermentation is carried out in a dedicated reaction apparatus, the fermentation reactor. The reaction mixture present in the fermentation reactor is also referred to as the fermentation broth. The fermentation in step (A) is carried out such that the pH in the fermentation broth is in the range of 6.0 to 8.5, preferably in the range of 6.6 to 8.0. If necessary, the pH can be regulated by adding a base, in particular by adding aqueous or gaseous ammonia, aqueous potassium hydroxide, or aqueous sodium hydroxide.At these pH values, the aminobenzoic acid is present predominantly to entirely in the form of its anion (the aminobenzoate anion, H2NC6H4COO-). Preferred microorganisms for carrying out step (A) are prokaryotes (such as, in particular, bacteria). Suitable microorganisms include, for example, Escherichia coli, Pseudomonas putida, Corynebacterium glutamicum, and Bacillus coagulans. Escherichia coli, Pseudomonas putida, and / or Corynebacterium glutamicum are preferably used. The use of mixtures of different microorganisms is possible, but preference is given to the use of microorganisms of a single species. The sole use of Corynebacterium glutamicum, in particular Corynebacterium glutamicum ATCC 13032, is particularly preferred.In this connection, particular reference is made to the patent applications WO 2015 / 124686 A1 and WO 2015 / 124687 A1, in which fermentation processes using bacteria are described (see, for example, WO 2015 / 124687 A1, page 15, line 8 to page 16, line 30, Example 1 (page 29, lines 4 to 26), Example 3 (especially page 34, lines 10 to 18) and Example 4 (especially page 55, lines 9 to 31). In particular, bacteria are used which are able to convert a reducing sugar, which can be used either in pure form or as a component of a fermentable carbon-containing compound, into aminobenzoate anions in the presence of a suitable nitrogen source, without these being immediately consumed again in cellular biochemical processes, so that aminobenzoate anions accumulate in the cell and are finally released into the Transfer to fermentation broth.

[0061] There are basically two ways to obtain such prokaryotes, which can also be combined in a preferred embodiment:

[0062] (i) The enzymatic reactions in the aminobenzoic acid pathway of the prokaryotic cell can be increased so that aminobenzoic acid is produced faster than it is consumed.

[0063] (ii) The subsequent reactions by which aminobenzoic acid is converted into further metabolites or products (e.g. tryptophan) can be reduced or eliminated, resulting in an accumulation of aminobenzoic acid in the cell.

[0064] Methods for obtaining prokaryotic organisms with the aforementioned properties are known in the art. Suitable prokaryotes can be identified, for example, by screening for mutants that release aminobenzoic acid into the surrounding medium. However, the targeted modification of key enzymes using genetic engineering techniques is preferred. Gene expression and enzyme activity can be enhanced, reduced, or even completely inhibited using conventional genetic engineering methods. This results in recombinant strains. A preferred embodiment for the particularly preferred ortho isomer is described below (for the para isomer, see, for example, [1]):

[0065] Particularly preferably, prokaryotes capable of converting a reducing sugar to aminobenzoic acid in the presence of a nitrogen-containing compound contain a modification of the anthranilate phosphoribosyltransferase activity, which reduces said enzyme activity. This modification reduces or completely prevents the conversion of ortho-aminobenzoate to N-(5-phospho-D-ribosyl)-anthranilate. This results in an accumulation of aminobenzoic acid in the cell. The term "anthranilate phosphoribosyltransferase activity" refers to an enzyme activity that catalyzes the conversion of ortho-aminobenzoate to N-(5-phospho-D-ribosyl)-anthranilate.

[0066] In the bacterium Corynebacterium glutamicum, the anthranilate phosphoribosyltransferase activity is encoded by the trpD gene (cg3361, Cgl3032, NCgl2929). In Pseudomonas putida, encoding occurs via the trpD gene (PP_0421) within the trpDC operon.

[0067] The described reduction of anthranilate phosphoribosyltransferase activity can in principle be achieved in three ways:

[0068] (i) The regulation of expression of the gene for anthranilate phosphoribosyltransferase activity can be modified so as to reduce or abolish transcription of the gene or subsequent translation.

[0069] (ii) The nucleic acid sequence of the gene for anthranilate phosphoribosyltransferase activity can be modified so that the enzyme encoded by the modified gene has a lower specific activity.

[0070] (iii) The native gene for anthranilate phosphoribosyltransferase activity can be replaced by another gene derived from a different organism and encode an enzyme with a specific anthranilate phosphoribosyltransferase activity lower than that of the previously mentioned native genes (e.g., TRP4, trpD or trpDC).

[0071] Aminobenzoic acid occurs in three isomeric forms (ortho-, meta-, and para-aminobenzoic acid). In principle, the process can be applied to all three isomers, either in isomerically pure form or as mixtures of different isomers. However, preference is given to the preparation of ortho-aminobenzoic acid or para-aminobenzoic acid, especially in isomerically pure form. Particular preference is given to the preparation of ortho-aminobenzoic acid, especially in isomerically pure form. "Isomerally pure" in this context means that the molar fraction of the desired aminobenzoic acid isomer, based on all aminobenzoic acid isomers present, is at least 99.0 mol%, preferably at least 99.9 mol%, particularly preferably 100 mol%. As is known in the art, the formation of the desired isomer can be controlled enzymatically. For example, in the shikimate pathway, chorismate can be enzymatically converted to anthranilate (= anion of ortho-aminobenzoic acid).Alternatively, there are also enzyme-catalyzed reactions from chorismate to para-aminobenzoate (= anion of para-aminobenzoic acid). Regardless of which microorganism is used and which isomer is desired, the fermentation broth at the beginning of the fermentation in step (A) comprises recombinant cells of the microorganism used, the reducible sugar (and optionally other carbon sources suitable for a fermentation process), and at least one nitrogen-containing compound as a nitrogen source. Particularly suitable reducing sugars are those from the group of monosaccharides (aldoses or ketoses), disaccharides, oligosaccharides (oligomers of three to ten linked monosaccharides), or mixtures of two or more of the aforementioned saccharides. Glucose, fructose, xylose, arabinose, mannose, galactose, ribose, maltose, lactose, or mixtures of two or more of the aforementioned reducing sugars.The preferred nitrogen source is ammonia gas, ammonia water, (at least) one ammonium salt, soy protein and / or urea.

