Sustainable fermentation

Abstract

The present invention relates to a process and an apparatus for cultivating a microorganism with pure oxygen in a fermenter. At least part of the gas leaving the headspace of the fermenter is recirculated into the fermentation broth. A gas containing at least 30% oxygen in volume is added to the recirculated gas.

Description

SUSTAINABLE FERMENTATIONI. FIELD OF THE INVENTION

[0001] The present invention relates to a process for cultivating a microorganism with pure O2, as well as to an apparatus for said method.II. BACKGROUND

[0002] The world's population is growing and so is the demand for food, including the demand for meat, dairy products and seafood. Thus, to meet the demand for food and feed, alternative protein sources are required. Additionally, energy consumption in the production of food and feed has become a major concern and new ways of producing food and feed are becoming of more and more interest.

[0003] Besides plant- and animal-derived protein, an alternative source of proteins has been identified, namely microbial or single cell-derived proteins (SCP). Said alternative proteins, as well as other fermentation products, can be produced using fungi, yeast, algae and / or bacteria that offer the ability of large-scale culturing at comparatively low cost. However, these fermentative productions still face several challenges. The productivity of industrial fermentation is, among other factors, limited by the transfer of oxygen from air to the broth. Hence, there is still a need to improve production of alternative proteins and to improve fermentation processes in general.III. SUMMARY

[0004] We surprisingly found that the utilization of the supplied pure O2may be improved through recirculation of at least a part of the offgas, and thereby drastically increasing the fermentation productivity and yield.

[0005] We found that O2utilization may be improved by adding pure O2to the air going into the fermenter and recycling the offgas.

[0006] Thus, it is an object of the invention to provide a more sustainable and more efficient route to prepare a protein-containing (microbial) biomass or SCP. In particular, it is an object to provide a method for preparing such a (microbial) biomass wherein the O2utilization is improved. The present invention further aims at providing an apparatus for performing the fermentation according to the present invention. Thus, the increasing demand for alternative feed and food solutions can be met, while ensuring sustainability.

[0007] In particular, the present invention relates to a method for cultivating and / or maintaining a microorganism with pure O2, comprising the steps of a. preparing and optionally sterilizing a fermentation substrate in or adding the substrate to an optionally sterilized fermenter, b. optionally heat-sterilizing the fermenter containing the fermentation substrate, c. optionally, saturating fermentation substrate by adding O2gas to the substrate in the fermenter, d. inoculating the fermenter containing fermentation substrate with a microorganism to produce a fermentation broth, e. optionally adding fermentation substrate or fermentation substrate ingredients continuously or during parts of the time of the fermentation process, f. adding gas, containing high levels of O2, optionally during parts of the time of the fermentation process, g. optionally harvesting partial volumes of the fermentation broth during the fermentation process, h. harvesting the complete fermentation broth at the end of the fermentation process, i. optionally concentrating or diluting and / or optionally purifying the fermentation broth to obtain targeted final product.

[0008] Said method is further characterized in that a. at least a part of the gas leaving the headspace of the fermenter is recycled back into the fermenter below the surface of the fermentation broth and b. gas containing high levels of O2, containing O2, at a level of more than 30%v / v is added to the remaining gas recycled into the fermenter below the surface of the fermentation broth.

[0009] Preferably, the level of O2in the gas containing high levels of O2is more than 50%, more preferably more than 90%, most preferably more than 95%. Preferably, the amount of CO2in the gas containing high levels of O2, is below 0.05% CO2.

[0010] Preferably, at least a part of the gas leaving the headspace of the fermenter is recycled back into the fermenter using a pump or compressor. The gas in the headspace of the fermenter may be dried before being recycled back into the fermenter. This may be achieved, for instance, by means of a condenser producing condensate, optionally recycled back into the fermenter. Further, the gas in the headspace of the fermenter maybe recycled back into the fermenter through a sparger to disperse the gas in the fermentation broth.

[0011] The rate of the gas containing high levels of O2added into the fermenter can be controlled by the amount of dissolved O2measured in the fermentation broth. Preferably, the measurement is given as percentage O2saturation and measured in the fermentation broth by a DO-probe. Optionally, the flow of the gas recycled back into the fermenter below the surface of the broth may be controlled by the heightlevel of the broth in the fermenter. Alternatively, the amount of dissolved O2measured in the fermentation broth may be controlled by any combination of the stirrer rate, the flowrate of the recycled offgas and the overhead pressure, and the rate of the gas containing high levels of O2added into the fermenter is controlled to compensate for the amount of O2that is consumed in the fermenter. In one embodiment of the invention, the fermentation process mixing of the broth is done solely by the gas recycled back into the fermenter below the surface of the broth. In another embodiment of the invention, mixing of the broth is done by the gas recycled back into the fermenter below the surface of the broth in combination with a stirring device. When a stirring device is used, it may be set at fixed or variable stirring rates during the whole fermentation process or parts of the time of the fermentation process.

[0012] The cultivation may be executed in a fermenter under non-atmospheric pressure.

[0013] Optionally, an inert gas may be introduced into the fermenter and / or to the gas recycled back into the fermenter.

[0014] The fermentation is further characterized by gas emissions with a flowrate that is controlled such that the overhead pressure of the fermentation is maintained at its setpoint.

[0015] The fermentation is preferably a fed-batch culture.

[0016] Preferably, the method further comprises the step of drying the biomass that is harvested from the broth produced during the fermentation.

[0017] Preferably, the the microorganism is a yeast, a bacteria, a fungus, plant cells, animal cells, or mammalian cells or any combination thereof. More preferably, the the microorganism is a Saccharomycetales yeast.

[0018] Preferably, the Saccharomycetales yeast is a yeast from the genus Cyberlindnera, Saccharomyces,Kluyveromyces, Wickerhamomyces, Pichia or Yarrowia, preferably from the genus Cyberlindnera or Saccharomyces or Kluyveromyces or Wickerhamomyces.

[0019] Preferably, the Saccharomycetales yeast is a yeast from Cyberlindnera jadinii, Saccharomyces cerevisiae, Kluyveromyces lactis, Wickerhamomyces anomalus, Pichia anomala or Yarrowia lipolytica, more preferably from Cyberlindnera jadinii or Saccharomyces cerevisiae or Kluyveromyces lactis or Wickerhamomyces anomalus.

