Sustainable biomass production

Abstract

The present invention relates to a method to prepare a high-protein (microbial) biomass or SCP from an alcohol feedstock. In particular, it is an object to provide a method for preparing such a high-protein (microbial) biomass wherein the oxygen transfer capacity is improved. The invention is directed to a method for the production of biomass, in particular single cell protein, wherein the yeast single cell protein product comprises Saccharomycetales yeast cells, as well as to an animal feed comprising such biomass.

Description

SUSTAINABLE BIOMASS PRODUCTIONI. FIELD OF THE INVENTION

[0001] The present invention relates to a method to prepare a high-protein (microbial) biomass or SCP from an alcohol feedstock. In particular, it is an object to provide a method for preparing such a high-protein (microbial) biomass wherein the oxygen transfer capacity is improved. The invention is directed to a method for the production of biomass, in particular single cell protein, wherein the yeast single cell protein product comprises Saccharomycetales yeast cells, as well as to an animal feed comprising such biomass.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. Animal feeding requires considerable amounts of protein for ensuring optimal growth of animals with a major source of protein being currently constituted by plants in traditional breeding. However, plant-derived proteins tend to be poorly converted into animal-derived protein. Furthermore, plant-derived protein production is associated with potential ethical conflicts between food and feed production. Thus, breeders and hence, the animal-protein derived protein production industry, need new protein sources to keep growing at high speed. However, health and welfare of the animals has to be ensured and their growth optimized, whilst moving away from traditional protein sources. Thus, to meet the demand for meat, dairy products and seafood in view of current and expected consumption levels, alternative protein sources in animal feeding are required. Additionally, new protein sources are becoming of more and more interest for human consumption as well.

[0003] Besides plant- and animal-derived protein, a further source of proteins has been identified, namely microbial-derived proteins. Said single cell proteins (SCPs) can be produced using fungi, algae and / or bacteria that offer the ability of large-scale culturing at comparatively low cost. However, SCP product production still faces 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 protein from an alternative protein source.III. SUMMARY

[0004] We surprisingly found that the oxygen transfer capacity may be improved, by increasing the oxygen level in the gas flow, preferably in combination with at least one of the additional measures selected from microsparging with oxygen and increasing the residence time of the gas in the fermentor.

[0005] Thus, it is an object of the invention to provide a more sustainable and more efficient route to prepare a high-protein (microbial) biomass or SCP from an alcohol feedstock. In particular, it is an object to provide a method for preparing such a high-protein (microbial) biomass wherein the oxygen transfer capacity is improved. The present invention further aims at providing an animal feed comprising such a single cell protein product. Thus, the increasing demand for alternative feed solutions can be met, while ensuring sustainability.

[0006] In particular, the present invention relates to a method for cultivating a microorganism capable of producing at least 40% protein, preferably at least 45% protein, more preferably at least 50% on dry cell weight, comprising the steps of (i) supplying microorganism to a reactor, (ii) feeding alcohol as feedstock, (iii) controlling the oxygen level in the gas flow. Preferably, further comprising the step of (iv) microsparging with oxygen, and / or (v) increasing the residence time of the gas in the fermentor. Preferably, the method comprises the further steps of (vi) controlling the feed rate, (vii) controlling the temperature, (viii) controlling pH and (x) controlling the growth rate.

[0007] Preferably, the oxygen uptake rate is at least 200 mmol / kg / h, preferably at least 300 mmol / kg / h, preferably at least 400 mmol / kg / h, preferably at least 500 mmol / kg / h, preferably at least 600 mmol / kg / h.

[0008] Preferably, the growth rate is at least 0.02 IT1, preferably at least 0.025 h-1, more preferably at least 0.03 h’1, more preferably at least 0.04 h’1, more preferably at least 0.05 h’1, more preferably at least 0.06 h’1, more preferably at least 0.07 h’1, more preferably at least 0.08 h-1, more preferably at least 0.09 h’1, more preferably at least 0.1 h’1, more preferably at least 0.15 h-1, more preferably at least 0.16 h’1, more preferably at least 0.17 h’1, more preferably at least 0.18 h-1, more preferably at least 0.19 h’1, more preferably at least 0.2 h’1, more preferably at least 0.21 h-1, most preferably at least 0.22 h-1.

[0009] Preferably, the feed rate is at least 5 gethanoi / kg / h, preferably at least 6.2 gethanoi / kg / h, more preferably at least 7.6 gethanoi / kg / h, more preferably at least 8.8 gethanoi / kg / h, more preferably at least 10 gethanoi / kg / h.

[0010] Preferably, the temperature is kept between 30 and 40°C, preferably between 30 and 38°C, more preferably between 30 and 36°C, most preferably between 30 and 34°C.

