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WO2015042114A1 - Fermentation process with distinct growth and production stages - Google Patents

Fermentation process with distinct growth and production stages Download PDF

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Publication number
WO2015042114A1
WO2015042114A1 PCT/US2014/056029 US2014056029W WO2015042114A1 WO 2015042114 A1 WO2015042114 A1 WO 2015042114A1 US 2014056029 W US2014056029 W US 2014056029W WO 2015042114 A1 WO2015042114 A1 WO 2015042114A1
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WIPO (PCT)
Prior art keywords
product
medium
microorganisms
certain embodiments
water
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PCT/US2014/056029
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French (fr)
Inventor
Emily H. GREENHAGEN
Andrew L. CONSIGLIO
Kyle M. MacEWEN
Arthur J. SHAW
William G. Latouf
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Novogy, Inc.
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Publication of WO2015042114A1 publication Critical patent/WO2015042114A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

Definitions

  • Man molecules are produced or have the potential to be produced via tandem fermentation and subsequent crystallization of an extracellular product.
  • U.S. Pat. No. 6,440,712 describes a process for crystallization of erythritol from fermentation medium.
  • Li et al. describes recovery of succinic acid from fermentation broth by crystallization (Li Q, et al. Separation and Purification Technology 2010, 72(3), 294-300),
  • a process for isolating 1 ,4-butanediol from a fermentation broth via crystallization has been described (U.S. Pat. Appl. Publ No. 201 1/0003355).
  • Okabe et al. describes a production and recovery process for itaconic acid (Okabe M. et al. Appl Microbiol Biotechnol 2009, 84, 597-606).
  • Product accumulation in microbes typically begins when a nutrient is exhausted in the medium, hut a surfeit of carbon, usually in the form of glucose, remains available.
  • the media are carefully designed to incorporate an unbalanced nutrient composition, such that cells propagate fast in the early- stage with a concurrent depletion of the controlling nutrient.
  • the cell propagation rate is significantly reduced but product anabolism remains active, leading to a net accumulation of intracellular product.
  • An early study disclosed a two-stage continuous culture system for product accumulation, while the entire output of the first stage was pumped directly into the second stage (Hall, M. I, et al Appl Environ. Microbiol.
  • die invention relates to a process, comprising the steps of: supplying a first medium;
  • the second medium consists essentiall of (i) water, a salt, and a substrate, or (ii) water and a substrate, thereby forming the product.
  • Figure 1 tabulates the presence ( ⁇ *-) or absence (empty box) of components in various fermentation media useful in eiythrito! production.
  • Figure 2 depicts erythrito! production under conditions outlined in Figure 1 (1 complete media without ( R J SQ*; 2 - complete media without (N3 ⁇ 4 >SO.*, Amberex, Amberferm; 3 ::: complete media. Thiamine and Trace Elements; 4 ::: complete media. Trace Elements only; and 5 ⁇ Sodium. Chloride only).
  • Figure 3 depicts data showin that erythritol production is a two-stage fermentation (squares) at ! -L scale demonstrates a higher production rate and erythritol titer than standard fermentation (diamonds).
  • the invention relates to a process for producing and purifying a small organic molecule, in certain embodiments, the costs associated with purification are minimized.
  • the invention relates to a two-stage fermentation process
  • the invention relates to a process, comprising the steps of: supplying a first medium; contacting a plural try of microorganisms with the first medium for a first period of time under conditions suitable for microorganism growth and reproduction, thereb forming an increased plurality of microorganisms; separating the increased plurality of microorganisms from the first medium, thereby forming a substantially pure plurality of microorganisms; contacting the substantially pure plurality of microorganisms with a second medium for a second period of time under conditions suitable for the production of a product, wherein the second medium consists essentially of (i) water, a salt buffer, and a substrate, or (ii) water and a substrate, thereby forming the product.
  • Dry weight and “dry cell weight” mean, weight determined in the relative absence of water.
  • dr weight means that the percentage is calculated based on the weight of the cell after substantially ail water has been removed
  • Microorganism is intended to mean a profcaryotic or eukaryotic cell or organism having a microscopic size.
  • the terra is intended to include bacteria of all species and eiikaryotie organisms such as yeast and fungi.
  • the term also includes ceil cultures of any species that can be cultured for the production of a biochemical or small organic molecule.
  • the second medium consists essentiaUv of (i) water, a salt, and a substrate, or (ii) water and a substrate, thereby forming the product
  • the invention relate to any one of the aforementioned processes, wheretn the microorganism is selected from the group consisting of Ctypiococcus curvatus, Ctypiococcus temcaius, Candida sp., Escherichia coU, L/pomyces sl rkayi, Lipomyces lipofor y Endomycopsis ve alis, Rhodoionria gl tmis, Mhodot nda gracilis., and Yarro a Upoiynca,
  • the invention relates to any one of the aforementioned processes, wherein the microorganism i selected from the group consisting of Mannheimia succ i iproducens, Anaerobiospirilltm succiniciproducens ⁇ Aciinob ciUus succinogems and Escherichia coli and the product is succinic acid.