[0020] Preferably, the Saccharomycetales yeast is a yeast from Cyberlindnera jadinii ATCC 26387, Cyberlindnera jadinii FERM-BP1656, Cyberlindnera jadinii CBS621, Cyberlindnera jadinii CBS841, Saccharomyces cerevisiae GHP1, Saccharomyces cerevisiae CEN.PK113-7D, Cyberlindnera jadiniiAQFM-009, Cyberlindnera jadinii AQFM-035, Cyberlindnera jadinii AQFM-036, Cyberlindnera jadinii AQFM-037, Cyberlindnera jadinii AQFM-038, Cyberlindnera jadinii AQFM-039, Cyberlindnera jadinii AQFM-041,Cyberlindnera jadinii AQFM-017, Cyberlindnera jadinii AQFM-042, Cyberlindnera jadinii AQFM-043,Cyberlindnera jadinii AQFM-044, Cyberlindnera jadinii AQFM-046, Cyberlindnera jadinii AQFM-048,Cyberlindnera jadinii AQFM-017, Cyberlindnera jadinii AQFM-049, Cyberlindnera jadinii AQFM-050,Cyberlindnera jadinii AQFM-051, Cyberlindnera jadinii AQFM-052, Cyberlindnera jadinii AQFM-053,Cyberlindnera jadinii AQFM-054, Cyberlindnera jadinii AQFM-055, Wickerhamomyces anomalus IFO 569,Wickerhamomyces anomalus CBS 1980, Cyberlindnera jadinii ATCC 9950, Kluyveromyces lactis CBS 2896,Wickerhamomyces anomalus CBS 2576 or Yarrowia lipolytica CBS 7504, preferably from Cyberlindnera jadinii ATCC 26387, Cyberlindnera jadinii FERM-BP1656, Cyberlindnera jadinii CBS621, Cyberlindnera jadiniiCBS841, Saccharomyces cerevisiae GHP1, Saccharomyces cerevisiae CEN.PK113-7D, Cyberlindnera jadiniiAQFM-009, Cyberlindnera jadinii AQFM-035, Cyberlindnera jadinii AQFM-036, Cyberlindnera jadinii AQFM- 037, Cyberlindnera jadinii AQFM-038, Cyberlindnera jadinii AQFM-039, Cyberlindnera jadinii AQFM-041,Cyberlindnera jadinii AQFM-017, Cyberlindnera jadinii AQFM-042, Cyberlindnera jadinii AQFM-043,Cyberlindnera jadinii AQFM-044, Cyberlindnera jadinii AQFM-046, Cyberlindnera jadinii AQFM-048,Cyberlindnera jadinii AQFM-017, Cyberlindnera jadinii AQFM-049, Cyberlindnera jadinii AQFM-050,Cyberlindnera jadinii AQFM-051, Cyberlindnera jadinii AQFM-052, Cyberlindnera jadinii AQFM-053,Cyberlindnera jadinii AQFM-054, Cyberlindnera jadinii AQFM-055, Wickerhamomyces anomalus IFO 569,Wickerhamomyces anomalus CBS 1980, Cyberlindnera jadinii ATCC 9950 or Kluyveromyces lactis CBS 2896.

[0021] In particular, the present invention relates to an apparatus for cultivating a microorganism by adding pure O2, comprising a fermenter, a means of feeding fermentation substrate to the fermenter, an outlet for gas from the headspace of the fermenter, a split of that outlet where part of the offgas is emitted towards the environment, and part is recycled back to the fermenter, a first inlet for gas into the fermenter below the surface of the fermentation broth, optionally a connection of the recycled offgas to the gas entering the fermentor via the first inlet, or, optionally, a second inlet for the recycled offgas gas into the fermenter below the surface of the fermentation broth.

[0022] Preferably, the first inlet for gas into the fermenter is positioned below the surface of the fermentation broth, preferably near the bottom of the fermentation vessel. Preferably, the first inlet for gas into the fermenter below the surface of the fermentation broth is below the stirring device (if present).

[0023] Optionally, the apparatus may further comprise an outlet for broth from the fermenter, in case of continuous cultures or in case of fed-batch cultures where broth is withdrawn from the fermenter before the end of fermentation.

[0024] The apparatus may further comprise a stirring device.

[0025] The apparatus may further comprise a DO-Probe for measuring O2saturation of the fermentation broth.IV. DETAILED DESCRIPTION

[0026] The O2transfer from the gaseous phase into the liquid phase in an aerobic fermentation will become limiting if media and substrate concentrations or the substrate feeding rate are maximized to increase the output of a fermentation. To increase the O2transfer, the surface of the gas-liquid interface is commonly increased by reducing the air-bubble size by increasing the stirrer rate, or using a porous, orifice or nozzle sparger. Implementing the former costs more energy and is therefore not desired. Implementing the latter improvements on large scale require adjustments of existing hardware and is therefore often costly and not desired. Moreover, use of porous, orifice or nozzle spargers often leads to blockages of the small opening of the sparger by the cultivated microorganism and salt complexes.

[0027] An alternative to increase the O2transfer is to increase the O2concentration difference between the gas phase and the liquid phase. A common way to do so is to increase the pressure in the fermenter. This requires a certain quality of equipment and material, and thus may be costly. It also requires extra pressure to be generated when compressing the supplied gas for the fermentation process which costs additional energy. Moreover, increasing the pressure in the system is beyond question dangerous, especially when working with common laboratory fermenters made of glass.

[0028] Another way to increase the O2concentration difference is to add pure O2to the air going into the fermenter. Because not all O2that flows into the fermenter is transferred from the gas phase to the broth,adding pure O2to expel N2and enrich the air significantly results in quite some O2losses to the off-gas; especially in large-scale systems.

[0029] This technical problem is solved by the subject-matter as defined in the claims, described in the description, exemplified in the Examples and illustrated in the Figures.

[0030] We surprisingly found that the utilization of the supplied O2may be improved through recirculation of at least part of the fermentation offgas, and thereby drastically increasing the fermentation productivity and yield. The part of the resulting offgas that is not recycled may be used to, among others, to produce fermentation substrates or other chemicals.

