WO2021216697A1 - Emulsion processing - Google Patents

Abstract

The present disclosure relates to methods for processing a fermentation broth, and more specifically, a fermentation broth comprising a stable emulsion. The present disclosure further describes effective methods for breaking emulsions that result in the efficient recovery of one or more product(s) of a fermentation process.

Description

IN THE UNITED STATES PATENT & TRADEMARK OFFICE PCT PATENT APPLICATION

EMULSION PROCESSING

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U. S. Provisional Application

No. 63/013,043 filed on April 21, 2020, which is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

[0002] The present disclosure relates to methods for processing a fermentation broth, and more specifically, a fermentation broth comprising a stable emulsion. The present disclosure further describes effective methods for breaking emulsions that result in the efficient recovery of one or more product(s) from a fermentation process.

BACKGROUND

[0003] Conventional methods for breaking emulsions, such as by addition of surfactants, adjustment of ionic strength, adjustment of pH, and adjustment of temperature have proven unsatisfactory in completely breaking stabilized emulsions generated during processes involving fermentation or fermentation-derived components, including processes for the production of nepetalactone. As a result, the purification of organic products from emulsions can be difficult and expensive. Accordingly, there remains a need in the art for more efficient and cost-effective methods for obtaining desired products from a fermentation broth.

SUMMARY

[0004] In one aspect, the present disclosure provides a method for processing a fermentation broth, the method comprising:

(a) centrifuging the fermentation broth; (b) removing water from the supernatant of step (a) (i.e., water removal step); and

(c) breaking an emulsion in the step (b) mixture (i.e., emulsion-breaking step), wherein the fermentation broth comprises an emulsion, an organic solvent (e.g., fatty acid methyl ester), water, biomass, and a terpene or terpenoid (such as a monoterpene, monoterpenoid, sesquiterpene, or sesquiterpenoid).

[0005] In some embodiments, the present disclosure provides a method for processing a fermentation broth, the method comprising:

(a) centrifuging the fermentation broth;

(b) removing water from the supernatant of step (a) (i.e., water removal step); and

(c) breaking an emulsion in the step (b) mixture (i.e., emulsion-breaking step), wherein the fermentation broth comprises an emulsion, an organic solvent, water, biomass, and nepetalactone.

[0006] In some embodiments, the water removal step (b) comprises filtering the supernatant of step (a) through a hydrophilic membrane.

[0007] In some embodiments, the emulsion-breaking step (c) comprises adding an adsorbent (e.g., a super absorbent polymer or silica-based adsorbent) to the retentate of the hydrophilic membrane filtration and filtering the absorbent-containing mixture.

[0008] In some embodiments, the emulsion-breaking step (c) comprises evaporating water from the retentate of the hydrophilic membrane filtration and filtering the solid- containing mixture.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Fig. 1 provides a schematic diagram showing a representative composition of a fermentation broth of the present disclosure.

[0010] Fig. 2 shows a method of the present disclosure for processing an emulsion, wherein the method includes filtering the supernatant through a hydrophilic membrane for aqueous removal and mixing the retentate with a super absorbent polymer (SAP)/silica- based adsorbent for emulsion breaking. [0011] Fig. 3 shows a method of the present disclosure for processing an emulsion, wherein the method includes filtering the supernatant through a hydrophilic membrane for aqueous removal and evaporation of water (batch or continuous) from the retentate for emulsion breaking.

[0012] Fig. 4 shows a method of the present disclosure for processing an emulsion, wherein the method includes filtering the supernatant through a hydrophilic membrane for aqueous removal and mixing the retentate with anhydrous salt for emulsion breaking.

[0013] Fig. 5 shows a method of the present disclosure for processing an emulsion, wherein the method includes filtering the supernatant through a hydrophilic membrane for aqueous removal and filtration of the retentate using a filter aid for emulsion breaking.

[0014] Fig. 6 shows a method of the present disclosure for processing an emulsion, wherein the method includes centrifugation of the supernatant for aqueous removal and mixing the light phase with super absorbent polymer (SAP)/silica-based adsorbent for emulsion breaking.

[0015] Fig. 7 shows a method of the present disclosure for processing an emulsion, wherein the method includes centrifugation of the supernatant for aqueous removal and evaporation of water (batch or continuous) from the light phase for emulsion breaking.

[0016] Fig. 8 shows a method of the present disclosure for processing an emulsion, wherein the method includes centrifugation of the supernatant for aqueous removal and mixing the light phase with anhydrous salt for emulsion breaking.

[0017] Fig. 9 shows a method of the present disclosure for processing an emulsion, wherein the method includes centrifugation of the supernatant for aqueous removal and filtration of the light phase using a filter aid for emulsion breaking.

[0018] Fig. 10 shows a method of the present disclosure for processing an emulsion, wherein the method includes membrane filtration (tangential or dead-end) of the supernatant for emulsion breaking and centrifugation of the permeate for aqueous removal.

[0019] Fig. 11 shows a method of the present disclosure for processing an emulsion, wherein the method includes tangential flow filtration (TFF) of the supernatant through a hydrophilic membrane for aqueous removal and emulsion-breaking along with filtration or centrifugation for solids removal.

[0020] Fig. 12 shows a method of the present disclosure for processing an emulsion, wherein the method includes tangential flow filtration (TFF) of the supernatant through a hydrophobic membrane for aqueous removal and emulsion-breaking.

[0021] Fig. 13 shows a schematic diagram for an embodiment of a tangential flow filtration process of the present disclosure.

Definitions

[0022] “Fermentation” as used herein, refers to a process in which a microorganism biochemically converts a carbon source (e.g., glucose, corn steep liquor, molasses, sugar cane juice, glycerol, ethanol, or sucrose) and sometimes a nitrogen source (e.g., ammonia, ammonium oxide, or nitrate salts) into a target molecule (e.g., monoterpenes/monoterpenoids and sesquiterpenes/sequiterpenoids) or a target macromolecule (such as enzymes).

[0023] “Aerobic fermentation” as used herein refers to a fermentation process that occurs with the presence of oxygen.

[0024] “Fermentation broth” as used herein, refers to a medium that comprises a biomass, a bioproduct (e.g., nepetalactone), water, organic solvent, and/or an emulsion. In embodiments of the present disclosure, the fermentation broth comprises an emulsion that is stabilized by a mixture of cell components (such as, proteins, lipids, and organelles), water, cell mass, and organic solvent, e.g., a fatty acid methyl ester (FAME).

[0025] The term “emulsion” as used here, refers to a stable mixture of two immiscible liquids, where a first liquid (the dispersed phase) is dispersed in the form of small droplets or particles in a second liquid (the continuous phase). The emulsions of the present disclosure result, for example, from processing a nepetalactone-containing fermentation broth with a solvent (such as, fatty acid methyl ester). Without being bound by any theory, the emulsions are thought to be stabilized by a mixture of cell components (such as, proteins lipids, and organelles), water, cell mass, and the solvent present in the fermentation broth. [0026] “Emulsion breaking”, as used herein, refers to the separation of a dispersed phase and continuous phase of an emulsion (e.g., an oil in water or water in oil emulsion) into separate phases. In some embodiments of the methods of the present disclosure, the separate phases comprise an organic phase and an aqueous phase.

DETAILED DESCRIPTION

[0027] The present disclosure relates to methods of processing of a terpene- or terpenoid-containing fermentation broth. In some embodiments, the terpene is a monoterpene or sesquiterpene and the terpenoid is a monoterpenoid or sesquiterpenoid. The fermentation broths described herein also contain a mixture of cell components (such as proteins, lipids, and organelles), water, and cell mass. It has been discovered that the aqueous portion (containing cell components that are water-soluble) of such broths often forms, in the presence of an organic solvent (e.g., a fatty acid methyl ester comprising methyl oleate), emulsions that are difficult to break using conventional methods. For example, the addition of surfactants, addition of salts, adjustment of pH, and adjustment of temperature have all proven unsuccessful in breaking the emulsion. The emulsions contain organic solvent, which entraps the terpene or terpenoid in the emulsion and reduces overall terpene or terpenoid recovery and process efficiency. Therefore, there is a need for methods of breaking emulsions in terpene- or terpenoid-containing fermentation broths to provide efficient recovery of terpene or terpenoid (e.g., nepetalactone) from an organic solvent such as FAME.

[0028] The present disclosure provides methods to break the emulsion by targeting one or more specific components of the emulsion to be removed in order to provide a broken emulsion.

[0029] In some embodiments, the emulsion of the present disclosure is a bio-based emulsion that is formed from mixtures that include water, an organic solvent (e.g., fatty acid methyl ester), and fermentation-derived components such as cells (in lysed or intact form), cell proteins, cell lipids, cell walls, plasmids, cell DNA, cell RNA, and cell organelles (e.g., nuclei, mitochondria, endoplasmic reticulum, Golgi apparatus, vacuoles, and ribosomes). Such emulsions can be formed in the fermentation step itself or after fermentation from fermentation-derived streams (such as supernatant, etc.). In some embodiments, the cells are yeast (e.g., Saccharomyces cerevisiae, Cryptococcus albidus, Candida albicans, etc.) or bacterial (e.g., Corynebacterium, Escherichia coli, Bacillus anthracis, etc.) cells.

[0030] In some embodiments, the emulsion of the present disclosure is a bio-based emulsion that is formed during an aerobic fermentation process in which an organic solvent is added to the fermentation broth before, during, or after fermentation.

[0031] In some embodiments, the emulsion of the present disclosure is a bio-based emulsion that is formed when significant lipid content (more than 1 g/L lipids in broth) is produced by the microorganism (yeast or bacterial) during an aqueous aerobic fermentation, and the cells are lysed either during or after the fermentation process.