[0072] Preferably, the fermentation broth also contains additional components selected from the group consisting of buffer systems, inorganic nutrients, amino acids, vitamins, and other organic compounds required for the growth or maintenance metabolism of the recombinant cells. The fermentation broth is water-based. After the fermentation process has begun, the fermentation broth also contains aminobenzoate anions, the desired target product of the fermentation.

[0073] As already mentioned, the reducing sugar can be used as such or as a component of a fermentable carbon-containing compound. A fermentable carbon-containing compound within the meaning of the present invention means a mixture of organic compounds that contains at least one reducing sugar and can be used by the recombinant cells of the microorganism used to produce aminobenzoate anions. The production of the aminobenzoate anions can take place in the presence or absence of oxygen. Preference is given to fermentable carbon-containing compounds that can also serve as an energy and carbon source for the growth of the recombinant cells of the microorganism used. Suitable examples include starch hydrolysate, sugar cane juice, sugar beet juice, and / or hydrolysates from lignocellulose-containing raw materials.

[0074] In one embodiment, step (A) is carried out continuously, i.e. the reactants are continuously fed to the fermentation reactor and the fermentation product is continuously removed from the fermentation reactor. In the simplest case, the fermentation product continuously removed from the fermentation reactor is the aqueous fermentation broth containing aminobenzoate anions, including the microorganisms present therein. The separation of the microorganisms from the fermentation product (step (B)) can take place outside the fermentation reactor using known separation techniques, such as, in particular, filtration, centrifugation or sedimentation. However, it is also conceivable to retain the microorganisms in the fermentation reactor by using known separation processes (in particular filtration) and to remove the already clarified fermentation product from it. In this embodiment, step (B) is therefore carried out in the fermentation reactor itself.

[0075] In another embodiment, step (A) is carried out in a discontinuous process (batch mode) in fermentation cycles. A fermentation cycle preferably comprises the initial introduction or addition of microorganisms into a nutrient medium, the introduction and / or addition of nutrients, the build-up of microorganisms, the formation of the desired product, i.e., the aminobenzoate anions, and the complete or partial emptying of the reactor after completion of the fermentation. In a variant of the discontinuous process (so-called "fed-batch mode"), the reactants are fed to the fermentation reactor (continuously or discontinuously [i.e., in portions]) for as long as the reactor volume allows, without products - possibly with the exception of gaseous components, which are discharged via a connection of the fermentation reactor to an exhaust system - being removed from the fermentation reactor.The reaction is stopped after the maximum possible amount of reactants has been added, and the product mixture is removed from the fermentation reactor. In batch processes, clarification of the fermentation product, particularly by filtration, centrifugation, or sedimentation, is preferred outside the fermentation reactor.

[0076] Separated microorganisms (biomass) can be returned to the fermentation, if necessary minus a discharged portion (4' in the figures).

[0077] In the case of a batch process, it is preferable to allow the fermentation in step (A) not to simply continue until no more aminobenzoate anions can be produced, but only until a previously determined specific minimum amount of reducing sugar ?RZ_MIN has been converted. The time at which the specific minimum amount of reducing sugar ?RZ_MIN has been converted for the first time is referred to as ti. In the context of the present invention, the specific minimum amount of converted reducing sugar ?RZ_MIN is given as the difference between the total mass m Rz i(ti) of reducing sugar added up to time ti and the mass of reducing sugar m Rz_2(ti) present in the fermentation product at time ti, based on the total volume Vpp(ti) of the fermentation product present in the fermentation reactor at time ti:

[0078] Depending on the conditions of the individual case, a value in the range from 20 g / L to 300 g / L, preferably from 40 g / L to 200 g / L, is set for the quantity ?RZ_MIN. In this embodiment, the mass of reducing sugar m Rz_2(t) present in the fermentation product at a specific time point is measured continuously or at intervals of 5 min to 240 min. When measuring at intervals, the time point at which the specific minimum amount of converted reducing sugar ?RZ_MIN was first determined to have been reached is considered ti. Therefore, if ?RZ_MIN is reached between two measurements, the time point of the later measurement is considered ti.For the purposes of the present invention, the value m Rz_2(t) (and thus the verification of whether ?RZ_MIN has been reached or not) is determined enzymatically and photometrically using analytical instruments known in the art for the determination of substrates, metabolites, and products in aqueous solution of cell culture and fermentation media. Cedex® Bio Analyzers are particularly suitable for low sample throughput, while Cedex BioHT® instruments are suitable for high sample throughput. The quantity m Rz i(ti) is known, and Vpp(ti) can be determined by continuously weighing the reactor and, using the resulting mass increase in combination with the density of the fermentation product at the time of sampling. Another option for determining the volume Vpp(ti) is to balance the volume difference between the liquids added and removed during the fermentation.

[0079] Regardless of the precise operating mode, the fermentation reactor preferably includes devices for measuring important process parameters such as temperature, pH, substrate and product concentration, dissolved oxygen content, and cell density of the fermentation broth. Particularly preferably, the fermentation reactor includes devices for adjusting at least one (preferably all) of the aforementioned process parameters.