[0031] The method of the invention is a fermentation process, preferably for cultivating a microorganism, with pure O2in an aerobic fermentation. This aerobic fermentation of the method for producing a biomass comprises cultivating a microorganism. The microorganism may be a yeast, a bacteria, a fungus, plant cells, animal cells, or mammalian cells or any combination thereof. More preferably, the the microorganism is a Saccharomycetales yeast. In one embodiment, the microorganism in the aerobic fermentation uses the ethanol, which is fed to the aerobic fermentation, as feedstock for the production of biomass.

[0032] It was found that the aerobic fermentation of the present invention allows fermentation using pure O2to take place with a minimum O2consumption. Further, the aerobic fermentation of the present invention has, besides the fermentation product, CO2as byproduct.

[0033] The method for cultivating a microorganism according to the present invention, allows use of pure O2in fermentation with minimal O2losses, even in existing laboratory-scale glass systems at atmospheric pressure. The method for cultivating a microorganism according to the present invention, does not require adjustment or replacement of existing equipment but its design can be implemented as add-on equipment, even at large scale.

[0034] In a preferred embodiment of the method for cultivating a microorganism according to the present invention, pure O2is supplied to the fermenter. This secures highest possible O2concentration at the bubble surface and thus in highest possible O2concentration difference between the gas and liquid phase. This will allow maximizing the O2transfer rate, OTR, under the used environmental conditions, improving fermentation yield.

[0035] In a preferred embodiment of the method for cultivating a microorganism according to the present invention, O2going into the fermenter is passing through the broth and is partly consumed. The O2leaving the fermenter unconsumed is partly recycled back into the fermenter. The O2that is consumed is converted into water, microbial biomass, optionally other fermentation products, and / or CO2. The latter remains partly dissolved in the broth and partly exits the fermenter with the unconsumed O2in the offgas and is also recycled back into the fermenter.

[0036] Pure O2, for use in the method for cultivating a microorganism according to the present invention, can be obtained from electrolysis of water. Next to O2, H2is formed during electrolysis which has an outlet in, for instance, generating electricity, or power and heat; as a reactant in thermophysical processes of petroleum refining and fertilizer production, and as an O2scavenger in the metal industry in heat treating of metals. Current production processes of H2are partly fossil based, either direct (made from fossil oil and natural gas) or indirect (made with fossil energy).

[0037] With the increasing demand for green H2originating from electrolysis of H2O, the production of O2will increase as well. Green H2and accompanied O2can both be used in fermentations with the method for cultivating a microorganism according to the present invention in which the H2supplies energy for electricity, heat production or the reduction of CO2to form the C-source for the fermentation (see below) while the O2is used as electron acceptor in the fermentation. The expected low energy consumption of the method for cultivating a microorganism according to the present invention contributes to a sustainable cradle to gate footprint of the method for cultivating a microorganism according to the present invention.

[0038] Emission of gasses from fermentations performed in the method for cultivating a microorganism according to the present invention are smaller than for conventional aerobic fermentation processes as a part of the gasses are recycled.

[0039] In the context of the present invention, "microsparging" or "microbubble sparging" refers to the technique of sparging micron-sized gas bubbles through the fermentation medium.

[0040] The process for cultivating a microorganism may comprise a sterilizing step, in which a fermentation substrate is prepared then sterilized or in which a fermentation substrate is added to an already sterilized fermenter. In case of, for instance, animal cell cultures, the fermenter and substrates are sterilized separately and not always by using heat

[0041] In the process for cultivating a microorganism, the flow rate of the gas recycled back into the fermenter below the surface of the broth may be controlled between 0.25-2 vvm. In the context of the present invention, the unit vvm ("Vessel Volume per Minute") refers to Vrecyciedgas*VFermentation1*min1.

[0042] The process for cultivating a microorganism may further comprise a step of recovering the biomass from the aerobic fermentation by suitable methods known in the art. Recovering the produced biomass may comprise centrifugation or filtration.

[0043] The process for cultivating a microorganism may further comprise a step of drying the harvested biomass by suitable methods known in the art. Drying biomass may comprise convective / direct drying technologies (like spray drying, fluidized bed) or contact / indirect technologies (like drum drying, vacuum drying, falling film) or supercritical drying (using superheated steam) or natural air / sun drying or even freeze drying.

[0044] In the context of the present invention, the term "DO-probe" refers to a Dissolved O2probe for measuring O2saturation in the fermentation broth. The probe is located below the surface of the fermentation broth.

[0045] In the context of the present invention, the term "fermentation substrate" refers to organic molecules that are converted into water, microbial biomass, optionally other fermentation products, and / or CO2 in the fermentation process. Fermentation substrates may be selected from, but not limited to, sugars, such as fructose, galactose, xylose, arabinose, sucrose, maltose, cellulose, and alcohols, such as methanol, ethanol and organic acids, such as formic acid, acetic acid, preferably glucose, and / or a mixture thereof.

[0046] In the context of the present invention, the protein yield is defined as the amount of protein produced per gram of substrate. A common manner, as stated in US3151038, of determining protein content is to analyze for total nitrogen by the Kjeldahl procedure and then multiply by 6.25, the standardfactor according to accepted practice. Hawk, Philip B., Oser, Bernard L, and Summerson, William H, 1947, Practical Physiological Chemistry, 12th edition, The Blakiston Company, Philadelphia and Toronto, state as follows on pages 213 and 214: The usual factor employed for the calculation of protein from the nitrogen content is 6.25 and is based on the assumption that proteins contain on the average 16 percent of nitrogen.

[0047] In the context of the present invention, the term "maintaining a microorganism" refers to the method of not cultivating (not growing) but only maintaining microorganisms metabolic active (= stay alive) under non-growing conditions. Such a fermentation will start with growth (cultivation) in or outside the described system and continue being metabolic active without growth (maintaining) in described system.

[0048] In the context of the present invention, the terms "microbial biomass" and "biomass" are used interchangeably.

[0049] In the context of the present invention, other fermentation products may be vitamins and vitamin precursors, enzymes, non-enzymatic proteins, microbial lipids, carotenoids, isoprenoids, flavonoids, hydrocolloids, human milk oligosaccharides.