  • the microorganism i selected from the group consisting of Mannheimia succ i iproducens, Anaerobiospirilltm succiniciproducens ⁇ Aciinob ciUus succinogems and Escherichia coli and the product is succinic acid.
  • the invention relates to any one of the aforementioned processes, wherein the microorganism is selected from the group consisting of Aspergillus terrms, Escherichia co!i, Vstilago zeae, Usidaga m ydis, Candida sp., Candida mutant, and Rh dot rula sp.; and the product is itaconic acid.
  • the invention relates to any one of the aforementioned processes, wherein the microorganism is a member of the genus " frigonop is, Momliella, Yarroma, Trichosporortoides, or Candida: and the product is erythritoL
  • the invention relates to any one of the aforementioned processes, wherein the microorganism is selected from the group consisting of Momliella fo mfosa, Momliella poHmi y Y rrawk) lipolyiica, T ' richosporomtides o docephalis, Trichosporonoides nigrescent; and Trichosporonoides megaehiUensis; and the product is erythritoL
  • the invention reiates to any one of the aforementioned processes, wherein the microorganism is Escherichia coii and the product is 1 ,4- butanedioi.
  • the invention reiates to any one of the aforementioned processes, wherein the pluraiity of microorganisms is contacted with the first medium: in a first reactor.
  • the invention relates to any one of the aforementioned processes, wherein the first medium is crude.
  • tiie invention reiates to any one of tiie aforementioned processes, wherein the first medium comprises iignoceiiulosie material glucose, xylose, sucrose, acetic acid, dextrose, glycerol, fructose, lactose, galactose, raannose, rhamnose, ara ' btnosc, yeast extract, urea, MgOs, CaC!3 ⁇ 4 M:nC3 ⁇ 4, NajHPO*. NaHaPO*. M COs, NaOH, NH4OH, ⁇ N3 ⁇ 4)?.HP0 4 , MgS0 , (NH )jS0 , NH 4 Cl. KH3PO4, K 2 HP0 4 , peptone, or a combination thereof.
  • the first medium comprises iignoceiiulosie material glucose, xylose, sucrose, acetic acid, dextrose, glycerol, fructos
  • the invention reiates to any one of the aforementioned processes, wherein conditions suitable for microorganism growth and reproduction are a first temperature, a first H, and a first inoculation volume.
  • the invention relates to any one of the aforementioned processes, wherein the first temperature is about 28 X to about 45 X. In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the first temperature is about 28 X to about 32 X. in certain embodiments, the invention relates to any one of the aforementioned processes, wherein the first temperature is about 28 X.. about 29 , about 30 X, about 31 , or about 32 X.
  • the invention relates to any one of the aforementioned processes, wherein the first pli is about 2,5 to about 7.5. n certain embodiments, the invention reiates to any one of the aforementioned processes, wherein the first pH is about 2.7 to about 4. In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the first pH is about 2.8, about 3.0, about 3.2, about 3.4, about 3.6 ' . about 3.8. or about 4.0, In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the first period of time is about- 12 h to about 48 h.
  • the invention relates to any one of the aforementioned processes, wherein the first period of time is about 24 h to about 48 h. in certain embodiments, the invention relates to an one of the aforementioned processes, wherein the first period of time is about 24 h, about 26 h, about 28 h, about 30 h, about 32 h, about 34 h, about 36 h, about 38 h, about 40 h, about 42 h, about 44 h, about 46 h, or about 48 h.
  • the invention relates to any one of the aforementioned processes, wherein the first inoculation volume is about ⁇ % to about 10% v/v, in certain embodiments, the invention relates to any one of the aforementioned processes, wherein the first inoculation volume is about 1 % to about 2% v/v. in certain embodiments, the in vention relates to any one of the aforementioned processes, wherein the first inoculation volume is about 1 %, about ⁇ .5%, or about 2% v/v.
  • the invention relates to any one of the aforementioned processes, wherein the increased plurality of microorganisms has a celi densit that is about 100 times larger to about iOOO times larger than the celi density of the plurality of microorganisms. In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the increased plurality of microorganisms has a ceil density that is about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, or about 1 00 times larger than the cell density of the plurality of micro organi sms .
  • the invention relates to any one of the aforementioned processes, wherein the increased plurality of microorganisms is separated from: the first medium by ceutrifugation.
  • the invention relates to any one of the aforementioned processes, further comprising the step of washing the substantially pure plurality of microorganisms, in certain embodiments, the invention relates to any one of the aforementioned processes, wherein the substantially pure pluralit of microorganisms is washed with water.
  • the invention relates to any one of the aforementioned processes, wherein the substantially pure plurality of microorganisms is contacted with the second medium in a second reactor. In certain embodiments, the invention relates to any one of die aforerae.ntio.ned processes, wherein the first reactor and the second reactor are not the same.
  • the invention relates to any one of the aforementioned processes, wherein the first reactor or the second reactor is a fermentor.