[0032] In some embodiments, the emulsions disclosed herein are stable emulsions that resist breaking using conventional methods, such as breaking by the addition of surfactants, addition of salts, adjustment of pH, and/or adjustment of temperature. In some embodiments, a stable emulsion of the present disclosure is an emulsion that does not break when subjected to centrifugation at about 8000 RCF (relative centrifugal force) for about 30 minutes at about 15 to 25 °C. In some embodiments, a stable emulsion is an emulsion that does not break when subjected to centrifugation at about 8000 RCF for about 20 minutes at about 15 to 25 °C. In some embodiments, a stable emulsion is an emulsion that does not break when subjected to centrifugation at about 8000 RCF for about 10 minutes at about 15 to 25 °C.

[0033] In some embodiments, cell mass is physically removed from the emulsion through filtration at elevated pressures, with or without filter aid. In some embodiments, a hydrophobic membrane filtration is used to remove a fatty acid methyl ester (FAME) from the emulsion. In some embodiments, a hydrophilic membrane filtration is used to remove water from the emulsion. In some embodiments, the physical removal of water is by evaporation or through the addition of a water absorbent, such as anhydrous salts (e.g. potassium carbonate), super absorbent polymers (e.g. cross-linked sodium polyacrylate), or silica-based adsorbents (e.g. diatomaceous earth, perlite, and the like). [0034] In some embodiments, the present disclosure provides a method for processing a stable bio-based emulsion formed from mixtures that include water, an organic solvent (e.g., fatty acid methyl ester), and fermentation-derived components such as cells (in lysed or intact form), cell proteins, cell lipids, cell walls, plasmids, cell DNA, cell RNA, and cell organelles (e.g., nuclei, mitochondria, endoplasmic reticulum, Golgi apparatus, vacuoles, and ribosomes). In some embodiments, the emulsion is formed during the fermentation process to produce a fermentation broth comprising of an emulsion, an organic solvent, water, and biomass. In some embodiments, the emulsion is isolated from the fermentation broth and then broken. In some embodiments, a stable emulsion is an emulsion that resists breaking when centrifuged at about 8000 RCF for about 20 min at about 15 to 25°C.

[0035] In some embodiments, the present disclosure provides a method for processing a fermentation broth, the method comprising:

(a) centrifuging the fermentation broth;

(b) removing water from the supernatant of step (a) (i.e., water removal step); and

(c) breaking an emulsion in the step (b) mixture (i.e., emulsion-breaking step), wherein the fermentation broth comprises an emulsion, an organic solvent, water, biomass, and a terpene and/or terpenoid. In some embodiments, following breaking of the emulsion, the terpene and/or terpenoid is recovered from the organic solvent using methods that are known to those skilled (such as evaporation).

[0036] In some embodiments, the present disclosure provides a method for processing a fermentation broth, the method comprising:

(a) centrifuging the fermentation broth;

(b) removing water from the supernatant of step (a) (i.e., water removal step); and

(c) breaking an emulsion in the step (b) mixture (i.e., emulsion-breaking step), wherein the fermentation broth comprises an emulsion, an organic solvent, water, biomass, and a monoterpene, monoterpenoid, sesquiterpene, sesquiterpenoid or combination thereof. In some embodiments, following breaking of the emulsion, the monoterpene, monoterpenoid, sesquiterpene, sesquiterpenoid or combination thereof is recovered from the organic solvent using methods that are known to those skilled (such as evaporation).

[0037] In some embodiments, the present disclosure provides a method for processing a fermentation broth, the method comprising:

(a) centrifuging the fermentation broth;

(b) removing water from the supernatant of step (a) (i.e., water removal step); and

(c) breaking an emulsion in the step (b) mixture (i.e., emulsion-breaking step), wherein the fermentation broth comprises an emulsion, an organic solvent, water, biomass, and a monoterpene or sesquiterpene. In some embodiments, following breaking of the emulsion, the monoterpene or sesquiterpene is recovered from the organic solvent using methods that are known to those skilled (such as evaporation).

[0038] In some embodiments, the monoterpene is selected from the group consisting of 8-oxogeranial, 8-hydroxygeraniol, a-pinene, b-pinene, myrcene, a-ocimene, b- ocimene, b-citronelle, limonene, a-phellandrene, b-phellandrene, 3-carene, camphene, isocamphene, sabinene, terpinolene, a-terpinene, and b-terpinene.

[0039] In some embodiments, the monoterpenoid is selected from the group consisting of bornyl acetate, camphor, carvone, citral, citronellal, citronellol, geranial, geraniol, eucalyptol, hinokitiol, linalool, menthane, menthol, neral, thymol, nepetalic acid, and iridoids. In some embodiments, the monoterpenoid is an iridoid. In some embodiments, the iridoid is nepetalactone.

[0040] In some embodiments, the monoterpene or monoterpenoid is selected from the group consisting of borneol, camphor, carene, carvone, citrol, citronellal, citronellol, eucalyptol, fenchol, geraniol, geranial, limonene, linalool, halomon, menthol, myrcene, nepetalactone, nerol, ocimene, pinene, sabinene, terpinene, terpinolene, thujene, and derivatives thereof.

[0041] In some embodiments, the sesquiterpene is selected from the group consisting of farnescene, farnesane, b-bisobolene, a-zingiberene, a-humulene, germacrene d, elemane, xanthane, chamazulene, seslinene, valencene, eremophilane, himalachane, chamigrane, b-caryophyllene, selinene, b-elemene, bicyclogermacrene, thujopsene, aromadederene, and bourbonane.

[0042] In some embodiments, the sesquiterpenoid is selected from the group consisting of farnesol, b-nerediol, abscisic acid, bisobolol, geosmin, humulone, b-santalol, nootkatone, khusimone, zizanal, cyperol, caryophyllene oxide, a-murool, khushimol, patchoulol, aromadederene oxide, matricin, and santonin.

[0043] In some embodiments, the sesquiterpene or sesquiterpenoid is selected from the group consisting of farnesene, farnesol, nerolidol, bisabolene, bergamotene, zingiberene, humulene, santalol, caryophyllene, cadinene, artemisinin, alantolactone, chamazulene, nootkatone, khusimol, thujopsene, cedrol, patchoulol, germacrene A, elemene, bourbonene, gurjunene, aristolochene, selinene, valencene, vetispiradiene, ylangene, and derivatives thereof

[0044] In some embodiments, the methods of the present disclosure are useful in processing a fermentation broth comprising a monoterpene, monoterpenoid, sesquiterpene, or sesquiterpene produced from one or more microbes that are sensitive to monoterpenes, monoterpenoids, sesquiterpenes and sesquiterpenoids.

[0045] In some embodiments, the present disclosure provides a method for processing a fermentation broth, the method comprising:

(a) centrifuging the fermentation broth;

(b) removing water from the supernatant of step (a) (i.e., water removal step); and

(c) breaking an emulsion in the step (b) mixture (i.e., emulsion-breaking step), wherein the fermentation broth comprises an emulsion, an organic solvent, water, biomass, and nepetalactone. In some embodiments, following breaking of the emulsion, the nepetalactone is recovered from the organic solvent using methods that are known to those skilled (such as evaporation).

[0046] Nepetalactone is an organic compound, first isolated from the plant Nepeta cataria. It is a bicyclic monoterpenoid derived from isoprene comprising ten carbons and two fused rings. Nepetalactone has the following structural formula:

The 4aa,7a,7aa-nepetalactone shown above is the active isomer isolated from the plant, although a number of isomers are known, each of which is included in the present disclosure. In humans, nepetalactone is known to act as a mild sedative and anti-spasmodic agent. It also can be used as an insect repellant and as a stimulant for cats.

[0047] Terpenes (e.g., monoterpenes and sesquiterpenes) and terpenoids (e.g., monoterpenoids and sesquiterpenoids) can be produced on large scale by fermentation. In some embodiments, fermentation results in a broth comprising an emulsion, which makes efficient isolation of terpene or terpenoid difficult. Accordingly, the present disclosure provides efficient methods for processing emulsions to provide efficient recovery of a terpene or terpenoid from a fermentation broth. One example of a monoterpenoid is nepetalactone. Fermentation methods to produce nepetalactone- containing fermentation broths are described in PCT Publication No. WO 2020/264400, which is incorporated herein by reference in its entirety for all purposes.

[0048] In some embodiments of the present disclosure, the fermentation broth comprises about 1% to about 9% (vol./vol.%) of an organic solvent, including about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, or about 9%, including all ranges and values therebetween. In some embodiments, the organic solvent comprises a fatty acid ester. In some embodiments, the organic solvent comprises a fatty acid ester selected from the group consisting of stearic acid esters, oleic acid esters, palmitic acid esters, lauric acid esters, sebacic acid esters, and mixtures thereof. In some embodiments, the fatty acid ester comprises an ester selected from the group consisting of butyl oleate, methyl oleate, isopropyl myristate, and mixtures thereof. In some embodiments, the fatty acid ester comprises methyl oleate. In some embodiments, the organic solvent comprises a fatty acid methyl ester. In some embodiments, the fatty acid methyl ester solvent comprises methyl palmitate, methyl palmitoleate, methyl stearate, methyl linoleate, methyl alpha linolenate, methyl arachidate, methyl eisosenoate, or mixtures thereof.

[0049] In some embodiments of the present disclosure, the fermentation broth comprises about 1% to about 30% of an emulsion, including about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, or about 30%, and all ranges and values therebetween. In some embodiments of the present disclosure, the fermentation broth comprises about 1% to about 20% of an emulsion, including about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20%, and all ranges and values therebetween. In some embodiments, the emulsion comprises a mixture of water, organic solvent (e.g., FAME), and/or cell debris.