[0050] In the context of the present invention, the term "single cell protein", optionally abbreviated herein also as "SCP", refers to a protein obtained by and / or derived from a (unicellular) microorganism, such as fungi, yeast, algae and / or bacteria. Thus, SCP may refer to a protein purified and / or isolated from a microorganism's cell culture for example. Alternatively or additionally, SCPs may refer to the dead dried cells of microorganisms. Hence, an "single cell protein product" or "SCP product" may or may not comprise one or more selected from the group of intact (unicellular) microorganism cells, disrupted (unicellular) microorganism cells, isolated proteins obtained from one or more (unicellular) microorganism(s), isolated proteins derived from one or more (unicellular) microorganism(s), purified proteins obtained from one or more (unicellular) microorganism(s), and purified proteins derived from one or more (unicellular) microorganism(s). An (unicellular) microorganism may relate to a bacterium, a fungus like yeast and / or an algae.

[0051] SCP products from yeast offer the advantage of providing comparatively high protein contents, while at the same time said products can be produced on industrial scale at comparatively low cost, independent from seasonal effects and with comparatively low harvesting efforts. Thus, yeast SCP products are highly advantageous.

[0052] In the context of the present invention, the term "yeast" refers to a eukaryotic, unicellular microorganism classified as a member of the fungus kingdom that mostly reproduce asexually by mitosis. Further herein, said term preferably relates to yeast cells, which can be grown under artificial and / or lab conditions, e.g. as in vitro culture conditions, and in particular under standard laboratory conditions. Said term preferably also encompasses yeast cells of a single type that have been grown in the laboratory for several generations and thus, said term preferably encompasses also potential mutants of a yeast cell and / or strain. Herein, yeast is preferably Saccharomycetales yeast.

[0053] A "yeast cell" is a cell of a yeast, preferably a cell of a yeast as described herein.

[0054] In the context of the present invention, the term "Saccharomycetales” refers to the order Saccharomycetales within the phylum Ascomycota. Members of Saccharomycetales are also known and sometimes referred to as budding yeasts.

[0055] In the context of the present invention, the term "v / v" is intended to be understood as "volume by volume" and thus refers to the proportion of a particular substance within a mixture, as measured by volume.

[0056] SCP product producer may vary in their ability to use and / or utilize ethanol as carbon source for SCP production. Thus, the yeast SCP product preferably comprises Saccharomycetales yeast cells, wherein said Saccharomycetales yeast cells are Saccharomycetales yeast cells from one or more Saccharomycetales yeast genera, species and / or strains that are capable of using ethanol as carbon source. For example, the Saccharomycetales yeast cells may be Saccharomycetales yeast cells from one or more genera selected from the group consisting of Cyberlindnera, Kluyveromyces, Wickerhamomyces, Yarrowia, Pichia and Saccharomyces.

[0057] More specifically, the yeast SCP product comprises Saccharomycetales yeast cells, wherein said Saccharomycetales yeast cells are preferably Saccharomycetales yeast cells selected from the group consisting of Pichia anomala, Yarrowia lipolytica, Wickerhamomyces anomalus, Cyberlindnera jadinii, Saccharomyces cerevisiae and / or Kluyveromyces lactis. Preferably, the strain is an improved strain.

[0058] (Unicellular) Microorganisms, such as fungi, yeast, algae and / or bacteria may also be used to produce other fermentation products, such as vitamins or vitamin intermediates, enzymes, carotenoids etc. (Unicellular) Microorganisms to produce such other fermentation products include, but are not limited toEscherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Yarrowia lipolytica, Bacillus subtilis, Bacillus amyloliquefaciens, Aspergillus spp., Trichoderma spp., Bacillus spp

[0059] Enzymes, which may be produced as such other fermentation products may include, but not be limited to one or more enzymes capable of modifying mycotoxin(s).

[0060] In some embodiments, said one or more polypeptide(s) capable of modifying mycotoxin(s) is / are one or more peptidase(s), hydrolase(s), esterase(s), lactonase(s), epoxidase(s), peroxidase(s), and / or peroxygenase(s). Preferably, said one or more further polypeptide(s) capable of modifying one or more mycotoxin(s) is / are e.g. fumonisin esterase (e.g. as disclosed in WO 2016 / 134387 Al), ochratoxin peptidase (e.g. as disclosed in WO 2023 / 025938 Al, or in WO 2022 / 129517 A2), zearalenone lactonase (e.g. as disclosed in WO 2020 / 025580 Al, or in WO 2022 / 073649 Al), ergopeptine hydrolase (e.g. as disclosed in WO 2014 / 056006 Al).V. BRIEF DESCRIPTION OF THE FIGURES

[0061] Figure 1: Concentrations (C). Solid lines are concentrations in broth, dotted lines are concentrations in supernatant. Glc=glucose, X=biomass, p=protein product; fermentation settings as in Example 1.1