[0011] Preferably, the pH is kept between 3.5 and 5.5, more preferably between 3.5 and 5.0, most preferably between 3.5 and 4.5.

[0012] Preferably, the method further comprises the step of (vii) drying the biomass.

[0013] The present invention also relates to a yeast single cell protein product, wherein the yeast single cell protein product comprising at least 40% protein, preferably at least 45% protein, more preferably at least 50% on dry cell weight obtained by the method of the resent invention.

[0014] Preferably, the the microorganism is a Saccharomycetales yeast.

[0015] Preferably, the Saccharomycetales yeast is a yeast from the genus Cyberlindnera, Saccharomyces, Kluyveromyces, Wickerhamomyces, Pichia, Komagataella, Candida boidinii or Yarrowia, preferably from the genus Cyberlindnera or Saccharomyces or Kluyveromyces or Wickerhamomyces.

[0016] Preferably, the Saccharomycetales yeast is a yeast from Cyberlindnera jadinii, Saccharomyces cerevisiae, Kluyveromyces lactis, Wickerhamomyces anomalus, Pichia anomala, Komagataella pastoris, Ogataea methanolica, Pichia pastoris or Yarrowia lipolytica, more preferably from Cyberlindnera jadinii or Saccharomyces cerevisiae or Kluyveromyces lactis or Wickerhamomyces anomalus.

[0017] 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 jadinii AQFM-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, Komagataella pastoris, Ogataea methanolica CBS8002, Komagataella pastoris CBS704, Ogataea methanolica CBS8002, Candida boidinii CBS5777, Ogataea philodendra CBS6075, Ogataea methanolica CBS6512, Ogataea methanolica CBS6515, Ogataea methanolica CBS8053, Ogataea saltuana CBS10795, Pichia pastoris NRRL- Y11430A, Komagataella pastoris CBS704, Ogataea methanolica CBS6512, Ogataea methanolica CBS8053, Pichia pastoris 86280 or Yarrowia lipolytica CBS 7504, preferably from Cyberlindnera jadinii ATCC 26387, Cyberlindnera jadinii FERM-BP1656, Cyberlindnera jadinii CBS621 , Cyberlindnera jadinii CBS841 , Saccharomyces cerevisiae GHP1 , Saccharomyces cerevisiae CEN.PK113-7D, Cyberlindnera jadinii AQFM-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, CBS704 or Kluyveromyces lactis CBS 2896.

[0018] In particular, the present invention relates to an animal feed comprising up to 20% (w / w), preferably up to 10% (w / w), yeast single cell protein (SCP) product, wherein the yeast single cell protein product comprises at least 40% protein, preferably at least 45% protein, more preferably at least 50% on dry cell weight.

[0019] Preferably, the yeast single cell protein product comprises ethanol fed Saccharomycetales yeast cells.

[0020] Preferably, the Saccharomycetales yeast cells are yeast cells from the genus Cyberlindnera, Saccharomyces, Kluyveromyces, Wickerhamomyces, Pichia or Yarrowia, preferably from the genus Cyberlindnera or Saccharomyces or Kluyveromyces or Wickerhamomyces, preferably wherein the Saccharomycetales yeast cells are from Cyberlindnera jadinii, Saccharomyces cerevisiae, Kluyveromyces lactis, Wickerhamomyces anomalus, Pichia anomala, Komagataella pastoris, Ogataea methanolica, Pichia pastoris or Yarrowia lipolytica, preferably from Cyberlindnera jadinii, Saccharomyces cerevisiae, Kluyveromyces lactis, Wickerhamomyces anomalus, preferably wherein the Saccharomycetales yeast cells are or are derived from Cyberlindnera jadinii ATCC 26387, Cyberlindnera jadinii FERM-BP1656, Cyberlindnera jadinii CBS621 , Cyberlindnera jadinii CBS841, Cyberlindnera jadinii AQFM-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, Saccharomyces cerevisiae GHP1 , Saccharomyces cerevisiae CEN.PK113-7D, Wickerhamomyces anomalus IFO 569, Wickerhamomyces anomalus CBS 1980, Cyberlindnera jadinii ATCC 9950, Kluyveromyces lactis CBS 2896, Wickerhamomyces anomalus CBS 2576, Komagataella pastoris, Ogataea methanolica CBS8002, Komagataella pastoris CBS704, Ogataea methanolica CBS8002, Candida boidinii CBS5777, Ogataea philodendra CBS6075, Ogataea methanolica CBS6512, Ogataea methanolica CBS6515, Ogataea methanolica CBS8053, Ogataea saltuana CBS10795, Pichia pastoris NRRL-Y11430A, Komagataella pastoris CBS704, Ogataea methanolica CBS6512, Ogataea methanolica CBS8053, Pichia pastoris 86280 or Yarrowia lipolytica CBS 7504,preferably from Cyberiindnera jadinii ATCC 26387, Cyberlindnera jadinii FERM-BP1656, Cyberlindnera jadinii CBS621 , Cyberlindnera jadinii CBS841, Cyberlindnera jadinii AQFM-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, Saccharomyces cerevisiae GHP1 , Saccharomyces cerevisiae CEN.PK113- 7D, Wickerhamomyces anomalus IFO 569, Wickerhamomyces anomalus CBS 1980, Cyberlindnera jadinii ATCC 9950 or Kluyveromyces lactis CBS 2896.