[0050] In some embodiments of the present disclosure, the fermentation broth comprises about 60% to about 85% of water, including about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, or about 80%, and all ranges and values therebetween. In some embodiments of the present disclosure, the fermentation broth comprises about 60% to about 75% of water, including about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, or about 75%, including all ranges and values therebetween. In some embodiments, the water includes dispersed emulsion. In some embodiments, the dispersed emulsion is about 0.1% to about 10% by volume of the water. In some embodiments, the dispersed emulsion is about 0.1% to about 5% by volume of the water. In some embodiments, the dispersed emulsion is about 1% to about 5% by volume of the water.

[0051] In some embodiments of the present disclosure, the fermentation broth comprises about 3% to about 30% of biomass, including about 3%, about 4.5%, about 6%, about 7.5%, about 9%, about 10.5%, about 12%, about 13.5%, about 15%, about 17.5%, about 18%, about 19.5%, about 21%, about 22.5%, about 24%, about 25.5%, about 27%, about 28.5%, or about 30%, including all ranges and values therebetween. In some embodiments, the biomass comprises cellular debris, trace water (less than about 1%), and/or trace emulsion (less than about 1%).

[0052] In some embodiments of the present disclosure, the fermentation broth comprises: about 1% to about 9% (vol./vol.%) of the organic solvent; about 1% to about 30% of the emulsion; about 60% to about 85% of water; and about 3% to about 30% of the biomass.

[0053] In some embodiments, the fermentation broth comprises: about 1% to about 9% (vol./vol.%) of the organic solvent; about 1% to about 20% of the emulsion; about 60% to about 75% of water; and about 3% to about 30% of the biomass.

[0054] Fig. 1 provides a schematic diagram of a fermentation broth with such a composition, as well as the layers that may be present.

[0055] The centrifugation process of step (a) can be carried out using any method known in the art, e.g., the processes disclosed in C. M. Todaro, “Centrifugation,” In: Henry C. Vogel and Celeste M. Todaro, Fermentation and Biochemical Engineering Handbook (Third Edition), William Andrew Publishing, 2014, pp. 267-281; S. O. Majekodunmi, “A Review on Centrifugation in the Pharmaceutical Industry,” Amer. J. of Biomed. Engin. , 2015, 5(2), pp. 67-78, or T. Beveridge, “Large-Scale Centrifugation,” In: I. D. Wilson, E. R. Adlard, M. Cooke and C. F. Poole, Encyclopedia of Separation Science, Academic Press, London, 2000, pp. 320-342., each of which is incorporated herein by reference in its entirety.

[0056] The centrifugation process of step (a) disclosed herein is typically used to separate sediment from the rest of the fermentation broth. In some embodiments, the sediment is a biomass (i.e., cellular fraction). In some embodiments, the biomass comprises trace amounts of water and emulsion. In some embodiments, the centrifuging the fermentation broth step (a) results in the formation of a biomass pellet, which is then separated from the rest of the fermentation medium to provide a supernatant.

[0057] Various types of centrifuges can be utilized in the disclosed method. In some embodiments, the centrifuging the fermentation broth step (a) is carried out in a hydrocyclone, tubular bowl centrifuge, chamber bowl centrifuge, imperforate basket centrifuge, decanter centrifuge or a disk stack centrifuge. In certain embodiments, the centrifuging the fermentation broth step (a) is carried out in a decanter centrifuge or a disk stack centrifuge. The centrifugation used in the methods of the present disclosure can be run in batch, intermittent, or continuous processes. When the centrifuging the fermentation broth step (a) is run as a continuous process, the fermentation broth can be fed into the centrifuge, and supernatant continuously removed. The continuous centrifugation processes for use in the methods disclosed herein provide efficient handling of the large volumes of fermentation broth often encountered in an industrial-scale fermentation process. In some embodiments, the centrifuging of step (a) processes from about 400 L/h to about 8,000 L/h of fermentation broth, including about 400 L/h, about 500 L/h, about 1,000 L/h, about 1,500 L/h, about 2,000 L/h, about 2,500 L/h, about 3,000 L/h, about 3,500 L/h, about 4,000 L/h, about 4,500 L/h, about 5,000 L/h, about 5,500 L/h, about 6,000 L/h, about 6,500 L/h, about 7,000 L/h, about 7,500 L/h, or about 8,000 L/h, including all ranges and values therebetween.

[0058] In some embodiments, the residence time of the fermentation broth in the centrifuge ranges from about 0.5 min (30 s) to about 10 min, including about 0.5 min, about 1 min, about 1.5 min, about 2 min, about 2.5 min, about 3 min, about 3.5 min, about 4 min, about 4.5 min, about 5 min, about 5.5 min, about 6 min, about 6.5 min, about 7 min, about 7.5 min, about 8 min about 8.5 min, about 9 min, about 9.5 min, or about 10 min, including all ranges and values therebetween. In some embodiments, the residence time of the fermentation broth in the centrifuge ranges from about 0.5 min (30 s) to about 3 min.

[0059] In some embodiments, the centrifuge rotates at about 200 rpm to about 3000 rpm. [0060] The centrifuging the fermentation broth step (a) can be carried out at any relative centrifugal force that is capable of providing a biomass sediment. In some embodiments, the centrifuging the fermentation broth step (a) is carried out at a relative centrifugal force ranging from about 1,000 RCF to about 15,000 RCF, including about

I,000, about 2,000, about 3,000 RCF, about 4,000 RCF, about 5,000 RCF, about 6,000 RCF, about 7,000 RCF, about 8,000 RCF, about 9,000 RCF, about 10,000 RCF, about

II,000 RCF, about 12,000 RCF, about 13,000 RCF, about 14,000 RCF, or about 15,000 RCF, including all ranges and values therebetween. In some embodiments, the relative centrifugal force ranges from 3,000 RCF to about 12,000 RCF.

[0061] After removing the biomass from the fermentation broth, the supernatant can be further processed by filtration or centrifugation to remove water according to step (b) of the disclosed process. In some embodiments, the water removal step (b) comprises filtering the supernatant of step (a) through a hydrophilic membrane. In some embodiments, the resulting retentate comprises organic solvent and nepetalactone. In some embodiments, the hydrophilic membrane comprises titanium dioxide (TiC ). In some embodiments, the hydrophilic membrane comprises silicon carbide. In some embodiments, the hydrophilic membrane comprises regenerated cellulose or sulfonated polysulfone. In some embodiments, the hydrophilic membrane comprises cellulose acetate, cellulose nitrate, polyethersulfone (PES), polyester (e.g., polyester track etch), or polycarbonate (e.g., polycarbonate track etch). In some embodiments, the hydrophilic membrane is a mixed cellulose ester membrane comprising cellulose acetate and cellulose nitrate. In some embodiments, the hydrophilic membrane comprises treated polytetrafluoroethylene (PTFE) or treated polyvinyl fluoride (PVDF). In some embodiments, the hydrophilic membrane is a nylon membrane. In some embodiments, the hydrophilic membrane is a water-permeable ceramic membrane, a spiral wound polymer membrane, or a hollow fiber membrane. In some embodiments, the hydrophilic membrane is a water-permeable ceramic membrane. In some embodiments, the hydrophilic membrane is a spiral wound polymer membrane. In some embodiments, the hydrophilic membrane is a hollow fiber membrane.

[0062] The hydrophilic membranes used in the membranes of the present disclosure separate water from the supernatant by allowing the aqueous layer and water-soluble components to pass through the filter material and be collected as permeate. In some embodiments, the hydrophilic membranes have a pore size of from about 300 Da to about 0.5 pm. In some embodiments, to facilitate filtration, a transmembrane pressure is provided. In some embodiments, the transmembrane pressure ranges from about 0 bar to about 10 bar, including about 0 bar, about 0.5 bar, about 1.0 bar, about 1.5 bar, about 2.0 bar, about 2.5 bar, about 3.0 bar, about 3.5 bar, about 4.0 bar, about 4.5 bar, about 5.0 bar, about 5.5 bar, about 6.0 bar, about 6.5 bar, about 7.0 bar, about 7.5 bar, about 8.0 bar, about 8.5 bar, about 9.0 bar, about 9.5 bar, or about 10.0 bar, including all ranges and values therebetween. In some embodiments, the transmembrane pressure ranges from about 1 bar to about 4 bar. In some embodiments, the resulting permeate comprises water-soluble proteins and/or water soluble salts. In some embodiments, the permeate comprises less than about 15%, less than about 10%, less than about 5%, or less than about 1% combined of the emulsion, dispersed emulsion, and organic solvent, e.g., FAME.

[0063] In some embodiments, the water removal step (b) comprises centrifuging the supernatant of step (a). The centrifugation for use in water removal step can be by any method known in the art, including those incorporated by reference above. In some embodiments, centrifuging the supernatant of step (a) is carried out at a relative centrifugal force ranging from about 3,000 RCF to about 15,000 RCF, including about 3,000 RCF, about 4,000 RCF, about 5,000 RCF, about 6,000 RCF, about 7,000 RCF, about 8,000 RCF, about 9,000 RCF, about 10,000 RCF, about 11,000 RCF, about 12,000 RCF, about 13,000 RCF, about 14,000 RCF, or about 15,000 RCF, including all ranges and values therebetween. In some embodiments, the relative centrifugal force ranges from 6,000 RCF to about 12,000 RCF.

