IL35521A - Method for separating fermentation products - Google Patents

Description

METHOD FOR SEPARATING FERMENTATION PRODUCTS This Invention relates to a method for separating a fermentation product which does not boil below about 200°C. from the aqueous fermentation broth which comprises adding to the broth an organic solvent which forms an azeotrope with water and in which water Is soluble to an extent of less than about 10 gc of water per 100 g. of solvent at 25°C; subjecting the mixture to azeotroplc distillation to remove water until the water remaining in the mixture Is soluble in the organic solvent remaining and recovering the fermentation product from the distillation residue, the amount of organic solvent added to the fermentation broth being in excess of the amount needed to azeotropically remove substantially all the water.

The method of recovering the fermentation product from the distillation residue will vary depending upon whether or not the product is soluble in the organic solvent as will be discussed in more detail below.

This process is one of general applicability to nonvolatile fermentation products. There is no competi-tion between the organic solvent and water for the fermentation product as is the case when an extraction procedure is used. In addition, waste disposal problems are minimized since the water taken overhead during the azeotroplc distillation Is free of contamination and other byproducts are obtained in concentrated form for easy handling.

It is well known to prepare a number of products by fermentation processes utilizing microorganisms. The mixture present at the conclusion of the fermentation process is known as the fermentation whole broth. This broth is a dilute aqueous solution of the desired product containing a number of other materials such as mycelium, spores, unused nutrients, and various impurities. The desired product must be separated from this broth and purified for use. i A number of different methods have been used in the past to separate the product from the broth. One such method of separation of nitrogen basic antibiotics described in United States Patent 2,736,831 involves con-tacting the broth, with or without prior filtration, with a particulate cation exchange resin and eluting the antibiotic from the resin with a dilute acid solution. This process has limited utility since it can be applied only to basic materials. Further, the treated whole broth has had only the antibiotic removed and must be subjected to further extensive treatment to remove other components before it can be discharged as a waste stream.

A second method of separation that can be used with water soluble, organic solvent soluble antibiotics involves a multistage solvent extraction of the broth.

The broth may or may not be filtered prior to the extraction. This process also has limited utility. Further, it is impossible to completely remove all the product from the broth regardless of the number of extraction steps employed, and therefore, there is loss of product with the aqueous waste stream. Both the filter cake and the extracted broth present troublesome waste disposal problems .

For convenience in describing the process of the present Invention, fermentation products are divided - into four Glasses depending upon the solubility of the product in water and the organic solvent used in the process. These four classes are: 1„ Water soluble, solvent soluble 2. Water soluble, solvent insoluble 3. Water insoluble, solvent soluble 4. Water insoluble, solvent insoluble This process, in its various embodiments, can be used to separate fermentation products falling into any of the above categories, but from a practical standpoint will be used primarily when a product falling into one of the first three classes is involved.

The basic process is the same regardless of the class into which the fermentation product falls. Simply stated, the process comprises adding to the aqueous fermentation broth an organic solvent which forms an azeo-trope with water and in which water is soluble to an extent of less than 10 g. per 100 g. of solvent at 25°C, subjecting the mixture to azeotropic distillation to re-move water until the water remaining is soluble in the organic solvent and recovering the fermentation product from the distillation residue. The manner in which recovery of the product from the residue is accomplished is a function of the solubility characteristics of the product o If the product is in class 1 or 3 it will be in solution in the organic solvent at the completion of the azeotropic distillation. Insoluble by-products can then be separated by some suitable means such as filtration or centrifugation to obtain a solution of the desired product in the organic solvent. The product can then be recovered from the solution by any well-known procedure such as evaporation of the solvent , precipitation by the addition of a nonsolvent or extraction» For those solvent insoluble products falling into classes 2 and , recovery is accomplished by filtration or centrifugation of the residue from the azeotroplc distillation. Solvent insoluble products are contaminated with other insoluble solid by-products .further complicat-ing their purification. For this reason this process is more likely to be used to recover those products that are solvent soluble.

In the case of water soluble products, whether solvent soluble or solvent insoluble, the aqueous broth may be filtered prior to the addition of the organic solvent to remove water insoluble by-products. Such a filtration step might simplify later purification procedures. This is particularly true in the. case of water soluble, solvent insoluble products, Subject to the limitations discussed in the preceding paragraphs this process can be used in the recovery of any nonvolatile fermentation product which does not boil below about 200°C. A large number of such products are presently produced on a commer-cial scale. A list of such products may be found in Biochemical Engineering by Aiba, Humphrey and MiIlls, page 11, Academic Press (I965). Included in such a list are antibiotics such as bacitracin, chloramphenicol, cephalosporins, erythromycin, neomycin, penicillins, streptomycin, tylosin and vancomy-cin; steroids such as cortisone, hydrocortisone, predniso- lones and methylprednisolone; enzymes such as amylase, catalase, glucose oxidase, lnvertase, lipase, and penicillinase; organic acids such as citric, lactic, gluconic, and glutamic acids j vitamins and related products such as riboflavin, gibberellin, xanthophylls, torula yeast, and vitamin B^gJ polymers such as dextran; and various miscellaneous fermentation products.