[0062] Figure 2: Total production and consumption rates in the vessel (R). glc=glucose, X=biomass, p=protein product; fermentation settings as in Example 1.1Figure 3: Respiration data (OUR, CER, RQ), flow rates of glucose and NH3(F), molar fractions in offgas of O2and CO2 (y), and gas flow in and out of the vessel; fermentation settings as in Example 1.1Figure 4: Yields of biomass, product and O2on glucose excluding inoculum production (Y), and volumetric protein productivity. S=glucose, X=biomass, p=protein product, O=O2; fermentation settings as in Example 1.1Figure 5: Dissolved concentrations of O2and CO2at the bubble surface (with *) and in the liquid phase (without *); fermentation settings as in Example 1.1Figure 6: Agitation speed; settings as in Example 1.1Figure 7: Concentrations (C). Solid lines are concentrations in broth, dotted lines are concentrations in supernatant. Glc=glucose, X=biomass, p=protein product; fermentation settings as in Example 1.11Figure 8: Total production and consumption rates in the vessel (R). glc=glucose, X=biomass, p=protein product; fermentation settings as in Example l.llFigure 9: Respiration data (OUR, CER, RQ), flow rates of glucose and NH3(F), molar fractions in offgas of O2and CO2(y), and gas flow in and out of the vessel; fermentation settings as in Example l.llFigure 10: Yields of biomass, product and O2on glucose excluding inoculum production (Y), and volumetric protein productivity. S=glucose, X=biomass, p=protein product, O=O2; fermentation settings as in Example l.llFigure 11: Dissolved concentrations of O2and CO2at the bubble surface (with *) and in the liquid phase (without *); fermentation settings as in Example l.llFigure 12: Agitation speed; settings as in Example l.llFigure 13: Concentrations (C). Solid lines are concentrations in broth, dotted lines are concentrations in supernatant. Glc=glucose, X=biomass, p=protein product; fermentation settings as in Example l.lllFigure 14: Total production and consumption rates in the vessel (R). glc=glucose, X=biomass, p=protein product; fermentation settings as in Example l.lllFigure 15: Respiration data (OUR, CER, RQ), flow rates of glucose and NH3(F), molar fractions in offgas of O2and CO2(y), and gas flow in and out of the vessel; fermentation settings as in Example l.lllFigure 16: Yields of biomass, product and O2on glucose excluding inoculum production (Y), and volumetric protein productivity. S=glucose, X=biomass, p=protein product, O=O2; fermentation settings as in Example l.lllFigure 17: Fresh and recycled gas flow, cumulative amount of O2fed to the reactor and bled from the reactor in the not-recycled offgas; fermentation settings as in Example l.lllFigure 18: Dissolved concentrations of O2and CO2at the bubble surface (with *) and in the liquid phase (without *); fermentation settings as in Example l.lllFigure 19: Agitation speed; fermentation settings as in Example l.lllFigure 20: Concentrations (C). Solid lines are concentrations in broth, dotted lines are concentrations in supernatant. Glc=glucose, X=biomass, p=protein product; fermentation settings as in Example 1.1VFigure 21: Total production and consumption rates in the vessel (R). glc=glucose, X=biomass, p=protein product; fermentation settings as in Example 1.1 VFigure 22: Respiration data (OUR, CER, RQ), flow rates of glucose and NH3(F), molar fractions in offgas of O2and CO2(y), and gas flow in and out of the vessel; fermentation settings as in Example 1.1 VFigure 23: Yields of biomass, product and O2on glucose excluding inoculum production (Y), and volumetric protein productivity. S=glucose, X=biomass, p=protein product, O=O2; fermentation settings as in Example 1.1VFigure 24: Fresh and recycled gas flow, cumulative amount of O2fed to the reactor and bled from the reactor in the not-recycled offgas; fermentation settings as in Example 1.1VFigure 25: Dissolved concentrations of O2and CO2at the bubble surface (with *) and in the liquid phase (without *); fermentation settings as in Example 1.1VFigure 26: Agitation speed; fermentation settings as in Example 1.1 VFigure 27: Concentrations (C). Solid lines are concentrations in broth, dotted lines are concentrations in supernatant. Glc=glucose, X=biomass; fermentation settings as in Example 2.1Figure 28: Total production and consumption rates in the vessel (R). glc=glucose, X=biomass; fermentation settings as in Example 2.1Figure 29: Respiration data (OUR, CER, RQ), flow rates of glucose and NH3(F), molar fractions in offgas of O2and CO2(y), and gas flow in and out of the vessel; fermentation settings as in Example 2.1Figure 30: Yields of biomass and O2on glucose excluding inoculum production (Y), and volumetric productivity. S=glucose, X=biomass, O=O2; fermentation settings as in Example 2.1Figure 31: Dissolved concentrations of O2and CO2at the bubble surface (with *) and in the liquid phase (without *); fermentation settings as in Example 2.1Figure 32: Agitation speed; fermentation settings as in Example 2.1Figure 33: Concentrations (C). Solid lines are concentrations in broth, dotted lines are concentrations in supernatant. Glc=glucose, X=biomass; fermentation settings as in Example 2.11Figure 34: Total production and consumption rates in the vessel (R). glc=glucose, X=biomass; fermentation settings as in Example 2.11Figure 35: Respiration data (OUR, CER, RQ), flow rates of glucose and NH3(F), molar fractions in offgas of O2and CO2(y), and gas flow in and out of the vessel; fermentation settings as in Example 2.11Figure 36: Yields of biomass and O2on glucose excluding inoculum production (Y), and volumetric productivity. S=glucose, X=biomass, O=O2; fermentation settings as in Example 2.11Figure 37: Dissolved concentrations of O2and CO2at the bubble surface (with *) and in the liquid phase (without *); fermentation settings as in Example 2.11Figure 38: Agitation speed; fermentation settings as in Example 2.11Figure 39: Concentrations (C). Solid lines are concentrations in broth, dotted lines are concentrations in supernatant. Glc=glucose, X=biomass; fermentation settings as in Example 2. IllFigure 40: Total production and consumption rates in the vessel (R). glc=glucose, X=biomass; fermentation settings as in Example 2. IllFigure 41: Respiration data (OUR, CER, RQ), flow rates of glucose and NH3(F), molar fractions in offgas of O2and CO2(y), and gas flow in and out of the vessel; fermentation settings as in Example 2. IllFigure 42: Yields of biomass and O2on glucose excluding inoculum production (Y), and volumetric productivity. S=glucose, X=biomass, O=O2; fermentation settings as in Example 2. IllFigure 43: Fresh and recycled gas flow, cumulative amount of O2fed to the reactor and bled from the reactor in the not-recycled offgas; fermentation settings as in Example 2. IllFigure 44: Dissolved concentrations of O2and CO2at the bubble surface (with *) and in the liquid phase (without *); fermentation settings as in Example 2. IllFigure 45: Agitation speed; fermentation settings as in Example 2. IllFigure 46: Concentrations (C). Solid lines are concentrations in broth, dotted lines are concentrations in supernatant. Glc=glucose, X=biomass; fermentation settings as in Example 2.1VFigure 47: Total production and consumption rates in the vessel (R). glc=glucose, X=biomass; fermentation settings as in Example 2.1VFigure 48: Respiration data (OUR, CER, RQ), flow rates of glucose and NH3(F), molar fractions in offgas of O2and CO2(y), and gas flow in and out of the vessel; fermentation settings as in Example 2.1VFigure 49: Yields of biomass and O2on glucose excluding inoculum production (Y), and volumetric protein productivity. S=glucose, X=biomass, O=O2; fermentation settings as in Example 2.1VFigure 50: Fresh and recycled gas flow, cumulative amount of O2fed to the reactor and bled from the reactor in the not-recycled offgas; fermentation settings as in Example 2.1VFigure 51: Dissolved concentrations of O2and CO2at the bubble surface (with *) and in the liquid phase (without *); fermentation settings as in Example 2.1VFigure 52: Agitation speed; fermentation settings as in Example 2.1VVI. EXAMPLES

[0063] Example 1: extracellular protein producing reference fermentation

[0064] I - fermentation with air as ingas and no offgas recycle

[0065] A yeast converts glucose as carbon source and NH3as nitrogen source to biomass (i.e. the production organism itself), an excreted protein (as a valuable product), and CO2in an aerobic fed batch process. The settings of the fermentation are given in table 1. The fermentation starts as a batch process and when the batched glucose is almost depleted, the carbon feed is started. In this reference fermentation, normal air, without pure O2addition is sparged into the fermentor and no offgas recycling takes place.Table 1: Settings of the fermentation

[0066] Selected results of the fermentation process are shown in figures 1-6.

