WO2010115074A1

WO2010115074A1 - Purification methods for bio-organic compounds

Info

Publication number: WO2010115074A1
Application number: PCT/US2010/029740
Authority: WO
Inventors: Pinar Tabur; Glenn Dorin
Original assignee: Amyris Biotechnologies, Inc.
Priority date: 2009-04-02
Filing date: 2010-04-02
Publication date: 2010-10-07
Also published as: WO2010115074A8

Abstract

Processes and systems for the purification of bio-organic compounds arc described. In certain embodiments, the purification method comprises adjusting the pH of the composition to a basic pH. In further embodiments, the purification method optionally comprises neutralizing the composition to reduce the total acid number to less than about 0.5 mg KOH/g of bio-organic compound.

Description

PURIFICATION METHODS FOR BIO-ORGANIC COMPOUNDS

RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional application no. 61/166,167 filed April 2, 2009. The disclosure of the above referenced application is incorporated by reference herein in its entirety.

FIELD

[0002] Provided herein are methods for purification of microbial-derived organic compounds.

BACKGROUND

[0003] Petroleum-derived compounds and compositions are found in a variety of products ranging from plastics to household cleaners as well as fuels. Given the environmental impact of these compositions, there is an increasing demand for more renewable and sustainable alternatives.

[0004] With recent advances in metabolic engineering, biology is providing viable alternatives to petroleum-derived compounds and compositions. For example, isoprenoids comprise a diverse class of compounds with over 50,000 members, and have a variety of uses including as specialty chemicals, pharmaceuticals and even fuels. Most isoprenoid compounds conventionally have been synthesized from petroleum sources or extracted from plant sources. Now, a third option exists which is capable of making a desired isoprenoid compound using microbial cells. Systems for making petroleum-derived compounds and compositions have been described, for example, by U.S. Patent No. 7,399,323; U.S. Patent Publication No. 2008/0274523; and PCT Publication Nos. WO 2007/140339, WO 2008/140492, WO 2008/133658, and WO 2009/014636.

[0005] However, in order for a microbial-derived compound to be competitive, it should be made more cost effectively than a comparable compound obtained from naturally occurring sources. As a result, methods for obtaining the most optimal yield of a desired compound are needed. Such methods are provided herein.

SUMMARY

[0006] Provided herein are purification methods for isolating a microbial-derived organic compound. In one aspect, provided herein are methods that comprise: a) clarifying a composition comprising host cells, an aqueous medium and a bio-organic compound produced by the host cells, wherein the pH of the mixture is neutral or acidic, by substantially separating the host cells from the composition; b) adjusting the pH of the composition to a basic pH; and, c) performing a liquid/liquid separation of the composition to provide a crude bio-organic composition.

[0007] In certain embodiments, performing the liquid/liquid separation at a basic pH reduces emulsion and allows for a cost-effective and scalable purification process.

BRIEF DESCRIPTION OF DRAWINGS

[0008] Figure 1 is a picture of a clarified fermentation broth in which the host cells were removed. When the broth is allowed to settle, three layers are observed: a top layer which comprises the bio-organic compound; a middle layer which comprises about 20% water, about 5% cells, with the balance being the bio-organic compound; and a bottom layer which is the aqueous fermentation medium.

[0009] Figure 2 is a picture of a disc stack separator when the liquid/liquid separation of the concentrated bio-organic composition is at acidic pH. A significant amount of emulsion can form and clog the apparatus.

[0010] Figure 3 is a picture of a disc stack separator when liquid/liquid separation of the concentrated bio-organic composition is at basic pH. Compared to the acidic processes, a significant reduction in emulsion is observed.

[0011] Figure 4 is a plot of total acid number (TAN) as a function of the amount of calcium hydroxide for a crude bio-organic composition that was obtained by liquid/liquid separation at a basic pH (produced by farnesene producing yeast strain A). Two different incubation times were investigated.

[0012] Figure 5 is a plot of total acid number (TAN) as a function of the amount of calcium hydroxide for a crude bio-organic composition that was obtained by liquid/liquid separation at an acidic pH (produced by farnesene producing yeast strain B). Two different incubation times were investigated.

[0013] Figure 6 is a plot of total acid number (TAN) as a function of time for a crude bio-organic composition that was obtained by liquid/liquid separation at an acidic pH (produced by farnesene producing yeast strain B). The amount of calcium hydroxide used was 0.356 % by weight.

10014] Figure 7 is a plot of total acid number (TAN) as a function of time for a crude bio-organic composition that was obtained by liquid/liquid separation at an acidic pH (produced by farnesene producing yeast strain B). In this experiment, instead of calcium hydroxide, two different amounts of sodium hydroxide were used.

DETAILED DESCRIPTION

Terminology

[0015] As used herein, "crude bio-organic composition" refers to a composition comprising a bio-organic compound wherein the bio-organic compound is present in the composition in an amount greater than about 75% by weight of the composition. [0016] As used herein, "bio-organic compound" refers to an immiscible compound that is made by microbial cells (both recombinant as well as naturally occurring). In certain embodiments, the bio-organic compound is a hydrocarbon. In certain embodiments, the bio- organic compound is an isoprenoid. In certain embodiments, the bio-organic compound is a C₅-C₂₀ isoprenoid. In certain embodiments, the bio-organic compound is a C₁₀-C₁₅ isoprenoid.

