Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
1999-05-25
2001-02-13
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S136000, C435S138000, C435S139000, C435S146000, C204S518000, C204S522000, C204S530000, C204S536000, C204S537000, C210S638000, C210S641000, C210S654000
Reexamination Certificate
active
06187570
ABSTRACT:
TECHNICAL FIELD
This invention relates generally to electrodialysis methods for separation and purification of preferred end products and in particular to electrodialysis methods for the purification and recovery of gluconic acid derivatives, such as 2-keto-L-gulonic acid from solutions removed from fermentation reactors and in-vitro reactors.
BACKGROUND OF THE INVENTION
A gluconic acid derivative, namely 2-keto-L-gulonic acid (hereinafter KLG), is a key intermediate in the production of valuable compounds including ascorbic acid (Vitamin C). However, to obtain high yields during the conversion of KLG to ascorbic acid, the KLG must be highly purified with a limited amount of impurities.
A convenient method for producing KLG is through a fermentation process. However, since most fermentation broths are maintained at neutral or near neutral pHs by the addition of basic substances, salts of KLG rather than KLG are produced. Furthermore, the fermentation broth also contains cells, neutrals and other undesirable materials. These additional components may interfere with the downstream chemistry used to convert KLG to ascorbic acid, therefore, the KLG must be isolated from the broth. Thus understood, any fermentation process for KLG must be integrated with an efficient recovery and purification process.
U.S. Pat. No. 5,747,306 discloses a method of separation using electrodialysis.
The viable fermentation broth is maintained at a near neutral or basic pH of between 5 and 9 by the addition of basic substances such as sodium hydroxide, potassium hydroxide or ammonia The broth is then passed through an electrodialysis tank which contains repeating cation and anion exchange membranes wherein the salts of KLG are removed from the broth. The viable fermentation broth containing neutrals, such as nutrients to insure survival of the microorganisms, is then recirculated into the fermentation system for reuse. However, the electrodialysis process produces a stream of purified KLG salt which gives lower yields during the conversion to ascorbic acid.
Additionally, the prior art process of converting organic sugars to the salt of KLG by using live and metabolically active microorganisms is complicated and demands constant vigilance to maintain a viable and active fermentation broth for the growth and/or metabolism of the microorganisms to ensure acceptable conversion of the substrate to the KLG salt.
To overcome the problems related with a high concentration of KLG salts during recovery of KLG from a fermentation medium, U.S. Pat. No. 4,990,441 discloses a method of acidifying the fermentation medium with sulfuric acid thereby precipitating the salt cation with the sulfate anion and protonating the KLG anion. However, it should be noted, that the medium containing salts of KLG also contains inorganic impurities such as phosphate and chloride anions which will also be converted to their corresponding acids with the addition of sulfuric acid. As a result, these inorganic acids can be concentrated during the KLG recovery processes, such as evaporative crystallization or direct drying and can cause acid catalyzed degradation of KLG. To rectify this problem the prior art contacts the medium containing the KLG and other impurities with a cation and anion exchange resin to remove any ionized impurities. But, neutral organics present in the fermentation broth which include simple and complex sugars may be unionized at the solution pH and thus not removed by passing the medium through cation and anion exchange resins. As such, the recovery of purified KLG is limited by the presence of these neutrals in the medium. The neutral sugars are concentrated during evaporative crystallization of KLG which causes increased viscosity of the mother liquor. As a result, multiple pass crystallization becomes difficult and KLG recovery is limited. Also, the neutrals interfere with direct drying which is a preferred method of recovery because of higher KLG yield with lower capital costs.
Accordingly, methods are needed for the concentration and purification of gluconic acid derivatives, such as KLG, which provide a higher recovery yield of KLG without the salts thereof, without contamination by inorganic impurities and neutral organics and/or without the need to maintain a fermentation medium for the growth and/or metabolism of a living and active culture of microorganisms.
SUMMARY OF THE INVENTION
For purposes of this invention, the terms and expressions below, appearing in the specification and claims, are intended to have the following meanings:
“Gluconic acid derivative” is defined as an organic acid derived from gluconic acid including, but not limited to, 2,4, keto-D-gluconic acid, 2,5, diketo-D-gluconic acid, idonic acid, 2-keto-L-gulonic acid (KLG), vanillic acid and ascorbic acid.
“Fermentation reactor” is defined as a classical fermentation reactor wherein live and viable microorganisms or cells such as bacteria are used for metabolizing carbohydrates.
“Fermentation medium ” is a medium or broth derived from a classical fermentation reactor.
“In vitro reactor” is a reactor wherein enzymes being substantially free of non-living, non-viable and non-metabolizing cell structures, enzymes attached to non-living, non-viable and non-metabolizing cell structures or enzymes immobilized on a substrate have the ability to chemically oxidize and/or chemically reduce substrates or intermediates on the path to synthesizing gluconic acid derivatives.
“In vitro reactor medium” is a medium or solution derived from an in vitro reactor comprising at least a salt of a gluconic acid derivative and a coenzyme.
“Coenzyme” is an organic molecule required for the catalytic functioning of an enzyme, such as nicotinamide, adenine dinucleotide, nicotinamide adenine dinucleotide phosphate and mixtures thereof.
“Neutrals” are defined as sugars and/or compounds that are substantially unionized at the solution's pH.
“Non-viable fermentation medium” means the conditions in the fermentation broth or medium, such as pH, are such that the microorganisms used in the fermentation process are incapable of survival therein and the medium or broth may not be recirculated back into the fermentation tank for reuse without pretreatment.
“Non-living medium” means a medium removed from a fermentation reactor or in-vitro reactor such as a non-viable fermentation medium or in-vitro reactor medium wherein no living cells or living microorganisms capable of growth and/or metabolic activity for producing gluconic acid derivatives and/or salts thereof are present.
It is the principal object of this invention to provide novel electrodialysis methods for the concentration and purification of gluconic acid derivatives from an acidified and/or non-viable fermentation medium or an in-vitro reactor medium.
It is another object of the present invention to provide electrodialysis purification methods that result in higher recovery of KLG with subsequent higher yields of ascorbic acid thereby realizing lower capital and operating costs in the production of ascorbic acid.
It is a further object of the present invention to provide an electrodialysis purification method that results in a recovered product that will not be subject to acid catalyzed degradation and can make multiple passes through an evaporation crystallization recovery process because of reduced viscosity of the mother liquid.
It is still further an object of the present invention to provide an electrodialysis purification method that concentrates the end product, such as KLG, to such a high level of purity that direct drying of KLG can be employed giving near quantitative recovery of same.
It is another object of the present invention to provide an electrodialysis purification method that allows the recovery of expensive coenzymes and/or enzymes used in an in vitro reactor system for chemically synthesizing the salts of a gluconic acid derivative such as KLG.
All of the above objects may be accomplished by an electrodialysis purification method comprising the following steps of:
a) providing a non-liv
Genders J. David
Gopal Ram
Hartsough Dan M.
Kendall Peter M.
Long William J.
Ellis Howard M.
Fuierer Marianne
Lilling Herbert J.
The Electrosynthesis Company, Inc.
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