WO1998054351A1

WO1998054351A1 - A combined process for the production of lysine and its salts and of a further weak acid and a salt thereof

Info

Publication number: WO1998054351A1
Application number: PCT/GB1998/001436
Authority: WO
Inventors: Aharon Meir Eyal; Robert Jansen; Pierre Cami
Original assignee: Tate and Lyle Europe NV; WHALLEY KEVIN
Current assignee: Tate and Lyle Europe NV; WHALLEY KEVIN
Priority date: 1997-05-27
Filing date: 1998-05-19
Publication date: 1998-12-03
Anticipated expiration: 1999-11-27
Also published as: IL120923A0; AU7443298A

Abstract

The invention provides a process for the combined production of products selected from the group consisting of lysine and its salts and products selected from the group consisting of at least one weak acid and a salt thereof, which weak acid is selected from the group consisting of organic acids and amino acids produced by a neutral bio-process, the process comprising: (a) acidulating an aqueous feed stream containing the weak acid and ammonium cations with an acidulating acid, which acidulating acid has at least one pKa lower than 4.2, (b) recovering at least part of the weak acid from the aqueous solution formed in step (a), forming thereby an aqueous solution of an ammonium salt comprising ammonium cations resulting from the feed solution in step (a) and anions of the acidulating acid, and (c) fermenting a medium containing at least one carbon source, at least one organic nitrogen source and ammonium salt resulting from step (b), utilizing a lysine producing microorganism, whereby there is formed a fermentation liquor containing at least 50 g/l of lysine values, whereby the fermentation liquor is suitable for use as a source of lysine in animal feed.

Description

A COMBINED PROCESS FOR THE PRODUCTION OF LYSINE AND ITS SALTS AND OF A FURTHER WEAK ACID AND A SALT THEREOF

The present invention relates to a fermentation process for the production of lysine and its salts. More particularly, the present invention relates to a process for the combined production of products selected from the group consisting of lysine and its salts and products selected from the group consisting of at least one weak acid and a salt thereof, which weak acid is selected from the group consisting of organic acids and amino acids produced by a neutral bio-process.

According to common industrial practice, dextrose is used as the main carbohydrate feed for lysine fermentation, with some sucrose from beet molasses. The inorganic nitrogen sources are ammonium sulfate and ammonia, the latter of which is also used for pH regulation. Typically, the fermentation medium also contains some free amino acids and bacteria hydrolysates as organic nitrogen sources. Lysine sulfate is formed in the fermentation liquor and reaches concentrations of 80 to 90 g/1. Typically, the fermentation yield is 0.34-0.36 kg lysine per kg of dextrose.

Lysine hydrochloride is formed out of the lysine sulfate containing broth in the following steps:

(a) acidulating with sulfuric acid;

(b) contacting with a Strong Acid Cation Exchanger (SACE) in its ammonium form (SACE-NH₄), whereby lysine in its (doubly charged) cationic form is absorbed on to the SACE and ammonium sulfate is formed in the effluent;

(c) stripping said adsorbed lysine with ammonia solution, using an excess of ammonia in order to complete the stripping, thereby, reforming the SACE-NH₄ and forming an aqueous lysine solution, which contains the excess of ammonia;

(d) concentration of said lysine solution and stripping the excess of ammonia which is reused in step (c);

(e) acidifying said lysine concentrate with hydrochloric acid;

(f) concentration of said lysine hydrochloride solution;

(g) crystallization of lysine hydrochloride dihydrate from said concentrated solution by cooling and separation of said crystals by.centrifugation; (h) dehydrating the crystals in a fluidized bed dryer to anhydrous lysine __ hydrochloride; and

(i) separating the bacteria cells from the effluent of step (b), which is then evaporated to give crystalline salts and a concentrated organic solution containing organics.

Due to the industrial importance of lysine hydrochloride formation, many patents were issued in the field. Thus, the fermentation and improvements to it are described in JP 50012292 claiming improvements of the fermentation productivity by adding a detergent, or an antioxidant; DD 298272 claiming high performance reactors, topping up high nutrient concentration with nutrient solution; SU 1647032, PT 91207 using a strain selected after mutagenesis of LNBT-20 fermentation strain with N-methyl N-nitro N-nitroso guanidine, which was in turn derived from the ATCC21513 strain. It was selected on the basis of its hyper-sensitivity to monofluoro-acetate and its "ability to produce a greater amount of lysine"; JP 2042995 using of biotin demanding, S-(2-aminoethyl)-L-(-) cysteine resistance Corynebactehum sp. bacterial cells; and JP 57063095 claiming lysine production for Brevibacterium or corynebacterium genus dissociant, which has resistance to vitamin P active compounds.

Many patents refer to the common practice of adsorbing lysine from the fermentation liquor on SACE in its NH₄ form, removal of the effluent and then stripping the lysine with NH₃. Thus, WO 9514002 claims adsorption at a pH< pi on a cation exchanger and elution at pH>pl, wherein pi is the isoelectric point. The improvement being the application of another cation exchanger for the removal of impurities. FR 2666996 claims purification of lysine on SACE-NH₄. The example given is for purification from sugar beet molasses ferment using Diaion SK-LB. According to CS 273122 the process can be applied without the separation of suspend bacterial biomass. Other patents related to this process are: US 4835309 where the eluted lysine was concentrated by reverse osmosis; JP 62065690 where the use of Duolite C-20 in NH₄ form is exemplified; JP 62061592, US 4691054 with an example, where the pH is adjusted to 2 and the solution is passed on diaion SK- 1 B-NH₄ which is then washed with water and eluted with 2N NH₄OH; JP 60256392 the improvement of which is that the acidulated broth is cooled to 10°C -32°C prior to the adsorption which reduces the rate of resin degradation; FR 2562067, using SACE (Duolite C-20)-NH₄, the improvement of which is that the broth is ultra-filtered prior to the acidulation and the effluent is used for the back wash of the resin US 4663048 using membrane treatment for the removal of high molecular weight impurities prior to resin treatment; US 4601829 wherein the lysine and ammonia comprising eluate from the resin process is concentration by reverse osmosis and the ammonia comprising solution, that passes through the membrane, is used as a regenerating agent for the resin or for elution in a subsequent elution; FR 2556980 where the effluent from the lysine adsorption is first passed on a semi-permeable membrane, then concentrated by reverse osmosis and then treated by electrodialysis to separate the organic matter (for animal feed) and the salt (ammonium sulfate for fertilizers); JP 59022608 claiming the pre-treatment of crude fermentation solution with UF membranes and then electrodialysis; and US 3565951.