[0017] As used herein, "neutralized crude composition" refers to a composition comprising a bio-organic compound wherein the bio-organic compound is present in the composition in an amount equal to or greater than about 75% by weight and has a total acid number that is less than 0.5 mg KOH/g as measured by ASTM D 664. In certain embodiments, the total acid number is zero mg KOH/g as measured by ASTM D 664. [0018] As used herein, "neutralized purified composition" is a composition comprising a bio-organic compound wherein the bio-organic compound is present in the composition in an amount equal to or greater than about 90% by weight and has a total acid number that is less than 0.5 mg KOH/g as measured by ASTM D 664. In certain embodiments, the bio-organic compound is present in an amount equal to or greater than about 95% by weight. In certain embodiments, the total acid number is zero mg KOH/g as measured by ASTM D 664.

[0019] As used herein, "phenolic antioxidant" refers to an antioxidant that is a phenol or a phenol derivative, wherein the phenol derivative contains an unfused phenyl ring with one or more hydroxyl substitutents. The term also includes polyphenols. Illustrative examples of a phenolic antioxidant include: resveratrol; 3-tert-butyl-4-hydroxyanisole; 2-tert- butyl-4-hydroxyanisole; 4-tert-butylcatechoI (which is also known as TBC); 2,4-dimethyl-6- tert-butylphenol; and 2,6-di-tert-butyl-4-methylphenol (which is also known as butylhydroxytoluene or BHT). Additional examples of phenolic antioxidants are disclosed in U.S. Patent No. 7,179,311.

[0020] As used herein, "purified bio-organic composition" refers to a composition comprising a bio-organic compound wherein the bio-organic compound is present in the composition in an amount equal to or greater than about 90% by weight. In certain embodiments, the bio-organic compound is present in an amount equal to or greater than about 95% by weight.

[0021] As used herein, "polished composition" refers to a purified bio-organic composition that is further treated, for example, to reduce formation of peroxides in the composition or to stabilize the composition with an anti-oxidant or treated with a chelating agent to reduce the amounts of metals in the compositions.

[0022] As used herein and unless otherwise indicated, the term "processes)" refers to a purification method(s) disclosed herein that is (are) useful for isolating a microbial-derived organic compound. Modifications to the methods disclosed herein (e.g., starting materials, reagents) are also encompassed.

[0023] In the following description, all numbers disclosed herein are approximate values, regardless whether the word "about" or "approximate" is used in connection therewith. Numbers may vary by 1 percent, 2 percent, 5 percent, or, sometimes, 10 to 20 percent. Whenever a numerical range with a lower limit, RL, and an upper limit, RU, is disclosed, any number falling within the range is specifically disclosed. In particular, the following numbers within the range are specifically disclosed: R=RL+k* (RU-RL), wherein k is a variable ranging from 1 percent to 100 percent with a 1 percent increment, i.e., k is 1 percent, 2 percent, 3 percent, 4 percent, 5 percent,..., 50 percent, 51 percent, 52 percent,..., 95 percent, 96 percent, 97 percent, 98 percent, 99 percent, or 100 percent. Moreover, any numerical range defined by two R numbers as defined in the above is also specifically disclosed.

[0024] The claimed subject matter can be understood more fully by reference to the following detailed description and illustrative examples, which are intended to exemplify non-limiting embodiments. Purification Methods

[0025] Provided herein are purification methods for bio-organic compounds. The microbial olefins can be made using any technique deemed suitable by one of skill in the art. Useful exemplary microbial methods for making olefinic isoprenoids are described in U.S. Patent No. 7,399,323; U.S. Patent Publication No. 2008/0274523; and PCT Publication Nos. WO 2007/140339, WO 2008/140492, WO 2008/133658, and WO 2009/014636, all incorporated by reference in their entireties. Useful exemplary microbial methods for making fatty-acid derived olefins are described in U.S. Patent Publication No. 2009/0047721; and PCT Publication Nos. WO 2008/113041 and WO 2008/151149, all incorporated by reference in their entireties.

[0026] In contrast, relatively few publications describe purification methods for such compounds from fermentation or other biological production systems. PCT Publication WO 2007/139924 relates to systems for making bio-organic compounds and describes purification methods which generally rely on the inherent tendency for the bio-organic compound to separate from an aqueous medium. However, although this separation does occur and purified bio-organic compounds can be obtained, there can be significant product losses due to emulsion.