This recovery process is also applicable to fermentation products that have been modified by chemical treatment in the fermentation broth prior to recovery, and as used herein the term "fermentation product" is intended to include such chemically modified materials.

Chemical modification might be done to aid · in the recovery of the product or to give a more desirable product „ Examples of chemical modification include the acidification of penicillin V whole broth to convert the penicillin V salt to penicillin V acid and the conversion of cephalosporin C to N-chloroacetylcephalospor.in C in the fermentation broth by treatment with chloroacetyl chloride or a mixed chloroacetyl anhydride.

The solvent to be used in the process is one that forms an azeotrope with water and which is a limited solvent for water. Thus, the solvent is preferably one in which water is soluble to the extent of no more than about 10 g„ per 100 g„ of solvent at 25°C. In general, aliphatic alcohols containing from about four to about eight carbon atoms are good solvents, as are the esters of the&e alcohols with lower aliphatic acids, particularly acetic acid. Other classes of suitable solvents include the halogenated lower alkanes, ketones containing at - - least five carbon atoms, and aromatic hydrocarbons.

Specific examples of suitable solvents include butyl alcohol, arayl alcohol, hexyl alcohol, benzyl alcohol, butyl acetate, amyl acetate, 1,2-dichloro'ethane, penta-none-3, hexanone-2, eyelohexanone, benzene, toluene, and the xylenes. This list is merely illustrative and is not intended to limit the class of solvents that may be used in any respect. Those skilled in the art are familiar with solvents forming azeotropes wi h, water . This inven-tion does not lie in which solvents will form such azeotropes but in the fact that these solvents may be used in our process for removing fermentation products from the broth. The particular solvent selected in any given case will depend upon the solubility characteristics of the product being removed.

The amount of solvent added will depend upon a number of factors, including the amount of water present in the broth, the composition of the water-solvent azeo-trope, and the solubility of water and the product in the solvent. Sufficient solvent should be added to azeotrop-ieally remove water until a single liquid phase remains. Thus, water should be azeotropically removed until the water remaining is soluble in the solvent present in the distillation vessel. Substantially all the water can be removed if desired. If the product being removed is soluble in the solvent, there should be sufficient solvent present after the azeotropic distillation to completely dissolve all the product. The solvent can be added all at once or can be added continuously or in increments over a period of time. Solvent taken overhead in the azeotrope can be recycled after separation from the water.

The azeotroplc distillation step can be carried out at atmospheric or reduced pressure, all In accordance with known chemical engineering techniques. If the desired product is heat-sensitive, it will be desirable to conduct the distillation at reduced pressure in order to achieve a lower temperature.

After the azeotrope has been condensed and allowed to cool it may be separated into the aqueous and solvent phases. The aqueous phase, after stripping to remove any dissolved solvent, can be discharged to the sewer. Because of the way in which the water is removed it is free of contaminants and does not need to be subjected to a waste disposal process. The solvent may be recycled to the process.

At the completion of the azeotroplc distillation the product may be recovered from the distillation residue in accordance with one of the procedures described above. Typical of those products which are sol-uble both in water and organic solvents are penicillin V, penicillin G, tylosin and erythromycin. These products are present in solution in the distillation residue.

An example of a product which is soluble in water but is insoluble in organic solvents is cephalosporin C, This product may be recovered from the residue by filtration, A typical example of a fermentation prod-uct that is insoluble in water but soluble in organic solvents is monensin. This ' product too, would be present in solution in the residue from the distillation, Once the product has been separated from the - - distillation residue, it can be purified in the manner heretofore employed for its purification. The purification processes for monensin and penicillin V are typical of the types of processes that are employed. In the case of monensinj, evaporation of the organic solvent from the distillation residue leaves a mixture of monensin and an oil. To this mixture is added methanol which dissolves the monensin but not the oil. The oil is removed from the solution by decantation and discarded. Addition of water to the methanol solution precipitates crystalline monensin. The product is then collected by filtration.

In the case of penicillin , the organic solvent solution from the distillation is treated with activated carbon to remove Impurities and aqueous potassium acetate solution is then added to the organic solution. The penicillin V in the organic solvent reacts with the potassium acetate to form the potassium salt of penicillin V which is insoluble in the organic solvent and precipitates. This potassium penicillin V is recovered by filtration or some other suitable means.

These two procedures are merely representative of purification processes employed in the prior art. The particular method of purification used is not a part of this invention and will vary with the particular product being purified.