[0021] Preferably, the Saccharomycetales yeast cells are capable of producing with ethanol as carbon source at least 60% protein, preferably at least 65% protein, more preferably at least 70% protein per gram dry weight of said Saccharomycetales yeast cells.

[0022] Preferably, the yeast SCP product comprises all essential amino acids.

[0023] Preferably, the Saccharomycetales yeast cells are not genetically engineered.

[0024] Preferably, the yeast SCP product comprises at least 40% protein, preferably at least 45% protein, more preferably at least 50% on dry cell weight of Saccharomycetales yeast cells.

[0025] Preferably, the yeast SCP product comprises dried Saccharomycetales yeast cells, preferably wherein said dried Saccharomycetales yeast cells are intact or disrupted or a mixture of intact and disrupted cells.

[0026] Preferably, the animal feed further comprises i) further less than 25% (w / w) plant-based protein products, ii) further 5% (w / w) or less fish meal, iii) no fish meal, and / or iv) from 1 to 25% (w / w) oil.

[0027] Preferably, the animal feed is a feed for poultry, pigs, horses, camels, cows, sheep or companion animals.

[0028] Preferably, the animal feed is a feed for aquatic species, wherein said aquatic species are preferably selected from crustaceans or fish, preferably wherein said crustaceans are shrimps and / or preferably wherein said fish are warm water fish or cold water fish, preferably wherein said warm water fish are selected from catfish, tilapia, seabream, seabass, or carp and / or preferably wherein said cold water fish are selected from cod, salmon or rainbow trout.

[0029] The present invention also relates to the use of the animal feed according to the present invention for feeding an animal.

[0030] The present invention also relates to the use of the animal feed according to the present invention for increasing body weight of an animal.

[0031] Preferably, the animal is a poultry, pig, horse, camel, cow, sheep or companion animal or said animal are aquatic species, preferably wherein said aquatic species are selected from crustaceans or fish, preferably wherein said crustaceans are shrimps and / or preferably wherein said fish are warm water fish or cold water fish, preferably wherein said warm water fish are selected from catfish, tilapia, seabream, seabass, or carp and / or preferably wherein said cold water fish are selected from cod, salmon or rainbow trout.IV. DETAILED DESCRIPTION

[0032] The 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.

[0033] The method of the invention is an aerobic fermentation for the production of biomass. This aerobic fermentation of the method for producing a biomass comprises cultivating a microorganism, a yeast. The microorganism in the aerobic fermentation uses the alcohol, which is fed to the aerobic fermentation, as feedstock for the production of biomass. Preferably, the alcohol feedstock (or “feedstock”) is selected from ethanol or methanol. Most preferably, the alcohol feedstock is ethanol.

[0034] It was found that the amount of biomass and the protein produced per hour from Saccharomycetales yeast cells can be greatly increased by increasing the oxygen transfer capacity.

[0035] The protein content of the cell is dependent on the growth rate. Cells growing at high growth rate have higher protein content compared to cells grown at a low growth rate. To improve the protein content, the fermentation must be halted at the appropriate growth rate. During the cultivation, the ethanol concentration inside the fermenter itself is always close to zero (<0.1 g / L), as any ethanol that is fed is virtually immediately consumed by the yeast. One parameter of the fermentation process, that determines the growth rate, is the ethanol feed rate. This feed rate has to be started low, as the biomass concentration at the start of fermentation is low and therefore can only consume a limited amount of ethanol fed per time unit, to prevent ethanol accumulation in the broth. The feed rate is subsequently increased exponentially to match the exponential growth rate of the biomass. At a certain point during the fermentation, this exponentially increasing feed rate is fixed to a constant feed rate, as the supply of oxygen in the fermenter becomes limiting. The maximum feed rate is limited by two main parameters in the fermentation process: 1) the biomass concentration, and 2) the oxygen transfer capacity.

[0036] In aerobic bioprocesses, oxygen is a key substrate employed to sustain the catabolic reaction which generates the cellular energy required for growth, maintenance and in other metabolic routes, including product synthesis. Due to its low solubility in broth, which are usually aqueous solutions, oxygen must be continuously provided by a gas phase. The concentration of dissolved oxygen in the broth, a suspension of respiring microorganisms, determines the oxygen transfer rate (OTR) from the gas to the liquid phase, and is dependent on the rate of its consumption by the microorganism, the oxygen uptake rate (OUR). In many processes oxygen transfer is a limitation on the maximum carbon feed rate, which is the controlling step for the microbial growth.