[0064] In some embodiments, the residence time of the supernatant from step (a) in the centrifuge ranges from about 0.5 min (30 s) to about 10 min, including about 0.5 min, about 1 min, about 1.5 min, about 2 min, about 2.5 min, about 3 min, about 3.5 min, about 4 min, about 4.5 min, about 5 min, about 5.5 min, about 6 min, about 6.5 min, about 7 min, about 7.5 min, about 8 min about 8.5 min, about 9 min, about 9.5 min, or about 10 min, including all ranges and values therebetween. In some embodiments, the residence time of the supernatant of step (a) in the centrifuge ranges from about 0.5 min (30 s) to about 3 mm. [0065] In some embodiments, the centrifuging (a) processes from about 400 L/h to about 8,000 L/h of supernatant from step (a), including about 400 L/h, about, about 500 L/h, about 1000 L/h, about 1500 L/h, about 2000 L/h, about 2500 L/h, about 3000 L/h, about 3500 L/h, about 4000 L/h, about 4500 L/h, about 5000 L/h, about 5500 L/h, about 6000 L/h, about 6500 L/h, about 7000 L/h, about 7500 L/h, or about 8000 L/h, including all ranges and values therebetween.

[0066] In some embodiments, centrifuging the supernatant of step (a) provides a heavy phase and a light phase. In some embodiments, the heavy phase comprises water and dispersed emulsion. In some embodiments, the dispersed emulsion is present in about 1% to about 15% by weight of the heavy phase, including about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%, about 10.0%, about 10.5%, about 11.0%, about 11.5%, about 12.0%, about 12.5%, about 13.0%, about 13.5%, about 14.0%, about 14.5%, or about 15.0%, including all ranges and values therebetween. In some embodiments, the dispersed emulsion is present in trace amounts (e.g., less than 500 ppm, less than 400 ppm, less than 300 ppm, less than 200 ppm, less than 100 ppm, less than 50 ppm, less than 25 ppm, or less than 10 ppm). In some embodiments, the light phase comprises organic solvent and nepetalactone.

[0067] In some embodiments, after removing water from the supernatant of step (a)

(i.e., the water removal step), a retentate or light phase (when the water removal step is conducted using hydrophilic membrane filtration and centrifugation, respectively) is provided comprising emulsion, dispersed emulsion, nepetalactone, and/or organic solvent, e.g., a fatty acid ester including, but not limited to, butyl oleate, methyl oleate, and isopropyl myristate.

[0068] In some embodiments, the emulsion-breaking step (c) comprises adding an adsorbent to the retentate of the hydrophilic membrane filtration and filtering the absorbent-containing mixture (Fig. 2). In some embodiments, the adsorbent is selected from the group consisting of a super absorbent polymer (SAP), a silica-based adsorbent, or mixtures thereof. In some embodiments, the adsorbent is a SAP. In some embodiments, the adsorbent is a silica-based adsorbent.

[0069] In some embodiments, the emulsion-breaking step (c) comprises adding an adsorbent to the light phase of the centrifugation and filtering the absorbent-containing mixture (Fig. 6). In some embodiments, the adsorbent is selected from the group consisting of a super absorbent polymer (SAP), a silica-based adsorbent, or mixtures thereof. In some embodiments, the adsorbent is a super absorbent polymer (SAP). In some embodiments, the adsorbent is a silica-based adsorbent.

[0070] Super absorbent polymers are useful for their ability to absorb and retain large volumes of water and aqueous solutions. In some embodiments, the SAP is a polyelectrolyte having a high molecular mass, e.g., a molecular mass of 150,000 Dalton or more. In some embodiments, the polyelectrolyte is a cross-linked polyelectrolyte. In some embodiments, the SAP has an absorption capacity for water of at least 10-times its own mass. In some embodiments, the SAP has an absorption capacity for water of at least 5- times its own mass. In some embodiments, the SAP has an absorption capacity for water of at least 2-times its own mass. In some embodiments, the SAP has an absorption capacity of water of from about 2-times to about 10-times its own mass. In some embodiments, the SAP has an absorption capacity of water of from about 2-times to about 5-times its own mass. In some embodiments, the SAP has an absorption capacity for water of 2-times, 3- times, 4-times, or 5-times its own mass. In some embodiments, the absorption of water is non-reversible or substantially non-reversible. In some embodiments, the SAP (e.g., sodium polyacrylate) is coarse grade (e.g., having >90% between 300 mM and 1200 mM). In some embodiments, the SAP is intermediate grade (e.g., having >90% between 150 mM and 600 mM). In some embodiments, the SAP is fine grade (e.g., having >90% between 75 mM and 300 mM). In some embodiments, the SAP comprises particles ranging in size from 5 pm to greater than about 1200 pm. In some embodiments, the SAP comprises particles ranging in size from 75 pm to about 1200 pm. In some embodiments, the SAP comprises >90% of particles ranging in size from 75 pm to about 300 pm. In some embodiments, the SAP comprises >90% of particles ranging in size from 150 pm to about 600 pm. In some embodiments, the SAP comprises >90% of particles ranging in size from 300 pm to about 1200 pm. [0071] In some embodiments of the present disclosure, the SAP is poly(acrylic acid). In some embodiments, the SAP is a sodium polyacrylate polymer. In some embodiments, the SAP is a potassium polyacrylate polymer. In some embodiments, the SAP is crosslinked, e.g., crosslinked poly (aery lie acid), crosslinked sodium polyacrylate, or crosslinked potassium polyacrylate. In some embodiments, the SAP is a polyacrylate/polyacrylamide copolymer. In some embodiments, the SAP is a sodium acrylate/acrylamide copolymer. In some embodiments, the SAP is a copolymer of 2- propanamide and sodium propionate, and /V,/V’-methylene-bis-2-propanamide. In some embodiments, the 2-propanamide and sodium propionate are crosslinked with the N,N’- methylene-bis-2-propanamdide. In some embodiments, the SAP is a copolymer of N,N’- diethylacrylamide and sodium methacrylate. In some embodiments, the SAP comprises polyacrylamide copolymer, ethylene maleic anhydride copolymer, crosslinked carboxymethylcellulose, polyvinyl alcohol copolymers, crosslinked polyethylene oxide, or starch grafted copolymer of polyacrylonitrile.

[0072] In some embodiments of the present disclosure, the adsorbent is a silica- based adsorbent, such as diatomaceous earth (diatomite) or perlite.

[0073] The amount of adsorbent used in the present methods can be readily determined by persons skilled in art. The amount of adsorbent may be adjusted, for example, depending on the water content and relative amount of emulsion present in the treated mixture. The amount of adsorbent can also depend on the capacity of the particular adsorbent to remove water. Accordingly, in some embodiments, the amount of adsorbent added to either the retentate of the hydrophilic membrane filtration or the light phase of the centrifugation ranges from about 1% to about 20% w/v of adsorbent-containing mixture, including about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20%, including all ranges and values therebetween.

[0074] As shown in Figs. 2 and 6, the spent adsorbent (e.g., a SAP or silica-based absorbent) can be removed from the suspension by a filter press. In some embodiments, the spent adsorbent comprises water-soluble proteins, water-soluble salts, cell debris, and trace organic solvent (e.g., FAME). The filtrate comprises a terpene or terpenoid, e.g., nepetalactone, which can be isolated cleanly after concentration of the FAME.

[0075] In some embodiments, the emulsion-breaking step (c) comprises removing water from the retentate of the hydrophilic membrane filtration by evaporation and filtering the evaporated mixture (Fig. 3).

[0076] In some embodiments, the emulsion-breaking step (c) comprises removing water from the light phase of the centrifugation by evaporation and filtering the evaporated mixture (Fig. 7).

[0077] The evaporation of water from either the retentate, or the light phase of the centrifugation, can be by any method known in the art, including, but not limited to the methods disclosed in H. L. Freese, “Evaporation,” In: Henry C. Vogel and Celeste M. Todaro, Fermentation and Biochemical Engineering Handbook (Third Edition), William Andrew Publishing, 2014, pp. 239-265, which is incorporated herein by reference in its entirety.

[0078] In some embodiments, the evaporation of water from either the retentate, or the light phase of the centrifugation, is carried out using multi-effect evaporation, thermal vapor recompression, or mechanical vapor recompression. In some embodiments, the retentate comprises a mixture of emulsion, dispersed emulsion, nepetalactone, and organic solvent, e.g., FAME. In some embodiments, the light phase comprises a mixture of emulsion, a terpene or terpenoid, e.g., nepetalactone and organic solvent, e.g., FAME.

[0079] In the case of emulsion-breaking through water removal, the emulsion breaking step can be preceded with an aqueous removal step whereby the majority of free water (water not present in the emulsion) and its water-soluble components are removed from the broth first in order to increase efficiency and effectiveness of the emulsion breaking step. In some embodiments, the aqueous removal step is a centrifugation step in which the emulsion and free oil is separated from the aqueous phase. In some embodiments, such as when the aqueous phase contains dispersed emulsion, a hydrophilic membrane filtration is used to remove the majority of the free water before the remaining free oil and emulsion is sent to the emulsion-breaking step. [0080] In some embodiments, the evaporation for use in the methods of the present disclosure is a continuous evaporation process. In some embodiments, the continuous evaporation is conducted using a wiped film evaporator, a falling film evaporator, a forced circulation evaporator, or a plate evaporator. In some embodiments, the forced circulation evaporator is an internal pump forced circulation evaporator. In some embodiments, the plate evaporator is a plate stripping evaporator. In some embodiments, the continuous evaporation is carried out a flow rate ranging from about 25 L/h/m² to about 150 L/h/m², including about 25 L/h/m², about 45 L/h/m², about 60 L/h/m², about 75 L/h/m², about 90 L/h/m², about 105 L/h/m², about 120 L/h/m², about 135 L/h/m², or about 150 L/h/m², including all ranges and values therebetween.

