WO1993016173A1

WO1993016173A1 - Decolorization and deodorization of fermentation broth by treatment with strongly basic extractants

Info

Publication number: WO1993016173A1
Application number: PCT/US1992/001048
Authority: WO
Inventors: Thomas J. Stierman; Karl-Heinz Maurer; Albrecht Weiss
Original assignee: Cognis, Inc.
Priority date: 1992-02-10
Filing date: 1992-02-10
Publication date: 1993-08-19

Abstract

Fermentation broths can be decolorized and deodorized by treatment with strongly basic extractants.

Description

DECOLORIZATION AND DEODORIZATION OF FERMENTATION BROTH BY TREATMENT WITH STRONGLY BASIC EXTRACTANTS

BACKGROUND OF THE INVENTION

1. Field Of The Invention:

The present invention relates to a process for the treatment of fermentation broth. In particular, the present invention relates to a process for the 5 decolorization and deodorization of fermentation broth which contains colored impurities and has a characteristic heavy odor. More particularly, it relates to the decolorization and deodorization of fermentation broths utilizing strongly-basic extractants.

10 Many microorganisms are cultured in a suitable fermentation broth to yield a variety of useful substances, e.g. enzymes, proteins, antibiotics and so on. In some cases the broth is dark in color and has a strong odor, neither of which are desirable in the final

15 product. For example, the organism Bacillus lentus or an appropriatelytransformedBacillus licheniformis produces in a suitable fermentation media a proteolytic enzyme known as Bacillus lentus alkaline protease (hereinafter BLAP) which has many industrial applications. The

20 fermentation is usually carried out, for example, in an aqueous solution with assimilable sources of carbon, hydrogen and nitrogen and a pH of 7 to 10 and a temperature range of from about 10-60° C. The BLAP enzyme is a polypeptide having 269 amino acid residues,

25 a molecular weight of 26,823 daltons and a calculated

* isoelectric point of 9.7. The amino acid sequence of this enzyme is described in copending application serial number 07/398,854 filed August 25, 1989, the disclosure of which is incorporated herein by reference. The organism B^ lentus DSM 5483 is on deposit with the American Type Culture Collection under ATCC number 53976 and Bacillus licheniformis under ATCC number 53926. Among the many industrial uses of BLAP there may be mentioned, for example as an ingredient in a detergent product. The enzyme thus produced has a distinct color and odor. These characteristics are therefore not desirable especially when the enzyme is to be incorporated into a detergent product. 2. Description Of The Related Art

Various approaches have been described for removing color or odor causing impurities from fermentation broths. German patent applications 3,911,099, 3,915,277, 3,917,645, and 3,930,284 describe adsorbents and precipitants, as well as masking and bleaching agents, for treating fermentation broths. However, none of them describe a liquid/liquid extraction process. Quaternary ammonium compounds (QUAT) have been used to extract amino acids from fermentation broth. There is for example, United States Patent 4,912,228 which teaches a process of extracting amino acids from aqueous solutions in which the amino acids are extracted with quaternary ammonium compounds. The amino acid containing solution is first treated with a tertiary a ine prior to extraction of the amino acid with the quaternary ammonium compound. This treatment results in the removal of impurities, or so called "poisons", from the aqueous solution.

Another example is U.S. Patent 4,661,606 which describes the purification of amino acid by extracting fermentation broth containing the desired amino acid with a water insoluble quaternary ammonium compound dissolved in a water immiscible organic solvent. U.S. Patent 4,886,888 discloses a process for the recovery of amino acid from aqueous mixtures. This process involves the treatment of the amino acid containing aqueous mixture prior to extraction of the amino acid with the water immiscible organic solution containing a water insoluble extractant. U.S. Patent 4,936,999 describes processes for recovering or stripping an amino acid from a water insoluble quaternary ammonium extractant on which the amino acid has been loaded. Finally, Applicants' copending application 07/490,122 filed March 6, 1990, allowed and the Issue Fee paid on July 31, 1991, describes a process of extracting an anion from an aqueous solution containing the anion utilizing a quaternary amine and a weak organic acid as the extractants.