[0027] An emulsion is a mixture of two immiscible liquids, such as water and a bio- organic compound. Mechanical energy from either fermentation (e.g. from agitators or fermentation gases produced by host cells) or downstream processing can also promote emulsion formation where a bio-organic compound is produced and extracted into, for example, an aqueous fermentation medium. Moreover, as described by various literature references, host cells as well as various bio-molecules therein promote or stabilize emulsion formation or both, thus making emulsion inevitable in a microbial production system. Consequently, a simple and scalable purification method that destabilizes an emulsion is useful for a cost-effective purification of a microbial derived, bio-organic compound. [0028] Provided herein are purification methods that destabilize an emulsion and provide cost-effective purification methods for a microbial derived, bio-organic compound. In certain embodiments, the emulsions are oil in water emulsions. The methods provided herein rely on destabilizing emulsions with basic pH. Because the prior art teaches that emulsions are destabilized by acids (adjusting the pH to an acidic pH, typically between about 3 and about 5), this is a surprisingly and unexpectedly result. For example, see, George Alther, Water Engineering & management, 2001, 27-29 and Pollution Prevention Guidance Manual for the PFPR Industry, Chapter 5, pages 41-46, Gallup et al, Energy Fuels, 2007, 21(3),pp 1741-1759, and Amalvy et al, Chem. Commun. 2003, 1826-1827. Moreover, the basic pH also can have the additional benefit of neutralizing some of the endogenous organic acids present in the biological composition. [0029] In one aspect, provided herein are methods that comprise: a) clarifying a composition comprising host cells, an aqueous medium and a bio-organic compound produced by the host cells, wherein the pH of the mixture is neutral or acidic, by substantially separating the host cells from the composition; b) adjusting the pH of the composition to a basic pH; and, c) performing a liquid/liquid separation of the composition to provide a crude bio-organic composition.

[0030J In certain embodiments, the bio-organic compound is a hydrocarbon. In certain embodiments, the bio-organic compound is a C₅-C₃₀ hydrocarbon. In certain embodiments, the bio-organic compound is an isoprenoid. In further embodiments, the bio- organic compound is a C5-C₂0 isoprenoid. In additional embodiments, the bio-organic compound is a C₁₀-C₁₅ isoprenoid. In certain embodiments, the bio-organic compound is a fatty acid or a fatty acid derivative. In certain embodiments, the bio-organic compound is a C₅-C₃₅ fatty acid or a fatty acid derivative. In additional embodiments, the bio-organic compound is selected from carene, geraniol, linalool, limonene, myrcene, ocimene, pinene, sabinene, terpinene, terpinolene, amorphadiene, farnesene, farnesol, nerolidol, valencene, and geranylgeraniol. In further additional embodiments, the bio-organic compound is myrcene, α-ocimene, β-ocimene, α-pinene, β-pinene, amorphadiene, α-farnesene, β-faraesene. In certain embodiments, the bio-organic compound is α-farnesene, β-farnesene, or a mixture thereof.

[0031] In certain embodiments, the microbial cells are bacteria. In certain embodiments, the microbial cells belong to the genera Escherichia, Bacillus, Lactobacillus. In certain embodiments, the microbial cells are E. coli. In further embodiments, the microbial cells are fungi. In still further embodiments, the microbial cells are yeast. In still further embodiments, the microbial cells are Kluyveromyces, Pichia, Saccharomyces, and Yarrowia. In additional embodiments, the microbial cells are S. cerevisiae. In certain embodiments, the microbial cells are algae. In certain embodiments, the microbial cells are Chlorella minutissima, Chlorella emersonii, Chloerella sorkiniana, Chlorella ellipsoidea, Chlorella sp., or Chlorella protothecoides.

[0032] In certain embodiments, the pH of the mixture is about 7. In certain embodiments, the pH of the mixture is between about 6 and about 7. In certain embodiments, the pH of the mixture is acidic. In certain embodiments, the pH of the mixture is below 6. In certain embodiments, the pH of the mixture is between about 4 and 6. [0033J 1° certain embodiments, the clarifying step occurs by liquid/solid separation.

In other embodiments, the clarifying step occurs by sedimentation followed by decantation. In still other embodiments, the clarifying step occurs by filtration. In certain embodiments, the clarifying step occurs by centrifugation. In certain other embodiments, the clarifying step occurs in a continuous disk stack nozzle centrifuge.

[0034] In certain embodiments, the pH of the composition is adjusted to a pH greater than about 7.5. In certain embodiments, the pH of the composition is adjusted to a pH between about 7.5 and about 10. In certain embodiments, the pH of the composition is adjusted to a pH between about 7.5 and about 9. In certain embodiments, the pH of the composition is adjusted to a pH between about 8 and about 8.5. In additional embodiments, pH of the composition is adjusted to a pH greater than 9.

[0035] The pH of the composition can be adjusted by using any base deemed suitable by one of skill in the art. Illustrative examples of suitable bases include: ammonia, potassium hydroxide, barium hydroxide, cesium hydroxide, sodium hydroxide, strontium hydroxide, calcium hydroxide, lithium hydroxide, rubidium hydroxide, and magnesium hydroxide. Highly soluble and economical bases are generally preferred for commercial scale operations. Illustrative examples of such bases include potassium hydroxide and sodium hydroxide. [0036] In certain embodiments, the composition is separated by liquid/liquid separation. In certain embodiments, the composition is separated by centrifugation that relies on the different densities between the bio-organic compound and the aqueous medium, m certain embodiments, the composition is separated by a continuous disk-stack centrifugation. In certain embodiments, the composition is separated by liquid/liquid extraction (also known as solvent extraction).