[0080] Suitable fermentation reactors include stirred tank reactors, membrane reactors, or loop reactors. Stirred tank reactors and loop reactors are particularly preferred for both aerobic and anaerobic fermentations (preferably airlift reactors, in which the circulation of the liquid in the reactor is achieved by gassing).

[0081] In addition to clarification (step (B)), the fermentation product from step (A) can be subjected to further pretreatment steps before being fed to step (C). Of particular note here is decolorization of the fermentation broth. Such decolorization is preferably carried out by passing the clarified fermentation product through a column with a fixed packing to remove colorants by adsorption. Diatomaceous earth or an ion exchange packing, for example, can be used as a possible solid phase. Such decolorization is preferably carried out when the fermentation broth contains colored substances that could interfere with the subsequent crystallization in step (C).

[0082] Crystallization and isolation of aminobenzoic acid from the clarified fermentation product (step (C))

[0083] In step (C) of the process, the aminobenzoic acid is precipitated (crystallized) from the clarified fermentation product. This step is carried out in a technical apparatus suitable for crystallization, known in the art as a crystallizer. Suitable crystallizers include, for example, stirred tanks or forced-circulation crystallizers such as those of the "Oslo" type. In the crystallizer, the pH is adjusted to a range of 3.0 to 4.7, preferably 3.2 to 3.7, particularly preferably 3.4 to 3.6, and most preferably 3.5. This is done by adding a protic acid, such as hydrochloric acid, sulfuric acid, and / or phosphoric acid. This pH adjustment converts the aminobenzoic acid anions (H2NC6H4COO) predominantly to completely into the electroneutral form (H2NC6H4COOH or H3N6H4COO) and causes them to crystallize out. This type of crystallization is also referred to as reactive crystallization.The crystallized aminobenzoic acid is then separated; this can be done by known methods such as filtration, sedimentation or centrifugation, leaving a mother liquor.

[0084] Depending on the concentrations present, it may be useful to separate off some of the water present to facilitate the isolation of the aminobenzoic acid in step (C) and / or to prevent the volume of mother liquor fed into the fermentation from becoming too large. Such a step can be carried out before and / or after, preferably at least before, the crystallization in step (C). This separation of water can be achieved by evaporation or by means of a membrane process. All evaporation apparatus known in the art are suitable for evaporation. To minimize thermal stress, it is preferable to carry out the evaporation at reduced pressure, in particular at 0.1 mbar to 900 mbar, particularly preferably at 100 mbar to 500 mbar. This enables gentle evaporation of water at temperatures from 45 °C to 97 °C, in particular up to 82 °C.

[0085] It has proven useful to feed the fermentation product and the protonic acid to the crystallizer via feed devices that are spatially (as far as possible) separated from one another. This ensures that the reactants are mixed as thoroughly as possible with the reactor contents before the acid-base reaction occurs. Suitable feed devices include, for example, pipelines, preferably with shut-off valves. In one embodiment, the feed device for the fermentation product and the feed device for the protonic acid are arranged at opposite points on the reactor wall (essentially) at right angles to the latter. In another embodiment, the feed device for the fermentation product and the feed device for the acid are arranged (essentially) parallel to the reactor wall, with the feed devices being opposite one another and located as close as possible, in particular directly, to the reactor wall.

[0086] It is possible to divide the crystallizer into chambers using suitable internals. The flow direction can be adjusted by selecting the stirrer geometry and operating mode. It is also possible to equip the crystallizer with an external pumping circuit, in which case one of the two reactants—fermentation broth or protonic acid—is fed into the pumping circuit and the other directly into the crystallizer. If a crystallizer is operated with a classifier and a pumping circuit, the pumping circuit is installed at the bottom of the classifier for fluidization or at the side of the classifier.

[0087] Crystallization in the crystallizer can be carried out continuously or batchwise. Continuous operation is preferred. Regardless of the operating mode (continuous or batchwise), the exact operating parameters are determined (among other things) by the desired crystal size, which can be adjusted by the residence time / reaction time and the degree of supersaturation (large crystal sizes are favored by long residence times / long reaction times and low degrees of supersaturation).

[0088] Crystallization is preferably carried out in the presence of seed crystals:

[0089] In a batch crystallization, the clarified fermentation product is preferably first introduced into the crystallizer and heated to a defined temperature (preferably 5 °C to 40 °C, for example, 20 °C). If the pH of the fermentation broth at the selected temperature is significantly above the pH at which the minimum solubility of aminobenzoic acid is reached, the clarified fermentation product is initially only slightly acidified to a pH at which the minimum solubility of aminobenzoic acid is not yet reached at the selected temperature and under the given boundary conditions, but which is significantly closer to this pH (preferably, acidification to pH 5.0 to 6.5 is achieved in this first step). This slight acidification can be carried out quickly.Seed crystals of the desired polymorph of aminobenzoic acid are then added (in the case of anthranilic acid, this is preferably polymorph (form) I). This polymorph has comparatively low solubility and therefore promotes the most complete recovery of the aminobenzoic acid possible. The amount of seed crystals added is preferably about 0.1% to 1% of the aminobenzoic acid dissolved in the fermentation broth. In this way, a suspension of seed crystals is obtained (see also WO 2017 / 085170 A1). The pH is then adjusted to 3.0 to 4.7, preferably 3.2 to 3.7, particularly preferably 3.4 to 3.6, and most preferably 3.5 by adding further protonic acid. The protonic acid is preferably added slowly; for example, with 1 kg of clarified fermentation product initially introduced and using 30% hydrochloric acid, the pH is adjusted over 2 hours.After the acid addition is complete, stirring is continued for a certain period, in particular for the same period of time that the acid addition took after the seed crystals were added. The precipitated aminobenzoic acid is then separated, in particular by filtration (if necessary under vacuum), sedimentation, or centrifugation (preferably by centrifugation), and preferably washed repeatedly (in particular twice) with an aqueous, in particular acidic (preferably acidified with the same protic acid that was used for crystallization) washing liquid with a pH of 3.0 to 4.7, preferably 3.2 to 3.7, particularly preferably 3.4 to 3.6, and most particularly preferably 3.5. Depending on the purity requirements of the intended subsequent application, the aminobenzoic acid can also be purified by recrystallization.In continuous crystallization, seed crystals generally only need to be added specifically during the start-up of the continuous process, since additional seed crystals later form spontaneously in situ (so-called secondary nucleation) or are available in sufficient quantities. The suspension of seed crystals required for start-up can be prepared as previously described for discontinuous crystallization. The processing (separation and washing of the crystallized aminobenzoic acid) can also be carried out as previously described.