SUMMARY OF THE INVENTION Broadly speaking, the present invention relates to a process useful for the concomitant decolorizing and deodorization of fermentation broth. In particular, it has been found that the fermentation broth from culturing certain Bacillus such as Bacillus lentus and Bacillus licheniformis has a distinct color and odor. This odor and color can be substantially reduced by treating the broth with certain strongly-basic extractants. The treatment results in the obtention of the desired proteolytic enzyme with little or no loss of its proteolytic activity. One of the features of this process is that the extractants can be recovered and reused. Generally, the extractants are recovered by stripping.

Suitable extractants are those which are compatible with the fermentation broth. Generally, they are soluble in water immiscible organic solvents and remain predominantly cationic at the pH of extraction and can be readily stripped. The extractants can be characterized as being "strongly-basic" compounds. Guanidines, including substituted guanidines, quaternary ammonium compounds (QUAT) and a combination of a QUAT and a weak acid are particularly advantageous to be employed in the process of this invention. "Modifiers" may be optionally included to aid the extraction process. Typically, they include water insoluble alcohol such as tridecanol.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, the microorganism is propagated in a fermentation broth by known methods. For example, the selected organism, e.g. a genetically transformed Bacillus licheniformis that is able to produce the alkaline protease of B^. lentus (BLAP) , is grown in a fermentation broth according to the teachings set forth in copending application serial number 07/398,854. When the growth of the organism has reached the stationary phase the fermentation is halted. The fermentation broth thus obtained has in the case of B. licheniformis the desired proteolytic enzyme (BLAP) . The fermentation broth contains unwanted color bodies as well as odor forming substances. In a typical practice of this invention to remove these unwanted color bodies and substances, the broth is treated with a strongly- basic extractant as described more fully below. Thus, the broth is allowed to come into intimate contact, e.g. by mixing, with a mixture of the selected strongly-basic extractant which has been previously dissolved in a water immiscible solvent. The treatment with the strongly- basic extractant is typically carried out at a pH which will not destabilize the enzyme and in the case of BLAP, a pH above 6, i.e. from about 6 to 10 and preferably at a pH of about 7 to 10. The extraction is preferably effected at ambient temperature, i.e. from about 18-20° C. The extraction results in the formation of two phases, i.e. an organic water immiscible phase and a decolorized and deodorized aqueous phase of the fermentation broth containing BLAP. The two phases are separated by procedure well known in the art. For example, separation may be by decanting as in a batch process. In a continuous process, the phases are separated by centrifugation or by flow through gravity settlers. The desired end product, e.g. BLAP, is recovered from the broth, clarified and processed by known procedures.

Prior to being recycled or returned for additional extraction purposes, the color and odor forming substances in the organic phase are stripped off. The stripping step is also a feature of the present process.

For guanidines or quat-weak acid extractants, which are pH-dependent, stripping may be done with either strongly acidic, i.e. at least 0.1 M strong acid (H₂S0₄,

HC1, etc), or strongly alkaline, i.e. 0.1 M base (NaOH, KOH, etc) aqueous solutions. Under strong acid conditions, the extracted substances are converted to their protonated non-ionic form, and go into the aqueous phase. Under strongly alkaline conditions, the extractant is deprotonated (becomes non-ionic) , and the extracted substances (having no cation in the organic phase to form an ion pair with) go into the aqueous phase.

The extractants useful in the present invention are those which are compatible with the media. They are soluble in water immiscible organic solvents and cationic at the pH of extraction. They are characterized as strongly-basic, meaning that the extractants remain predominantly cationic at pH values up to at least 10. These strongly basic extractants include for example guanidines having the structural formula (1) , quaternary ammonium compounds (QUAT) having the structural formula (2) and a combination of a water insoluble weak organic acid and the QUAT.