[0037] We found that the oxygen transfer capacity may be improved by increasing the oxygen concentration in the gas flow, by microsparging with oxygen and / or increasing the residence time of the gas in the fermentor.

[0038] Preferably, the oxygen level in the gas flow is increased to at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%.

[0039] Bubble coalescence may be suppressed by addition of salts, proteins, alcohols, stabilizes small-diameter bubbles, and thus suppressing coalescence, allowing improved oxygen transfer.

[0040] 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.

[0041] The residence time of gas in the fermenter may be increased through the installation of bioreactor internals, such as porous plates. This is counterintuitive, as normally increased gas residence time results in excessive foaming. Surprisingly, the oxygen bubbles do not contribute to foaming.

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

[0043] The method for producing biomass may further comprise a step of drying the 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] Accordingly, when using such Saccharomycetales yeast cells as single cell protein product in animal feed, it was found that animal feed with up to 20% (w / w) yeast single cell protein product was well eaten by animals, particularly by aquatic species, such as fish or crustaceans. Moreover, when using such Saccharomycetales yeast cells as single cell protein product in animal feed, it was found that animal feed with up to 10% (w / w) yeast single cell protein product has a beneficial effect on the increase of the body weight of animals, particularly aquatic species, such as fish or crustaceans. Also, animal feed comprising up to 20% (w / w) or 10 % (w / w) yeast single cell protein product can advantageously fully replace animal-derived protein, such as fish meal.

[0045] In the context of the present invention, the term “animal” refers to any animal except humans. Examples of animals are non-ruminants and ruminants. Ruminant animals include, for example, animals such as horses, sheep, goats, cattle, e.g. beef cattle, cows, dairy cows, and young calves, deer, yank, camel, llama and kangaroo. Non-ruminant animals include monogastric animals, including but not limited to companion animals (including, but not limited to cats and dogs), pigs or swine (including, but not limited to, piglets, growing pigs, and sows); poultry such as turkeys, ducks, quail, guinea fowl, geese, pigeons (including squabs) and chicken (including but not limited to broiler chickens (referred to herein as broilers), chicks, layer hens (referred toherein as layers)); horses (including but not limited to hotbloods, coldbloods and warm bloods); crustaceans (including but not limited to shrimps and prawns); and fish including but not limited to warm water fish and cold water fish and thus, fish include but not limited to amberjack, arapaima, barb, bass, bluefish, bocachico, bream, bullhead, cachama, carp, catfish, catla, chanos, char, cichlid, cobia, cod, crappie, dorada, drum, eel, goby, goldfish, gourami, grouper, guapote, halibut, java, labeo, lai, loach, mackerel, milkfish, mojarra, mudfish, mullet, paco, pearlspot, pejerrey, perch, pike, pompano, roach, salmon, sampa, sauger, seabass, seabream, shiner, sleeper, snakehead, snapper, snook, sole, spinefoot, sturgeon, sunfish, sweetfish, tench, terror, tilapia, trout (preferably rainbow trout)), tuna, turbot, vendace, walleye and whitefish, with preferred warm water fish being selected from catfish, tilapia, seabream, seabass, or carp and / or preferred cold water fish being cod, salmon or rainbow trout.

[0046] In the context of the present invention, the microbial biomass is a yeast and the terms “single cell protein product” and “yeast single cell protein product” are therefore used interchangeably.

[0047] In the context of the present invention, the protein yield is defined as the amount of protein produced per gram of ethanol. 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 standard factor 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.

[0048] In the context of the present invention, the protein content is defined as the amount of protein in the biomass based on dry matter.

[0049] Of note, any animal referred to herein is preferably not a wildlife animal. Thus, said animal is preferably a farming animal and / or a livestock animal. In case of aquatic species, the animal is preferably an animal of an aquaculture. Herein, the term “aquaculture" relates to aquafarming and thus, the farming of aquatic species such as fish or crustaceans in a variety of environments, including but not limited to tanks, lakes, ponds, or any other natural or man-made aquatic reservoirs that may be suitable for breeding, hatchery, rearing and harvesting of the aquatic species.