[0067] It is observed that:• The batch phase lasts 35h and is followed by a carbon limited fed-batch phase that ends when the vessel is full (=10 kg of broth weight) at ~83h.• NH3never becomes limiting• O2never becomes limiting. After 45h the agitation speed increases above its setpoint of 600 rpm to control the DO level at the chosen setpoint of 0.1 mmol / kg.• The process ends with the following KPI's: a protein titer of 7.8 g / kg broth (11.0 g / kg supernatant), an amount of protein produced of 77.5 g, a product yield of 0.038 g protein / g glucose and a productivity of 0.062 g / l / h (@83h).• The OUR peaks at 100 mmol / kg / h at the end of the exponential increase of the feed rate profile (~60h) and then gradually decreases towards a final value of 71 mmol / kg / h at end of fermentation due to a fixed carbon feed rate and an increasing broth mass.

[0068] II - fermentation with pure O2as ingas and no offgas recycle

[0069] A fermentation process was executed that was identical to the process in Example 1.1, except for the changed settings that are given in Table 2. This fermentation process uses 100% pure O2as ingas and does not apply offgas recycling.Table 2: Fermentation settings of Example l.ll when different from those in Example 1.1.

[0070] Selected results of the fermentation process are shown in figures 7-12.

[0071] It is observed that:All KPI's of the fermentation are close to those of Example 1.1• The cumulative amount of O2sparged into the fermentor equals ~2,000 mol or 50 Nm3. The O2fraction in the offgas is always above 0.97, so the large majority of O2sparged into the fermentor is wasted.

[0072] III - fermentation with pure O2as ingas and offgas recycle

[0073] A fermentation process was executed that was identical to the process in Example 1.1, except for the changed settings that are given in Table 3. This fermentation process uses 100% pure O2as ingas and applies offgas recycling.Table 3: Fermentation settings of Example 1.1 II when different from those in Example 1.1.

[0074] Selected results of the fermentation process are shown in figures 13-19.

[0075] It is observed that:• All KPI's of the fermentation are close to those of Example 1.1 and 3.11• The cumulative amounts of O2entering and leaving the fermentor via the feed and bleed are 34.7 respectively 8.7 mol O2. This means that less than 1 Nm3of pure O2is consumed during the entire fermentation of which only 25% is lost via the offgas. For comparison: the total gas flowing through the fermentor (equal to the cumulative amount of air sparged in Example 1.1 and the cumulative amount of O2sparged in Example 1.11) equals ~2,000 mol or 50 Nm3.• The fraction of CO2in the offgas rises to a value around 0.8 in this example, which is beneficial for CCS / CCU purposes. At the same time, the dissolved CO2concentration becomes as high as 40 mmol / kg, while it increased to 1.5-2 mmol / kg in Examples 1.1 and 1.11.

[0076] IV - fermentation with pure O2as ingas and offgas recycle and intensified feed rate profile

[0077] A fermentation process was executed that was identical to the process in Example 1.1, except for the changed settings that are given in Table 4. Similarly to Examples 1. II and 1.111, this fermentation process uses 100% pure O2as ingas and similarly to Example l.lll it applies offgas recycling. Deviating from Example l.lll, this Example has an intensified glucose feed profile to demonstrate the benefit that fermentations with higher O2in the ingas can have a higher productivity.Table 4: Fermentation settings of Example 1.1V when different from those in Example 1.1.

[0078] Selected results of the fermentation process are shown in figures 20-26.

[0079] It is observed that:• The intensified feed rate leads to a faster filling of the vessel and thus a reduced fermentation time (from 83h to 67h)• The use of pure O2to control the dissolved O2at its setpoint level also works well for this Example. A higher pure O2inflow is required than in Example l.lll. In this scenario close to 36.8 mol of pure O2is consumed. This is only slightly higher than in Example l.lll (34.7 mol) but the duration of the fermentation of Example 1.1 V is substantially shorter as well. The higher pure O2inflow leads to a higher driving force for O2transfer by an increased O2fraction of the gas in the fermentor of up to 0.4. As a consequence, the intensification leads to an O2bleed of 13.2 mol (i.e. 36% of the fed O2).• The higher intensity of this scenario is clearly visible in the OUR, which peaks at 180 mmol / kg / h in this Example, compared to a peak of ~ 100 mmol / kg / h for Examples 1.1, 3.11 and l.lll.• The KPI's of this intensified fermentation are slightly lower in terms of protein titer (7.0 g / kg broth; 10 g / kg supernatant) and yield (0.036 g protein / g glucose), but higher in terms of volumetric productivity (0.070 g / l / h at end of fermentation).

[0080] Example 2: SCP producing reference fermentation

[0081] I - fermentation with air as ingas and no offgas recycle

[0082] A yeast converts glucose as carbon source and NH3 as nitrogen source to biomass (i.e. the production organism itself) as the main product (also called 'single cell protein'), and CO2in an aerobic fed batch process. The settings of the fermentation are identical to those given in table 1 in Example 1.1. The fermentation starts as a batch process and when the batched glucose is almost depleted, the carbon feed is started. In this reference fermentation, normal air, without pure O2addition is sparged into the fermentor and no offgas recycling takes place.

[0083] Selected results of the fermentation process are shown in figures 27-32.