[0037] In certain embodiments, the method further comprises concentrating the bio- organic compound in the composition into a concentrated composition thereby reducing the volume for subsequent downstream processing. Thus, if the concentration step occurs, then the pH adjustment step and the liquid-liquid separation step are performed on the concentrated composition instead of on the composition. [0038] Thus in another aspect, the method comprises: a) clarifying a composition comprising host cells, an aqueous medium and a bio-organic compound produced by the host cells, wherein the pH of the mixture is neutral or acidic, by substantially separating the host cells from the composition; b) concentrating the composition to form a concentrated composition wherein the concentrated composition comprises substantially all of the bio-organic compound and the volume of the concentrated composition is less than the volume of the composition; c) adjusting the pH of the concentrated composition to a basic pH; and, d) performing a liquid/liquid separation of the concentrated composition to provide a crude bio-organic composition.

[0039] In certain embodiments, the concentrated composition comprises about 50 percent of the volume of the composition. In certain embodiments, the concentrated composition comprises about 40, 35, 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1 percent of the volume of the composition. In certain embodiments, the concentrated composition comprises about 25 percent or less of the volume of the composition. In further embodiments, the concentrated composition comprises about 10 percent or less of the volume of the composition. In still further embodiments, the concentrated composition comprises about 5 percent or less of the volume of the composition.

[0040] In certain embodiments, the concentration step occurs by tangential flow filtration ("TFF"). For example the clarified composition (which is substantially free of host cells) is dewatered using TFF to produce a concentrated composition. In certain other embodiments, the clarification and concentration steps occur simultaneously. For example, when the clarifying step occurs by sedimentation of the host cells, the top portion of the mixture, containing substantially all of the bio-organic compound, can be decanted. This top layer then becomes the concentrated composition. In another example, if the clarifying step occurs from using a continuous disk stack nozzle centrifuge, then the portion of the mixture that includes the bio-organic compound can be separated based on the different densities between the bio-organic compound and the aqueous medium. The portion containing the bio- organic compound then becomes the concentrated composition.

[0041] In certain embodiments, the pH of the concentrated composition is adjusted to a pH greater than about 7.5. In certain embodiments, the pH of the concentrated composition is adjusted to a pH between about 7.5 and about 10. In certain embodiments, the pH of the concentrated composition is adjusted to a pH between about 7.5 and about 9. In certain embodiments, the pH of the concentrated composition is adjusted to a pH between about 8 and about 8.5. In additional embodiments, pH of the concentrated composition is adjusted to a pH greater than 9.

[0042] In certain embodiments, the concentrated composition is separated by liquid/liquid separation to provide a crude bio-organic composition. In certain embodiments, the concentrated composition is separated by centrifugation that relies on the different densities between the bio-organic compound and the aqueous medium. In certain embodiments, the concentrated composition is separated by a continuous, three-phase, disk- stack centrifugation. In certain embodiments, the concentrated composition is separated by liquid/liquid extraction (also known as solvent extraction).

[0043] In certain embodiments, the method further comprises purifying the crude bio- organic composition to yield a purified bio-organic composition. Any suitable method may be used and is likely to depend on the desired level of purity of the bio-organic compound or the acceptable levels of impurities in the final composition. Suitable methods include, but are not limited to: fractional distillation, adsorption, and liquid chromatography. In certain embodiments, the purification is by flash distillation. In certain embodiments, the purification is by silica gel filtration. In additional embodiments, the purification is by alumina filtration.

[0044] In certain embodiments, the method further comprises either neutralizing the crude bio-organic composition thereby forming a neutralized crude composition or neutralizing the purified bio-organic composition to yield a neutralized purified composition. Neutralization occurs by the removal of organic acids (e.g. by precipitation) or by conversion of the acid functionality to a non-acidic group, and is characterized as a reduction in total acid number to 0.5 mg KOH/g or less. In one method, the organic acids are neutralized by the addition of a base. Although any base will generally work, alkali bases were found to be less effective than alkaline earth metal hydroxides such as calcium hydroxide and magnesium hydroxide. In another method, the organic acids are neutralized by adsorption. Suitable adsorbents are materials that sequester polar compounds such as alumina. [0045] In another aspect, the method comprises: a) clarifying a composition comprising host cells, an aqueous medium and a bio-organic compound produced by the host cells wherein the pH of the mixture is less than 6, by substantially separating the host cells from the composition; b) concentrating the composition to form a concentrated composition wherein the concentrated composition comprises substantially all of the bio-organic compound and the volume of the concentrated composition is less than the volume of the composition; c) adjusting the pH of the concentrated composition with NaOH or KOH to a pH between about 7.5 and about 9; and, d) centrifuging the concentrated composition to separate the bio-organic compound from the aqueous medium thereby forming a crude bio-organic composition; and, e) neutralizing the crude bio-organic composition to yield a neutralized crude composition; and f) flash distilling the neutralized crude composition to yield a neutralized purified composition.