Several patents and other publications describe preferred equipment and process embodiments: Weiss S. in Ion Exch. Adv., Proc. IEX '92 2266-63 and Rossiter and Tolbert in AICHE Conf. Nov. 1991 describe the use of ISEP contractors for the recovery of lysine from fermentation liquors. Kawakita T. (Ajinomoto), Sep. Sci. Technol. (1991 ), 26(6), 869-83 describes a method for determining optimum conditions of counter-current multi-column adsorption of lysine; US 5279744 claims lysine and glutamic acid recovery by passing the acidified solution through a cation exchange resin counter-currently and elution with NH₃; SU 1664399 and 1650244 claim lysine recovery by passing the acidified solution through a cation exchange resin counter-currently and elution with NH3 while both the solutions and the resin are flowing; and US 4714767 claims an improved contractor operation for lysine adsorption.

Separation and purification of lysine is taught by Kikuchi, Ken-ichi et. al. J. Chem. Eng. Jpn. who separate basic (or acidic) amino acids from neutral ones by electrodialysis (ED), Shaposhnik V. A. et. al. Zh. Prikl. Khim (Leningrad) (1990), 63 (1), 206-9; JP 62195351 contacting amino acid solutions, containing foreign inorganic cations with acid solutions via cation exchange membranes, to exchange the foreign cations with H⁺ , followed by adsorption of the decationized amino acid solution by cation exchange resin; Khleborodova R. T. et. al., Zh. Prikl. Khim_

(Leningrad) 19969), 42(5), 1053-8; SU 891643 which claim purification of technical grade lysine mono hydrochloride, SU 722904 claiming production of lysine hydrochloride for use in dietary food and parental feeding in medical practice by treating with active carbon, ion exchanger resin, crystallization and washing the crystals with ethanol, JP 52001092 teaching purification by electrodialysis at relatively elevated temperatures (47-70^βC ) at which temperature it is claimed that the selectivity in separation of neutral amino acid is improved and the membrane fouling is reduced. FR 2097381 also teaches such a purification.

Resin selection for the lysine process, kinetics fouling and deterioration are also widely dealt with, e.g. by Kawakita T et. al. (Ajinomoto), React. Polym. (1991), 14(2), 169-76, describing the mechanism of deterioration of SACE/NH₄ (diaion sk- 1 b) in cyclic separation for lysine recovery. Capacity loss results from loss of SO₃ groups and from both cleavage of the network structure of the resin by oxidation and fouling, induced mainly by color substances derived from the broth. Regenerants such as Na₂SO₃, NaNO₃ and NaOH are not capable of complete regeneration of the capacity. The optimum number of cycles was determined. Other relevant articles are: Churbanov V., Kim. Farm. Zh. (1989), 23(4), 468071; Yan X. et. al. Weishengwuxue Tongbao (1987), 14(2), 54-8; Churbanov, V.G., Khim. Farm. Zh. (1987) 21 (2), 216-21 ; Churbanov, V.G., Khim. Farm. Zh. (1986), 20(12), 1485- 90, Villan J. et. al., Rev. Scienc. Biol. (Havana), 1984), 15(1 ), 81-93; and Bergk K. et. al, Pharmazie (1975), 30(11), 750-1.

All these processes suffer from a common major problem related to the consumption of sulfuric acid and ammonia and to the formation of ammonium sulfate as an undesired by-product salt. These problems of consumption and formation are illustrated in the following presentation of the current industrial process:

(1) 1/2 (LyH)₂SO₄ + 1/2H₂SO₄ = LyH₂SO₄

(2) LyH₂SO₄ + 2SACE-NH₄ = (SACE-) ₂LyH₂+(NH₄)₂S0₄.

(3) (SACE)₂-LyH₂ + 2NH₃ = 2SACE-NH₄ + Ly

(4) Ly + HCI = LyHCI

(5) 1/2 (LyH)₂SO₄ + 1/2H₂SO₄ + 2NH₃ + HCI = LyHCI + (NH₄)₂S0₄ Per mole of lysine hydrochloride formed, two moles of ammonia and two- equivalents of sulfuric acid are consumed and a mole of ammonium sulfate is formed.

In many industrial processes an acid needs to be recovered from its salt. Examples of particular interest for the present invention are salts of carboxylic acids and those of amino acids, specifically acidic amino acids. In many cases those are formed in a bio-process such as fermentation of a carbohydrate source (e.g. the production of glutamic acid or fumaric acid) or an enzymatic conversion of a salt (as in the case of converting ammonium fumarate to ammonium aspartate). Fermentation media, typically, contain a complex nitrogen source such as molasses, yeast extract, corn steep liquor or cell hydrolysate and other nutrients such as phosphate and some micro-element. In the case of amino acid , production a stoichiometric amount of nitrogen is required, usually inorganic nitrogen such as ammonia. Most of those fermentations are neutral (the micro-organism is not productive at low pH, which means that a base needs to be added to the fermentation medium to neutralize the formed acid). Most of the enzymatic reactions are conducted at about neutral pH, hence their products are typically neutral too.

In those cases where the desired product is the acid form of the bio- process product a combination of purification and conversion (from salt to acid) is required. In many cases an acidulant (acidulating acid) is used to displace the product acid from its salt. Typically the acidulating acid is stronger than the product acid. Therefore protons of the acidulant are bound to the product acid, which is converted to its free acid form and could be separated as such by methods known per se, e.g. crystallization or extraction. In some cases the acidulation is direct, i.e. the acidulant is directly added to the solution of the product salt. In other cases an indirect acidulation is applied. One way for achieving that is using a cation exchanger which is at least partially in its acid form. Contacting the salt containing fermentation liquor with such cation exchanger results in a cation exchange process wherein cations form the solution are adsorbed on the cation exchanger and protons are transferred from the cation exchanger into the solution forming the corresponding acid therein. This acid can then be separated by known methods, e.g. by crystallization, if its solubility in water is limited. The cation exchanger is regenerated by contact with an aqueous solution of the acidulant whereby protons- from the solution are adsorbed on the cation exchanger and the cations adsorbed previously on it transfer into the aqueous solution forming therein a by-product salt.