[0050] In the context of the present invention, the term “animal feed” (e.g., fish feed) refers to any compound, preparation, or mixture suitable for, or intended for intake by an animal (e.g., a fish). Animal feed for a monogastric animal typically comprises concentrates as well as vitamins, minerals, enzymes, direct fed microbial, amino acids and / or other feed ingredients (such as in a premix) whereas animal feed for ruminants generally comprises forage (including roughage and silage) and may further comprise concentrates as well as vitamins, minerals, enzymes direct fed microbial, amino acid and / or other feed ingredients (such as in a premix). An animal feed additive (e.g., fish feed additive) is a formulated enzyme product which may further comprise e.g. vitamins, minerals, enzymes, amino acids, preservatives and / or antibiotics; i.e. a premix. The animal feed additive / premix is typically mixed in a feed mill with concentrates and / or forage such as vegetable protein, legumes or other plant material. Further, the animal feed is typically fed as a pelleted feed to mono-gastric animals.

[0051] 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. Thus, an 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). While an (unicellular) microorganism may relate to a bacterium, a fungus like yeast and / or an algae, said (unicellular) microorganism is yeast according to the present invention. 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 standardlaboratory conditions. Said term preferably also embraces yeast cells of a single type that have been grown in the laboratory for several generations and thus, said term preferably embraces 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 “gas flow” refers to gas entering the fermenter. This may be achieved through one or more, preferably two or more, separate gas streams. The gas being feed to the fermenter I preferably selected from air, oxygen enriched air or oxygen.

[0055] In one embodiment, at least one gas stream supplies pure oxygen and at least one gas stream supplies air to the fermentation and the oxygen level in the gas flow can be adjusted by adjusting the flow rate of the oxygen. Additionally, the oxygen gas stream is microsparged.

[0056] The purity of the oxygen being feed to the fermenter may be at least at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%.

[0057] 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.

[0058] In the context of the present invention, the term “w / w” is intended to be understood as "weight by weight" and thus refers to the proportion of a particular substance within a mixture, as measured by weight or mass.

[0059] 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 Cyberiindnera, Kluyveromyces, Wickerhamomyces, Yarrowia, Pichia and Saccharomyces.

[0060] 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, Komagataella pastoris, Ogataea methanolica, Pichia pastoris, Yarrowia lipolytica, Wickerhamomyces anomalus, Cyberlindnera jadinii, Saccharomyces cerevisiae and / or Kluyveromyces lactis. Preferably, the strain is an improved strain.

[0061] Thus, it is particularly preferred that the animal feed according to the present invention comprises up to 20% (w / w) or up to 10% (w / w) yeast SCP product, wherein the yeast SCP product comprises Saccharomycetales yeast cells, and wherein said Saccharomycetales yeast cells are yeast cells from the genus Wickerhamomyces, Cyberlindnera, Saccharomyces, Kluyveromyces, Yarrowia and / or Pichia, preferably from Cyberlindnera, Saccharomyces, Kluyveromyces and / or Wickerhamomyces. This is advantageous as yeast cells from said genera are capable of growing on a culture medium comprising ethanol as carbon source as also shown herein in the Examples (see, e.g., Figure 1).

[0062] Preferably, that the animal feed according to the present invention comprises up to 20% (w / w) or up to 10% (w / w) yeast SCP product, wherein the yeast SCP product comprises Saccharomycetales yeast cells, and wherein said Saccharomycetales yeast cells are preferably from Cyberlindnera jadinii, Saccharomyces cerevisiae, Kluyveromyces lactis, Wickerhamomyces anomalus, Pichia anomala, Komagataella pastoris, Ogataea methanolica, Pichia pastoris, and / or Yarrowia lipolytica, more preferably from Wickerhamomyces anomalus, Cyberlindnera jadinii, Saccharomyces cerevisiae and / or Kluyveromyces lactis. This is especially advantageous as SCP products from said species can fully replace animal-derived protein, such as fish meal, in an animal feed according to the present invention.

[0063] Preferably, that the animal feed according to the present invention comprises up to 20% (w / w) or up to 10% (w / w) yeast SCP product, wherein the yeast SCP product comprises Saccharomycetales yeast cells, and wherein said Saccharomycetales yeast cells are derived and / or are from Cyberlindnera jadinii ATCC 26387, Cyberlindnera jadinii FERM-BP1656, Cyberlindnera jadinii CBS621 , Cyberlindnera jadinii CBS841, Cyberlindnera jadinii AQFM-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, Cyberlindnerajadinii 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, Saccharomyces cerevisiae GHP1 , Saccharomyces cerevisiae CEN.PK113- 7D, Wickerhamomyces anomalus IFO 569, Wickerhamomyces anomalus CBS 1980, Cyberlindnera jadinii ATCC 9950, Kluyveromyces lactis CBS 2896, Wickerhamomyces anomalus CBS 2576, Komagataella pastoris, Ogataea methanolica CBS8002, Komagataella pastoris CBS704, Ogataea methanolica CBS8002, Candida boidinii CBS5777, Ogataea philodendra CBS6075, Ogataea methanolica CBS6512, Ogataea methanolica CBS6515, Ogataea methanolica CBS8053, Ogataea saltuana CBS10795, Pichia pastoris NRRL-Y11430A, Komagataella pastoris CBS704, Ogataea methanolica CBS6512, Ogataea methanolica CBS8053, Pichia pastoris 86280 and / or Yarrowia lipolytica CBS 7504, preferably from Cyberlindnera jadinii ATCC 26387, Cyberlindnera jadinii FERM-BP1656, Cyberlindnera jadinii CBS621 , Cyberlindnera jadinii CBS841, Cyberlindnera jadinii AQFM-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, Saccharomyces cere visiae GHP1 , Saccharomyces cerevisiae CEN.PK113-7D, Wickerhamomyces anomalus CBS 1980, Cyberlindnera jadinii ATCC 9950 and / or Kluyveromyces lactis CBS 2896.