  • the invention relates to any one of the aforementioned processes, wherein the substrate is glycerol, glucose, xylose, sucrose, acetic acid f dextrose, fructose, lactose, galactose, mannose, rhamrtose f arabinose, starch, molasses, hydrosylates of corn syrup, hydrosylates of wood, or a combination thereof.
  • the invention relates to any one of the aforementioned processes, wherein, the concentration of the substrate in the second medium is about ⁇ 00 g L to about 400 g/L. In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the concentration of the substrate in the second medium is about 100 g/L, about 150 g/L, about 200 g/L, about 250 g/L f about 300 g/L, about 350 g/L, or about 400 g/L.
  • the invention relates to any one of the aforementioned processes, wherein the second .medium consists essentially of water, a salt, and a substrate; and the salt is a buffer.
  • the invention relates to any one of the aforementioned processes, wherein the second medium consists essentially of water, a salt, and a substrate; and the salt is sodium chloride.
  • the invention relates to any one of the aforementioned processes, wherein the water is distilled water. In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the water is undistilled water.
  • the invention relates to any one of the aforementioned processes, wherein conditions suitable for the production of the product arc a. second temperature, a second H, and a second inoculation volume.
  • the invention relates to any one of the aforementioned processes, wherein the second temperature is about 28 °C to about 45 ⁇ C. in certain embodiments, the invention relates to any one of the aforementioned processes, wherein the second temperature is about 28 °C to about 32 °C. In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the second temperaiitre is about 28 C, about 29 X, about 30 °C, about 31 5 C f or about 32 X.
  • the invention relates to any one of the aforementioned processes, wherein the second pH is about 2.5 to about 7.5- in certain embodiments, the invention relates to any one of the aforementioned processes, wherein the second pH is about 2.7 to about 4. In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the second pH is about 2.8, about 3.0, about 3.2, about 3,4, about 3,6, about 3.8, or about 4,0.
  • the invention relates to any one of the aforementioned processes, wherein the second period of time is about 24 h to about 120 h. In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the second period of time is about 48 h to about 120 h. in certain embodiments, the invention relates to any one of the aforementioned processes, wherein the second period of time is about 48 h, about 54 h, about 60 h, about 66 h, about 72 h, about 78 h, about 84 b f about 90 h, about 96 h, about 102 h, about 108 h, about 1 14 h, or about 120 h.
  • the invention relates to any one of the aforementioned processes, wherein the second inoculation volume is about 1 % to about 10% v/v. In certain embodiments, the invention relates to any one of the aforementioned, processes, wherein the second inoculation volume is about 1% to about 2% v v. In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the second inoculation volume is about I %, about 1,5%, or about 2% v/v.
  • the invention relates to any one of the aforementioned processes, wherein the product is selected from the group consisting of erythritol, succinic acid, 1 t 4-butanediol, and ttacontc acid.
  • the invention relates to any one of the aforementioned processes, wherein the product is not a triacylgiyceroi
  • the invention relates to any one of the aforementioned processes, further comprising the step of; separating the product from the substantially pure plurality of microorganisms.
  • the invention relates to any one of the aforementioned processes, wherein the product is separated from the substantially pure plurality of microorganisms by centrifuga-ion, thereb forming an aqueous broth comprising the product.
  • the invention relates to any one of the aforementioned processes, wherein the product is separated from the substantially pure plurality of microorganisms by solid-liquid separation.
  • the invention relates to any one of the aforementioned processes, further comprising the step of: crystallizing the product from the aqueou broth, thereby forming a crystallized product.
  • the invention relates to any one of the aforementioned processes, wherein the concentration of the product in the second medium is about 150 g L to about 300 g/L. In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the concentration of the product in the second medium is about 150 g/L, about 200 g/L, about 250 g/L, or about 300 g/L.
  • the invention relates to any one of the aforementioned processes, further comprising the step of: crystallizing the product, thereby forming a crystallized product. In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the crystallization is carried out in the second reactor. In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the crystallization involves exposing the product to a third temperature.
  • the invention relates to any one o the aforementioned processes, wherein the process does .not involve subjecting the product to calcium hydroxide, vacuum distillation, ultrafiltration, ion exchange, column chromatography, or activated carbon.
  • the invention relates to any one of the aforementioned processes, wherein the product is substantially mote pure than a product produced under substantially the same conditions by the same microorganism, but without the steps of separating the increased plurality of microorganisms from: the first medium and contacting the substantially pure pluralit of microorganisms to the second medium:.
  • the invention relates to any one of the aforementioned processes, wherein the crystallized product is substantially more pure than a crystallized product produced under substantially the same conditions by the same microorganism, but without the steps of separating the increased plurality of microorganisms from the first medium and contacting the substantially pure plurality of microorganisms to the second medium.
  • the invention relates to any one of the aforementioned processes, wherein the yield of the product or the crystallized product is about 50% to about 99%. In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the yield of the product or the crystallized product is about 70% to about 99%. In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the yield of the product or the crystallized product is about 80% to about 99%. In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the yield of the product or the crystallized product is about 90% to about 99%.