(2) (1)

In the above structural formulas, R_{l f} R₂, R₃, R and R₅ which could be equal or different each represents hydrogen or alkyl of sufficient length to render the resulting compounds immiscible with water and X is an anion, e.g. chloride sulfate, etc. Preferably R₁, R₂, R₃ and R₄ taken together have at least about 16 carbon atoms and more preferably 25 carbon atoms, preferably at least three of the R groups have at least 4 carbons. As stated

_SUBSTITUTESHEET above, the use of these compounds, i.e. the quaternary amines, and the guanidines as set forth above, are particularly advantageous since quaternary amines are always cationic and guanidines are basic enough to be protonated under the pH conditions where extraction occurs. On the other hand, weak bases such as tertiary amines would not be significantly protonated at pH 6 to 10 in an organic phase and would not be expected to be efficient extractants in the process of this invention. However, a combination of the QUAT and the water insoluble weak organic acid wherein the acid will provide an acidic portion and having a pKa as measured in water in the range of about 8 to 12 is particularly advantageous. These weak acids include for example alkylphenols, alkylnaphthols, hydroxyaryl aldehydes, alkyl-benzenesulfonomides, hydroxyaryl aldoximes, hydroxyarylketoximes, hydroxyaryl ketones, and beta- diketones. A typical weak acid is for example nonylphenol or dodecylphenol. This combination provides an extractant which is cationic at pH values up to at least 10, but which becomes a non-extracting ion pair under strongly-basic conditions.

The concentration of the extractant in the water immiscible solvent should be such that is sufficient to remove the color and odor forming substances. 1% w/v to

30% w/v is preferred and 10 to 20 % w/v most preferred.

As described above, the extractant is dissolved in a water immiscible solvent. These solvents are aliphatic and aromatic hydrocarbons. Aliphatic hydrocarbons such as alkanes including cycloalkanes and halogenated alkanes may be used. These aliphatic hydrocarbons having at least 5 carbon atoms in the chain are preferred. Low odor kerosene is especially preferred. The preferred halogenated alkanes have at least two carbon atoms. Typical aromatic hydrocarbons useful include benzenes. alkyl benzenes, e.g. toluene, xylene and so on. Other water immiscible solvents include water immiscible esters, ethers, alcohols and the like. These solvents may be employed alone or in admixtures. In particular any fluid of these substances with kerosene may be advantageously employed.

Modifiers may be optionally added to the solvent to enhance the extraction. Preferred modifiers are alcohols having 10 to 13 carbons. For example, mixtures of water immiscible alcohols such as tridecanol with hydrocarbon solvents such as kerosene may be employed to increase the solubility of the strongly-basic extractant in the organic phase.

As an illustration, a broth obtained from the fermentation of Bacillus lentus is treated with N,N'-bis- cyclohexyl-N"-tridecylguanidine ("BCTG") . The results obtained are set forth in Table 1 below.

150 g/1 Aliquat 336 10 0.015 8% 150 g/1 TDA in

Escaid 110 kerosene 5 0.162 85%

150 g/1 BCTG 10 0.031 16%

150 g/1 TDA in Escaid 110 kerosene 6 0.110 58% In Table 1, Aliquat™ 336 is methyltri(C₈C₁₀ alkyl) ammonium chloride available from Henkel Corp. The foregoing results indicate that substantial color reduction in BLAP fermentation broth was obtained as reflected by the reduced absorption at 440 nanometer. Concomitant with the observation of color reduction, the characteristic odor which is associated with the broth is also reduced.

The resulting organic phase can be readily stripped of the extracted substances. Thus, the organic phase is first clarified by centrifugation to remove suspended solids. The stripping of the loaded organic phases is illustrated in Table 2 set forth below.

TABLE 2: Stripping Of Loaded Organic Phases

Loaded Aqueous Absorption Color

Organic Strip at 440 nm (% of init)

Phase Solution (diluted)

150 g/1 BCTG None 0.167 100% 150 g/1 TDA in Escaid 110 kerosene (loaded with BLAP 10% NaOH broth at pH 10)

2N HC1 0%

150 g/1 Aliquat 336 None 0.048 100? 150 g/1 TDA in Escaid 110 kerosene (loaded with BLAP 2N HCl The following examples serve to illustrate, but not limit the invention. All parts and percentages are by weight, unless otherwise noted. In the examples, the fermentation broth used was the fermentation broth obtained by fermenting the Bacillus licheniformis ATCC 53926.