[0064] In the context of the present invention, the term “derived from” preferably refers to yeast cells, which were originally obtained from a given yeast strain and thus originate from said given yeast strain. Such derived cells may differ from said given yeast strain due to naturally occurring and / or artificially introduced alterations like genetic mutations, but preferably have similar characteristics as cells from the yeast strain they originated from. Such similar characteristics are preferably the capability to produce with ethanol as carbon source 60% or more protein, preferably from protein, more preferably 70% or more, most preferably 75% or more protein per gram dry weight of yeast cells. A skilled person can readily test such a capability by culturing yeast cellswith ethanol as carbon source, whereby a range of ethanol concentrations as carbon source are tested. Accordingly, cells that are derived from a given strain may have, preferably on genome level, a sequence identity of 80% or more, preferably of 85% or more, more preferably of 90% or more, even more preferably of 95% or more to the respective strain that can be seen as reference. Thus, a derived cell may have a sequence identity of at least, e.g., 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to the respective reference, preferably on genome level.

[0065] As used herein, the term “sequence identity” or “identity” denotes a property of sequences that measures their similarity or relationship. The term “sequence identity” or “identity” as used in the present disclosure means the percentage of pair-wise identical residues - following (homologous) alignment of a sequence of nucleotide and / or amino acids with a respective sequence in question - with respect to the number of residues in the longer of these two sequences. Sequence identity is measured by dividing the number of identical nucleotides and amino acid residues, respectively, by the total number thereof and multiplying the product by 100. A skilled artisan will recognize available computer programs, for example BLAST (Altschul et al., 1997), BLAST2 (Altschul et al., 1990), FASTA (Pearson and Lipman, 1988), GAP (Needleman and Wunsch, 1970), Smith-Waterman (Smith and Waterman, 1981), and Wisconsin GOG Package, for determining sequence identity using standard parameters. The percentage of sequence identity can, for example, be determined herein using the program BLASTP, version 2.2.5, November 16, 2002 (Altschul et al., 1997), calculating the percentage of numbers of “positives” (homologous amino acids) from the total number of amino acids selected for the alignment.

[0066] Accordingly, "percent (%) sequence identity" with respect to cells and / or strains described herein is preferably defined on nucleic acid level and thus, as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent nucleotides sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared. The same is applicable to amino acid sequences, mutatis mutandis.

[0067] For example, an appropriate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, (1981), Advances in Applied Mathematics 2: 482-489. This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov (1986), Nucl. Acids Res. 14(6): 6745-6763. An exemplary implementation of this algorithm to determine percent identity of a sequence is provided by the Genetics Computer Group (Madison, Wis.) in the "BestFit" utility application. The default parameters for this method are described in the Wisconsin Sequence Analysis Package Program Manual, Version 8 (1995) (available from Genetics Computer Group, Madison, Wis.). A preferred method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, Calif). From this suite of packages the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the "Match" value reflects "sequence identity." Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters.

[0068] In the context of the present invention, the following illustrative example may be considered. When an obtained yeast strain, which may have been an officially deposited yeast strain initially, has been cultivated and propagated for some generations, e.g. under laboratory conditions, the resulting yeast cells may be genetically identically to the deposited genetic material of the initial yeast strain. In said case, the cells “are” the cells from the deposited strain and have 100% sequence identity with the deposited material on genome level. However, a portion of the cells or even all cells may show some degree of genetic and / or epigenetic variation, for example due to one or more mutations. In said case, the cells under study “are derived” from the initial strain the genetic material and / or cells of which is deposited. Such mutations may be naturally occurring during cultivation and propagation of cells. Alternatively or additionally, though preferred, such mutations may be artificially introduced, e.g., by genetic engineering. Consequently, derived cells may potentially exhibit, e.g., a variation in the protein per dry matter (%) (w / w), the amount of essential amino acids and / or composition of essential amino acids, compared to the initial yeast strain the cells are derived from. The obtained yeast cells may also be referred to as mutants compared to the cells and / or strain they are derived from.