[0090] Feeding the mother liquor into the fermentation

[0091] The mother liquor obtained in step (C) is now fed to step (A).

[0092] In a batch process, a further fermentation is initiated by feeding the mother liquor obtained in step (C) into step (A), followed by further steps (B) and (C). Due to the salt content of the mother liquor, this process cannot be carried out indefinitely, so that the total number of fermentations that can be carried out using the inventive procedure is limited in practice to a value n, which, based on current knowledge, can be up to 6 depending on the specific conditions of the individual case. However, possible yield losses of aminobenzoic acid via the mother liquor can only occur in the nth implementation of steps (A) to (C) using the inventive procedure (apart from a possibly required purge; see below for details). The aminobenzoic acid obtained in each of the n steps (C) is preferably combined to form an aminobenzoic acid product fraction.In many cases, n will be 2 or 3. Further fermentation with addition of the mother liquor can be carried out in the same fermentation reactor or in a different fermentation reactor. In total, steps (A) to (C) are therefore only carried out n times before the entire process has to be started again. The number n corresponds to the total number of fermentations, i.e. including the first fermentation for which no mother liquor is present. A suitable measure for determining a reasonable value for n is the so-called osmolality (the molarity of osmotically active particles in a solution), as explained in more detail below. The effects of osmolality on Corynebacterium glutamicum are described by Cristian A. Varela et al.in Applied and Environmental Microbiology 2004, 70 (7), 4222 - 4229 ("Osmotic Stress Response: Quantification of Cell Maintenance and Metabolic Fluxes in a Lysine-Overproducing Strain of Corynebacterium glutamicum") [2] beschrieben.

[0093] In a continuous process, one cannot, of course, speak of individual (discrete) fermentations and therefore cannot specify a value n as in a discontinuous process. However, here too, osmolality is a suitable criterion for how long the process can be carried out overall. In fermentation reactors connected in series as shown in FIG. 2, osmolality is a suitable tool for determining a reasonable number m of fermentation reactors - for example, in preliminary tests. In this embodiment, the number m of fermentation reactors is somewhat comparable to the number n of fermentations in a discontinuous process. The aminobenzoic acid obtained in each of the m fermentation reactors operated continuously in series is preferably combined to form an aminobenzoic acid product fraction.

[0094] Regardless of whether the process is carried out continuously or batchwise, it is therefore preferable not to feed the mother liquor obtained in step (C) into a fermentation step if the osmolality of the fermentation product obtained in the corresponding step (A) exceeds a certain, predetermined limit. The technical background for this is as follows:

[0095] The salts of aminobenzoic acid formed during fermentation (e.g. sodium, potassium or ammonium aminobenzoate) become toxic to the microorganisms with increasing concentration, so that for this reason alone the maximum product concentration that can be achieved in fermentation is limited. Furthermore, everything indicates that the use of reducing sugars during fermentation leads to a reaction between the aldehyde group of the reducing sugar and the amino group of the aminobenzoate anions, forming a glycosylamine. The fact that the fermentation product becomes toxic to the microorganisms with increasing concentration and that the use of reducing sugars leads to the formation of a glycosylamine justifies the advantage of the inventive recycling of mother liquor into the fermentation.On the one hand, this mother liquor contains the (not inconsiderable) proportion of reducing sugar released in step (C), which can be used again as a nutrient source for the microorganisms when the mother liquor is returned. On the other hand, the aminobenzoic acid remaining after crystallization is returned with the mother liquor, thus reducing the effort required to extract the aminobenzoic acid from the mother liquor.

[0096] In addition to the sugar released due to the low pH values ​​and the dissolved aminobenzoic acid, the mother liquor naturally also contains the salts of the protonic acid used (e.g., sodium, potassium, or ammonium chloride when hydrochloric acid is used) formed during crystallization, which are also not tolerated by the microorganisms at any concentration. However, all indications indicate that, at the same osmolality, the salts of protonic acid are less toxic to microorganisms than the salts of aminobenzoic acid. This makes it possible to determine the end of a fermentation not on the maximum tolerable product concentration, but on the maximum salt concentration of the protonic acid. The osmolality limit for the salt of protonic acid that can still be tolerated by the microorganisms is significantly higher than the osmolality limit for the salt of aminobenzoic acid.

[0097] This osmolality limit for protonic acid salts lies in the range of 1.0 osmol / kg to 3.0 osmol / kg, preferably in the range of 1.5 osmol / kg to 2.5 osmol / kg, and most preferably in the range of 2.0 osmol / kg to 2.2 osmol / kg. Osmolality can be determined using commercially available analyzers such as the Osmomat 3000 from Gonotec. The measurement principle is based on determining the freezing point of the sample compared to the freezing point of pure water. For example, a solution with a salt concentration of 1 osmol / kg has a freezing point of -1.858 °C, while water has a freezing point of 0 °C. By comparing the freezing points of the sample and water, the osmolality value of the sample can be determined. The unit "osmol / kg" refers to the ratio of the amount of osmotically active particles to the mass of the solvent.