  • the invention relates to any one of the aforementioned processes, wherein the purity of the product or the crystallized product is about 70% to about 99%. in certain embodiments, the invention relates to any one of the aforementioned processes, wherein the purity of the product or the crystallized product is about 80% to about 99%. in certain embodiments, the invention relates to any one of the aforementioned processes, wherein the purity of the product or the crystallized product is about 90% to about 99%.
  • the invention relates to any one of the aforementioned processes, wherein an aspect of the process is described in U.S. Patent No. 5,902,739, U.S. Patent No. 6,440,712, or U.S, Patent Application Publication No. 201 1/0003355, the contents of each of which are hereby incorporated by reference in their entirety.
  • the invention relates to a product made by any one of the af remen tioned processe .
  • Figure 2 demonstrates equivalent erythritol production in the salt-buffered water and substrate solution (Flask 5) as in the full medium formulation (Flask 1 ⁇ .
  • Flask 5 conditions were then scaled up to a 1-L stirred tank reactor. Both erythritol production rate and final titer were improved in the two-stage process (Figure 3).
  • the products of formed by a two-stage process of the invention are qualitativel and quantitatively more pare than products formed by standard fermentation.

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Abstract

In certain embodiments, the invention relates to a process, comprising the steps of: supplying a first medium; contacting a plurality of microorganisms with the first medium for a first period of time under conditions suitable for microorganism growth and reproduction, thereby forming an increased plurality of microorganisms; separating the increased plurality of microorganisms from the first medium, thereby forming a substantially pure plurality of microorganisms; contacting the substantially pure plurality of microorganisms with a second medium for a second period of time under conditions suitable for the production of a product, wherein the second medium consists essentially of (i) water, a salt, and a substrate, or (ii) water and a substrate, thereby forming the product.

Description

Fermentation Process with Distinct Growth and
Production Stages
RELATED APPLICATIONS
This application claims the benefit of priority to United States Provisional Patent
Application serial number 61 /879,243, filed September .18, 2013, which is hereby incorporated by reference.
BACKGROUND
Man molecules are produced or have the potential to be produced via tandem fermentation and subsequent crystallization of an extracellular product. For example, U.S. Pat. No. 6,440,712 describes a process for crystallization of erythritol from fermentation medium. Li et al. describes recovery of succinic acid from fermentation broth by crystallization (Li Q, et al. Separation and Purification Technology 2010, 72(3), 294-300), In addition, a process for isolating 1 ,4-butanediol from a fermentation broth via crystallization has been described (U.S. Pat. Appl. Publ No. 201 1/0003355). Finally, Okabe et al. describes a production and recovery process for itaconic acid (Okabe M. et al. Appl Microbiol Biotechnol 2009, 84, 597-606).
Product accumulation in microbes typically begins when a nutrient is exhausted in the medium, hut a surfeit of carbon, usually in the form of glucose, remains available. To realize product accumulation in a batch culture, the media are carefully designed to incorporate an unbalanced nutrient composition, such that cells propagate fast in the early- stage with a concurrent depletion of the controlling nutrient. During the middle and the late stage of the culture, the cell propagation rate is significantly reduced but product anabolism remains active, leading to a net accumulation of intracellular product. An early study disclosed a two-stage continuous culture system for product accumulation, while the entire output of the first stage was pumped directly into the second stage (Hall, M. I, et al Appl Environ. Microbiol. 1977, 33, 577-584). Because residua! nutrients as well as potential extracellular metabolites were carried over, the second stage was essentially similar to that of the batch culture described above. Therefore, current microbial product technology incorporates cell propagation and product accumulation in the same culture broth, yet both cellular processes are compromised due largely to unbalanced nutrient availability. In addition, impurities that cany over from the fermentation stage can affect isolation, crystallization, or recovery of the desired product, in certain embodiments, crude components of the medium, such as yeast extract f corn steep liquor, are among the impurities; however, those crude components of the medium are often the most economical means to optimize biomass production. Therefore, fermentation processes requiring crystallization typically utilize downstream processing, such as ultrafiltration, ion exchange, chromatographic separation columns, or activated carbon, to improve product purity.
So, there exists a need for a process for the production of small organic molecules by fermentation that minimizes or decreases the need for downstream processing, thereby reducing costs or increasing throughput or both.
SUMMARY OF THE INVENTION
In certain embodiments, die invention relates to a process, comprising the steps of: supplying a first medium;
contacting a plurality of microorganisms with the first medium: for a first period of time under conditions suitable for microorganism growth and reproduction, thereby forming an increased plurality of microorganisms;
separating the increased plurality of microorganisms from the first medium, thereby forming a substantially pure plurality of microorganisms;
contacting the substantially pure plurality of microorganisms with a second medium for a second period of time under conditions suitable for the production of a product, wherein the second medium consists essentiall of (i) water, a salt, and a substrate, or (ii) water and a substrate, thereby forming the product.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 tabulates the presence (·*-) or absence (empty box) of components in various fermentation media useful in eiythrito! production.
