(12) Patent Application Publication (10) Pub. No.: US 2016/0168595 A1 JANSSEN Et Al
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US 2016O168595A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0168595 A1 JANSSEN et al. (43) Pub. Date: Jun. 16, 2016 (54) TWO-PHASE FERMENTATION PROCESS (86). PCT No.: PCT/NL2014/OOOO2O FOR THE PRODUCTION OF AN ORGANIC S371 (c)(1) COMPOUND (2) Date: Jan. 4, 2016 (71) Applicants: Antonius Cornelis Johannes Matheus (30) Foreign Application Priority Data JANSSEN, Geleen (NL); Johannes Gerardus Theodorus KIERKELS, Jul. 4, 2013 (EP) ............................. EP 130O3384.8 Geleen (NL); Georg Friedrich LENTZEN, Geleen (NL); Publication Classification ISOBIONICS B.V., Geleen (NL) (51) Int. Cl. (72) Inventors: Antonius Cornelis Johannes Matheus CI2P 5/00 (2006.01) JANSSEN, Geleen (NL); Joannes (52) U.S. Cl. Gerardus Theodorus KIERKELS, CPC ...................................... CI2P5/007 (2013.01) Geleen (NL); Georg Friedrich LENTZEN, Geleen (NL) (57) ABSTRACT The invention relates to a two phase fermentation process for (21) Appl. No.: 14/902,707 producing an organic compound, in particular an isoprenoid and to a bioreactor comprising a two phase fermentation (22) PCT Fled: Jul. 4, 2014 system for producing an organic compound. Patent Application Publication Jun. 16, 2016 US 2016/0168595 A1 F.G. 1 FIG. 2 US 2016/01 68595 A1 Jun. 16, 2016 TWO-PHASE FERMENTATION PROCESS the product concentration does not rise to a point where FOR THE PRODUCTION OF AN ORGANIC product biosynthesis is inhibited, thus ensuring a Sustained COMPOUND period of high rate of productivity. One such attempt utilizes 0001. The invention relates to a two phase fermentation a liquid that is immiscible with the aqueous fermentation process for producing an organic compound, in particular an medium but is an extractant for the desired product. The target isoprenoid and to a bioreactor comprising a two phase fer product partitions between the extractant and the aqueous mentation system for producing an organic compound. fermentation medium when the two are brought into contact, 0002) Isoprenoids (also known as terpenoids or terpenes) thereby reducing the concentration of the product in the aque are a large and diverse class of naturally occurring organic ous medium. In-situ separation of the released product being compounds that find potential utility, interalia in the produc performed in a two-phase fermentation using an organic Sol tion of pharmaceuticals, cosmetics, perfumes, flavors, animal vent as the secondary phase has been attempted to mitigate feed Supplements and nutraceuticals. the drawbacks of conventional fermentation methods. (Mali 0003 Conventional production methods involve, e.g., nowski, Biotech Advances 2001, 19: 525-538). extraction of these compounds from plants, microbes and 0008. In practice, two-phase extractive fermentation sys animals. However, these extraction methods suffer from tems are complex owing to the unpredictable nature of these numerous limitations such as low yield of extraction, lack of systems, particularly for large scale production. One of the amenability of the source organisms to large scale cultivation most significant challenges is the selection of the right solvent and complicated production methods. Also, chemical synthe system for a given product/microorganism combination. A sis methods for producing isoprenoids are not lucrative due to frequently encountered difficulty, for instance is that most the high cost of starting materials and the requirement of common water-immiscible solvents are toxic to the microor extensive product purification steps. In-vitro enzymatic ganisms and/or hazardous rendering them unsuitable for approaches have also been explored but the exploitation of commercial scale production of products Such as isoprenoids this approach is for instance restricted by the limited avail via fermentation. It has also been encountered that certain ability of the precursors. Solvents form stable emulsions with the aqueous fermenta 0004 Metabolic engineering of microorganisms for iso tion medium causing difficulties of separation, equipment prenoid production is believed to be most promising for the blockage etc. The selection of a biocompatible organic carrier production of large amounts of isoprenoids from cheap car solvent with favorable partition coefficients is thus crucial for bon Sources in fermentation processes, although some the implementation of an effective bioconversion in an aque hurdles still have to be taken (reviewed in Ajikumaretal, Mol. ous-organic biphasic system (Cruz et al.2004; León et al. Pharmaceutics, 2008, 5 (2), 167-190). Production of iso 1998). Preferably, the solvent has other desirable carrier sol prenoid through fermentation of microorganisms is deemed vent characteristics, such