[0081] In some embodiments, the evaporation for use in the methods of the present disclosure is a batch process. In some embodiments, the batch evaporation process occurs inside a reactor that is optionally connected to a vacuum system. In some embodiments, the reactor is an industrial-scale reactor.

[0082] In some embodiments, the continuous evaporation involves removing water by distillation. In some embodiments, the mixture that remains after removing the water (e.g., organic solvent comprising nepetalactone and solid material such as precipitated proteins, precipitate salts, and cell debris) is also removed continuously. In some embodiments, the mixture is then filtered using a filter press in order to separate the organic solvent comprising nepetalactone from the solid impurities.

[0083] In some embodiments, the evaporation is conducted at an absolute pressure of no more than 250 mbar. In some embodiments, the evaporation is conducted at an applied external temperature of from about 20 °C to about 150 °C, including about 20 °C, about 30 °C, about 40 °C, about 50 °C about 60 °C, about 70 °C, about 80 °C, about 90 °C, about 100 °C, about 110 °C, about 120 °C, about 130 °C, about 140 °C, or about 150 °C, including all ranges and values therebetween.

[0084] As shown in Figs. 3 and 7, the solids remaining after evaporation of water can be removed from the organic solvent suspension by a filter press. In some embodiments, the removed solids comprise precipitated proteins, precipitated salts, cell debris, and trace organic solvent (e.g., FAME). The filtrate comprises organic solvent and a terpene or terpenoid, e.g., nepetalactone. The terpene or terpenoid may be isolated using methods known to those skilled in the art, such as concentration of the organic solvent (e g., FAME).

[0085] In some embodiments, the emulsion-breaking step (c) comprises adding an anhydrous salt to the retentate of the hydrophilic membrane filtration and centrifuging the salt-containing mixture (Fig. 4).

[0086] In some embodiments, the emulsion-breaking step (c) comprises adding an anhydrous salt to the light phase of the centrifugation and centrifuging the salt-containing mixture (Fig. 8).

[0087] According to the methods of the present disclosure, anhydrous salts can be used to remove water remaining in either the retentate or light phase after the water- removal step (b) described above. The added salts can be mixed with retentate or light phase by any known method in the art suitable for agitation. In some embodiments, mixing is provided by a mechanical stirrer, such as a propeller, blade, or the like.

[0088] In some embodiments, the anhydrous salt is selected from the group consisting of calcium sulfate (CaSCH), magnesium sulfate (MgSCE), sodium sulfate (Na2S04), potassium carbonate (K2CO3), sodium carbonate (Na2CC ), calcium chloride (CaCh), or mixtures thereof. In some embodiments, the anhydrous salt is potassium carbonate.

[0089] The amount of anhydrous salt used in the present methods can be readily determined by persons skilled in art. The amount of anhydrous salt may be adjusted, for example, depending on the water content and relative amount of emulsion present in the treated mixture. The amount of anhydrous salt can also depend on the capacity of the particular salt to remove water. Accordingly, in some embodiments, the amount of anhydrous salt added to either the retentate of the hydrophilic membrane filtration or the light phase of the centrifugation ranges from about 1% to about 50% w/v of anhydrous salt- containing mixture, including about 2%, about 4%, about 6%, about 8%, about 10%, about 12%, about 14%, about 16%, about 18%, about 20%, about 22%, about 24%, about 26%, about 28%, about 30%, about 32%, about 34%, about 36%, about 38%, about 40%, about 42%, about 44%, about 46%, about 48%, or about 50%, including all ranges and values therebetween.

[0090] In some embodiments, the emulsion-breaking step (c) comprises adding a filter aid to the retentate of the hydrophilic membrane filtration and filtering the filter aid- containing mixture (Fig 5).

[0091] In some embodiments, the emulsion-breaking step (c) comprises adding a filter aid to the light phase of the centrifugation and filtering the filter aid-containing mixture (Fig. 9).

[0092] The filter aids used in the methods of the present disclosure can be used to prevent blockage or binding of the filter by the solids intended for removal. In some embodiments, filtration is carried out with a depth filter or a body-feed filter. In some embodiments, the filter aid is used to pre-coat the filter medium. The amount of filter aid used in the present methods can be readily determined by persons skilled in art. The amount of filter aid may be adjusted, for example, depending on the relative amount of emulsion present in the treated mixture.

[0093] In some embodiments, the filter aid for use in the present methods is selected from the group consisting of diatomaceous earth (dicalite, diatomite or Celite), perlite, cellulose-based filter aids (e.g., Jelucel), activated carbon, activated alumina, clays (e.g., acid-activated clay, Fuller’s earth, etc.), molecular sieves, anthracite, sand, and resins. In some embodiments, the filter aid is diatomaceous earth, perlite, or a cellulose-based filter aid. In some embodiments, the filter aid is diatomaceous earth (e.g., dicalite) or perlite. In some embodiments, the filter aid is a cellulose-based filter aid comprising potato starch particles or rice hull ash. In some embodiments, the filter aid is a flocculant. In some embodiments, the flocculant is a polymer. In some embodiments, the polymer is a polyelectrolyte, e.g., a cationic polyelectrolyte such as cationic starch, chitosan, and poly- y-glutamic acid.

[0094] In some embodiments, the process of the present disclosure further comprises centrifuging the filtrate from the emulsion break step (c). For example, as shown in Figs. 4, 5, 8, and 9, further centrifugation provides a heavy phase comprising water, water-soluble protein, water-soluble salts, and/or cell debris. The remaining light phase comprises a terpene or terpenoid, e.g., nepetalactone, and an organic solvent, which can be isolated by methods known to those skilled in the art, such as concentration of the organic solvent (e.g., FAME). In some embodiments, about 40% to 80% by weight of the total amount of FAME is recovered from the light phase, including about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or about 80% by weight, and all ranges and values therebetween.

[0095] In some embodiments, the present disclosure provides a method for processing a fermentation broth, the method comprising:

(a) centrifuging the fermentation broth; and

(b) breaking an emulsion in the supernatant of step (a) (i.e., emulsion-breaking step); wherein the fermentation broth comprises an emulsion, an organic solvent, water, biomass, and nepetalactone; and wherein the emulsion-breaking step comprises filtration of the supernatant of step (a) (see Fig. 10).

[0096] In some embodiments, filtration of the supernatant of step (a) comprises dead-end filtration. In some embodiments, the dead-end filtration is through a nonspecific membrane. In some embodiments, the dead-end filtration is used to remove cell debris from the supernatant. Accordingly, in some embodiments, after breaking the emulsion, the resulting filtrate comprises FAME, water, water-soluble proteins, and water-soluble salts.

[0097] In some embodiments, the method further comprises centrifuging the filtrate from dead-end filtration to remove water, water-soluble proteins, and water-soluble salts. In some embodiments, centrifuging the filtrate from the dead-end filtration provides a heavy phase comprising water, water-soluble proteins, and water-soluble salts and a light phase comprising FAME and a terpene or terpenoid, e.g., nepetalactone. The terpene or terpenoid can be recovered following removal of the organic solvent, e.g., FAME. In some embodiments, about 40% to 80% by weight of the total amount of FAME is recovered from the light phase, including about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or about 80% by weight, and all ranges and values therebetween. [0098] In some embodiments, filtration of the supernatant of step (a) comprises tangential flow filtration. Tangential flow filtration (TFF) is a process of separation that can be used for removal of water, cells, cellular debris, etc. from a fermentation broth. In TFF, a feed stream flows parallel to the membrane face (Fig. 13). Applied pressure causes one portion of the flow stream to pass through the membrane (filtrate) while the remainder (retentate) is recirculated back to the feed reservoir. Among the advantages of TFF is the reduction in membrane-fouling that can occur in a dead-end filtration process. Reducing membrane fouling can facilitate a process that involves continuous filtration of a fermentation broth disclosed herein.

[0099] In some embodiments, tangential flow filtration is through a nonspecific membrane. In some embodiments, tangential flow filtration is through a hydrophilic or hydrophobic membrane. In some embodiments, tangential flow filtration is used to remove cell debris from the supernatant. Accordingly, in some embodiments, after breaking the emulsion, the resulting permeate comprises FAME, water, water-soluble proteins, and water-soluble salts.

[0100] In some embodiments, the method further comprises centrifuging the permeate from tangential flow filtration to remove water, water-soluble proteins, and water-soluble salts. In some embodiments, centrifuging the permeate from the tangential flow filtration provides a heavy phase comprising water, water-soluble proteins, and water- soluble salts and a light phase comprising FAME and a terpene or terpenoid, e.g., nepetalactone. The terpene or terpenoid can be recovered following removal of the organic solvent, e.g., FAME. In some embodiments, about 40% to 80% by weight of the total amount of FAME is recovered from the light phase, including about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or about 80% by weight, and all ranges and values therebetween.

[0101] As noted above, after removal of the biomass, for example, using the methods described herein, the emulsion is processed by tangential flow filtration through a hydrophilic membrane. Accordingly, in some embodiments, the tangential flow filtration comprises filtration of the supernatant of step (a) through a hydrophilic membrane (Fig. 11). The hydrophilic membrane can be any such membrane known in the art, including those described above. In some embodiments that use TFF with a hydrophilic membrane, the water, any water-soluble proteins, and any water-soluble salts pass through the membrane, resulting in removal of the aqueous permeate. The retentate comprising the organic solvent, a terpene or terpenoid, e.g., nepetalactone, and cell debris is recirculated back to the reservoir for further processing.