Figure 2 depicts erythrito! production under conditions outlined in Figure 1 (1 complete media without ( R JSQ*; 2 - complete media without (N¾ >SO.*, Amberex, Amberferm; 3 ::: complete media. Thiamine and Trace Elements; 4 ::: complete media. Trace Elements only; and 5 ~ Sodium. Chloride only). Figure 3 depicts data showin that erythritol production is a two-stage fermentation (squares) at ! -L scale demonstrates a higher production rate and erythritol titer than standard fermentation (diamonds).
'DETAILED DESCRIPTION OF THE INVENTION
Overview
in certain embodiments, the invention relates to a process for producing and purifying a small organic molecule, in certain embodiments, the costs associated with purification are minimized.
in certain embodiments, the invention relates to a two-stage fermentation process
(e.g., a tandem process comprising a "growth stage" and a "fermentation stage"). In certain embodiments, the invention relates to a process, comprising the steps of: supplying a first medium; contacting a plural try of microorganisms with the first medium for a first period of time under conditions suitable for microorganism growth and reproduction, thereb forming an increased plurality of microorganisms; separating the increased plurality of microorganisms from the first medium, thereby forming a substantially pure plurality of microorganisms; contacting the substantially pure plurality of microorganisms with a second medium for a second period of time under conditions suitable for the production of a product, wherein the second medium consists essentially of (i) water, a salt buffer, and a substrate, or (ii) water and a substrate, thereby forming the product.
Definitions
"Dry weight" and "dry cell weight" mean, weight determined in the relative absence of water. For example, refere nce to oleaginous cells as comprising a specified percentage of a particular component by dr weight means that the percentage is calculated based on the weight of the cell after substantially ail water has been removed,
"Microorganism" is intended to mean a profcaryotic or eukaryotic cell or organism having a microscopic size. The terra is intended to include bacteria of all species and eiikaryotie organisms such as yeast and fungi. The term also includes ceil cultures of any species that can be cultured for the production of a biochemical or small organic molecule. *
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contacting the substantially pure plurality of microorganisms with a second medium for a second period of time under conditions suitable for die production of a product, wherein the second medium consists essentiaUv of (i) water, a salt, and a substrate, or (ii) water and a substrate, thereby forming the product
In certain embodiments, the invention relate to any one of the aforementioned processes, wheretn the microorganism is selected from the group consisting of Ctypiococcus curvatus, Ctypiococcus temcaius, Candida sp., Escherichia coU, L/pomyces sl rkayi, Lipomyces lipofory Endomycopsis ve alis, Rhodoionria gl tmis, Mhodot nda gracilis., and Yarro a Upoiynca,
in certain embodiments, the invention relates to any one of the aforementioned processes, wherein the microorganism i selected from the group consisting of Mannheimia succ i iproducens, Anaerobiospirilltm succiniciproducens^ Aciinob ciUus succinogems and Escherichia coli and the product is succinic acid.
In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the microorganism is selected from the group consisting of Aspergillus terrms, Escherichia co!i, Vstilago zeae, Usidaga m ydis, Candida sp., Candida mutant, and Rh dot rula sp.; and the product is itaconic acid.
in certain embodiments, the invention relates to any one of the aforementioned processes, wherein the microorganism is a member of the genus "frigonop is, Momliella, Yarroma, Trichosporortoides, or Candida: and the product is erythritoL In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the microorganism is selected from the group consisting of Momliella fo mfosa, Momliella poHmi y Y rrawk) lipolyiica, T ' richosporomtides o docephalis, Trichosporonoides nigrescent; and Trichosporonoides megaehiUensis; and the product is erythritoL
In certain embodimeftts, the invention reiates to any one of the aforementioned processes, wherein the microorganism is Escherichia coii and the product is 1 ,4- butanedioi.
In certain embodiments, the invention reiates to any one of the aforementioned processes, wherein the pluraiity of microorganisms is contacted with the first medium: in a first reactor.
in certain embodiments, the invention relates to any one of the aforementioned processes, wherein the first medium is crude.
In certain embodiments, tiie invention reiates to any one of tiie aforementioned processes, wherein the first medium comprises iignoceiiulosie material glucose, xylose, sucrose, acetic acid, dextrose, glycerol, fructose, lactose, galactose, raannose, rhamnose, ara'btnosc, yeast extract, urea, MgOs, CaC!¾ M:nC¾, NajHPO*. NaHaPO*. M COs, NaOH, NH4OH, <N¾)?.HP04, MgS0 , (NH )jS0 , NH4Cl. KH3PO4, K2HP04, peptone, or a combination thereof.
In certain embodiments, the invention reiates to any one of the aforementioned processes, wherein conditions suitable for microorganism growth and reproduction are a first temperature, a first H, and a first inoculation volume.
In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the first temperature is about 28 X to about 45 X. In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the first temperature is about 28 X to about 32 X. in certain embodiments, the invention relates to any one of the aforementioned processes, wherein the first temperature is about 28 X.. about 29 , about 30 X, about 31 , or about 32 X.