[0098] The aim is to feed as much of the mother liquor as possible into a fermentation step in order to maximize the advantages of the invention. However, it may be necessary to exclude a smaller portion of the mother liquor from fermentation in order to avoid an accumulation of impurities in the process (so-called purge of a portion of the mother liquor for the purpose of removing impurities; 8' in FIG. 1, not shown in FIG. 2). Such a purge can be carried out periodically or at intervals and, if carried out, preferably comprises 0.5% to 45.0%, particularly preferably 1.0% to 25%, most preferably 1.5% to 5.0% of the amount of mother liquor obtained in step (C). The remainder, i.e. preferably 55.0% to 99.5%, particularly preferably 75.0% to 99.0%, very particularly preferably 95.0% to 98.5%, of the mother liquor obtained in step (C) is then passed into a fermentation according to step (A).The discharged portion of mother liquor is depleted of aminobenzoic acid and fed to a wastewater treatment plant. The depletion of aminobenzoic acid can be carried out by processes known per se in the prior art. Examples include extraction with an organic solvent and adsorption on an adsorbent followed by desorption. The organic solvent used is preferably an alkanol having 8 to 12, preferably 9 to 11, carbon atoms, in particular 1-decanol. The extraction is preferably carried out as described in WO 2023 / 117756 A1 on page 19, line 15 to line 33. The dissolved aminobenzoic acid can be obtained by evaporating the extractant or by back-extraction as described in WO 2023 / 117756 A1 on page 20, line 1 to page 21, line 8.Activated carbon or a polymeric adsorbent, particularly based on polystyrene and / or polydivinylbenzene, is preferred as the adsorbent. Typical pore sizes range from 1.5 to 65 nm, e.g., 4.5 to 10 nm. Adsorbent types with micro- and macropores are also available. Commercially available products are available under the brand names Lewatit (e.g., OC 1064 MD PH or AF 5), Macronet (e.g., MN 270, MN 202, MN 100, or MN 102), PuroSorb (e.g., PAD600), AmberSorb (e.g., L493 or 560), and Amberlite (e.g., XAD4). These (and other adsorbents such as activated carbon) are suitable for both acidic (as described in WO 2018 / 114841 Al) and basic (as described in WO 2015 / 124687 Al) desorption. If desorption is performed in acidic conditions, it is recommended to perform an additional regeneration of the adsorbent with base from time to time (e.g., after five acidic desorptions).In this way, the adsorber bed is freed of organic contaminants. Regardless of the type of desorption, the desorbate is preferably fed into the crystallization process from step (C). The methods described above are also suitable for treating the mother liquor from the fermentation product of the last fermentation reactor in series-operated fermentation reactors (FIG. 2), provided it is not recycled to the first fermentation reactor.

[0099] Regardless of its size, the portion of the mother liquor obtained in step (C) fed into the fermentation according to step (A) is preferably previously mixed with nutrients such as the aforementioned carbon and nitrogen sources, or salts such as potassium phosphate, calcium chloride, or trace elements such as iron sulfate, manganese sulfate, copper sulfate, zinc sulfate, or nickel chloride. In addition, a foam-reducing agent such as polypropylene glycol can be added during the fermentation.

[0100] As already mentioned, it is preferable to increase the pH of the mother liquor before introducing it into the fermentation reactor by adding a base (e.g., by adding aqueous or gaseous ammonia, aqueous potassium hydroxide, or aqueous sodium hydroxide) to a pH in the range of 6.0 to 8.5, preferably to a pH that deviates by a maximum of 0.2, particularly preferably by a maximum of 0.1, from the pH maintained during fermentation and, in particular, corresponds to this pH. Alternatively, the mother liquor can also be added to the fermentation reactor without pH adjustment; however, in this procedure, the addition of the mother liquor should be sufficiently slow so that the pH control in step (A) can compensate for the unavoidable initial pH reduction at the point of addition of the mother liquor quickly enough.

[0101] USE OF AMINOBENZOIC ACID IN THE PRODUCTION OF OTHER VALUED PRODUCTS

[0102] The aminobenzoic acid obtained in step (C) is suitable, optionally after further purification by known methods (e.g. recrystallization), for all applications of aminobenzoic acid known in the prior art.

[0103] For example, ortho-aminobenzoic acid (anthranilic acid) plays an important role as a starting material for the synthesis of anthranilic acid esters, which are important fragrances, and indigo, as well as for pharmaceuticals and pesticides (acaricides). Anthranilic acid produced according to the invention is suitable for all of these purposes.

[0104] The use of aminobenzoic acid prepared according to the invention is preferred for the production of polymeric compounds. Here, the process according to the invention can make a valuable contribution to the more sustainable production of plastics, which are often required in large quantities. For example, anthranilic acid can be converted to a poly(anthranilic amide) as described in WO 2022 / 008449 A1 (after conversion to isatoic anhydride) or WO 2022 / 008450 A1 (after conversion to an anthranilic acid ester). Anthranilic acid obtained according to the invention can also be converted to an anthranilic acid derivative selected from anthranilic acid halide, isatoic anhydride, or a mixture thereof as described in WO 2022 / 122906 A1 and then reacted with a polyol to form a polyamine, which in turn is suitable for phosgenation to the corresponding polyisocyanate.

[0105] Aminobenzoic acid obtained according to the invention can also be decarboxylated to aniline, which is an important raw material, particularly in the polyurethane industry.

[0106] Decarboxylation can be carried out as is generally known in the art. A catalyst may, but is not required.