Some of the indirect acidulations are more complicated as recovery of the product acid is assisted by ion-exchange operations. Yet, the overall reaction is the same. A salt of the required product is introduced and an acidulant is used to yield the product in its acid form and a salt of the acidulant. A particular example of such process is that for the production of monosodium glutamate (MSG).

According to PCT GB97/00177 corresponding to Israel specification 116848, the teachings of which are incorporated herein by reference, there is described and claimed an indirect acidulation process for producing glutamic acid from an aqueous feed containing glutamate resulting from fermentation, comprising the steps of:

(a) contacting said aqueous feed stream at an elevated temperature with a weak acid cation exchanger (WACE) which is at least partially in its acid form whereby part of the cations in the solution is taken up by the cation exchanger and protons are introduced into the solution;

(b) contacting a second aqueous feed containing glutamate and cations at an elevated temperature with a strong acid cation exchanger (SACE) that is obtained from a subsequent step and carries cationic glutamate, whereby the cationic glutamate is transferred into the solution and most of said cations in said second aqueous feed are taken up by the SACE;

(c) crystallizing glutamic acid from the effluent of step (b);

(d) contacting the fermentation liquor of step (c) with SACE which is at least partially in its acid form whereby cationic glutamate is bound;

(e) utilizing the SACE obtained in step (d) in step (b);

(f) regenerating the SACE from step (b) to its at least partially acid form by a solution of a strong acid and utilizing the SACE in its at least partially acid form in step (d) while forming an effluent containing an acidic solution of salts, comprising cations bound to the cation exchanger in step (b) and the anions of the strong acid; (g) regenerating the WACE from step (a) to its at least partially acid forrr by the effluent from step (f) and utilizing the WACE in its at least partially acid form in step (a) while forming an effluent containing a solution of salts, comprising cations bound to the cation exchangers in steps (a) and (b) and the anions of the strong acid; and

(h) directing the salt solution obtained as the effluent of step (g) for commercial use.

Theoretically one mole of nitrogen is required for the formation of a mole of glutamic acid to form the amino group therein. Yet, in current industrial practice at least two moles of ammonia are consumed in the fermentation per mole of glutamic acid since one mole is consumed for neutralization of the glutamic acid formed.

Therefore the fermentation liquor contains ammonium glutamate. In the process of said PCT application the acidulation results in half a mole of ammonium sulfate per mole of product glutamic acid or per mole of monosodium glutamate.

The by-product salt formed on the direct or indirect acidulation is composed of the anions of the acidulating acid and the cations of the acidulated salt. If said acidulated salt is a product of neutral fermentation, the cations of the by-product salts are typically those of the base used as a neutralizing agent in the fermentation. In many cases said base is ammonia, as in the case of glutamic acid fermentation. That is because ammonia is, in most cases, the cheapest base available (except for lime, which is undesired due to its low solubility in its base form as well as in the form of many of its salts). Typically the acidulant is sulfuric acid, also due to its low cost. Thus in many cases the by-product salt is ammonium sulfate.

The main reason for using the cheapest base and acid is the fact that typically the by-product salt contains many impurities resulting from the solution of the salt to be acidulated. That is particularly true when that salt is a product of a bio-process. In the case of fermentation, significant amounts of impurities (including those resulting from the complex nitrogen source, other nutrients added and fermentation by-product such as amino acids, etc.) find their way to the by-product salt solution. In the case of enzymatic conversion the purity of the product is dependent on the purity of the reagent (which could be a fermentation product as in the case of converting fermentatively formed ammonium fumarate to ammoniurr . aspartate) as well as on the efficiency and selectivity of the conversion.

Typically, the by-product salt is partially purified, in many cases by crystallization, which is expensive in energy. Due to the content of impurities, usually the yield on the by-product salt crystallization is limited so that some of it ends up in a waste solution representing reagents loss and a waste management problem. Furthermore, the partially purified by-product salt is still oτ very low value and is useful only as a fertilizer. In many cases the price obtained for that fertilizer is less than one half of the cost of the ammonia contained therein.

Surprisingly, it was found that the solution of the by-product ammonium salt, formed in the direct or indirect acidulation of ammonium salts formed by neutral bio- processes, is pure enough, as is or after a very low cost polishing process, to serve as an inorganic nitrogen source and optionally also as an anion source for lysine fermentation. Furthermore, it was found that some of the impurities can be utilized in the fermentation, for example, as a nitrogen source.

With this state of the art in mind, there is now provided, according to the present invention, a process for the combined production of products selected from the group consisting of lysine and its salts and products selected from the group consisting of at least one weak acid and a salt thereof, which weak acid is selected from the group consisting of organic acids and amino acids produced by a neutral bio-process said process comprising:

(a) acidulating an aqueous feed stream containg said weak acid and ammonium cations with an acidulating acid, which acidulating acid has at least one pKa lower than 4.2;

(b) recovering at least part of said weak acid from the aqueous solution formed in (a), forming thereby an aqueous solution of an ammonium salt comprising ammonium cations resulting from said feed solution in step (a) and anions of said acidulating acid; and

(c) fermenting a medium containing at least one carbon source, at least one organic nitrogen source and ammonium salt resulting from step (b), utilizing a lysine producing microorganism, whereby there is formed a fermentation liquor containing at least 50 g/1 of lysine values; whereby said fermentation liquor is suitable for use as a source of lysine in animal- feed.