The invention will be further illustrated by the following examples: Example 1 To 2 1. of penicillin V whole broth which had been assayed at 9.84 mg. of penicillin activity and 30 mg. - - of insoluble solids per ml. was added 1 1. of n-hexyl alcohol. While agitating, 20 ml. of 20 percent sulfuric acid was added to the mixture lowering the pH of the aqueous phase to 3. 9. An additional 600 ml. of hexyl alcohol was added to the mixture and the resulting slurry was distilled at a temperature of about 35°C, and a pressure of about 1 inch mercury absolute until only the solvent phase remained. Near the end of the distillation 400 ml. additional n-hexyl alcohol was added to the boil-ing slurry to replace the solvent which had been removed in the azeotrope. The dry slurry was filtered and the solids washed with 200 ml. of fresh hexyl alcohol to remove the occluded activity-laden n-hexyl alcohol. The total solvent phase contained 14 .2 g. of penicillin activity.

To illustrate the method of purifying penicillin removed from the broth in this manner, a portion of the filtrate containing 7. 90 g. of penicillin activity was treated with 10 ml. of 80 percent potassium acetate. The mixture was slurried for 30 minutes and filtered to remove precipitated penicillin V potassium salt. Recovery was 6. 13 go of penicillin activity.

Example 2 To 900 ml. of penicillin V whale broth having a pH of 3 »2 and a potency of 9.56 mg. of penicillin activity and 27 mg. insoluble solids per ml. was added 3 1. of amyl acetate. The mixture was azeotropically distilled at about 28°C. and a vacuum of about 1-inc mercury absolute until only the solvent phase remained. The dry slurry was filtered and the solids washed with 200 ml. of - - fresh am l acetate. The total solvent phase measured I050 ml. and the potency was 4.43 mg. of penicillin activity per ml. of solution. A portion of the solution was purified by crystallization as in Example 1.

Example 3 To 1 1. of monensin whole broth assayed at 2.26 mg. of activity per ml. was added I5OO ml. of n-hexyi alcohol. The mixture was azeotropically distilled at 98°C, at atmospheric pressure until only the solvent phase re-mained. The slurry was cooled, filtered, and the solids washed with 100 ml. of fresh n-hexyl alcohol. . The filtrate was evaporated under vacuum at about 30°C. until only an oil phase remained. To the oil was added 500 ml. of methanol and the mixture separated into an oil phase and a methanol phase. . The methanol contained I.89 g. of monensin activity. A portion of the monensin was recovered by concentrating the methanol solution and adding water. The crystalline monensin was recovered by filtration.

Example 4 To a continuous falling film evaporator was charged 35 -1. of benzyl alcohol. The evaporator was operated under vacuum of 27.5 inches of mercury at a temperature of 50°C. while the solvent was circulated in the system at a rate of four gallons per minute. To the evaporator were continuously changed 50 1. per hour of monensin whole broth containing 8.75 mg. of activity per ml. and 27 1. per hour of fresh benzyl alcohol. The azeo-trope was condensed overhead, and the slurry of dry solids and monensin-rich solvent was discharged periodically from the evaporator so as to maintain a constant volume of about 35 1. in the evaporator body. The solvent was de- for canted from the solids and assayed -ftrom-monensin activity. The overall recovery of monensin by this process was 90 percent from whole broth to the solvent extract.

Example 5 To a 300-gallon distillation pot containing 200 gallons of n-hexyl alcohol was slowly charged 100 gallons of fermentation broth containing the extracellular bac-terial polysaccharide, dextran, which had been produced by the microorganism Xanthomonas campestris, NRRL B-1459.

The broth contained about three percent solids by weight, and had a viscosity of 7800 cp. Viscosity is an indie. tor of the polysaccharide concentration and molecular weight.

The mixture was azeotropically distilled at 98°C. while the azeotrope was condensed overhead. At the end of the run the temperature was allowed to increase to 102 °C. in The dextran, which is insoluble /n-hexyl alcohol, was collected by filtration and dried with hot air at 160°F. The dry, oil-free polysaccharide product was stable during prolonged storage. On reconstituting the product with water to the Initial volume the viscosity measured 7,100 cp.

Example 6 This example illustrates the recovery of cephalosporin P from cephalosporin C whole broth. Cephalosporin P is a microbial steroid present in trace amounts in cephalosporin C whole broth. This steroid is difficult to purify and recover. A mixture of 150 ml. of cephalo-sporin C whole broth whose pH had been adjusted to 10.5 - - using 5 N sodium hydroxide and 250 ml. of n-hexyl alcohol was azeotroplcally distilled under vacuum at a temperature of about 35°C„ until a single liquid -phase remained. The solvent phase was decanted from the solids and assayed for cephalosporin P by paper chromatography. Within the accuracy of the assay the cephalosporin P had been entirely recovered in the solvent. Separation of the cephalosporin P from the solvent was accomplished by evaporation.