[0102] In some embodiments, the TFF process disclosed herein further comprises filtering the retentate from the hydrophilic membrane filtration to remove cell debris and provide a filtrate comprising a terpene or terpenoid, e.g., nepetalactone. The terpene or terpenoid can be recovered following removal of the organic solvent, e.g., FAME. In some embodiments, about 40% to 80% by weight of the total amount of FAME is recovered from the retentate, including about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or about 80% by weight, and all ranges and values therebetween.

[0103] In some embodiments, the TFF process disclosed herein further comprises centrifuging the retentate from the hydrophilic membrane filtration to remove cell debris. In some embodiments, the resulting heavy phase comprising the cell debris is separated from the light phase comprising a terpene or terpenoid, e.g., nepetalactone. The terpene or terpenoid can be recovered following removal of the organic solvent, e.g., FAME. In some embodiments, about 40% to 80% by weight of the total amount of FAME is recovered from the light phase, including about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or about 80% by weight, and all ranges and values therebetween.

[0104] In some embodiments, the tangential flow filtration comprises filtration of the supernatant of step (a) through a hydrophobic membrane (Fig. 12). In some embodiments, the hydrophobic membrane comprises polytetrafluoroethylene (PTFE), polyvinyl fluoride (PVDF), or polypropylene (PF). In this case, the permeate comprises the bioproduct, e.g., nepetalactone, which can be recovered following removal of the organic solvent, e.g., FAME. In some embodiments, about 40% to 80% by weight of the total amount of FAME is recovered from the permeate, including about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or about 80% by weight, and all ranges and values therebetween.

[0105] In some embodiments, the hydrophobic membrane is a ceramic membrane, a spiral wound polymer membrane, or a hollow fiber membrane. In some embodiments, the hydrophobic membrane is a ceramic membrane. In some embodiments, the hydrophobic membrane is a spiral wound polymer membrane. In some embodiments, the hydrophobic membrane is a hollow fiber membrane.

[0106] In some embodiments, the hydrophobic membrane for use in the present methods has a pore size of from about 300 Da to about 0.5 pm. In some embodiments, to facilitate filtration, a transmembrane pressure is provided. In some embodiments, the transmembrane pressure ranges from about 0 bar to about 10 bar, including about 0 bar, about 0.5 bar, about 1 bar, about 1.5 bar, about 2 bar, about 2.5 bar, about 3 bar, about 3.5 bar, about 4 bar, about 4.5 bar, about 5 bar, about 5.5 bar, about 6 bar, about 6.5 bar, about 7 bar, about 7.5 bar, about 8 bar, about 8.5 bar, about 9 bar, about 9.5 bar, or about 10 bar, including all ranges and values therebetween.

[0107] In some embodiments, the method of processing a fermentation broth is the method disclosed in Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7, Fig. 8, Fig. 9, Fig. 10. Fig. 11, or Fig. 12. As described herein, these methods are useful for processing emulsions and enabling efficient recovery of one or more desired products (e.g., nepetalactone) from the fermentation broth.

EXAMPLES

[0108] The present disclosure is further illustrated by reference to the following

Examples. However, it is noted that these Examples, like the embodiments described above, are illustrative and are not to be construed as restricting the scope of the invention in any way.

[0109] Example 1: Processing Fermentation Broth Without Emulsion Breaking

(Comparative Example)

[0110] Biomass Removal [0111] 873 kg of fermentation broth, including fatty acid methyl ester (FAME), emulsion, an aqueous phase, biomass, and nepetalactone was processed through a solids- ejecting disc stack centrifuge in clarifier mode. The broth was fed at room temperature at 400 to 800 L/hr through the centrifuge, and 480 kg of supernatant was produced. The remaining 372 kg of sediment consisted of biomass, trace aqueous phase and trace emulsion.

[0112] Aqueous Removal with Centrifugation

[0113] The 372 kg of supernatant, consisting of FAME, aqueous, emulsion, and nepetalactone was further processed through a solids-ejecting disc stack centrifuge in concentrator mode at 400 to 1000 L/hr at room temperature. 33 kg of light phase, consisting of FAME and emulsion was produced. The remaining heavy phase, consisting of an aqueous phase with some finely dispersed emulsion, was removed as waste.

[0114] Draining

[0115] No emulsion-breaking was performed on the light phase, which was instead allowed to settle in 20 L buckets before 26 kg of nepetalactone-containing FAME was decanted from the top of the buckets. The remaining 7 kg of emulsion and aqueous phase were removed as waste.

Table 1.

[0116] Example 2: Emulsion Breaking with SAP (Aqueous Removal with

Hydrophilic Membrane)

[0117] Biomass Removal

[0118] 667 kg of fermentation broth, including fatty acid methyl ester (FAME), emulsion, an aqueous phase, biomass, and nepetalactone was processed through a solids- ejecting disc stack centrifuge in clarifier mode. The broth was fed at room temperature at 800 L/hr through the centrifuge, and 566 kg of supernatant was produced. The remaining 101 kg of sediment consisted of biomass, trace aqueous phase and trace emulsion.

[0119] Aqueous Removal with Hydrophilic Membrane

[0120] 399 kg of the supernatant, consisting of FAME, aqueous, emulsion, and nepetalactone was fed through a tangential flow filtration unit, which was fitted with 1.05 m² of 0.1 pm hydrophilic silicon carbide membranes. The operation was run at room temperature with a transmembrane pressure between 1 and 3 bar. 380 kg of aqueous permeate and 19 kg of retentate (emulsion and FAME) was produced.

[0121] Emulsion-Breaking with SAP

[0122] 2.8 g of >90% granular, cross-linked sodium polyacrylate, a super absorbent polymer (SAP), was added to 40.6 g of the retentate material in a 50 mL conical tube. The tube was vortexed at room temperature until the two components were well-mixed. The tube was then centrifuged at 4816 RCF for 5 minutes in a tabletop centrifuge installed with a swinging bucket rotor. 19.7 g of nepetalactone- containing free FAME was recovered alongside 23.7 g of solids.

Table 2.

[0123] Example 3: Emulsion Breaking by Evaporation (Aqueous Removal with

Hydrophilic Membrane)

[0124] Biomass Removal

[0125] 885 kg of fermentation broth, including fatty acid methyl ester (FAME), emulsion, an aqueous phase, biomass, and nepetalactone was processed through a solids- ejecting disc stack centrifuge in clarifier mode. The broth was fed at room temperature at 800 L/hr through the centrifuge, and 623 kg of supernatant was produced. The remaining 262 kg of sediment consisted of biomass, trace aqueous phase and trace emulsion.

[0126] Aqueous Removal with Hydrophilic Membrane

[0127] 584 kg of the supernatant, consisting of FAME, aqueous, emulsion, and nepetalactone was fed through a tangential flow filtration unit, which was fitted with 1.05 m² of 0.1 pm hydrophilic silicon carbide membranes. The operation was run at room temperature with a transmembrane pressure between 1 and 4 bar. 507 kg of aqueous permeate and 60 kg of retentate (emulsion and FAME) was produced.

[0128] Emulsion-Breaking with Evaporation

[0129] The 60 kg of retentate was added to an 80 L jacketed 316Ti stainless steel reactor. Evaporation was performed at a temperature between 50 and 120°C under a pressure of between 50 to 250 mbar until 27% was removed as condensate.

[0130] The concentrate from the process was removed and filtered through a filter press fitted with 3.7 L of chamber volume and pressurized with 0.5 to 1.5 bar air. 39 kg of nepetalactone-containing free FAME was recovered, and the remaining 3 kg of solids was removed as waste.

Table 3.

[0131] Example 4: Emulsion Breaking by Evaporation or Addition of Additives

(Aqueous Removal by Centrifugation)

[0132] Biomass Removal

[0133] 8200 kg of fermentation broth, including fatty acid methyl ester (FAME), emulsion, an aqueous phase, biomass, and nepetalactone was processed through a solids- ejecting disc stack centrifuge in clarifier mode. The broth was fed at room temperature at 1000 L/hr through the centrifuge, and 4950 kg of supernatant was produced. The remaining 3250 kg of sediment consisted of biomass, trace aqueous phase and trace emulsion.

[0134] Aqueous Removal with Centrifugation

[0135] The 4950 kg of supernatant, consisting of FAME, aqueous, emulsion, and nepetalactone was further processed through a solids-ejecting disc stack centrifuge in concentrator mode at 1000 L/hr at room temperature. 648 kg of light phase, consisting of FAME and emulsion was produced. The remaining heavy phase, consisting of an aqueous phase with some finely dispersed emulsion, was removed as waste.

[0136] Emulsion-Breaking with SAP

[0137] 0.96 g of >99% granular, cross-linked sodium polyacrylate was added to

23.36 g of light phase material in a 50 mL conical tube. The tube was vortexed at room temperature until the two components were well-mixed. The tube was then centrifuged at 10,000 RCF for 3 minutes in a floor-standing centrifuge installed with a fixed-angle rotor. 8.74 g of nepetalactone-containing free FAME was recovered, and the remaining 15.58 g of solids was removed as waste.

[0138] Emulsion-Breaking with Evaporation

[0139] 91.75 g of light phase material was added to a rotovap. Evaporation was performed at 40 to 50 Torr until 31% was removed in condensate. The bath temperature was between 55°C and 60°C. The concentrate from the process was removed and centrifuged in a floor-standing centrifuge at 10,000 RCF for 3 minutes. 25.82 g of nepetalactone-containing free FAME was recovered, and the remaining 37.25 g of solids was removed as waste.

[0140] Emulsion-Breaking with Anhydrous Salt

[0141] 7.91g of anhydrous potassium carbonate was added to 23.15 g of light phase material in a 50mL conical tube. The tube was vortexed at room temperature until the two components were well-mixed. The tube was then centrifuged at 10,000 RCF for 3 minutes in a floor-standing centrifuge installed with a fixed-angle rotor. 20.13 g of aqueous liquid was discarded as waste. 10.93 g of the resulting liquid was nepetalactone-containing free FAME.