EXAMPLE 1 Removal of Color and Odor from a Protease Broth

Organic extractant solutions were prepared by dissolving 150g/L of tridecanol (available from Exxon) and specified quantities of either Aliquat™ 336 (tri-C₈_ ₁₀alkylmethyl quaternary ammonium chloride, available from Henkel Corp.) or N,N'-biscyclohexyl-N"- tridecylguanidine (BCTG) in Escaid 110 (available from Exxon Corp.) . BCTG was prepared by melting 981 g of 1,3- dicyclohexylcarbodiimide (available from Aldrich) and transferring to a 20 L Buchi Rotovap flask. A solution of 1928 g tridecylamine (mixed isomers, available from Fluka) in 612 g molten t-butanol was sucked from a second flask into the Buchi flask using vacuum. The second flask and the transfer line was washed by addition of another 600 g of molten t-butanol. Vacuum was released, and the contents were heated to 97°C while rotating for 2 1/2 hours. Vacuum was re-applied, stripping t-butanol to 55-60° at 10 mbar vacuum. The residue was distilled in two passes through a wiped film evaporator. The first pass distillate, at 600 microns pressure and 64° wall temperature, contained mostly amine and some product guanidine; the second pass distillate, at 90 microns pressure and 192° wall temperature, contained 1433 g of the product guanidine. Protease broth was obtained by fermentation of Bacillus licheniformis ATCC 53926 using complex media; the broth was dark brown with a strong characteristic odor. Broth was used either at its natural pH of 9, or after adjustment with acid to pH 8. A 10 ml portion of organic solution and a 10 ml portion of protease broth were added to a 2 oz. bottle and shaken in a reciprocating box at 120 cycles/minute for the indicated time. After shaking, the separated aqueous phase was centrifuged to remove traces of organic, and was analyzed for protease activity, color and odor. Protease activity was determined by standard methods, see R. Schϋlein, et al. Mol. Gen. Genet. (1991) 227:137-143, (standard deviation + 5%), and results are given as a percentage of the activity before extraction. Color intensity was determined by measuring absorbance at 440 mμ, and results are given as a percentage of the absorbance for the same sample before extraction. Odor was determined independently by a panel of two persons on randomly numbered samples. Odor scoring as follows: heavy - 4, medium = 3, light = 2, only kerosene odor = 1. Results given are an average of the two scores.

*QUAT = Aliquat™ 336; BCTG = N,N'-Biscyclohexyl-N"-tridecylguanidine

EXAMPLE 2 Organic Stripping and Recycle

Organic extractant solutions were prepared by dissolving

150 g/L of tridecanol and 150g/L of either Aliquat™ 336 or BCTG in Escaid 110. 50 m/L or organic and 50 ml of protease broth (pH adjusted to 6.4) were shaken for 5 minutes in horizontal bottles. The pH of each broth aqueous after extraction was determined. The loaded organic was separated and clarified by centrifugatio . 10 ml portions of clarified loaded organic were stripped by shaking for 5 minutes with either 10 ml of 2N HC1 or 10 ml of 10% NaOH. 5 ml of each stripped organic was then contacted with 5 ml of fresh protease broth for 5 minutes, and the phases centrifuged. Similarly, 5 ml of unstripped loaded organic was contacted with another 5 ml of fresh protease broth for 5 minutes, and the phases centrifuged. The color intensity of each phase was determined by measuring the absorbance at 440 mμ. Results for extracted aqueous phases are given as a percentage of the absorbance of the broth before extraction. Results for the color intensity of the organic phases are given as a percentage of the absorbance of the loaded organic.