Example 7 A mixture of 200 ml. of cephalosporin C whole broth containing 6.8Ο mg. activity per ml. and 200 ml. of n-hexyl alcohol was azeotroplcally distilled under vacuum at a temperature of about 30°C. until there remained only a suspension of solids in a single liquid phase. The slurry was filtered and the solids dried at 120°F. Analysis of the solids for cephalosporin C activity showed that 78 percent of the solvent insoluble cephalosporin C had been recovered in the dry solid product.

Example 8 A mixture of 200 ml. of tylosin whole broth containing 150 mg. of activity per ml. and whose pH had been adjusted to 9.5 using 1 N sodium hydroxide and 250 ml. of n-hexyl alcohol was azeotropically distilled under vacuum at a temperature of about 30°C. until only one liquid phase remained.' The distillation residue was filtered, and the filtrate was shown by analysis to contain the tylosin activity.

Example 9 Citric acid is an organic acid produced by fer-mentation. Its solubility in water at 20°C. is 207 mg. per ml. while it is only 65 mg, per ml, in benzyl alcohol. This makes recovery by extraction difficult due to the poor distribution coefficient of citric acid between water and benzyl alcohol. However, Citric acid can be recovered from broth into benzyl alcohol following the procedure of Example 8 using citric acid whole broth rather than tylo-sin whole broth and benzyl alcohol rather than hexy alcohol.

Example .10 The exoenzyme, Arylamidase III, produced by the microorganism Cephalosporium acremonlum is soluble in water but highly insoluble in /Morgan!c solvents. A whole broth containing a specific activity of 670 units per milligram of protein can be azeotropically distilled with n-hexyl alcohol until there is only one liquid phase. The insoluble enzyme is separated from the distillation residue by filtration.

A similar separation can be conducted using benzyl alcohol rather than n-hexyl alcohol, Example 11 Twenty-five hundred milliliters of cephalosporin C whole broth was assayed at a potency of 5. 2 mg./ml.

The N-chloroacetyl cephalosporin C derivative was prepared by treating the whole broth with nine equivalents of the mixed proplonic-chloroacetic anhydride. To the broth was then added 2500 ml. of cyclohexanone and the mixture was azeotropically distilled at 0°C. and 20 mm. mercury pressure. When only one liquid phase remained, the slurry was filtered and the solids were washed with one liter of cyclohexanone to remove the N-chloroacetyl cephalosporin C adsorbed on the solids. There was obtained 1 90 ml. of solvent with antibiotic activity, representing a recovery of 82.4 percent.

Example 12 Two hundred -fifty gallons of cephalosporin C whole broth was assayed at a potency of 5.61 mg./ml. The n-chloroacetyl cephalosporin C derivative was prepared by treating testing- the whole broth with seven equivalents of the mixed propionic -chloroacetic anhydride. Two hundred - fifty gallons of eyelohexanone was then added and the mixture was azeotropically distilled in a recirculation evaporator at 50°C. and 60 mm. mercury pressure. When only one liquid phase remained the slurry was filtered . Seventy-five gallons of solvent with antibiotic activity was obtained, a recovery of 5 percent.

Claims (10)

non -volatile

1. A method for separating a / fermentation product which does not boil below about 200°C . from the aqueous fermentat ion broth characterized by add ing to the broth an organic solvent which f orms an azeotrope with water and in which water is soluble to an extent of less than about 10 g o per 100 g „ of solvent at 5° C , s ubject ing t he mixture to azeotrdplc distillation to remove water unt il t he water remaining is soluble in the organic sol vent , and recovering the fermentation product from the d istillation residue, the amount of organic solvent added to the fermentation broth being in excess of the amount needed t o azeotropically remove the water .

2. „ The method of claim 1 characterized in that the fermentation prod uct is in solution in t he broth.

3. . The method as in claim 2 characterized in that the fermentation product is soluble in the organic solvent .

4. The method as in claim 3 wherein the fermentation product is penicillin V, penicillin G, tylos in or erythromycin.

5. The method of claim 3 characterized in that the fermentation product is N-chloroacetyl cephalosporin C .

6. The method of claim 2 character ized in that the fermentation product is insoluble in the organic solvent .

7. The method of claim 6 characterized in that the fermentation product is cephalosporin C .

8. The method of claim 1 characterized, in that the fermentation product is insoluble In the aqueous fer - mentation broth .

9. The method of claim 8 characterized in that the fermentation product is soluble in the organic solvent .

10. The method as in claim 9 characterized in that the fermentation product is monensin. AGENTS FOR APPLICANTS