[0069] Herein, the term “genetic engineering” is used in its broadest sense for methods known to the person skilled in the art to modify desired nucleic acids in vitro and in vivo, e.g. by targeted mutagenesis and / or recombinant DNA technology. Accordingly, said methods may comprise cloning, sequencing and transformation of recombinant nucleic acids, and appropriate vectors, primers, enzymes, host cells and the like are known by the skilled artisan. Preferably, genetically engineered cells are genetically engineered in view of high protein per dry matter (%), suitable essential amino acid composition, efficient ethanol usage as carbon source and the like in the context of the present invention.

[0070] Furthermore, herein the terms "mutated" and “mutant” mean permanent (epi-) genetic modification(s) of genetic material, i.e. nucleic acids, caused, for example, naturally or by physical means or chemical compounds / substances / agents such as EMS. Said modifications include point mutations, transitions, transversions, deletion / insertion / addition of one or more bases within a nucleic acid / gene / chromosome thereby optionally modifying the nucleic acid / gene / chromosome which can cause, inter alia, phenotypic effects like varying protein per dry matter (%) (w / w). Furthermore, such modification(s) may be induced by methods known to the person skilled in the art. The skilled person is also aware of suitable methods to select cells in view of one or more favorable and / or desired phenotypic trait(s) like an increase of protein per dry matter (%) (w / w) and / or utilization of ethanol as carbon source.

[0071] Preferably, the Saccharomycetales yeast cells are not genetically engineered. This is also advantageous for a constant SCP product quality.

[0072] Additionally or alternatively, it is preferred that the yeast SCP product comprised in the animal feed according to the present invention comprises all essential amino acids. This is advantageous, as an animal feed comprising a yeast SCP product comprising all essential amino acids is capable of fully replacing currently used animal- and / or plant-derived protein sources in animal feed. Thus, said SCP product can represent an alternative protein source that does not require any supplementation in view of essential amino acids.

[0073] The animal feed according to the present invention is preferably a feed for poultry, horses, camels, pigs, cows, such as beef cattle or dairy cows, sheep or companion animals, such as cats or dogs. Alternatively, or additionally, said animal feed is preferably a feed for aquatic species. In case of the animal feed being a feed for aquatic species said species are preferably selected from crustaceans or fish. Thus, the animal feed may be a feed for crustaceans and / orfish. When the animal feed is a feed for crustaceans, said crustaceans are preferably shrimps. Additionally, or alternatively, in case the animal feed is a feed for fish, said fish may preferably be warm water fish or cold water fish. When said fish are warm water fish, said fish are preferably selected from the group consisting of catfish, tilapia, seabream, seabass, and carp. Preferably, said fish are cold water fish with said fish being selected from cod, salmon, or rainbow trout. It is particularly preferred that the animal feed according to the present invention is a feed for shrimps, salmon and / or rainbow trout.

[0074] It is to be noted that in case of any definition given herein, the respective definition of a term, phrase, and / or abbreviation applies vice versa throughout the specification. Furthermore, all definition given herein are intended to encompass all grammatical forms.

[0075] Additional objects, advantages, and features of this disclosure will become apparent to those skilled in the art upon examination of the following Examples and the attached Figures thereof, which are not intended to be limiting. Thus, it should be understood that although the present disclosure is specifically disclosed by exemplary embodiments and optional features, modification and variation of the disclosures embodied therein herein disclosed may be resorted to by those skilled in the art and that such modifications and variations are considered to be within the scope of this disclosure.V. BRIEF DESCRIPTION OF THE FIGURES

[0076] Figure 1 shows the protein content in the biomass (g / kg) for Cyberlindnera jadinii FERM- BP1656 with ethanol as feedstock produced with air (with an oxygen concentration of 21 %) and with 02 enriched air with an oxygen concentration of 50%.

[0077] Figure 2 shows the biomass (g / kg) produced for Cyberlindnera jadinii FERM-BP1656 with ethanol as feedstock produced with air (with an oxygen concentration of 21 %) and with 02 enriched air with an oxygen concentration of 50%.

[0078] Figure 3, shows the ethanol feed rate (g / h) used for Cyberlindnera jadinii FERM-BP1656 with air (with an oxygen concentration of 21%) and with 02 enriched air with an oxygen concentration of 50% and 90%.