[0046] In certain embodiments, the host cells are yeast cells.

[0047] In certain embodiments, the neutralization step occurs from the use of an adsorbent. In other embodiments, the neutralization step occurs from the use of alumina. In certain embodiments, the neutralization step occurs from the use of an alkaline earth metal hydroxide. In certain other embodiments, the neutralization step occurs from the use of calcium hydroxide.

[0048] In certain embodiments, the purified composition (whether neutralized or not) is further polished. For example, when the bio-organic compound is an olefin, the method can further comprise adding an antioxidant to the purified bio-organic composition. The addition of the antioxidant can retard the formation of peroxides and stabilizes the purified bio-organic composition. Any anti-oxidant deemed suitable by one of skill in the art can be used. However, if the olefin is to be subsequently hydrogenatcd, a phenolic antioxidant which does not interfere with hydrogenation reactions under mild conditions like certain commonly used antioxidants such as α-tocopherol is preferred. Illustrative examples of suitable anti-oxidants include: resveratrol; 3-tert-butyl-4-hydroxyanisole; 2-tert-butyl-4- hydroxyanisole; 2,4-dimethyl-6-tert-butylphenol; 2,6-di-tert-butyl-4-methylphenol; and 4- tert-butylcatechol.

[0049] In another example, the purified compositions can be further polished by the addition of a chelating agent to reduce the amounts of metals in the compositions. Ln certain embodiments, the purification step also includes removing metals present in the crude bio- organic composition by the addition of a chelating agent. Any suitable chelating agent can be used. Illustrative examples of suitable chelating agents include ascorbic acid, citric acid, malic acid, oxalic acid, succinic acid, dicarboxymethyllutamic acid, ethylenediaminedisuccinic acid (EDDS), ethylenediaminetetraacetic acid (EDTA) and the like.

[0050] While the processes and systems provided herein have been described with respect to a limited number of embodiments, the specific features of one embodiment should not be attributed to other embodiments of the processes or systems. No single embodiment is representative of all aspects of the methods or systems, hi certain embodiments, the processes may include numerous steps not mentioned herein. In other embodiments, the processes do not include any steps not enumerated herein. Variations and modifications from the described embodiments exist.

[0051] It is noted that the purification methods are described with reference to a number of steps. In certain embodiments, these steps can be practiced in any sequence. In certain embodiments, one or more steps may be omitted or combined but still achieve substantially the same results. The appended claims intend to cover all such variations and modifications as falling within the scope of the claimed subject matter. [0052] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Although the claimed subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings herein that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. EXAMPLES

Example 1

[0053] This example describes the purification of a bio-organic compound from fermentation broth when the liquid/liquid separation of the concentrated bio-organic composition was performed at pH 5.0 (which is also the fermentation pH). In this instance the bio-organic compound was farnesene that was produced by farnesene producing yeast strain A.

[0054] The yeast cells were separated from the fermentation broth using a continuous disk stack nozzle centrifuge (Alfa Laval DX 203 B-34). In addition to removing the cells, this step also served to concentrate the bio-organic compound in a smaller volume. For this particular yeast strain, a twenty fold concentration resulted in a composition that was approximately half farnesene and half fermentation medium. So for a twenty fold concentration, approximately 95% of the volumetric flow exited the centrifuge from the nozzles (cells + liquid) as waste while approximately 5% of the volumetric flow was captured as concentrated bio-organic composition.

[0055] This composition when allowed to settle or centrifuged, separated into three distinct phases. As shown in Figure 1 , the top layer comprised primarily the bio-organic compound. The middle layer comprised emulsion formed by the cells, the bio-organic compound and water. The bottom layer comprised the aqueous fermentation medium. [0056] The concentrated bio-organic composition (pH ~ 5) was then subjected to liquid/liquid separation using the same centrifuge. When large volumes were processed, a continuous centrifugation procedure could not be used due to the emulsion fouling the separating discs. For example, when 3.5 liters of the concentrated farnesene composition was processed (feed rate 130 niL/min at 30 ⁰C), due to the fouling of the discs, the carryover of the bio-organic compound into the aqueous output increased resulting in substantial product loss. Figure 2 shows the separation disc covered with emulsion. When enough emulsion forms, then the centrifuge becomes ineffective in separating the bio-organic compound from the aqueous phase.

[0057] Table 1 details the amounts of the bio-organic compound in each of the three phases (bio-organic compound layer; emulsion; and the aqueous layer) of the 3.5 liters of a concentrated farnesene composition prepared consistent with this example. Table 1 : liquid/liquid recovery of the concentrated bio-organic composition at pH 5.

Layer % of farnesene bio-organic 24% (338 mL)

Emulsion 63%

Aqueous 13%

[0058] The bio-organic compound recovered from the bio-organic layer is referred to as the crude bio-organic composition and its purity was 94.5 % (w/w). The total acid number of the crude bio-organic composition as measured by ASTM D 664 was 1.2 mg KOH/g.