[0084] It is observed that:• The batch phase lasts 15h and is followed by a carbon limited fed-batch phase that ends when the vessel is full (=10 kg of broth weight) at ~60h.• NH3never becomes limiting.• O2never becomes limiting. After lOh, 26h and 30h and the agitation speed increases above its setpoint of 600 rpm to control the DO level at the chosen setpoint of 0.1 mmol / kg.• The process ends with the following KPI's: a dry biomass (SCP) titer of 98.5 g / kg broth, an amount of protein produced of 967 g, a product yield of 0.51 g dry biomass / g glucose and a productivity of 1.09 g / l / h (@60h).• The OUR peaks at 114 mmol / kg / h at the end of the batch phase and peaks at 95 mmol / kg / h at the end of the exponential increase of the feed rate profile (~39h) and then gradually decreases towards a final value of 68 mmol / kg / h at end of fermentation due to a fixed carbon feed rate and an increasing broth mass.

[0085] II - fermentation with pure O2as ingas and offgas recycle

[0086] A fermentation process was executed that was identical to the process in Example 2.1, except for the changed settings that are given in Table 2 in Example 1.11. This fermentation process uses 100% pure O2as ingas and does not apply offgas recycling.

[0087] Selected results of the fermentation process are shown in figures 33-38.

[0088] It is observed that:• All KPI's of the fermentation are close to those of Example 2.1• The cumulative amount of O2sparged into the fermentor equals ~l,500 mol or 36 Nm3. The O2fraction in the offgas is always above 0.97, so the large majority of O2sparged into the fermentor is wasted.

[0089] III - fermentation with pure O2as ingas and offgas recycle

[0090] A fermentation process was executed that was identical to the process in Example 2.1, except for the changed settings that are given in Table 3 in Example 1.1 II. This fermentation process uses 100% pure O2as ingas and applies offgas recycling.

[0091] Selected results of the fermentation process are shown in figures 39-45.

[0092] It is observed that:• All KPI's of the fermentation are close to those of Example 2.1 and 4. II.• The cumulative amounts of O2entering and leaving the fermentor via the feed and bleed are 32 respectively 8.2 mol O2. This means less than 1 Nm3 of pure O2is consumed during the entire fermentation of which only 25% is lost via the offgas. For comparison: the total gas flowing through the fermentor (equal to the cumulative amount of air sparged in Example 2.1 and O2sparged in Example 2.11) equals ~l,500 mol or 36 Nm3.• Since the fermentation in this Example is fed with pure O2only, the fraction of CO2in the offgas rises to a value around 0.8, which is beneficial for CCS / CCU purposes. At the same time, the dissolved CO2concentration becomes as high as 40 mmol / kg, while it increased to 1.5-2 mmol / kg in Examples 2.1 and 2.11.

[0093] IV - fermentation with pure O2as ingas and offgas recycle and intensified feed rate profile

[0094] A fermentation process was executed that was identical to the process in Example 2.1, except for the changed settings that are given in Table 4 in Example 1.1V. Similarly to Examples 2.11 and 2. Ill, this fermentation process uses 100% pure O2as ingas and similarly to Example 2. Ill it applies offgas recycling.Deviating from Example 2. Ill, this Example has an intensified glucose feed profile to demonstrate the benefit that fermentations with higher O2in the ingas can have a higher productivity.

[0095] Selected results of the fermentation process are shown in figures 46-52.

[0096] It can be observed that:• The intensified feed rate leads to a faster filling of the vessel and thus a reduced fermentation time (from 60h to 45h)• The use of pure O2to control at setpoint level also works well for this Example. A higher pure O2inflow is required than in Example 2. III. In this scenario close to 35 mol of pure O2is consumed. This is only slightly higher than in Example 2. Ill (32 mol) but the duration of the fermentation of this Example is substantially shorter as well. The higher pure O2inflow leads to a higher driving force for O2transfer by an increased O2fraction of the gas in the fermentor of up to 0.4. As a consequence, the intensification leads to an O2bleed of 12.6 mol (i.e. 36% of the fed 02).• The higher intensity of this Example is clearly visible in the OUR, which peaks at close to 160 mmol / kg / h, compared to a peak of ~100 mmol / kg / h for Examples 2.1, 2.11 and 2. III.• The KPI's of this intensified fermentation scenario are unchanged in terms of dry biomass titer (98.5 g / kg) and yield (0.52 g dry biomass / g glucose), but higher in terms of volumetric productivity (1.45 g / l / h at end of fermentation).* * *

[0097] The present invention may also be summarized in the following items:1. A process for cultivating a microorganism with pure O2, comprising the following steps: a. Preparing fermentation substrate in or adding fermentation substrate to a fermenter b. Optionally heat-sterilizing the fermenter containing the fermentation substrate c. Optionally adding gas containing O2to the medium in the fermenter d. Inoculating the fermenter containing fermentation substrate with a microorganism to produce a fermentation broth e. Optionally adding fermentation substrate or fermentation substrate ingredients continuously or during parts of the time of the fermentation process f. Adding gas, containing O2, during parts of the time of the fermentation process g. Optionally harvesting partial volumes of the fermentation broth during the fermentation processh. Harvesting the complete fermentation broth at the end of the fermentation process i. Optionally concentrating or diluting and / or purifying the fermentation broth to obtain the targeted final productWherein during the fermentation process j. At least part of the gas leaving the headspace of the fermenter is recycled back into the fermenter below the surface of the fermentation broth; k. A gas, containing O2at a level of more than 30%v / v, preferable more than 50%, more preferable more than 90%, most preferable more than 95%, is added to the gas that is recycled into the fermenter below the surface of the fermentation broth2. The process according to any of the preceding claims, wherein at least a part of the gas leaving the headspace of the fermenter is recycled back into the fermenter using a pump or compressor.3. The process according to any of the preceding claims, wherein at least part of the gas leaving the headspace of the fermenter is recycled back into the fermenter after drying of the gas by, for instance, means of a condenser.4. The process according to any of the preceding claims, wherein at least part of the gas leaving the headspace of the fermenter is recycled back into the fermenter using a sparger to disperse the gas in the fermentation broth.5. The process according to any of the preceding claims, wherein the flow rate of the gas containing high levels of O2is controlled by the amount of dissolved O2measured in the fermentation broth.6. The process according to any of claims 1 to 5, wherein the flow rate of the gas recycled back into the fermenter below the surface of the broth is controlled between 0.25-2 vvm.7. The process according to any of the preceding claims, wherein during the fermentation process mixing of the broth is done solely by the gas recycled back into the fermenter plus the fresh gas added into the fermenter.8. The process according to any of the claims 1-6, wherein during the fermentation process mixing of the broth is done by the gas recycled back into the fermenter below the surface of the broth in combination with a stirring device9. The process according to claim 8, wherein a stirring device is used at fixed or variable stirring rates during the whole fermentation process or parts of the time of the fermentation process10. The process according to any of the preceding claims, wherein the cultivation is executed in a fermenter under non-atmospheric pressure11. The process according to any of the preceding claims 1 to 10, wherein the process is a fed-batch culture.12. The process according to any of the preceding claims, wherein the microorganism is a yeast, bacteria, fungus, plant, animal, or mammalian cells or any combination thereof.13. The process according to any of the preceding claims, wherein the microorganism is a Saccharomycetales yeast.14. The process according to any of the preceding claims, wherein the fermentation substrate may be selected from sugars, alcohols, organic acids and / or a mixture thereof.15. A single cell protein product, as produced by the methods as claimed in any of the preceding claims.16. An apparatus for cultivating a microorganism with pure O2, comprising a. a fermenter b. a means of feeding fermentation substrate to the fermenter c. an outlet for gas from the headspace of the fermenter d. a first inlet for gas into the fermenter, preferably below the surface of the fermentation broth e. a second inlet for gas into the fermenter, preferably below the surface of the fermentation broth, or means of feeding a gas into the gas stream, optionally below the surface of the fermentation broth17. The apparatus according to claim 16, further comprising an outlet for broth from the fermenter.18. The apparatus according to claim 16 or 17, further comprising a stirring device.19. The apparatus according to any of claims 16 to 18, further comprising a DO-Probe for measuring the level of 02 saturation of the fermentation broth.* * *

