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WO2015042114A1 - Procédé de fermentation comportant des étages distincts de croissance et de production - Google Patents

Procédé de fermentation comportant des étages distincts de croissance et de production 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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WO
WIPO (PCT)
Prior art keywords
product
medium
microorganisms
certain embodiments
water
Prior art date
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Ceased
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PCT/US2014/056029
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English (en)
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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Novogy Inc
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Filing date
Publication date
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Publication of WO2015042114A1 publication Critical patent/WO2015042114A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Dans certaines formes de réalisation, l'invention concerne un procédé comprenant les étapes de fourniture d'un premier milieu ; de mise en contact d'une pluralité de microorganismes avec le premier milieu pendant un premier laps de temps dans des conditions convenant à la croissance et la reproduction des microorganismes, en formant de ce fait une pluralité accrue de microorganismes ; de séparation de la pluralité accrue de microorganismes du premier milieu, en formant de ce fait une pluralité sensiblement pure de microorganismes ; de mise en contact de la pluralité sensiblement pure de microorganismes avec un deuxième milieu pendant un deuxième laps de temps dans des conditions convenant à la production d'un produit, le deuxième milieu consistant essentiellement en (i) de l'eau, un sel et un substrat, ou (ii) de l'eau et un substrat, en formant de ce fait le produit.
PCT/US2014/056029 2013-09-18 2014-09-17 Procédé de fermentation comportant des étages distincts de croissance et de production Ceased WO2015042114A1 (fr)

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US61/879,243 2013-09-18

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11155808B2 (en) 2015-12-07 2021-10-26 Zymergen Inc. HTP genomic engineering platform
US11208649B2 (en) 2015-12-07 2021-12-28 Zymergen Inc. HTP genomic engineering platform
WO2022083526A1 (fr) 2020-10-19 2022-04-28 中国石油化工股份有限公司 Catalyseur d'hydrocraquage chimique, son procédé de préparation, et son application
CN114456959A (zh) * 2022-01-26 2022-05-10 山东星光首创生物科技有限公司 一种耐高渗的解脂亚罗酵母菌株、选育方法及应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0136805B1 (fr) * 1983-08-24 1991-10-09 Cpc International Inc. Procédé de production de polyols par fermentation de sucres à l'échelle industrielle
US7198936B2 (en) * 2000-10-31 2007-04-03 Novozymes Biopharma Ab Method for growth of bacteria, minimising the release of endotoxins from the bacteria into the surrounding medium
US7695949B2 (en) * 2000-08-08 2010-04-13 Dsm Ip Assets B.V. Process for producing a target fermentation product
WO2012061653A2 (fr) * 2010-11-03 2012-05-10 The Regents Of The University Of California Production de biocarburant et de produits chimiques par des microorganismes recombinants par fermentation d'une biomasse protéinique
US8497105B2 (en) * 2009-06-26 2013-07-30 Cobalt Technologies, Inc. Integrated system and process for bioproduct production

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0136805B1 (fr) * 1983-08-24 1991-10-09 Cpc International Inc. Procédé de production de polyols par fermentation de sucres à l'échelle industrielle
US7695949B2 (en) * 2000-08-08 2010-04-13 Dsm Ip Assets B.V. Process for producing a target fermentation product
US7198936B2 (en) * 2000-10-31 2007-04-03 Novozymes Biopharma Ab Method for growth of bacteria, minimising the release of endotoxins from the bacteria into the surrounding medium
US8497105B2 (en) * 2009-06-26 2013-07-30 Cobalt Technologies, Inc. Integrated system and process for bioproduct production
WO2012061653A2 (fr) * 2010-11-03 2012-05-10 The Regents Of The University Of California Production de biocarburant et de produits chimiques par des microorganismes recombinants par fermentation d'une biomasse protéinique

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11155808B2 (en) 2015-12-07 2021-10-26 Zymergen Inc. HTP genomic engineering platform
US11155807B2 (en) 2015-12-07 2021-10-26 Zymergen Inc. Automated system for HTP genomic engineering
US11208649B2 (en) 2015-12-07 2021-12-28 Zymergen Inc. HTP genomic engineering platform
US11312951B2 (en) 2015-12-07 2022-04-26 Zymergen Inc. Systems and methods for host cell improvement utilizing epistatic effects
US11352621B2 (en) 2015-12-07 2022-06-07 Zymergen Inc. HTP genomic engineering platform
WO2022083526A1 (fr) 2020-10-19 2022-04-28 中国石油化工股份有限公司 Catalyseur d'hydrocraquage chimique, son procédé de préparation, et son application
CN114456959A (zh) * 2022-01-26 2022-05-10 山东星光首创生物科技有限公司 一种耐高渗的解脂亚罗酵母菌株、选育方法及应用

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