Example 2

[0059] This example describes the purification of a bio-organic compound from fermentation broth when the liquid/liquid separation of the concentrated bio-organic composition was performed at pH 8.3.

[0060] A concentrated bio-organic composition was obtained as described by

Example 1. However, instead of proceeding directly with liquid/liquid separation, the composition was treated with various conditions and agents for their ability to break up the emulsion or release the bio-organic compound from the emulsion or both. Some of the conditions and agents tested include: pH, salt; polymers (cationic, anionic, and non-ionic); chelators; antifoams; temperature; and shear. Of these, adjusting the pH of the composition from acidic to basic pH was the most effective.

[0061] The pH of the concentrated bio-organic composition was adjusted to pH 8.3 using 5 N NaOH, followed by incubation at about 3O⁰C for approximately one hour. The concentrated bio-organic composition (pH ~ 8.3) was then subjected to liquid/liquid separation as in Example 1. In contrast to the acidic concentrated bio-organic composition, the basic concentrated bio-organic composition resulted in significantly less emulsion. Figure 3 is a picture of the separation disc after the liquid/liquid separation. In contrast to the acidic process, a significantly less emulsion is seen on the discs.

[0062] Table 2 details the amounts of the bio-organic compound in each of the three phases (bio-organic compound layer; emulsion; and the aqueous layer) of the basic concentrated bio-organic composition.

Table 2: liquid/liquid recovery of the concentrated bio-organic composition at pH 8.3 Layer % of farnesene bio-organic 82% (1.15L)

Emulsion 8%

Aqueous 10%

[0063] The purity of the bio-organic compound recovered from the bio-organic layer was 94.9 % (w/w). The total acid number as measured by ASTM D 664 was 0.9 mg KOH/g. Adjusting the pH of the concentrated bio-organic composition also served to reduce the total acid number of the crude bio-organic composition.

[0064] Because of the significant reduction of emulsion that is seen by performing the liquid/liquid separation at a basic pH, this step can be performed using a continuous three phase desludging centrifuge (e.g. Alfa-Laval LAPX-404 Continuous Centrifuge) which can be used at commercial scale. When a continuous three phase desludging centrifuge is used, solids gradually accumulate within the bowl, and need to be discharged periodically to maintain separation efficiency.

Example 3

[0065] This example describes further purification of the crude bio-organic composition of Example 2.

[0066] The crude bio-organic composition of Example 2 was incubated with 0.4% w/w calcium hydroxide (e.g., Acros Organics, >98% pure, Cat. No. 21918000) for 2.5 hours at ambient temperature. This results in precipitation of various impurities which can be removed by various methods including centrifugation and filtration to yield a neutralized composition. If desired, the neutralized composition can be further purified, for example, by distillation. Table 3 describes the total acid number and glycerin content of the various compositions.

Table 3: TAN & Glycerin content

Table 4: Hydrocarbon quantification

Table 5: Trace Metals

Following metals are < 1 ppm: Silver, aluminum, barium, copper, iron, molybdenum, tin, and vanadium.

[0067] The calcium hydroxide treatment reduces the total acid number without significantly affecting the impurity profiles of the other components of the composition with the exception of increased calcium. However, the high levels of calcium can be fully removed by other purification methods such as flash distillation.

Example 4

[0068] This example describes a dose response of the total acid number as a function of the amount of calcium hydroxide of the crude bio-organic composition derived from yeast strain A.

[0069] The crude bio-organic composition from Example 2 was treated with varying amounts of calcium hydroxide. In one set of experiments, the mixture was allowed to incubate for four hours. In another set of experiments, the mixture was allowed to incubate over night. The results are shown by Figure 4. As illustrated by the plot, the total acid number can be reduced to zero mg KOH/g with smaller amounts of calcium hydroxide in this experiment if the mixture is incubated for longer periods of time. Example 5

[0070] This example describes a dose response of the total acid number as a function of the amount of calcium hydroxide of the crude bio-organic composition derived from yeast strain B.

[0071] The crude bio-organic composition was obtained as described by Example 1 except a different farnesene producing strain was used (strain B). The crude bio-organic composition was treated with varying amounts of calcium hydroxide and the results are shown in Figure 5. As it can be seen, the crude bio-organic composition has a higher total acid number (equal to or greater than 2 versus 1.2 mg KOH/g) than was seen for the crude bio-organic composition from strain A. Nevertheless, the total acid number is reduced to zero with the calcium hydroxide treatment.

Example 6

[0072] This example describes a dose response of the total acid number as a function of the amount of calcium hydroxide of the crude bio-organic composition derived from yeast strain B .

[0073] The crude bio-organic composition was obtained as described by Example 5 and it was treated with 0.356 weight percent of calcium hydroxide. Figure 6 is a plot of the total acid number (TAN) as a function of time.

Example 7

[0074] This example describes a dose response of the total acid number as a function of the amount of sodium hydroxide of the crude bio-organic composition derived from yeast strain B.