TABLE 4: Organic Stripping and Recycle

First Second Loaded Stripped Extracted Extracte

Organic Strip Organic Broth Broth

Extractant* Color Agent Color pH Color pH Cblα

BCTG 100% 10 8% 10 9

2N HCL 0% 5 85 10% NaOH 0% 10 10

QUAT 100% 58% 6.5 51

2N HC1 0% 4.5 63 10% NaOH 127% 10 16

*QUAT = Aliquat™ 336; BCTG = N,N^f-biscyclohexyl-N"-tridecylguanidine

Claims

What is claimed is:

1. A process for the concomitant deodorizing and decolorizing of a fermentation broth containing color and odor forming substances resulting from fermenting a microorganism comprising a.) contacting said fermentation broth with a water insoluble strongly-basic extractant in a water immiscible organic solvent thereby forming a separate organic phase from the fermentation broth; b.) allowing the organic phase to be in contact with said fermentation broth for a sufficient period of time to allow for the extraction of the color and odor forming substances into said organic phase and; maintaining the pH of said fermentation broth between about 6 and 10; and c.) separating the organic phase from the fermentation broth.

2. A process according to claim 1 wherein said microorganism is a member selected from the genus Bacillus.

3. A process according to claim 2 wherein said microorganism is Bacillus licheniformis.

4. A process according to claim 2 wherein said microorganism is Bacillus lentus.

5. A process according to claim 1 wherein the strongly- basic extractant is a member selected from the group consisting of compounds of the formula

(2) (1)

wherein R_lf R₂, R₃, R₄ and R₅ which may be equal or different is a member selected from the group consisting of hydrogen or alkyl of sufficient carbon length to render the strongly-basic extractant immiscible with water, and X^" is an anion or a combination of said compound (2) with a water insoluble weak organic acid.

A process according to claim 1 wherein the fermentation broth after extraction has a pH from about 7 to 10.

SUBSTITUTE SHEET 7. A process according to claim 1 wherein said extractant is a substituted guanidine (1) in which the combined R groups have a total of at least 16 carbon atoms.

8. A process according to claim 7 wherein said extractant is N, N ' -bis-cyclohexy1-N"- tridecylguanidine.

9. A process according to claim 1 wherein said strongly-basic extractant is a quaternary amine (2) in which at least three of the R groups have at least 4 carbon atoms and the combined R groups have a total of at least 16 carbon atoms.

10. A process according to claim 1 wherein said strongly-basic extractant is a combination of a quaternary amine with a water insoluble organic weak acid.

11. A process according to claim 10 wherein said water insoluble organic weak acid is selected from the group consisting of nonylphenol and dodecylphenol.

12. A process according to claim 1 further comprising the steps of stripping the organic phase color and odor bodies into an aqueous stripping solution and recycling the organic phase to step a.

13. A process according to claim 12 wherein the strongly-basic extractant is a member selected from the group consisting of compounds of the formula

(2) (1)

wherein R_{l f} R₂, R₃, R₄ and R₅ which may be equal or different is a member selected form the group consisting of hydrogen or alkyl of sufficient carbon length to render the strongly-basic extractant immiscible with water, and X is an anion or a combination of said compound (2) with a water insoluble weak organic acid.

A process according to claim 13 wherein the strongly-basic extractant is selected from the

S_UB_STITUTESHEET group consisting of a substituted guanidine (1) and a combination of a quaternary amine with a water insoluble organic weak acid, and the aqueous stripping solution is a strong acid solution or a strong base solution.

15. A process according to claim 13 wherein the strongly-basic extractant is quaternary amine (2) and the aqueous stripping solution is a strong acid solution.

16. A process according to claim 13 wherein the aqueous stripping solution contains at least 0.1 M of a strong mineral acid.

17. A process according to claim 13 wherein the aqueous stripping solution contains at least 0.1 M of a strong base.

18. A process according to claim 1 wherein said extractant is employed at a range of about 1% w/v to 30% w/v.

19. A process according to claim 18 wherein said extractant is employed at 10 to 20% w/v.

20. A process according to claim 1 wherein a modifier selected from water immiscible alcohol is included in the extractant.

21. A process according to claim 20 wherein the alcohol is tridecanol.