In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the first pli is about 2,5 to about 7.5. n certain embodiments, the invention reiates to any one of the aforementioned processes, wherein the first pH is about 2.7 to about 4. In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the first pH is about 2.8, about 3.0, about 3.2, about 3.4, about 3.6'. about 3.8. or about 4.0, In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the first period of time is about- 12 h to about 48 h. In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the first period of time is about 24 h to about 48 h. in certain embodiments, the invention relates to an one of the aforementioned processes, wherein the first period of time is about 24 h, about 26 h, about 28 h, about 30 h, about 32 h, about 34 h, about 36 h, about 38 h, about 40 h, about 42 h, about 44 h, about 46 h, or about 48 h.
In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the first inoculation volume is about \ % to about 10% v/v, in certain embodiments, the invention relates to any one of the aforementioned processes, wherein the first inoculation volume is about 1 % to about 2% v/v. in certain embodiments, the in vention relates to any one of the aforementioned processes, wherein the first inoculation volume is about 1 %, about ί .5%, or about 2% v/v.
In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the increased plurality of microorganisms has a celi densit that is about 100 times larger to about iOOO times larger than the celi density of the plurality of microorganisms. In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the increased plurality of microorganisms has a ceil density that is about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, or about 1 00 times larger than the cell density of the plurality of micro organi sms .
In certatn embodiments, the invention relates to any one of the aforementioned processes, wherein the increased plurality of microorganisms is separated from: the first medium by ceutrifugation.
In certain embodiments, the invention relates to any one of the aforementioned processes, further comprising the step of washing the substantially pure plurality of microorganisms, in certain embodiments, the invention relates to any one of the aforementioned processes, wherein the substantially pure pluralit of microorganisms is washed with water.
In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the substantially pure plurality of microorganisms is contacted with the second medium in a second reactor. In certain embodiments, the invention relates to any one of die aforerae.ntio.ned processes, wherein the first reactor and the second reactor are not the same.
In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the first reactor or the second reactor is a fermentor.
In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the substrate is glycerol, glucose, xylose, sucrose, acetic acidf dextrose, fructose, lactose, galactose, mannose, rhamrtosef arabinose, starch, molasses, hydrosylates of corn syrup, hydrosylates of wood, or a combination thereof.
In certain embodiments, the invention relates to any one of the aforementioned processes, wherein, the concentration of the substrate in the second medium is about Ϊ 00 g L to about 400 g/L. In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the concentration of the substrate in the second medium is about 100 g/L, about 150 g/L, about 200 g/L, about 250 g/Lf about 300 g/L, about 350 g/L, or about 400 g/L.
In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the second .medium consists essentially of water, a salt, and a substrate; and the salt is a buffer.
In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the second medium consists essentially of water, a salt, and a substrate; and the salt is sodium chloride.
In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the water is distilled water. In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the water is undistilled water.
In certain embodiments, the invention relates to any one of the aforementioned processes, wherein conditions suitable for the production of the product arc a. second temperature, a second H, and a second inoculation volume.
In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the second temperature is about 28 °C to about 45 ~C. in certain embodiments, the invention relates to any one of the aforementioned processes, wherein the second temperature is about 28 °C to about 32 °C. In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the second temperaiitre is about 28 C, about 29 X, about 30 °C, about 31 5Cf or about 32 X.
In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the second pH is about 2.5 to about 7.5- in certain embodiments, the invention relates to any one of the aforementioned processes, wherein the second pH is about 2.7 to about 4. In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the second pH is about 2.8, about 3.0, about 3.2, about 3,4, about 3,6, about 3.8, or about 4,0.
In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the second period of time is about 24 h to about 120 h. In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the second period of time is about 48 h to about 120 h. in certain embodiments, the invention relates to any one of the aforementioned processes, wherein the second period of time is about 48 h, about 54 h, about 60 h, about 66 h, about 72 h, about 78 h, about 84 bf about 90 h, about 96 h, about 102 h, about 108 h, about 1 14 h, or about 120 h.
In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the second inoculation volume is about 1 % to about 10% v/v. In certain embodiments, the invention relates to any one of the aforementioned, processes, wherein the second inoculation volume is about 1% to about 2% v v. In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the second inoculation volume is about I %, about 1,5%, or about 2% v/v.
In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the product is selected from the group consisting of erythritol, succinic acid, 1 t4-butanediol, and ttacontc acid.
In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the product is not a triacylgiyceroi
In certain embodiments, the invention relates to any one of the aforementioned processes, further comprising the step of; separating the product from the substantially pure plurality of microorganisms.
In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the product is separated from the substantially pure plurality of microorganisms by centrifuga-ion, thereb forming an aqueous broth comprising the product.
In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the product is separated from the substantially pure plurality of microorganisms by solid-liquid separation.
In certain embodiments, the invention relates to any one of the aforementioned processes, further comprising the step of: crystallizing the product from the aqueou broth, thereby forming a crystallized product.
In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the concentration of the product in the second medium is about 150 g L to about 300 g/L. In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the concentration of the product in the second medium is about 150 g/L, about 200 g/L, about 250 g/L, or about 300 g/L.
In certain embodiments, the invention relates to any one of the aforementioned processes, further comprising the step of: crystallizing the product, thereby forming a crystallized product. In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the crystallization is carried out in the second reactor. In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the crystallization involves exposing the product to a third temperature.