[0075] The crude bio-organic composition was obtained as described by Example 5 and it was treated with two differing amounts of sodium hydroxide. The results are shown in Figure 7. Although sodium hydroxide was able to reduce the total acid number, it was less effective than the calcium hydroxide. One reason appears to be that sodium was not able to partition as efficiently into the bio-organic composition like calcium. Moreover, sodium hydroxide appears to promote saponification of residual impurities in the bio-organic composition.

Example 8

[0076] This example describes purification of the crude olefin composition to a purified olefin composition using alumina. This purification method also serves to neutralize the organic acids that are present in the crude olefin composition.

[0077] Alumina sorbent is regenerated prior to use by heating at 250 ⁰C for at least two hours. The crude olefin composition is brought to room temperature and is mixed with granular alumina (e.g., Selexsorb CDX) at 10% w/v of the bio-organic compound and mixed overnight. The mixture is then filtered by 0.45um filter and treated with 0.01% phenolic antioxidant such as 4-tert-butylcatechol.

[0078] When this method is used to purify the crude farnesene composition from either strain Λ or B, the resulting purified farnesene composition has a total acid number of 0 mg KOH/g.

Example 9

[0079] The whole cell broth from the fermentation of a farnesene producing yeast strain was fed to an Alfa Laval continuous centrifuge model DX203-34B. The centrifuge was equipped with 3 x 0.5mm nozzles and 3 blank nozzles. The feed flowrate for the operation was ~ 2.3L/min. Two output streams (heavy phase and light phase) were produced. The heavy phase (aka cell slurry) was a mixture of water and yeast cells. A ImL microfuge spin (10,000g for 5 minutes) of the cell slurry revealed that the yeast cells comprised 60-70% of the cell slurry, with the remainder being aqueous components. The light phase (clarified broth) was comprised of emulsion and aqueous components. A ImL microfuge spin (10,000g for 5 minutes) revealed -95% aqueous components. The remaining 5% was an emulsion of oil, water, and biomass which contained the farnesene product. The clarified broth was split and processed two different ways: Clarified Broth Process and TFF Process. [0080] For the Clarified Broth Process, 17.5 liters of clarified broth was treated in the following order:

• The pH titrated to 9.50 with 5N NaOH;

• NaCl was added at a concentration of 1.2M in the aqueous phase; and • 0.5% (v/v) L-81 Tergitol Surfactant was added (the volume of L-81 Tergitol added is 0.5% of the initial clarified broth volume).

[008 IJ The clarified broth was allowed to mix for 1 hour at room temperature. A

ImL microfuge spin (10,000g for 5 minutes) revealed that 90-95% of the emulsion had broken to release crude farnesene product. The clarified broth was sent through the liquid separation unit operation (see details in section below).

[0082] For the TFF Process (tangential flow filtration), approximately 200 liters of clarified broth was used. At ambient temperature, the clarified broth was processed in series through 4 Industrial TFF cartridges (316L Stainless Steel, 1.5" diameter tube, 0.1 micrometer pore size, 0.35m² each). The purpose of the TFF processing is to remove water and aqueous components, leaving behind a concentrated clarified broth. The initial processing conditions for the TFF were lόpsig inlet pressure, 5psig outlet pressure, and 880mL/min of permeate flux (38L/m²/hr). Time course samples of permeate and retentate streams were obtained. Throughout the course of processing, the permeate flux gradually dropped off due to accumulation of solids in the cartridges and increased viscosity of the retentate. [0083] Approximately 14 liters of concentrated clarified broth was recovered from the

TFF process and was treated in the following order:

• The pH titrated to 9.50 with 5N NaOH;

• NaCl was added at a concentration of 1.2M in the aqueous phase; and

• 0.5% (v/v) L-81 Tergitol Surfactant was added (the volume of L-81 Tergitol added is 0.5% of the initial clarified broth volume).

[0084] The clarified broth was allowed to mix for 1 hour at room temperature. A

ImL microfuge spin (10,000g for 5 minutes) revealed that 90-95% of the emulsion had broken to release crude farnesene product. The concentrated clarified broth was sent through the liquid separation unit operation (see details in section below).

[0085] Both the clarified broth and the concentrated broth were then processed in the same way (in the liquid separation unit) to recover the crude farnesene from the aqueous and solids debris. The feed material was fed at ambient temperature to the Alfa Laval Gyrotester centrifuge, equipped with 4 x 6.5mm heavy phase screws. The feed flowrate was ~200mL/min. The Liquid Separation operation produced two output streams (aqueous phase and a farnesene Phase). The aqueous Phase contains solids, debris, and aqueous components. At the conclusion of the process it is discarded as waste. The farnesene phase is a clear, slightly yellow oil phase. At the conclusion of the process it is filtered with a 1.0 micron glass fiber filter. An antioxidant (4-tertbutylcatechol) is added at a concentration of 100 ppm. [0086] The farnesene mass balance across the cell clarification and liquid separation process steps are shown in Table 6:

Table 6: % Farnesene Recovery

[0087] The examples set forth above are provided to give those of ordinary skill in the art with a complete disclosure and description of how to make and use the claimed embodiments, and are not intended to limit the scope of what is disclosed herein. Modifications that are obvious to persons of skill in the art are intended to be within the scope of the following claims. All publications, patents, and patent applications cited in this specification are incorporated herein by reference as if each such publication, patent or patent application were specifically and individually indicated to be incorporated herein by reference.