[0107] Suitable catalysts include, for example, aqueous acids such as sulfuric acid, nitric acid, and hydrochloric acid; solid acids such as zeolites and Si-Ti molecular sieves; solid bases such as hydroxyapatites and hydrotalcites; and polymeric acids such as ion exchange resins (preferably Amberlyst). Particularly preferred catalysts are those described in WO 2022 / 253890 A1. The catalysts disclosed therein are characterized by a high aluminum oxide mass fraction (at least 40%). The aluminum oxide is preferably γA1O3 or γ-Al2O3, especially when no other metal oxides are present besides aluminum oxide. In addition to aluminum oxide, other metal oxides may in principle also be present, in particular magnesium oxide (MgO) in a mass fraction of 1.0% to 60.0%, preferably 2.0% to 50.0%, particularly preferably 5.0% to 35.0%, based on the total mass of the metal oxides.Furthermore, the catalyst may contain SiO? in a mass fraction of 1.0% to 30.0%, preferably 2.0% to 20.0%, particularly preferably 2.0% to 10.0%, based on its total mass.

[0108] As for the reaction conditions, the decarboxylation can be carried out over a wide range of temperature and pressure. A suitable reaction temperature is preferably in the range of 150°C to 300°C, more preferably 160°C to 280°C, and most preferably 180°C to 240°C. The (absolute) reaction pressure can be 0.05 bar to 300 bar, preferably 1.0 bar to 100 bar, and most preferably 1.0 bar to 60 bar.

[0109] The decarboxylation can also be carried out in the presence of aniline, i.e., the aminobenzoic acid is dissolved in aniline. Since aniline exerts a catalytic effect with respect to the decarboxylation of aminobenzoic acid, thus accelerating its own formation (autocatalytic effect), the use of a catalyst external to the system is not absolutely necessary in this variant; see also WO 2020 / 020919 A1, which describes such a process. In contrast to the procedure disclosed in WO 2020 / 020919 A1, purified aniline can also be used as a solvent for the aminobenzoic acid. Furthermore, in contrast to the procedure disclosed in WO 2020 / 020919 A1, external catalysts can also be used.When the reaction is carried out discontinuously, a mass fraction of aniline, based on the total mass of aniline and aminobenzoic acid, of 0.1% to 90%, preferably 1.0% to 70%, particularly preferably 5.0% to 50%, is preferably set before the start of the decarboxylation. When the reaction is carried out continuously, a mass fraction of aniline, based on the total mass of aniline and aminobenzoic acid, of 0.1% to 90%, preferably 1.0% to 70%, particularly preferably 5.0% to 50%, is always set during the decarboxylation.

[0110] In addition to aniline, other solvents or diluents can of course also be used, especially water. Furthermore, organic, polar, or protic solvents are preferably suitable, such as halogenated aliphatic or aromatic hydrocarbons, linear or cyclic ethers, linear or cyclic esters, linear or cyclic amides, alcohols, ketones, nitriles, phenol derivatives, benzanilides, sulfonamides, or sulfolane, which preferably have a boiling point that, under the selected conditions, is higher than the selected reaction temperature and, at this temperature, preferably forms a homogeneous reaction mixture with the reaction components.

[0111] As far as the reaction procedure is concerned, both the gas phase and the liquid phase are suitable. The reaction can be carried out continuously (preferred) or discontinuously.

[0112] Preferred procedures include carrying out the decarboxylation of the aminobenzoic acid

[0113] • in the liquid or gas phase in a reactor, in particular in a tubular reactor, with an integrated fixed bed of the catalyst (including a bed of the catalyst as a shaped body (extrudates) or a design of the catalyst as a monolithic structure),

[0114] • in the liquid or – preferably – gas phase in a fluidized bed reactor or

[0115] • in the liquid phase in a stirred tank containing a suspension (slurry) of the catalyst.

[0116] A tubular reactor is understood here as a tubular reactor through which the reacting reaction mixture flows during operation in a continuous reaction (which is preferred). Tubular reactors with small length-to-diameter ratios are also referred to as tower reactors; these are also encompassed by the underlying understanding of the term "tubular reactor," as are special designs of tubular reactors such as bubble column reactors.

[0117] The use of catalyst moldings (extrudates) or monolithic catalyst structures allows easy reuse of the catalyst after decarboxylation.

[0118] The catalyst remaining after decarboxylation is preferably regenerated before reuse. This can be done by washing the catalyst with organic solvents or aqueous solutions and / or burning it out at elevated temperatures in the presence of O2 to remove organic deposits.

[0119] The resulting aniline can be isolated and purified using standard techniques, especially by distillation, and then used for further applications. In particular, the (acid-catalyzed) reaction with formaldehyde to form methylenediphenylenediamine and polymethylenepolyphenylenepolyamine, the phosgenation of which to

[0120] Methylenediphenylene diisocyanate and / or polymethylenepolyphenylene polyisocyanate and the subsequent reaction with polyols to form polyurethanes are mentioned. Of course, aniline obtained by decarboxylation of aminobenzoic acid prepared according to the invention can also be used for other applications, such as the production of azo compounds. One example is the production of methyl red, which is accessible by diazotization of the amino group of anthranilic acid with sodium nitrite and hydrochloric acid, followed by azo coupling with N,N-dimethylaniline.

[0121] Examples:

[0122] Production master

[0123] All experiments were performed using the microbial strain described below.

[0124] Based on the bacterium Corynebacterium glutamicum ATCC13032, a microbial strain was induced to produce anthranilic acid through targeted chromosomal modifications. All genetic modifications, i.e., chromosomal deletions and gene integration, were performed by double homologous recombination using corresponding pK19mohsacß derivatives (Schäfer et al., 1994: "Small mobilizable multipurpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum." Gene 145(l):69-73. doi: 10.1016 / 0378-1119(94)90324-7) [3].

[0125] The activity of the anthranilate phosphoribosyltransferase TrpD was reduced by first deleting the native trpD-A\\e\ (SEQ ID NO. 1) and replacing it with an allele (called trpD5) with GTG instead of ATG start codon and ribosome binding site with reduced distance to the start codon (SEQ ID NO. 2).