as low emulsion-forming tendency, more desirable than the traditional methods as it meets the chemical, thermal, and biological stability. requirement of sustainable production in a more economical, 0009 Typically, addition of solvents during the fermenta industrially scalable and productive way. Production of iso tion process involves addition of large Volumes of an organic prenoids via fermentation methods represents an alternate solvent within a short time interval which, e.g., involves the process technology that utilized lower cost of feedstock and risk of introducing potential contaminants into the medium. A higher productivity, affording a potential for lower cost of loss of sterility of fermentation has serious consequences manufacture. impacting production costs, schedules and affecting the prod 0005 One of the most frequently encountered problems in uct quality and quantity. Apart from the risk of introducing fermentation procedures is end-product inhibition, that is, the contamination, extractants seriously affect the stability of the microorganisms responsible for the fermentation may be signals from crucial probes, like e.g., pH-electrode and dis impaired by the fermentation product, e.g. because the fer Solved oxygen probe, interfering with accurate measurement mentation product is cytotoxic. Accumulation of the product of important parameters such pH and dissolved oxygen con beyond a critical concentration inactivates the microorganism tent of the medium. This is more common during the employ and substantially diminishes the rate of productivity. This ment of organic solvents as extractants since oxygen usually phenomenon is particularly relevant in the production of iso has a higher solubility in organic solvents. Therefore, addi prenoids as most microbes are destroyed or inactivated in the tion of organic solvents during fermentation has a serious presence of these cytotoxic compounds posing a severe limi impact on the accurate measurement of crucial parameters tation to obtain highly productive strains. Owing to these Such as pH and dissolved oxygen that are required to control compounds not being obtained beyond a critical concentra the fermentation. tion, additional steps of concentration and purification may be required rendering the process cumbersome and expensive. 0010. These limitations have hindered the possibilities of 0006 Another major drawback is that isoprenoids, being using water-immiscible solvents during fermentation for highly volatile organic compounds, are poorly soluble in industrial scale production of the target products. There is aqueous Solutions. Thus, loss of product during fermentation thus a need for an industrially scalable fermentation process through the off-gas is a major problem in the development of for the production of organic compounds, in particular iso an economically feasible process (Asadollahi et al. Biotech prenoids in the presence of a water-immiscible solvent. Fur Bioengin 2007, 99(3): 666-677). In previous studies, terpe ther, there is a need to produce these compounds with a good noids synthesized in E. coli were partly lost by evaporation yield and productivity with a low tendency to build up toxic due to their highly volatile character (Newman et al., Biotech levels of metabolic intermediates. nol. Bioeng. (2006).95: 684-691). 0011. It is therefore an objective of the present invention to 0007 To overcome the above mentioned drawbacks, provide a fermentation method for producing organic com attempts have been made to remove the product from the pounds, in particular isoprenoids, that fulfills this need. It is fermentation medium as the fermentation advances such that one objective of the present invention to specifically address US 2016/01 68595 A1 Jun. 16, 2016 the challenges associated with utilization of water-immis ried out as follows: in a first step, a Zero current measurement cible solvents in industrial scale fermentation process. is performed by using Zeroing gel or nitrogen (N2) or carbon 0012. It is a particular objective of the present invention to dioxide (CO2) calibration gases, alternatively in a sample provide an industrially scalable and robust method for the medium Saturated with one of these gases. The probe is then production of organic compounds which permits the utiliza mounted into the fermenter and autoclaved, dodecane is tion of organic solvents as an extractant. added and the 100% value is determined (second calibration 0013. It is a further objective of the present invention to step) after Saturating with air. In this particular embodiment, provide an efficient fermentation process for the industrial the oxygen probe is thus provided to the fermenter before scale production of an organic compound, preferably an iso addition of the solvent. After addition of the solvent