Claims

What is claimed is:

1. A method comprising: a) clarifying a composition comprising host cells, an aqueous medium and a bio-organic compound produced by the host cells, wherein the pH of the mixture is neutral or acidic, by substantially separating the host cells from the composition; b) adjusting the pH of the composition to a basic pH; and, c) performing a liquid/liquid separation of the composition to provide a crude bio-organic composition.

2. The method of claim 1, wherein the bio-organic compound is a hydrocarbon.

3. The method of claim 1, wherein the bio-organic compound is an isoprenoid.

4. The method of claim 1, wherein the bio-organic compound is a C₅-C₂₀ isoprenoid.

5. The method of claim 1 , wherein the bio-organic compound is a fatty acid or a fatty acid derivative.

6. The method of claim 1 , wherein the host cells are bacteria.

7. The method of claim 1, wherein the host cells belong to the genera Escherichia, Bacillus or Lactobacillus.

8. The method of claim 1 , wherein the host cells are fungi.

9. The method of claim 1, wherein the host cells are Kluyveromyces, Pichia, Saccharomyces, or Yarrowia.

10. The method of claim 1, wherein the host cells are S. cerevisiae.

11. The method of claim 1 , wherein the host cells are algae.

12. The method of claim 1, wherein the host cells are Chlorella minutissima, Chlorella emersonii, Chloerella sorkiniana, Chlorella ellipsoidea, Chlorella sp., or Chlorella protothecoides.

13. The method of claim 1, wherein the pH is adjusted to a pH between about 7.5 and about 10.

14. The method of claim 1, wherein the pH is adjusted to a pH between about 8 and about 8.5.

15. A method comprising: a) clarifying a composition comprising host cells, an aqueous medium and a bio-organic compound produced by the host cells, wherein the pH of the mixture is neutral or acidic, by substantially separating the host cells from the composition; b) concentrating the composition to form a concentrated composition wherein the concentrated composition comprises substantially all of the bio-organic compound and the volume of the concentrated composition is less than the volume of the composition; c) adjusting the pH of the concentrated composition to a basic pH; and, d) performing a liquid/liquid separation of the concentrated composition to provide a crude bio-organic composition.

16. The method of claim 15, wherein the concentrated composition comprises about 25% or less of the volume of the composition.

17. The method of claim 15, wherein the method further comprises purifying the crude bio-organic composition to yield a purified bio-organic composition.

18. The method of claim 17, wherein the method further comprises neutralizing the purified bio-organic composition to yield a neutralized purified composition.

19. The method of claim 18, wherein the neutralization occurs by treating the purified bio-organic composition with calcium hydroxide.

20. The method of claim 18, wherein the neutralization occurs by treating the purified bio-organic composition with alumina.

21. The method of claim 15, wherein the method further comprises neutralizing the crude bio-organic composition to yield a neutralized crude composition.

22. The method of claim 21 , wherein the neutralization occurs by treating the crude bio-organic composition with calcium hydroxide.

23. The method of claim 21 , wherein the neutralization occurs by treating the crude bio-organic composition with alumina.

24. The method of claim 21 , further comprising further purifying the neutralized crude composition to yield neutralized purified composition.

25. The method of claim 24, wherein the purification method is flash distillation.

26. A method comprising: a) clarifying a composition comprising host cells, an aqueous medium and a bio-organic compound produced by the host cells wherein the pH of the mixture is less than 6, by substantially separating the host cells from the composition; b) concentrating the composition to form a concentrated composition wherein the concentrated composition comprises substantially all of the bio-organic compound and the volume of the concentrated composition is less than the volume of the composition; c) adjusting the pH of the concentrated composition with NaOH or KOH to a pH between about 7.5 and about 9; and, d) centrifuging the concentrated composition to separate the bio-organic compound from the aqueous medium thereby forming a crude bio-organic composition; and, e) neutralizing the crude bio-organic composition to yield a neutralized crude composition; and t) flash distilling the neutralized crude composition to yield a neutralized purified composition.

27. The method of claim 26, wherein the neutralization step is by adsorption.

28. The method of claim 27, wherein the neutralization step is by treating the crude bio-organic composition with alumina.

29. The method of claim 26, wherein the neutralization step is by treating the crude bio-organic composition with an alkaline earth metal hydroxide.

30. The method of claim 29, wherein the alkaline earth metal hydroxide is calcium hydroxide.

31. The method of claim 26 further comprising treating the neutralized purified composition with an anti-oxidant.

32. The method of claim 31 , wherein the anti-oxidant is a phenolic antioxidant.

33. The method of claim 26 further comprising treating the neutralized purified composition with a chelating agent.