In certain embodiments, the invention relates to any one o the aforementioned processes, wherein the process does .not involve subjecting the product to calcium hydroxide, vacuum distillation, ultrafiltration, ion exchange, column chromatography, or activated carbon.
In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the product is substantially mote pure than a product produced under substantially the same conditions by the same microorganism, but without the steps of separating the increased plurality of microorganisms from: the first medium and contacting the substantially pure pluralit of microorganisms to the second medium:.
In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the crystallized product is substantially more pure than a crystallized product produced under substantially the same conditions by the same microorganism, but without the steps of separating the increased plurality of microorganisms from the first medium and contacting the substantially pure plurality of microorganisms to the second medium.
In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the yield of the product or the crystallized product is about 50% to about 99%. In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the yield of the product or the crystallized product is about 70% to about 99%. In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the yield of the product or the crystallized product is about 80% to about 99%. In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the yield of the product or the crystallized product is about 90% to about 99%.
In certain embodiments, the invention relates to any one of the aforementioned processes, wherein the purity of the product or the crystallized product is about 70% to about 99%. in certain embodiments, the invention relates to any one of the aforementioned processes, wherein the purity of the product or the crystallized product is about 80% to about 99%. in certain embodiments, the invention relates to any one of the aforementioned processes, wherein the purity of the product or the crystallized product is about 90% to about 99%.
In certain embodiments, the invention relates to any one of the aforementioned processes, wherein an aspect of the process is described in U.S. Patent No. 5,902,739, U.S. Patent No. 6,440,712, or U.S, Patent Application Publication No. 201 1/0003355, the contents of each of which are hereby incorporated by reference in their entirety.
Figure imgf000011_0001
In certain embodiments, the invention relates to a product made by any one of the af remen tioned processe . EXEMPLIFICATION
"This invention is further illustrated b the following examples, which should not be construed as limiting.
Example t
To determine the nutrients necessary for the "production stage'1 a reduction of medium components was evaluated in shake flasks as outlined in Figure I . Figure 2 demonstrates equivalent erythritol production in the salt-buffered water and substrate solution (Flask 5) as in the full medium formulation (Flask 1 }.
Flask 5 conditions were then scaled up to a 1-L stirred tank reactor. Both erythritol production rate and final titer were improved in the two-stage process (Figure 3).
Example 2 (Prophetic)
After crystallization, the products of formed by a two-stage process of the invention are qualitativel and quantitatively more pare than products formed by standard fermentation.
INCORPORATION BY REFERENCE
All of the U.S. patents and U.S. published patent applications cited herein are hereby incorporated by reference.
EQUIVALENTS
Those ski fled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
l i

Claims

We claim:
1 . A process, comprising the steps of:
supplying a first medium;
contacting a plurality of raicroorgaiiisms with the first medium for a first period of time under conditions suitable for microorganism growth and reproduction, thereby forming an increased plurality of microorganisms;
separating the increased plurality of raicroorgaiiisms from the first medium, thereby forming a substantially pure plurality of microorganisms;
contacting the substantially pure plurality of microorganisms with a second medium for a second period of time under conditions suitable for die production of a product, wherein the second medium consists essentiaUv of (i) water, a salt, and a substrate, or (ii) water and a substrate, thereby forming the product
2. The process of claim I, wherein the microorganism is selected from the group consisting of Mamketmia sucemiciproducens, Anaerobiospirillum s cciniciproducens, Actinohacill s succinogenes, and Escherichia coir, and the product is succinic acid,
3. The process of claim J , wherein the microorganism is selected from the group consisting of Aspergillus hitrens, Escherichia colt, Ustilago zeae, Ustilago maydis, Candida sp,, Candida mutant, and Rhodoioruki sp.; and the product is itaconic acid,
4. The process of claim 1 , wherein the microorganism is a member of the genus iVigonopsis, Moniliella, Yarrowia, Trichosporonoides, or Candida; and the product is erythritol.
5. The process of claim 1 , wherein the microorganism is selected from the group consisting of Moniliella lamenfma, Moniliella pollittis, Yarrowia Hpolytica, Trichosporonoides oedttcephalis, Trichosporonoid s nigrescem, and Trichosporonoides megachiliensis; and the product is erythritol.
6. The process of claim, t , wherein the microorganism is Escherichia, colt and the product is 1 ,4-butanediol.
7. The process of any one of claims 1 -6, wherein the plurality of microorganisms is contacted with the first medium in a first reactor.
8. The process of any one of claims i-7, wherein the first medium is crude.
9. The process of an one of claims 1 -8, wherein the first medium comprises lignoeeliuiosic material, glucose, xylose, sucrose, acetic acid, dextrose, glycerol, fructose, lactose, galactose, mannose, rhamnose, arahinose, yeast extract, urea, MgC¾, CaCI¾ Mn<¾, NajHPO*. Na¾?at. MgC03, NaOH, NH4OH, ( H^HPO^ gSO*. CNH^SO*, NR<Q, KH2PO4, iHPO.i, peptone, or a combination thereof.