[0142] Emulsion-Breaking with Filter Aid

[0143] 0.88 g of Dicalite 4408 filter aid was added to 45 mL of de-ionized water in a 50 mL conical tube to form a pre-coat mixture. The mixture was poured into a high- pressure membrane cell fitted with 12-25 pm filter paper, and 3 bar air was applied to the cell to form the pre-coat. 1.43 g of the filter aid was added to 55.89 g of the light phase material. The light phase mixture was added before applying 3 to 5 bar air to the cell. The process was run at room temperature, and the resulting filtrate consisted of 4.59 g of nepetalactone-containing free FAME. 58.2 g of remaining material was removed as waste.

Table 4.

[0144] Example 5: Emulsion Breaking using a Hydrophilic Membrane

[0145] Biomass Removal

[0146] 399 kg of fermentation broth, including fatty acid methyl ester (FAME), emulsion, an aqueous phase, biomass, and nepetalactone was processed through a continuous solids-discharging disc stack centrifuge. The broth was fed at room temperature at 300 to 450 L/hr through the centrifuge. 163 kg of supernatant, consisting of FAME, aqueous, emulsion, and nepetalactone was produced. The remaining 266 kg of waste consisted of biomass, an aqueous phase and trace emulsion. [0147] Emulsion-Breaking with Membrane Filtration

[0148] 399 kg of the supernatant was fed through a tangential flow filtration unit, which was fitted with 1.05 m² of 0.08 pm silicon carbide membranes. The operation was run at room temperature with a transmembrane pressure between 0.5 and 2 bar. 12 kg of retentate was recovered alongside the permeate. 23 kg of nepetalactone-containing free FAME was recovered from the permeate.

Table 5.

[0149] Example 6: Emulsion Breaking using Multiple Hydrophilic Membranes

[0150] Biomass Removal

[0151] 45 kg of fermentation broth, including fatty acid methyl ester (FAME), emulsion, an aqueous phase, biomass, and nepetalactone was processed through a solids- ejecting disc stack centrifuge in clarifier mode. The broth was fed at room temperature at 100 to 120F/hr through the centrifuge, and 35 kg of supernatant was produced. The remaining 10 kg of sediment consisted of biomass, trace aqueous phase and trace emulsion.

[0152] Emulsion-Breaking with Hydrophilic Membrane

[0153] The 35 kg of supernatant, consisting of FAME, aqueous, emulsion, and nepetalactone was fed through a tangential flow filtration unit, which was fitted with 1.05 m² of 0.1 pm hydrophilic silicon carbide membranes. The collected retentate was further processed through a 0.14 pm titanium oxide membrane of 0.032 m². The filtrations were run at room temperature with a transmembrane pressure of between 1 and 5 bar. 47 kg of aqueous permeate and 5 kg of retentate (consisting of cell debris and free FAME) were produced. 4 kg of free FAME was recovered from the retentate after filtering through a Buchner funnel. Table 6.

[0154] Numbered Embodiments of the Disclosure

[0155] Other subject matter contemplated by the present disclosure is set out in the following numbered embodiments:

[0156] 1. A method for processing a fermentation broth, the method comprising:

(a) centrifuging the fermentation broth;

(b) removing water from the supernatant of step (a); and

(c) breaking an emulsion in the step (b) mixture, wherein the fermentation broth comprises an emulsion, an organic solvent, water, biomass, and/or terpene or terpenoid.

[0157] 2. The method of embodiment 1, wherein the emulsion is formed during a fermentation process.

[0158] 3. The method of embodiment 1, wherein the emulsion is formed after a fermentation process.

[0159] 4. The method of any one of embodiments 1-3, wherein the terpene is a monoterpene or sesquiterpene and the terpenoid is a monoterpenoid or sesquiterpenoid.

[0160] 5. The method of embodiment 4, wherein the monoterpene is selected from the group consisting of 8-oxogeranial, 8-hydroxygeraniol, a-pinene, b-pinene, myrcene, a- ocimene, b-ocimene, b-citronelle, limonene, a-phellandrene, b-phellandrene, 3-carene, camphene, isocamphene, sabinene, terpinolene, a-terpinene, and b-terpinene.

[0161] 6. The method of embodiment 4 or 5, wherein the monoterpenoid is selected from the group consisting of bornyl acetate, camphor, carvone, citral, citronellal, citronellol, geranial, geraniol, eucalyptol, hinokitiol, linalool, menthane, menthol, neral, thymol, nepetalic acid, and iridoids. [0162] 7. The method of embodiment 6, wherein the iridoid is nepetalactone or nepetalactol.

[0163] 8. The method of any one of embodiments 4-7, wherein the sesquiterpene is selected from the group consisting of farnescene, farnesane, b-bisobolene, a-zingiberene, a-humulene, germacrene d, elemane, xanthane, chamazulene, seslinene, valencene, eremophilane, himalachane, chamigrane, b-caryophyllene, selinene, b-elemene, bicyclogermacrene, thujopsene, aromadederene, and bourbonane.

[0164] 9. The method of any one of embodiments 4-8, wherein the sesquiterpenoid is selected from the group consisting of farnesol, b-nerediol, abscisic acid, bisobolol, geosmin, humulone, b-santalol, nootkatone, khusimone, zizanal, cyperol, caryophyllene oxide, a-murool, khushimol, patchoulol, aromadederene oxide, matricin, and santonin.

[0165] 10. The method of any one of embodiments 1-9, wherein the fermentation broth comprises an emulsion, an organic solvent, water, biomass, and/or nepetalactone.

[0166] 11. The method of any one of embodiments 1-10, wherein the organic solvent comprises a fatty acid ester.

[0167] 11a. The method of embodiment 11, wherein the organic solvent comprises a fatty acid methyl ester.

[0168] 12. The method of embodiment 11, wherein the fatty acid ester is selected from the group consisting of butyl oleate, methyl oleate and isopropyl myristate, and mixtures thereof.

[0169] 13. The method of embodiment 11 or 12, wherein the fatty acid ester comprises methyl oleate.

[0170] 14. The method of any one of embodiments 1-13, wherein the fermentation broth comprises: about 1% to about 9% (vol./vol.%) of the organic solvent; about 1% to about 30% of the emulsion; about 60% to about 85% of water; and about 3% to about 30% of the biomass. [0171] 15. The method of any one of embodiments 1-14, wherein the emulsion comprises a mixture of water, organic solvent, and fermentation-derived components.

[0172] 16. The method of embodiment 15, wherein the fermentation-derived components comprise cell proteins, cell lipids, cell walls, plasmids, cell DNA, cell RNA, and/or cell organelles.

[0173] 17. The method of any one of embodiments 1-16, wherein the emulsion is a stable emulsion that resists breaking when centrifuged at about 8000 RCF for about 20 min at 15- 25°C.

[0174] 18. The method of any one of embodiments 1-17, wherein the water removal step (b) comprises filtering the supernatant of step (a) through a hydrophilic membrane.

[0175] 19. The method of any one of embodiments 1-17, wherein the water removal step (b) comprises centrifuging the supernatant of step (a).

[0176] 20. The method of embodiment 18, wherein the emulsion-breaking step (c) comprises adding an adsorbent to the retentate of the hydrophilic membrane filtration and filtering the absorbent-containing mixture.

[0177] 21. The method of embodiment 19, wherein the emulsion-breaking step (c) comprises adding an adsorbent to the light phase of the centrifugation and filtering the absorbent-containing mixture.

[0178] 22. The method of embodiment 20 or 21, wherein the adsorbent is selected from the group consisting of a super absorbent polymer (SAP), a silica-based adsorbent, and mixtures thereof.

[0179] 23. The method of any one of embodiments 20-22, wherein the adsorbent is a SAP.

[0180] 24. The method of embodiment 22 or 23, wherein the SAP is cross-linked sodium polyacrylate.

[0181] 25. The method of embodiment 22, wherein the silica-based adsorbent is selected from the group consisting of diatomaceous earth and perlite. [0182] 26. The method of embodiment 18, wherein the emulsion-breaking step (c) comprises removing water from the retentate of the hydrophilic membrane filtration by evaporation and filtering the evaporated mixture.

[0183] 27. The method of embodiment 19, wherein the emulsion-breaking step (c) comprises removing water from the light phase of the centrifugation by evaporation and filtering the evaporated mixture.

[0184] 28. The method of embodiment 26 or 27, wherein the evaporation is a batch process.

[0185] 29. The method of embodiment 28, wherein the batch process is conducted using a batch reactor.

[0186] 30. The method of embodiment 26 or 27, wherein the evaporation is a continuous process.

[0187] 31. The method of embodiment 30, wherein the continuous process is conducted using a wiped film evaporator, a falling film evaporator, a forced circulation evaporator, or a plate evaporator.

[0188] 32. The method of embodiment 18, wherein the emulsion-breaking step (c) comprises adding an anhydrous salt to the retentate of the hydrophilic membrane filtration and centrifuging the salt-containing mixture.

[0189] 33. The method of embodiment 19, wherein the emulsion-breaking step (c) comprises adding an anhydrous salt to the light phase of the centrifugation and centrifuging the salt-containing mixture.

[0190] 34. The method of embodiment 32 or 33, wherein the anhydrous salt is potassium carbonate.

[0191] 35. The method of embodiment 18, wherein the emulsion-breaking step (c) comprises adding a filter aid to the retentate of the hydrophilic membrane filtration and centrifuging the filter aid-containing mixture. [0192] 36. The method of embodiment 19, wherein the emulsion-breaking step (c) comprises adding a filter aid to the light phase of the centrifugation and centrifuging the filter aid-containing mixture.