[0126] Furthermore, phosphoenolpyruvate carboxylase was inactivated by largely deleting the corresponding gene as described in (SEQ ID NO. 3).

[0127] To enhance the aromatic biosynthesis pathway, an artificial, polycistronic P tU f- aroLAC operon (SEQ ID NO. 4), consisting of the genes aroL (b0388), encoding a shikimate kinase from Escherichia coli, aroA (cg0873), encoding the 3-phosphoshikimate-l-carboxyvinyltransferase from C. glutamicum, and aroC (cgl829), encoding the chorismate synthase from C. glutamicum, was integrated downstream of cg2563 under the control of the constitutive promoter of the elongation factor Tuf. In addition, the construct P t uf-aroG new (SEQ ID NO. 5), which encodes a feedback-resistant variant of DAHP synthase from E. coli under control of P tU f promoter, downstream of cg3132 into the genome of the strain.

[0128] To conduct the experiments, a clarified fermentation product was first synthetically reproduced. Anthranilic acid was precipitated from this clarified fermentation product by acidification and separated. The remaining mother liquor was subjected to fermentation. Reproducing a clarified fermentation product

[0129] First, the media components listed in Table 1 were mixed.

[0130] Table 1: Media components of the synthetically prepared fermentation broth; data per liter The pH of the mixture prepared according to Table 1 was adjusted to pH 7.0 with potassium hydroxide solution. The volume was then made up to 1.0 L with water. The resulting solution was stirred at room temperature for 60 hours, which corresponds to a typical fermentation time. Crystallization

[0131] The pH of the solution was then adjusted to 3.5 by adding 30% hydrochloric acid, which caused the anthranilic acid to precipitate as a solid. The anthranilic acid crystals were separated by filtration, leaving the mother liquor. The mother liquor (1.8 L) was supplemented by adding the media components listed in Table 2. The pH of the thus supplemented mother liquor was adjusted to pH = 7.0 with potassium hydroxide solution. Table 2: Addition of media components to the 1.80 L of mother liquor obtained in the crystallization.

[0132] Culture medium and preculture

[0133] For pre-cultivation of the C. glutamicum strain, it was first transferred from a cryo-culture into sterile SY medium (10 g / L yeast extract, 16 g / L soy peptone, 5 g / L NaCl, 16 g / L glucose) and incubated in 1 L Erlenmeyer flasks with 50 mL liquid culture at 30 °C and 200 rpm until an OD600 of approximately 30 was reached. 5 mL / flask of this first preculture was transferred into sterile modified CGXII medium (5 g / L yeast extract, 10 g / L (NH^2SC, 1 g / L KH2PO4, 1 g / L K2HPO4, 0.25 g / L MgSO4x 7 H2O, 0.01 g / L CaCl2x 2 H2O, 5 g / L urea, 62 g / L MOPS, 10 mg / L MnSO4x H2O, 10 mg / L FeSO4x 7 H2O, 1 mg / L ZnSO4x 7 H2O, 0.2 mg / L CuSO4 x 5 H2O, 0.02 mg / L NiCl2 x 6 H2O, 2 mg / L biotin, 40 g / L glucose) and incubated under otherwise identical conditions until an OD600 of approx. 40. The final cultivation stage ("seed"), like the main cultivation, was carried out in stirred bioreactors (BioBlock, Eppendorf) under aerobic conditions (dissolved oxygen concentration > 30%).80 mL of the second preculture were dissolved in 520 mL of seed medium (8.33 g / L yeast extract, 5 g / L (NH4)2SO4, 6.67 g / L KH2PO4, 6.67 g / L K2HPO4, 3.33 g / L MgSO4 x 7 H2O, 0.07 g / L CaCl2 x 2 H2O, 4.17 g / L PPG 2000, 167 mg / L MnSO4 x H2O, 167 mg / L FeSO4 x 7 H2O, 16.7 mg / L ZnSO4 x 7 H2O, 3.34 mg / L CuSO4 x 5 H2O, 0.334 mg / L NiCl2 x 6 H2O, 3 mg / L biotin, 50 g / L granulated corn steep liquor, 33 g / L glucose). Seed fermentation was carried out at 30 °C and pH 7.0 with the addition of a glucose-tryptophan mixture (480 g / L glucose, 1.6 g / L tryptophan) and a mixture of NaOH and NH3 solution (1.55 mol / L NaOH, 8 mass% NH3) for pH control.

[0134] Cultivation of the preculture

[0135] During the cultivation, sodium anthranilate was added at two points in time (after approximately 13 h and 16 h) by adding 57 mL of a sterile-filtered stock solution (150 g of anthranilic acid was dissolved in 1.00 L of water with the addition of sodium hydroxide solution, adjusting the pH to 7.0). (This addition of sodium anthranilate was performed because the fermentation broth described above was prepared synthetically; this is not necessary in real processes.) The addition of the sodium anthranilate solution increased the concentration in the preculture reactor by approximately 10 g / L. After 18 hours of fermentation, 85 mL each were transferred from the preculture reactor to a main reactor.

[0136] Main crop

[0137] The main culture experiments were conducted in triplicate in parallel. For this purpose, 320 mL of the supplemented mother liquor was transferred to each of three main reactors (= three parallel fermentation reactors for step (A) of the invention) and inoculated with 85 mL of the preculture containing microorganisms. The main reactors were cultured aerobically at 30 to 33 °C at a pH of approximately 7 (regulated by the addition of sodium hydroxide solution) for 70 hours by adding air (30% dissolved oxygen; regulated by the stirrer speed).