The lysine produced is utilized as a supplement of an essential amino acid in animal feed. It could be used for that purpose in a solid or in a liquid form (solution). Presently most of the solid product is lysine hydrochloride (LyHCI), but that could change. Lysine solutions could contain the lysine in its free base form (typically at pH levels of 9 and higher) or in a salt form. Typically, the anions in those solutions are sulfate or chloride (less preferred due to the low solubility of LyHCI). Lysine sources where the anion is of propionic acid or of polycarboxylic acids were suggested recently. Those solutions could be process streams, such as the fermentation liquor or crystallization mother liquor (as they are or after a treatment such as some purification and concentration). Alternatively, they are produced from free base lysine or from a lysine salt separated from the fermentation liquor. In a preferred embodiment of the present invention said process comprises a further recovery step wherein lysine is recovered from said fermentation liquor in a form selected from the group consisting of lysine salt of the acidulating acid, lysine salt of another acid and free base lysine.

The weak acid produced according to the present invention in acid or in salt form is an organic or an amino acid. Preferably they carry at least one acidic function with a pKa higher than 1.8, most preferably higher than 3. Preferably the weak acid is selected from the group consisting of glutamic acid, aspartic acid, fumaric acid, malic acid, succinic acid, oxoglutaric acid and gluconic acid. Said weak acid is a product of a bio-process .preferably selected from the group consisting of fermentation and enzymatic conversion. Examples for fermentation products are glutamic acid, succinic acid, gluconic acid and fumaric acid. Examples for products of enzymatic conversion are aspartic acid, malic acid and oxoglutaric acid. In a most preferred embodiment the substrate for the enzymatic conversion is a fermentation product, e.g. the cases of converting ammonium fumarate resulting from fermentation into ammonium aspartate or ammonium malate.

Said bio-process is conducted in a neutral bio-process. The term neutral in the present case is not intended to mean pH 7, but a pH of about neutrality. Thus, neutral pH here means that through most of the process and particularly at the end of it, the pH of the bio-process medium is between 4.5 and 9.5, most preferably- between 5 and 8.5. As a result the product is obtained in a salt form, rather than in the free acid form, i.e. a significant part of it is negatively charged. In the case of enzymatic conversion neutrality is preferably achieved by using a neutral substrate, while in fermentation a neutralizing agent is used. For the purpose of the present invention the bio-process product is introduced in a feed containing ammonium ions. It could be formed in the form of an ammonium salt due to enzymatic conversion of an ammonium salt substrate or due to using ammonia as a neutralizing agent in the fermentation. Alternatively, it is formed in the form of another salt and converted to the ammonium form prior to use in the present process.

An aqueous feed stream containing said weak acid and ammonium cations is acidulated with an acidulating acid. The pH of the aqueous feed is about neutral, having a pH in ranges similar to those given for the bio-process. Said feed could be the product of said bio-process or result from it. Being about neutral means that the weak acid is present in said feed solution, mostly in its salt form. Thus, here and hereinafter, if not specified otherwise, the term weak acid could mean the acid, its salts and combinations thereof. The acidulating acid could be an organic or an inorganic acid of relatively high acidity, i.e., having at least one pKa lower than 4.2, more preferably lower than 3.2, most preferably smaller than 2.5. Preferably the acidulating acid is selected from the group consisting of H₂S0 , HCI, H₃P0₄ and dicarboxylic acids. The acidulation could be direct, i.e. by the addition of the acidulating acid into the aqueous feed solution or indirectly, e.g. through the use of a cation exchanger.

Thus in a preferred embodiment of the present invention, there is now provided a process for the combined production of products selected from the group consisting of lysine and its salts and products selected from the group consisting of at least one weak acid and a salt thereof; which weak acid is selected from the group consisting of organic acids and amino acids produced by a neutral bio-process said process comprising:

(a) contacting an aqueous feed stream containing said weak acid and ammonium cations with a cation exchanger, which is at least partially in its acid form whereby at least part of the ammonium cations in the solution is adsorbed ort- the cation exchanger and protons introduced into the aqueous solution;

(b) recovering at least part of said weak acid from the aqueous solution formed in (a);

(c) regenerating the cation exchanger from step (a) to its at least partially acid form by a solution of acidulating acid, which acid has at least one pKa lower than 4.2, while forming an aqueous solution of an ammonium salt comprising ammonium cations bound to the cation exchanger in step (a) and the anions of said acidulating acid;

(d) recycling the cation exchanger in its at least partially acid form for reuse in step (a); and

(e) fermenting a medium containing at least one carbon source, at least one organic nitrogen source and ammonium salt resulting from step (c), utilizing a lysine producing microorganism, whereby there is formed a fermentation liquor containing at least 50 g 1 of lysine values; whereby said fermentation liquor is suitable for use as a source of lysine in animal feed.

The cation exchanger could be a strong one, a weak one or a combination thereof, as, e.g. in the case of said PCT/GB97/00177.

Thus, in an especially preferred embodiment of the present invention there is provided a process for the combined production of products selected from the group consisting of glutamic acid and its salts and products selected from the group consisting of lysine and its salts, said process comprising:

(a) contacting an aqueous feed stream containing glutamate resulting from fermentation and ammonium cations at an elevated temperature with a Weak Acid Cation Exchanger (WACE) which is at least partially in its acid form whereby part of the ammonium cations in the solution is taken up by the cation exchanger and protons are introduced into the solution;

(b) contacting a second aqueous feed containing glutamate and ammonium cations at an elevated temperature with a strong acid cation exchanger (SACE) that is obtained from a subsequent step and carries cationic glutamate, whereby the cationic glutamate is transferred into the solution and most of sai ammonium cations in said second aqueous feed are taken up by the SACE;

(c) crystallizing glutamic acid from the effluent of step (b);

(d) contacting the mother liquor of step (c) with SACE which is at least partially in its acid form whereby cationic glutamate is bound;

(e) utilizing the SACE obtained in step (d) in step (b);

(f) regenerating the SACE from step (b) to its at least partially acid form by a solution of an acidulating acid and utilizing the SACE in its at least partially acid form in step (d) while forming an aqueous solution of an ammonium salt, comprising ammonium cations bound to the cation exchanger in step (b) and the anions of said acidulating acid;

(g) regenerating the WACE from step (a) to its at least partially acid form by effluent from step (f) and recycling the WACE in its at least partially acid form for re-use in step (a) while forming an aqueous solution containing an ammonium salt, comprising ammonium cations bound to the cation exchangers in steps (a) and (b) and the anions of said acidulating acid; and

(h) fermenting a medium containing at least one carbohydrate source, at least one organic nitrogen source and ammonium salt from steps (f) and (g) or a combination thereof, utilizing a lysine producing microorganism whereby there is formed a fermentation liquor containing at least 50 g 1 of lysine values; whereby said fermentation liquor is suitable for use as a source of lysine in animal feed.

