Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
1999-06-30
2001-06-12
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S142000, C435S145000, C210S637000, C210S639000, C210S651000, C210S741000, C562S580000, C562S582000
Reexamination Certificate
active
06245538
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a process for recovering a carboxylic acid made by the biological oxidation of a substrate by a microorganism from a fermentation broth.
2. Statement of the Related Art
Standard methods for recovering carboxylic acids in general and polycarboxylic acids in particular from fermentation broths are based on the physical separation of the spent microorganism cells from the aqueous phase such as by centrifugation followed by precipitation of the carboxylic acid as a result of pH reduction of the aqueous phase. This method is unsatisfactory for a number of reasons, the most notable of which include the problem of physically separating the spent cells and then acidifying the cell-free broth to effect the precipitation of the carboxylic acid. The precipitation of the carboxylic acid is time consuming and the separation and isolation of the precipitated carboxylic acid is not always clean.
SUMMARY OF THE INVENTION
The present invention is an improved process for the recovery of a carboxylic acid made by the biological oxidation of a substrate by a microorganism such as a yeast. After employing a standard fermentation procedure to produce a carboxylic acid, the pH of a fermentation broth which contains one or more carboxylic acids is adjusted to a value of at least about 6.0. The pH-adjusted broth is then heated to a temperature sufficient to effect the formation of three immiscible phases. The top phase is a clear, aqueous phase. The middle phase is an oily phase which is rich in the carboxylic acid. The bottom phase is an aqueous phase containing the spent microorganism cells. This method allows the easy isolation of the carboxylic acid by means of a simple phase separation.
Another aspect of the present invention relates to an improved process for making a dicarboxylic acid. This process comprises fermenting a microorganism in a culture medium comprised of a nitrogen source, an organic substrate which is a compound having one carboxyl group and one methyl group or is a compound having one methyl group and a functional group that can be at least partially hydrolyzed to a carboxyl group and a cosubstrate. The substrate is partially neutralized with an alkaline earth metal hydroxide prior to the addition of the substrate to the fermentation broth.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Other than in the claims and in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term “about”.
It is understood that a carboxylic acid is any compound containing one or more carboxyl groups. A polycarboxylic acid is any compound having two or more carboxyl groups.
The process for the recovery of a carboxylic acid according to the invention comprises fermenting a microorganism in a culture medium which is comprised of a nitrogen source, and at least an organic substrate. The organic substrate can be any compound which can be oxidized to a compound having at least one carboxyl group by biooxidation. The microorganism can be any microorganism that is capable of biologically oxidizing an organic substrate as set forth above to a compound having at least one carboxyl group.
The process for the recovery of a carboxylic acid is particularly applicable to the production of polycarboxylic acids by fermentation and most particularly to the production of dicarboxylic acids. In such situations, the substrate can be any compound having at least one methyl group, a terminal carboxyl group and/or a terminal functional group which is oxidizable to a carboxyl group by biooxidation. The substrate can also contain one or more carbon-carbon multiple bonds and/or one or more carbocyclic or heterocyclic aromatic rings. There is an advantage to adding the organic substrate in increments as opposed to an all-in method. In the incremental addition method, the total charge of organic substrate is divided into a plurality of smaller amounts each of which is added to the fermentation broth on a regular basis. The advantage gained by the incremental addition is that the rate of production of carboxylic acid remains essentially constant as opposed to an ever decreasing rate observed with the all-in method. The amount of organic substrate added in each increment and the time between additions will vary depending upon the fermentation conditions, the nature of the substrate, cosubstrate, the microorganism and the carboxylic acid formed in the fermentation. The exact incremental addition parameters can be readily determined by those skilled in the art.
The microorganism can be any microorganism capable of biooxidizing the substrate as defined herein. Typically, such a microorganism will be a yeast. Several strains of yeast are known to excrete alpha, omega-dicarboxylic acids as a byproduct when cultured on alkanes or fatty acids as the carbon source. These strains are set forth in U.S. Pat. No. 5,254,466, the entire contents of which are incorporated herein by reference. Preferably, the microorganism is a beta-oxidation blocked
C. tropicalis
cell which has been genetically modified so that the chromosomal POX4A, POX4B and both POX5 genes have been disrupted. The substrate flow in this strain is redirected to the omega-oxidation pathway as the result of functional inactivation of the competing &bgr;-oxidation pathway by POX gene disruption. The strain may also have one or more reductase genes amplified which results in an increase in the amount of rate-limiting omega-hydroxylase through P450 gene amplification and an increase in the rate of substrate flow through the &ohgr;-oxidation pathway. Preferred strains are H5343, AR40 and R24. Strain H5343 has the ATCC accession number ATCC 20962 and is described in U.S. Pat. No. 5,254,466. Strain AR40 is a
C. tropicalis
cell which is an amplified H 5343 strain wherein all four POX4 genes and both copies of the chromosomal POX5 genes are disrupted by a URA3 selectable marker and which also contains 3 additional copies of the cytochrome P450 gene and 2 additional copies of the reductase gene, the P450RED gene. Strain AR40 has the ATCC accession number ATCC 20987. Strain R24 is an amplified H 5343 strain in which all four POX4 genes and both copies of the chromosomal POX5 genes are disrupted by a URA3 selectable marker and which also contains multiple copies of the reductase gene. Strains AR40 and R24 are described in copending application Ser. No. 07/1975,154, filed on Nov. 12, 1992, the entire contents of which are incorporated herein by reference.
The first step of the process for the recovery of a carboxylic acid according to the invention is the adjustment of the pH of the fermentation broth to at least 6.0. Typically, the pH value will fall in the range of from 6.0 to 7.5 but may be higher than 7.5 depending upon the fermentation conditions, the nature of the substrate, cosubstrate, the microorganism and the carboxylic acid formed in the fermentation. The optimum pH value will be readily determinable by those skilled in the art. The pH adjustment can take place at any point in the fermentation process.
The second step of the process for the recovery of a carboxylic acid according to the invention involves heating the pH-adjusted fermentation broth to a temperature sufficient to effect the formation of three immiscible phases, one of which is an oily phase rich in the carboxylic acid product. The temperature required to effect the phase separation will typically range in the 60° C.-75° C. range but may be higher than 75° C. depending upon the fermentation conditions, the nature of the substrate, cosubstrate, the microorganism and the carboxylic acid formed in the fermentation. The optimum temperature will be readily determinable by those skilled in the art.
The improved process for making a dicarboxylic acid comprises fermenting a microorganism in a culture medium comprised of a nitrogen source, an organic substrate and a cosubstrate wherein said substrate is a comp
Anderson Kevin W.
Wenzel J. Douglas
Drach John E.
Henkel Corporation
Lilling Herbert J.
Trzaska Steven J.
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