[0138] As can be seen from Figures 3 to 8, the supplemented mother liquor, with its dissolved glucose and anthranilic acid content, could be reused as a fermentation medium. Figures 3 and 8 show the dry microbial biomass during the fermentation as a concentration value in the fermentation broth (Figure 3) and as an absolute amount in the reactor (Figure 8), since concentration values ​​are influenced by the addition of substrate and base. Both figures demonstrate that even when mother liquor is used, microbial growth occurs to an extent that allows for industrially relevant biomass concentrations of approximately 15–20 g / L during the fermentation. This allows the concentration and absolute values ​​of aminobenzoic acid shown in Figures 4 and 7 to be produced by fermentation. Since part of the aminobenzoic acid reacts with the glucose present, the concentration of free, unbound aminobenzoic acid (FIG.5) is lower than the total aminobenzoic acid concentration present (FIG. 4). At the same time, the concentration of bound glucose shown in FIG. 6 is also not accessible for direct metabolism. By recycling the mother liquor, the loss of glucose and anthranilic acid can be reduced accordingly. Furthermore, unused media components can be returned to the fermentation.

Claims

Patent claims:

1. A process comprising the preparation of aminobenzoic acid, the process comprising the following steps: (A) Fermentation of a raw material containing a reducing sugar and a nitrogen-containing compound in the presence of microorganisms in a fermentation reactor to form a fermentation product containing aminobenzoate anions, wherein the pH value during the fermentation is maintained in the range of 6.0 to 8.5 by adding an inorganic base, (B) separating the microorganisms from the fermentation product to obtain a clarified fermentation product and (C) Precipitating aminobenzoic acid from the clarified fermentation product by adjusting the pH to 3.0 to 4.7 by adding a protic acid and separating the precipitated aminobenzoic acid to leave a mother liquor, wherein the mother liquor obtained in step (C) is fed to step (A).

2. Process according to claim 1, in which steps (A) to (C) are carried out discontinuously, wherein a further fermentation is started by feeding the mother liquor obtained in step (C) into step (A), followed by a further implementation of steps (B) and (C), wherein steps (A) to (C) are carried out a total of n times, where n is a natural number in the range from 2 to 6.

3. The method according to claim 2, wherein step (B) is carried out in each case after reaching a time ti, wherein the time ti denotes the time at which a specific minimum amount of reducing sugar / JRZ MIN was converted for the first time, wherein the specific minimum amount of converted reducing sugar / JRZ MIN is given as the difference between the total mass m Rz i(ti) of reducing sugar added up to the time ti and the mass of reducing sugar m Rz z(ti) present in the fermentation product at the time ti (determined by an enzymatic-photometric measuring method as described in the description), based on the total volume Vpp(ti) of the fermentation product present in the fermentation reactor at the time ti, / ?RZ_MIN = [lD RZ_l(tl) — ID RZ_2(tl)] / VFp(ti), where the specific minimum amount of converted reducing sugar has a predetermined value in the range of 20 g / L to 300 g / L and where the The mass of reducing sugar m RZ_2(t) present in the fermentation product is measured continuously or at intervals of 5 min to 240 min.

4. The process according to claim 1, wherein steps (A) to (C) are carried out continuously.

5. A process according to any one of claims 1 to 4, wherein the mother liquor obtained in step (C) is returned to the same fermentation reactor from which the fermentation product originates, by the treatment of which in steps (B) and (C) said mother liquor was obtained, or wherein a plurality of fermentation reactors are operated in series, the mother liquor obtained in step (C) being fed to a fermentation reactor which is arranged downstream in the direction of flow from the one from which the fermentation product originates, by the treatment of which in steps (B) and (C) said mother liquor was obtained.

6. A process according to any one of the preceding claims, wherein the nitrogen-containing compound is selected from ammonia gas, ammonia water, an ammonium salt, soy protein, urea or a mixture of two or more of the aforementioned nitrogen-containing compounds.

7. A process according to any one of the preceding claims, wherein the reducing sugar is selected from the group consisting of monosaccharides, disaccharides, oligosaccharides or mixtures of two or more of the aforementioned saccharides.

8. A process according to any one of the preceding claims, wherein the reducing sugar is selected from glucose, fructose, xylose, arabinose, mannose, galactose, ribose, maltose, lactose or mixtures of two or more of the aforementioned reducing sugars.

9. A process according to any one of the preceding claims, wherein the reducing sugar is a component of a fermentable carbon-containing compound selected from starch hydrolysate, sugar cane juice, sugar beet juice, hydrolysates from lignocellulosic raw materials or a mixture of two or more of the aforementioned carbon-containing compounds.

10. A method according to any one of the preceding claims, wherein the microorganisms are selected from Escherichia coli, Pseudomonas putida, Corynebacterium glutamicum, Bacillus coagulans or a mixture of two or more of the aforementioned microorganisms.

11. A process according to any one of the preceding claims, wherein ortho-aminobenzoic acid or para-aminobenzoic acid is produced.

12. A process according to any one of the preceding claims, wherein the inorganic base is selected from sodium hydroxide solution, potassium hydroxide solution, aqueous ammonia, gaseous ammonia, or a mixture of two or more of the aforementioned inorganic bases.

13. A process according to any one of the preceding claims, wherein the protonic acid is selected from hydrochloric acid, sulfuric acid, phosphoric acid or a mixture of two or more of the aforementioned protonic acids.

14. A process according to any one of the preceding claims, wherein the feeding of the mother liquor obtained in step (C) into step (A) is carried out until the fermentation product containing aminobenzoate anions reaches or exceeds a predetermined osmolality limit for the first time, the osmolality limit being in the range from 1.0 osmol / kg to 3.0 osmol / kg.

15. A process according to any one of the preceding claims, wherein the mother liquor obtained in step (C) is adjusted to a pH in the range from 6.0 to 8.5 by adding an inorganic base before being fed to step (A).

Images