[0193] 37. The method of embodiment 35 or 36, wherein the filter aid is selected from the group consisting of diatomaceous earth, perlite, or cellulose.

[0194] 38. The method of any one of embodiments 35-37, further comprising centrifuging the filtrate from the emulsion-breaking step.

[0195] 39. A method for processing a fermentation broth, the method comprising:

(a) centrifuging the fermentation broth; and

(b) breaking an emulsion in the supernatant of step (a), wherein the fermentation broth comprises an emulsion, an organic solvent, water, biomass, and nepetalactone and wherein the emulsion-breaking step comprises filtration of the supernatant of step (a).

[0196] 40. The method of embodiment 39, wherein the emulsion is formed during a fermentation process.

[0197] 41. The method of embodiment 39, wherein the emulsion is formed after a fermentation process.

[0198] 42. The method of any one of embodiments 39-41, wherein filtration of the supernatant of step (a) comprises dead-end filtration.

[0199] 43. The method of embodiment 42, further comprising centrifuging the filtrate from dead-end filtration.

[0200] 44. The method of any one of embodiments 39-41, wherein filtration of the supernatant of step (a) comprises tangential flow filtration.

[0201] 45. The method of embodiment 44, further comprising centrifuging the permeate from tangential flow filtration.

[0202] 46. The method of embodiment 44, wherein the tangential flow filtration comprises filtration of the supernatant of step (a) through a hydrophobic membrane. [0203] 47. The method of embodiment 44, wherein the tangential flow filtration comprises filtration of the supernatant of step (a) through a hydrophilic membrane.

[0204] 48. The method of embodiment 47, further comprising centrifuging the retentate from hydrophilic membrane filtration.

[0205] 49. The method of embodiment 47, further comprising filtering the retentate from hydrophilic membrane filtration.

INCORPORATION BY REFERENCE

[0206] The entire disclosure of each of the patent documents and scientific articles referred to herein is incorporated by reference for all purposes.

EQUIVALENTS

[0207] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and the range of equivalency of the claims are intended to be embraced therein.

Claims

WHAT WE CLAIM IS:

1. A method for processing a fermentation broth, the method comprising:

(a) centrifuging the fermentation broth;

(b) removing water from the supernatant of step (a); and

(c) breaking an emulsion in the step (b) mixture, wherein the fermentation broth comprises an emulsion, an organic solvent, water, biomass, and/or terpene or terpenoid.

2. The method of claim 1, wherein the emulsion is formed during a fermentation process.

3. The method of claim 1, wherein the emulsion is formed after a fermentation process.

4. The method of any one of claims 1-3, wherein the terpene is a monoterpene or sesquiterpene and the terpenoid is a monoterpenoid or sesquiterpenoid.

5. The method of claim 4, wherein the monoterpene is selected from the group consisting of 8-oxogeranial, 8-hydroxygeraniol, a-pinene, b-pinene, myrcene, a- ocimene, b-ocimene, b-citronelle, limonene, a-phellandrene, b-phellandrene, 3- carene, camphene, isocamphene, sabinene, terpinolene, a-terpinene, and b- terpinene.

6. The method of claim 4 or 5, wherein the monoterpenoid is selected from the group consisting of bornyl acetate, camphor, carvone, citral, citronellal, citronellol, geranial, geraniol, eucalyptol, hinokitiol, linalool, menthane, menthol, neral, thymol, nepetalic acid, and iridoids.

7. The method of claim 6, wherein the iridoid is nepetalactone or nepetalactol.

8. The method of any one of claims 4-7, wherein the sesquiterpene is selected from the group consisting of farnescene, farnesane, b-bisobolene, a-zingiberene, a- humulene, germacrene d, elemane, xanthane, chamazulene, seslinene, valencene, eremophilane, himalachane, chamigrane, b-caryophyllene, selinene, b-elemene, bicyclogermacrene, thujopsene, aromadederene, and bourbonane.

9. The method of any one of claims 4-8, wherein the sesquiterpenoid is selected from the group consisting of farnesol, b-nerediol, abscisic acid, bisobolol, geosmin, humulone, b-santalol, nootkatone, khusimone, zizanal, cyperol, caryophyllene oxide, a-murool, khushimol, patchoulol, aromadederene oxide, matricin, and santonin.

10. The method of any one of claims 1-9, wherein the fermentation broth comprises an emulsion, an organic solvent, water, biomass, and/or nepetalactone.

11. The method of any one of claims 1-10, wherein the organic solvent comprises a fatty acid ester.

12. The method of claim 11, wherein the fatty acid ester is selected from the group consisting of butyl oleate, methyl oleate and isopropyl myristate, and mixtures thereof.

13. The method of claim 11 or 12, wherein the fatty acid ester comprises methyl oleate.

14. The method of any one of claims 1-13, wherein the fermentation broth comprises: about 1% to about 9% (vol./vol.%) of the organic solvent; about 1% to about 30% of the emulsion; about 60% to about 85% of water; and about 3% to about 30% of the biomass.

15. The method of any one of claims 1-14, wherein the emulsion comprises a mixture of water, organic solvent, and fermentation-derived components.

16. The method of claim 15, wherein the fermentation-derived components comprise cell proteins, cell lipids, cell walls, plasmids, cell DNA, cell RNA, and/or cell organelles.

17. The method of any one of claims 1-16, wherein the emulsion is a stable emulsion that resists breaking when centrifuged at about 8000 RCF for about 20 min at 15- 25°C.

18. The method of any one of claims 1-17, wherein the water removal step (b) comprises filtering the supernatant of step (a) through a hydrophilic membrane.

19. The method of any one of claims 1-17, wherein the water removal step (b) comprises centrifuging the supernatant of step (a).

20. The method of claim 18, wherein the emulsion-breaking step (c) comprises adding an adsorbent to the retentate of the hydrophilic membrane filtration and filtering the absorbent-containing mixture.

21. The method of claim 19, wherein the emulsion-breaking step (c) comprises adding an adsorbent to the light phase of the centrifugation and filtering the absorbent-containing mixture.

22. The method of claim 20 or 21, wherein the adsorbent is selected from the group consisting of a super absorbent polymer (SAP), a silica-based adsorbent, and mixtures thereof.

23. The method of any one of claims 20-22, wherein the adsorbent is a SAP.

24. The method of claim 22 or 23, wherein the SAP is cross-linked sodium polyacrylate.

25. The method of claim 22, wherein the silica-based adsorbent is selected from the group consisting of diatomaceous earth and perlite.

26. The method of claim 18, wherein the emulsion-breaking step (c) comprises removing water from the retentate of the hydrophilic membrane filtration by evaporation and filtering the evaporated mixture.

27. The method of claim 19, wherein the emulsion-breaking step (c) comprises removing water from the light phase of the centrifugation by evaporation and filtering the evaporated mixture.

28. The method of claim 26 or 27, wherein the evaporation is a batch process.

29. The method of claim 28, wherein the batch process is conducted using a batch reactor.

30. The method of claim 26 or 27, wherein the evaporation is a continuous process.

31. The method of claim 30, wherein the continuous process is conducted using a wiped film evaporator, a falling film evaporator, a forced circulation evaporator, or a plate evaporator.

32. The method of claim 18, wherein the emulsion-breaking step (c) comprises adding an anhydrous salt to the retentate of the hydrophilic membrane filtration and centrifuging the salt-containing mixture.

33. The method of claim 19, wherein the emulsion-breaking step (c) comprises adding an anhydrous salt to the light phase of the centrifugation and centrifuging the salt-containing mixture.

34. The method of claim 32 or 33, wherein the anhydrous salt is potassium carbonate.

35. The method of claim 18, wherein the emulsion-breaking step (c) comprises adding a filter aid to the retentate of the hydrophilic membrane filtration and centrifuging the filter aid-containing mixture.

36. The method of claim 19, wherein the emulsion-breaking step (c) comprises adding a filter aid to the light phase of the centrifugation and centrifuging the filter aid-containing mixture.

37. The method of claim 35 or 36, wherein the filter aid is selected from the group consisting of diatomaceous earth, perlite, or cellulose.

38. The method of any one of claims 35-37, further comprising centrifuging the filtrate from the emulsion-breaking step.

39. A method for processing a fermentation broth, the method comprising:

(a) centrifuging the fermentation broth; and

(b) breaking an emulsion in the supernatant of step (a), wherein the fermentation broth comprises an emulsion, an organic solvent, water, biomass, and nepetalactone and wherein the emulsion-breaking step comprises filtration of the supernatant of step (a).

40. The method of claim 39, wherein the emulsion is formed during a fermentation process.

41. The method of claim 39, wherein the emulsion is formed after a fermentation process.

42. The method of any one of claims 39-41, wherein filtration of the supernatant of step (a) comprises dead-end filtration.

43. The method of claim 42, further comprising centrifuging the filtrate from dead end filtration.

44. The method of any one of claims 39-41, wherein filtration of the supernatant of step (a) comprises tangential flow filtration.

45. The method of claim 44, further comprising centrifuging the permeate from tangential flow filtration.

46. The method of claim 44, wherein the tangential flow filtration comprises filtration of the supernatant of step (a) through a hydrophobic membrane.

47. The method of claim 44, wherein the tangential flow filtration comprises filtration of the supernatant of step (a) through a hydrophilic membrane.

48. The method of claim 47, further comprising centrifuging the retentate from hydrophilic membrane filtration.

49. The method of claim 47, further comprising filtering the retentate from hydrophilic membrane filtration.