[0098] Those skilled in the art will recognize, or be able to ascertain, using not more than routine experimentation, many equivalents to the specific embodiments of the present invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0099] Unless otherwise stated, the following terms used in this document, including the description and claims, have the definitions given below.

[0100] It is to be noted that as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a reagent" includes one or more of such different reagents and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[0101] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain usingno more than routine experimentation, many equivalents to the specific embodiments of the present invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0102] The term "and / or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[0103] The term "about" or "approximately" as used herein means within 20%, preferably within 10%, and more preferably within 5% of a given value or range. It includes, however, also the concrete number, e.g., about 20 includes 20.

[0104] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term "comprising" can be substituted with the term "containing" or "including" or sometimes when used herein with the term "having".

[0105] When used herein "consisting of" excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0106] In each instance herein any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms.

[0107] It should be understood that this invention is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0108] All publications cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.) are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

Claims

Claims1. A process for cultivating a microorganism with pure O2, comprising the following steps: a. Preparing fermentation substrate in or adding fermentation substrate to a fermenter b. Optionally heat-sterilizing the fermenter containing the fermentation substrate c. Optionally adding gas containing O2to the medium in the fermenter d. Inoculating the fermenter containing fermentation substrate with a microorganism to produce a fermentation broth e. Optionally adding fermentation substrate or fermentation substrate ingredients continuously or during parts of the time of the fermentation process f. Adding gas, containing O2, during parts of the time of the fermentation process g. Optionally harvesting partial volumes of the fermentation broth during the fermentation process h. Harvesting the complete fermentation broth at the end of the fermentation process i. Optionally concentrating or diluting and / or purifying the fermentation broth to obtain the targeted final productWherein during the fermentation process j. At least part of the gas leaving the headspace of the fermenter is recycled back into the fermenter below the surface of the fermentation broth; k. A gas, containing O2at a level of more than 30%v / v, preferable more than 50%, more preferable more than 90%, most preferable more than 95%, is added to the gas that is recycled into the fermenter below the surface of the fermentation broth2. The process according to any of the preceding claims, wherein at least a part of the gas leaving the headspace of the fermenter is recycled back into the fermenter using a pump or compressor.

3. The process according to any of the preceding claims, wherein at least part of the gas leaving the headspace of the fermenter is recycled back into the fermenter after drying of the gas by, for instance, means of a condenser.

4. The process according to any of the preceding claims, wherein at least part of the gas leaving the headspace of the fermenter is recycled back into the fermenter using a sparger to disperse the gas in the fermentation broth.

5. The process according to any of the preceding claims, wherein the flow rate of the gas containing high levels of O2is controlled by the amount of dissolved O2measured in the fermentation broth.

6. The process according to any of claims 1 to 5, wherein the flow rate of the gas recycled back into the fermenter below the surface of the broth is controlled between 0.25-2 vvm.

7. The process according to any of the preceding claims, wherein during the fermentation process mixing of the broth is done solely by the gas recycled back into the fermenter plus the fresh gas added into the fermenter.

8. The process according to any of the claims 1-6, wherein during the fermentation process mixing of the broth is done by the gas recycled back into the fermenter below the surface of the broth in combination with a stirring device9. The process according to claim 8, wherein a stirring device is used at fixed or variable stirring rates during the whole fermentation process or parts of the time of the fermentation process10. The process according to any of the preceding claims, wherein the cultivation is executed in a fermenter under non-atmospheric pressure11. The process according to any of the preceding claims 1 to 10, wherein the process is a fed-batch culture.

12. The process according to any of the preceding claims, wherein the microorganism is a yeast, bacteria, fungus, plant, animal, or mammalian cells or any combination thereof.

13. The process according to any of the preceding claims, wherein the microorganism is aSaccharomycetales yeast.

14. The process according to any of the preceding claims, wherein the fermentation substrate may be selected from sugars, alcohols, organic acids and / or a mixture thereof.

15. A single cell protein product, as produced by the methods as claimed in any of the preceding claims.

16. An apparatus for cultivating a microorganism with pure O2, comprising a. a fermenter b. a means of feeding fermentation substrate to the fermenter c. an outlet for gas from the headspace of the fermenter d. a first inlet for gas into the fermenter, preferably below the surface of the fermentation broth e. a second inlet for gas into the fermenter, preferably below the surface of the fermentation broth, or means of feeding a gas into the gas stream, optionally below the surface of the fermentation broth17. The apparatus according to claim 16, further comprising an outlet for broth from the fermenter.

18. The apparatus according to claim 16 or 17, further comprising a stirring device.

19. The apparatus according to any of claims 16 to 18, further comprising a DO-Probe for measuring the level of 02 saturation of the fermentation broth.