Ammonium ions from the solution are adsorbed on the cation exchanger and protons are released into the solution. The anions of the weak acid present in the solution are strong bases. Therefore they bind the protons and convert thereby to their free acid form. At least a part of this free acid is then recovered by known methods, such as solvent extraction and crystallization, which is useful particularly for low solubility acids such as glutamic acid and fumaric acid. In the case of an indirect acidulation of salts of these acids through the use of a cation exchanger, the contact with the cation exchanger is preferably done at an elevated temperature to avoid crystallization in the resin. In the case of direct acidulation separation of the product acid forms a— solution containing an ammonium salt of the acidulating acid. In the case of an indirect acidulation the ammonium ion carrying cation exchanger is regenerated to its at least partially acid form and recycled to the contact with said aqueous feed, in said regeneration it is contacted with a solution of an acidulating acid, protons are transferred from the solution to the resin and ammonium ions are transferred to the solution. A solution of an ammonium salt is formed, comprising ammonium cations bound previously to the cation exchanger and anions of the acidulating acid.

The ammonium salt is used as a source of inorganic nitrogen and a source of anions in lysine fermentation. In some cases the solution of the ammonium salt obtained in the previous stage could be used as such or after some concentration to maintain the water balance in the fermentation. In other cases some polishing of the solution or some crystallization might be required. Optionally, the ammonium salt of the acidulating acid is converted to a salt of another acid, which is more desired in the lysine fermentation, in the downstream processing of the fermentation liquor or in the application.

According to the present invention, an acidulating acid is used for the acidulation of the bio-process product salt and the ammonium salt of the acidulant is used as both a source of inorganic nitrogen and a source of anions in the process for the production of lysine or its salts. This results in some important advantages for integrated production of lysine or its salts (i) and said carboxylic or amino acid and their salt (ii);

(a) High yields on ammonia are obtained as the ammonia used as a neutralizing agent in (ii) forms an amino group in the lysine molecule formed in (i). This high yield on ammonia provides significant economic savings.

(b) The costs related to crystallization and purification of the ammonium salt are avoided or, at least, reduced;

(c) Salt rejection in the effluents and the related water treatment costs are reduced.

(d) Lysine is a basic amino acid. In most cases it is used in its salt form. In the overall process, an acid is usually consumed to provide the anion for the lysine salt. In the integrated process of the present invention that acid is utilized twice: first in (i) for acidulating the salt and then provides the anion for the lysine- salt resulting from (i).

(e) Some of the impurities in the ammonium salt resulting from (ii) are consumed in the lysine formation of (i), for example, as organic nitrogen sources or as nutrients.

The invention also provides a lysine source prepared according to the processes as hereinbefore defined as well as an animal feed containing lysine values prepared according to the processes of the present invention.

In preferred embodiments of the present invention, the lysine fermentation uses at least one carbon source selected from the group consisting of sucrose, molasses from sucrose production, starch and starch hydroiysates, which starch could result from various sources, such as corn and wheat, including low grade starches, sucrose, molasses, and a combination thereof. In some cases byproducts of said bio-process might serve as a carbon source as well.

The concentration of the lysine in the fermentation liquor is preferably between 70 and 250 g/l.

At least one organic nitrogen source is used. Sources for such organic nitrogen are preferably selected from the group consisting of molasses, proteins, peptides and amino acids formed as a product of fermentation, bacteria or soybean acid-hydrolysate, corn steep liquor, yeast extracts, proteins of effluents from wheat processing, hydroiysates thereof and combinations thereof.

The fermentation is preferably effected by a lysine producing microorganism belonging to the genus Brevibacterium or Corynebacterium. It is most preferably conducted in air-lift fermentors. Preferably the temperature in the fermentation medium is kept between 30°C and 35°C and the pH is between 5 and 8.5, most preferably between 6.0 and 7.5. The fermentation is preferably conducted for a period of between 40 and 100 hours, at the end of which the lysine content in the fermentation liquor is at least 50 g/l of lysine values. Preferably, the content of lysine values in said fermentation liquor is at least 60% of the total solutes in said liquor.

Lysine has two amino groups and one carboxylic group. Therefore, depending on the pH, it could carry a single negative charge, no net charge (the zwitterionic or free base form), one positive charge and two negative charges. If noL.. otherwise specified, the term lysine values will refer to any of those and to combinations thereof.

The positively charged lysine (cationic lysine) in a neutral or acidic solution is balanced by negatively charge ions (anions) in the solution, which anions. in the present invention are preferably selected from mineral anions, carboxylic acid anions and other.

The fermentation liquor could be used as such or first treated to recover lysine values therefrom. Ion exchangers could be used for that purpose, preferably cation exchangers and most preferably a strong acid cation exchanger, possibly in a combination with a weak acid cation exchanger. Preferably said ion exchanger or ion exchangers are used in a counter current mode, most preferably in a chromatographic operation mode.

In a preferred embodiment, said cation exchanger is at least partially in its acid form, lysine is adsorbed and an acid is liberated into the effluent solution. The lysine is adsorbed in its cationic form carrying a single positive charge, a double positive charge or a combination thereof, most preferably carrying a single positive charge. The lysine carrying cation exchanger is eluted, preferably after washing with water, by a solution of an acid to regenerate said cation exchanger to its at least partial acid form and to form an eluate containing LyHA, wherein A is the anion of a regenerating acid, which eluate can be used as such or after some concentration or treated for crystallization of LyHAI in methods known per se. The acid containing effluent is treated for acid recovery by known methods, preferably selected from the group consisting of solvent extraction, acid retardation, ion exclusion and distillation. The recovered acid is reused, preferably as an acidulating acid according to the present invention, for elution of lysine adsorbed on a cation exchanger, for hydrolysis of bacteria, hydrolysis of wheat processing effluents and a combination thereof.