10. The process of any one of claims 1 -9, wherein conditions suitable for microorganism growth and reproduction are a first temperature, a first pH, and a first inoculation volume.
1 1. The process of claim 10, wherein the first temperature is about 28 °C to about 45 T.
12. The process of claim 10 or claim 1 1 , wherein the first pH is about 2,5 to abotit 7.5,
13. The process of any one of claims 10- 12, wherein the first -period of time is about 12 h to about 4$ h.
14. The process of any one of claims 10-13, wherein the first inoculation volume is about 1¾ to about 10% y/v.
15. The process of any one of claims 1- 14, wherein the increased plurality of microorganisms has a cell density that is about 100 times larger to about 1000 times larger than the cell densi ty of the pl uralit of microorganisms.
16. The process of any one of claims 1 - 15, wherein the increased plurality of microorganisms is separated from the first medium by centxitngation.
17. The process of any one of claims 1 - 6, further comprising the step of washing the substantially pure plurality of microorganisms.
18. The process of claim 1 7, wherein the substantially pure plurality of microorgani ms is washed with water.
19, The process of any one of claims 1-18, wherein the substantially pure plurality of microorganisms is contacted with the second medium in a second reactor.
20. The process of any one of claims 1-19, wherein the first reactor and the second reactor are not the same.
21. The process of any one of claims 1-20, wherein the first reactor or the second reactor is a fermentor.
22. The process of any one of claims .1-21, wherein the substrate is glycerol, glucose, xylose, sucrose, acetic acid, dextrose, fructose, lactose, galactose, mannosef rhamnose, arabinose, starch, molasses, hydrosylates of com syrup, hydrosylates of wood, or a combination thereof.
23. The process of any one of claims 1-22, wherein the concentration of ihe substrate in the second medium is about 500 g/L to about 400 g L.
24. The process of any one of claims 1-22, wherein the concentration of the substrate in the second medium is about 150 g/L,
25. The process of any one of claims 1-24, wherein the second medium consists essentially of water, a salt, and a substrate; and the salt is a buffer.
26. The process of any one of claims 1 -24, wherein the second medium consists essentially of water, a salt and a substrate; and the sal t is sodium chloride.
27. The process of any one of claims 1-26, wherein the water is distilled water.
28. The process of any one of claims 1-26, wherein the water is undistii!ed water.
29. The process of any one of claims 1-28, wherein conditions suitable for the production of the product are a second temperature, a second H, and a second inoculation volume,
30. The process of claim 29, wherein the second temperature is about 28 °C to about 45 X.
31. The process of claim 29 or claim 30, wherein the second pH is about 2.5 to about 7,5.
32. The process of any one of claims 29-31 , wherein, the second period of time is about 24 h to about 120 h.
33. The process of any one of claims 29-32, wherein the second inoculation volume is about 1 % to about 1 % v/v.
34. The process of any one of claims 1-33, wherein the product is selected from the group consisting of erythritol, succinic acid, 1,4-butancdiol, and iiaconic acid.
35. The process of any one of claims 1-34, wherein the product is not a triacylglyceroL
36. The process of any one of claims i-35, further comprising the step of: separating the product from the substantially pure plurality of microorganisms.
37. The process of claim 36, wherein the product is separated from the substantially pure plurality of microorganisms by centrifugatiorr, thereby forming an aqueous broth comprising the product.
38. The process of any one of claims 1-37, further comprising the step of: crystallizing the product from the aqueous broth, thereby forming a crystallized product.
39. The process of claim 37, wherein the concentration of the product in the aqueous broth is about 150 g/L to about 300 g/L.
40. The process of any one of claims 1-39, wherein the concentration of the product in the second medium is about 150 g L to about 300 g/L.
41. The process of any one of claims 1-37, further comprising the step of: crystallizing the product, thereby forming a crystallized product.
42. Hie process of claim 41, wherein the crystallization is carried out in the second reactor.
43. The process of any one of claims 38, 39, 4.1 , or 42, wherein the crystallization involves exposing the product to a third temperature.
44. The process of any one of claims 1-43, wherein the process does not involve subjecting the product to calcium hydroxide, vacuum distillation, ultrafiltration, ion exchange, column chromatography, or activated carbon.
45. The process of any one of claims 1-44, wherein the product is substantially more pure than a product produced under substantially the same conditions by the same microorganism, but without the steps of separating the increased plurality of microorganisms from the first medium and contacting the substantially pure plurality of microorganisms to the second medium.
46. The process of any one of claims 38, 39, 41, or 42, wherein the crystallized product is substantially more pure than a crystallized product produced under substantially the same conditions by the same microorganism, but without the steps of separating the increased plurality of niteroorganisnis from the first medium and contacting the substantially pure plurality of microorganisms to the second medium.
47. The process of any one of claims 1 -46, wherein the yield of the product or the crystallized product is about 50% to about 99%,
48, The process of an one of claims 1-47, wherein the purity of the product or die crystallized product is about 70% to about 99%,
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