In a preferred embodiment, said cation exchanger used in said recovery of lysine from the fermentation liquor is carrying lysine cations, preferably in double positive charge form (LyH₂++). Upon contacting of said Lysine salt containing fermentation liquor with said LyH₂ carrying cation exchanger lysine is bound to said cation exchanger without eiuting any significant amount of the previously bound-, lysine. The lysine bound from said fermentation liquor is preferably bound in its salt form. Other resins that adsorb lysine in its salt form could also be used. The adsorbed lysine salt is then eluted, preferably by water at ambient or higher temperature with no acid or base, but optionally in the presence of CO₂.

In a most preferred embodiment of the above described process, lysine, preferably in its salt form, is adsorbed from said fermentation-liquor on an ion exchanger and eluted with water at a temperature higher than that of readsoption. Particularly suitable ion exchangers are those know as amphoteric resins, thermally regeneratable resins, sirotherm resins and snake-cage resins.

According to the present process and according to some of its preferred embodiments, various process streams are formed. Those process streams as they are or after some treatment provide large freedom in preparing lysine and/or lysine salt containing products of desired compositions, e.g. those compositions referred to as liquid lysine. Thus, in a preferred embodiment, at least two of said process stream preferably selected from the group consisting of a crystalline lysine salt, said free base lysine or a lysine salt recovered from said fermentation-liquor and said fermentation-liquor prior to said lysine recovery or after it, as they are or after some adjustment, are combined to form a solution containing at least 30% of lysine, lysine salt, or a combination thereof, which solution is used as a lysine source.

In another preferred embodiment, a reagent ammonium salt and free base lysine formed in said recovery are reacted to form lysine salt and ammonia.

In another preferred embodiment lysine in its free base form, formed in the recovery step of the above defined process, is reacted with a process stream, which stream contains at least one reagent ammonium salt in which reaction ammonia is stripped out of said stream into the vapor phase, separated and reused as an inorganic nitrogen source or in the recovery step and said ammonium salt in said stream is converted to a lysine salt.

In a variation of said embodiment, lysine in its free base form, formed in said recovery step, is reacted with a stream obtained by a combination of process streams, which streams contain at least one reagent ammonium salt in which reaction ammonia is stripped out of said stream into the vapor phase, separated and reused as an inorganic nitrogen source or in said recovery step and said- ammonium salt in said stream is converted to a lysine salt.

In a preferred embodiment, said reagent ammonium salt is a salt of an acid selected from the group consisting of hydrochloric acid, sulphuric acid, phosphoric acid, organic acids and amino acids and mixtures thereof. In a further preferred embodiment said reagent salt is of an acid, which is a product of a bio-process. In a most preferred embodiment said reagent salt is selected from the group consisting of salts of said weak acid, products thereof and their combination. Thus, according to these preferred embodiments a free base lysine is formed in the recovery step and reacted with ammonium chloride (preferably formed as a by-product of another process or as a by-product of acidulation according to the present process) to form lysine hydrochloride and ammonia. Such operation regenerates the ammonia from the NH₄CI and uses a cheap chloride source, rather than HCI, for the production of LyHCI. Another example is the production of the lysine salt of an organic acid or an amino acid obtained in its ammonium form, preferably by a bio-process. Free base lysine is reacted with a stream containing said ammonium salt, whereby ammonia is displaced (and collected for reuse, e.g. as a nitrogen source or in the recovery step) and the lysine salt of said acid is formed.

In a preferred embodiment at least a part of the lysine is utilized as a salt of said weak acid or products thereof.

According to one of the preferred embodiments the acidulating acid is H₂S0₄, lysine sulfate is formed in said fermentation liquor and is converted, at least partially into lysine hydrochloride. In a further preferred embodiment, said conversion utilizes as a reagent a chloride salt, selected from the group consisting of NH₄CI, NaCI, KCI, CaCI₂ and combinations thereof., rather than HCI saving thereby on reagent costs. In a most preferred embodiment lysine hydrochloride formed thereby is recovered by crystallization, preferably directly from said fermentation liquor.

Examples for the most preferred embodiment of operating the process of the present invention are related to the cases in which glutamic acid is the weak acid as described above and those in which fumaric acid is the weak acid. Ammonium fumarate is formed in fermentation, either directly or by conversion of fermentatively produced calcium fumarate (e.g. in the process of Israel PatenV- Application 116,849 the teaching of which is incorporated here by reference). Part of the ammonium fumarate is acidulated according to the present invention, while another part is reacted with free base lysine to displace ammonia (collected and reused as above) and lysine fumarate. In another highly preferred embodiment ammonium fumarate, preferably resulting from fermentation is converted in an enzymatic reaction to ammonium aspartate. The latter is acidulated with sulfuric acid according to the present invention to form aspartic acid and ammonium sulfate. Said ammonium sulfate is used as a source of nitrogen and sulfate anions in the fermentation step that yields lysine sulfate. Similarly, ammonium fumarate can be converted in an enzymatic process to other products such as ammonium malate that would be processed as above, through acidulation or through reaction with free base lysine. Thus lysine sulfate is preferably formed in the fermentation, which uses ammonium sulfate resulting from the acidulation of ammonium fumarate, glutamate, aspartate or malate. This lysine sulfate could be used for animal feed as such, after conversion to LyHCI (as a part of , or after, the recovery process), recovered as a free base or any combination thereof. In a most preferred embodiment said lysine is used for the production of concentrated aqueous solutions containing lysine and at least one polycarboxylic acid according to Israel Patent Application No. 120,491, the teaching of which is incorporated herein by reference. Most preferably, said polycarboxylic acid is malic acid and said concentrated solution is obtained by reacting free base lysine formed according to the present invention with ammonium malate formed from ammonium fumarate, preferably by enzymatic conversion.

While the invention will now be described in connection with certain preferred embodiments in the following examples so that aspects thereof may be more fully understood and appreciated, it is not intended to limit the invention to these particular embodiments. On the contrary, it is intended to cover all alternatives, modifications and equivalents as may be included within the scope of the invention as defined by the appended claims. Thus, the following examples which include preferred embodiments will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purposes of illustrative discussion of preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be-. the most useful and readily understood description of formulation procedures as well as of the principles and conceptual aspects of the invention.

EXAMPLES Example 1:

A 10 liter sterile solution containg 1270g dextrose, 17.5g (NH₄)₂SO₄, 4g.

MgS0₄.7H₂O, 3g KH₂P0₄, 0.5g. ZnS0₄.7H₂O, 0.1g. FeCl₃.6H₂0, 5g. com steep liquor and 960g of CaCO₃ in suspension is fermented at 34 °C using an Rhizopus arrhizus strain. After 54 hours of fermentation the broth was analyzed. Per Kg. it contained 66g. fumarate, 8g. malate, 4.8 g. succinate, 4.1 g. alpha keto glutarate and 25.7 g. glycerol. 5 Kg. of the broth were filtered and the solids were washed with water to yield 1.4 Kg. of wet cake containing 283g. fumarate, 4.2g. malate,

8.8g. succinate, less than 9g. of alpha keto glutarate and less than 3g. glycerol. 1.4

Kg. cake is suspended in 1.5 liter of deionised water at 30 °C and 344 g. of a 33% ammonia solution is added. Gaseous C0₂ is bubbled through the solution until the pH is 8.7. After cooling to ambient temperature the suspension is filtered and the cake is washed with 1.2 Kg. water. The wash water is combined with the filtrate.

The combined solution contains 257 g. fumarate, more than 90% conversion, 2.2 g. malate, 8.2g succinate, 0.4g g. alpha keto glutarate, 0.18 g. calcium and 90 g. ammonia. The ammonium fumarate solution is concentrated. Sulfuric acid in a stoichiometric amount is added and the solution is cooled to 5°C, whereby most of the fumaric acid crystallizes out. The ammonium sulfate solution formed is used, after further concentration as a source of an inorganic nitrogen and as a source of sulfate in lysine formation by fermentation of dextrose using a corynebacterium strain at 35 °C. Ammonia is used for regulating the pH to about neutral. The analysis of the broth is 21.6 g/L bacteria cells, 102.9 g/L lysine, 28.8 g/L organic matter, 35.9 g/L sulfate and small amounts of ammonia, phosphate, sodium, potassium chloride and other ions. This lysine sulfate containing fermentation liquor is filtered by a Kerasep ceramic membrane at an average flow rate of 110 L/h at

70°C for the separation of the bio-mass. The permeate is acidulated by the addition of sulfuric acid and contacted with a strong acid cation exchanger (SACE) in ammonium form. 99% of the lysine present in the solution is bound and ammonium sulfate is formed in the effluent. The lysine carrying SACE is eluted with an_ aqueous solution of ammonia. The excess of ammonia is distilled out of the eluate to form a solution of free base lysine. A stoichiometric amount of HCI is added to a part of the free base solution, which is then concentrated under reduced pressure of 0.3 bar and at 70°C to reach 60% DS. This concentrated solution is cooled to 5°C under agitation after the addition of lysine hydrochloride dihydrate seed crystals and lysine hydrochloride crystallizes. Another part of the free base lysine is reacted with a solution of ammonium malate. Water and ammonia are distilled out of the combined solution to form lysine malate therein. Example 2:

A solution of diammonium fumarate is prepared as in Example 1. The pH of this solution is adjusted to 8 and the diammonium fumarate is converted at 58°C to monoammonium aspartate by means of the bacterium Pseudomonas fluorescens. The molecular yield of conversion is higher than 95%. The solution of ammonium aspartate is concentrated and reacted with a stoichiometric amount of sulfuric acid. On cooling to 10°C, aspartic acid crystallizes out of the solution. The ammonium sulfate solution formed thereby is used, after further concentration as a source of an inorganic nitrogen and as a source of sulfate as in Example 1 with similar results. Example 3:

Ammonium glutamate is formed by fermentation of dextrose, using ammonia as a nitrogen source and a neutralizing agent. The average analysis of the broth is: 17.2 g/L bacteria cell, 115 g/L glutamic acid, 40.5 g/L organic matter, 14.4 g/L ammonia and smaller amounts of sulfate, phosphate, chloride, sodium, potassium, magnesium and calcium ions. The broth is filtered on a PCL membrane with a 200,000 dalton cut off. 1.5 liters of the permeate is fed at 75 °C on a 1 liter column of the cation exchanger IMAC HP 336 of Rohm & Haas at a rate of 6 lit/h. Ammonium ions are bound to the cation exchanger and protons are released into the solution. The resin is then washed with water and the solution obtained is combined with the glutamic acid containing effluent. 2.25 liters of solution of pH=3.5 are obtained. This aqueous solution is concentrated and cooled down to 20°C. 123 g. Glutamic acid crystals of purity >98% are obtained. The ammonium ion carrying cation exchanger is treated with a solution of sulfuric acid to regenerate the cation_^ exchanger to acid form and to form a solution of ammonium sulfate. This solution is concentrated and used as a source of inorganic nitrogen and of sulfate in lysine fermentation as in Example 1 with similar results.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative examples and that the present invention may be embodied in other specific forms without departing from the essential attributes thereof, and it is therefore desired that the present embodiments and examples be considered in all respects as illustrative and not restrictive, reference being made to the appended claims, rather than to the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims

What is Claimed is: _

1 . , A process for the combined production of at least one product selected from the group consisting of lysine and its salts, and at least one product selected from the group consisting of at least one weak acid and a salt thereof, which weak acid is selected from the group consisting of organic acids and amino acids produced by a neutral bio- process, said process comprising:

(a) acidulating an aqueous feed stream containing said weak acid and ammonium cations with an acidulating acid, which acidulating acid has at least one pKa lower than 4.2;

(c) fermenting a medium containing at least one carbon source, at least one organic nitrogen source and ammonium salt resulting from step (b), utilizing a lysine producing microorganism, whereby there is formed a fermentation liquor containing at least 50 g/l of lysine values; whereby said fermentation liquor is suitable for use as a source of lysine in animal feed.

2. A process according to claim 1 for the combined production of products selected from the group consisting of lysine and its salts and products selected from the group consisting of at least one weak acid and a salt thereof; which weak acid is selected from the group consisting of organic acids and amino acids produced by a neutral bio-process, said process comprising:

(a) contacting an aqueous feed stream containing said weak acid and ammonium cations with a cation exchanger, which is at least partially in its acid form whereby at least part of the ammonium cations in the solution is adsorbed on the cation exchanger and protons introduced into the aqueous solution;

(c) regenerating the cation exchanger from step (a) to its at least partially acid form by a solution of acidulating acid, which acid has at least one pKa lower than 4.2, while forming an aqueous solution of an ammonium salt comprising _. ammonium cations bound to the cation exchanger in step (a) and the anions of said acidulating acid;

(e) fermenting a medium containing at least one carbon source, at least one organic nitrogen source and ammonium salt resulting from step (c), utilizing a lysine producing microorganism, whereby there is formed a fermentation liquor containing at least 50 g/l of lysine values; whereby said fermentation liquor is suitable for use as a source of lysine in animal feed.

3. A process according to Claim 1 , comprising a first recovery step wherein lysine values are recovered from said fermentation liquor in step (c) in a from selected from the group consisting of a lysine salt of the acidulating acid, a lysine salt of another acid and free base lysine.

4. A process according to Claim 1 , wherein said weak acid is selected from the group consisting of glutamic acid, aspartic acid, fumaric acid, malic acid, succinic acid, oxoglutaric acid and gluconic acid.

5. A process according to Claim 1 wherein said acidulating acid is selected from the group consisting of H₂S0₄, HCI, H₃P0₄ and dicarboxylic acids.

6. A process according to Claim 1 , wherein said bio-process is selected from the group consisting of fermentation and enzymatic conversion.

7. A process according to Claim 6, wherein said enzymatic conversion is of a product of a fermentation process.

8. A process according to Claim 1 for the combined production of products selected from the group consisting of glutamic acid and its salts and products selected from the group consisting of lysine and its salts, said process comprising:

(a) contacting an aqueous feed stream containing glutamate resulting from fermentation and ammonium cations at an elevated temperature with a Weak Acid Cation Exchanger (WACE) which is at least partially in its acid form whereby part of the ammonium cations in the solution is taken up by the cation exchanger and protons are introduced into the solution; (b) contacting a second aqueous feed containing glutamate and_ ammonium cations at an elevated temperature with a strong acid cation exchanger (SACE) that is obtained from a subsequent step and carries cationic glutamate, whereby the cationic glutamate is transferred into the solution and most of said ammonium cations in said second aqueous feed are taken up by the SACE;

(c) crystallizing glutamic acid from the effluent of step (b);

(e) utilizing the SACE obtained in step (d) in step (b);

(h) fermenting a medium containing at least one carbohydrate source, at least one organic nitrogen source and ammonium salt from steps (f) and (g) or a combination thereof, utilizing a lysine producing microorganism whereby there is formed a fermentation liquor containing at least 50 g/l of lysine values; whereby said fermentation liquor is suitable for use as a source of lysine in animal feed.

9. A process according to Claim 1 wherein said at least one carbon source is selected from the group consisting of hydroiysates of high grade or low grade starch, sucrose, molasses and combinations thereof.

10. A process according to Claim 1 , wherein the concentration of said lysine in said fermentation liquor is between 70 and 250 g/l.

11. A process according to Claim 1 , wherein said at least one organic nitrogen source is selected from the group consisting of molasses, proteins, peptides and amino acids formed as a product of fermentation, bacteria or soybean acid^ hydrolysate, corn steep liquor, yeast extracts, proteins of effluents from wheat processing, hydroiysates thereof and combinations thereof.

12. A process according to Claim 1 , wherein the temperature of said medium is between 30 °C and 35 °C.

13.. A process according to Claim 1 , wherein the pH of said medium is kept between 5 and 8.5.

14. A process according to Claim 1 , wherein said lysine producing microorganism is selected from the genus consisting of Brevibacterium and Corynebacterium.

15. A process according to Claim 1 wherein the content of lysine in said fermentation liquor is at least 60% of the total solutes in said liquor.

16. A process according to Claim 3, wherein an ion exchanger is used in said recovery.

17. A process according to Claim 16 wherein said ion exchanger is a cation exchanger.

18. A process according to Claim 17 wherein said cation exchanger is a strong acid cation exchanger.

19. A process according to Claim 3 wherein a combination of a weak acid cation exchanger and a strong acid cation exchanger is used in said recovery.

20. A process according to Claim 17, wherein said cation exchanger is contacted with said fermentation liquor in a counter-current mode.

21. A process according to Claim 18, wherein said cation exchanger is at least partially in its acid form.

22. A process according to Claim 3, wherein lysine is adsorbed from said fermentation-liquor on an ion exchanger and eluted with water at a temperature higher than that of readsoption.

23. A process according to Claim 3, wherein lysine in its neutral form, formed in said recovery step is reacted with a process stream, which stream contains at least one reagent ammonium salt in which reaction ammonia is stripped out of said stream into the vapor phase, separated and reused as an inorganic nitrogen source and said ammonium salt in said stream is converted to a lysine salt.

24. A process according to Claim 23, wherein said reagent ammonium salt is a- salt of an acid selected from the group consisting of hydrochloric acid, organic acids and amino acids.

25. A process according to Claim 23, wherein said reagent salt is of an acid which acid is a product of a bio-process.

26. A process according to Claim 23, wherein said reagent salt is selected from the group consisting of salts of said weak acid, products thereof and combinations thereof.

27. A process according to Claim 1 , wherein the acidulating acid is H₂S0₄ and the lysine sulfate in said fermentation liquor is converted into lysine hydrochloride.

28. A process according to Claim 27, wherein said conversion utilizes as a reagent a chloride salt selected from the group consisting of NH₄CI, NaCI, KCI, CaCI₂ and a combination thereof.

29. A process according to Claim 27, wherein lysine hydrochloride is crystallized from said fermentation liquor.

30 A process according to Claim 1 , wherein at least part of the lysine is utilized as a salt of said weak acid or product thereof.

31. A lysine source prepared according to claim 1.

32. An animal feed containing lysine prepared according to claim 1.