[0079] Figure 4 shows the oxygen uptake rate (mmol / kg / h) for Cyberlindnera jadinii FERM- BP1656 with ethanol as feedstock produced with air (with an oxygen concentration of 21 %) and with 02 enriched air with an oxygen concentration of 50% and 90%.VI. EXAMPLES

[0080] Example 1 : Production of Cyberlindnera jadinii single cell protein (SCP) using air enriched with oxygen (50%)Yeast strain Cyberlindnera jadinii FERM-BP1656 was cultivated in a shake flask (100 ml) for24h at 32 °C and 280 rpm. The shake flask medium was based on Verduyn (Verduyn et al., 1992), an overview of which is shown in Table 1. From the shake flask, a seed fermenter was inoculated with 100 ml material, resulting in a starting weight of 4.5 kg. The medium composition of this fermenter is described in Table 1. During the fermentation, pH was controlled at 5.0 by addition of 25% (w / w) ammonia. Temperature was controlled at 32 °C. Airflow was controlled at 3.5 nL / min. Dissolved oxygen concentration was controlled at 20% using the agitation rate. After all glucose in the seed fermenter was consumed, the ethanol feed rate was started at 6 g / h. At t = 36 h, this feed rate was subsequently exponentially increased with an exponent of 0. 1 h~1. After a feed rate of 20 g / h was reached, the feed rate was kept constant at this value. The feed consisted of 96 % (v / v) ethanol. This cultivation was run for 68 h, after which biomass was harvested for inoculation of the main fermenter. At the end of this fermentation, a biomass concentration of 65 g dry weight / kg was obtained.From the seed fermenter, two main fermenters were inoculated to produce the SCP product. One fermenter was aerated with air (with an oxygen concentration of 21%), whereas the other fermenter was aerated with air enriched with pure oxygen leading to a final oxygen concentration of 50%.The 6 L main fermenter was inoculated with 25 g dry matter or approximately 390 ml culture, resulting in a starting weight of 4.6 kg. The medium composition of this fermenter is described in Table 1. During the fermentation, pH was controlled at 5.0 by addition of 25% (w / w) ammonia. Temperature was controlled at 32 °C. Gas flow into the fermenter was controlled at 6 nL / min. Thisgasflow contained 21% oxygen for the reference air condition, or 50% oxygen for the oxygen enriched air condition. Dissolved oxygen concentration was controlled at 20% using the agitation rate. The ethanol feed rate was started upon inoculation at 7.18 g / h. This feed rate was subsequently exponentially increased with an exponent of 0.2 h1. After a feed rate of 18 g / h, for the reference air condition, or 42 g / h, for the oxygen enriched air condition, was reached, the feed rate was kept constant at this value. The feed consisted of 96 % (v / v) ethanol. The oxygen uptake rate (OUR) reached at this feed rate was 140 mmol / kg / h for the reference air condition, and 280 mmol / kg / h for the oxygen enriched air condition. At the end of fermentation, after 46h, a biomass concentration of 88 g / kg was reached for the reference air condition. For the oxygen enriched air condition, a biomass concentration of 102 g / kg was reached at the end of fermentation, after 28 h.Table 1. Medium composition of the preculture shake flask, seed fermenter and main fermenter.

[0081] Example 2: Production of Cyberlindnera jadinii single cell protein (SCP) using air enriched with oxygen (92%)Yeast strain Cyberlindnera jadinii FERM-BP1656 was cultivated in a shake flask (100 ml) for 24h at 32 °C and 280 rpm. The shake flask medium was based on Verduyn (Verduyn et al., 1992), an overview of which is shown in Table 1. From the shake flask, a seed fermenter was inoculated with 100 ml material, resulting in a starting weight of 3.0 kg. The medium composition of this fermenter is described in Table 1. During the fermentation, pH was controlled at 5.0 by addition of 25% (w / w) ammonia. Temperature was controlled at 32 °C. Airflow was controlled at 3.5 nL / min. Dissolved oxygen concentration was controlled at 20% using the agitation rate. After all glucose in the seed fermenter was consumed, the ethanol feed rate was started at 6 g / h. At t = 36 h, this feed rate was subsequently exponentially increased with an exponent of 0.1 h’1. After a feed rate of 20 g / h was reached, the feed rate was kept constant at this value. The feed consisted of 96 % (v / v) ethanol. This cultivation was run for 68h, after which biomass was harvested for inoculation of the main fermenter. At the end of this fermentation, a biomass concentration of 87 g dry weight / kg was obtained.From the seed fermenter, the main fermenter was inoculated to produce the SCP product. The 6 L main fermenter was inoculated with 50 g dry matter or approximately 574 ml culture, resulting in a starting weight of 4.0 kg. The medium composition of this fermenter is described in Table 1. During the fermentation, pH was controlled at 5.0 by addition of 25% (w / w) ammonia. Temperature was controlled at 32 °C. Gas flow into the fermenter was controlled at 5 nL / min. This gasflow contained 91 .78% oxygen. Dissolved oxygen concentration was controlled at 20% using the agitation rate. The ethanol feed rate was started upon inoculation at 12.0 g / h. This feed rate was subsequently exponentially increased with an exponent of 0.3 h’1. After a feed rate of 78 g / h was reached, the feed rate was kept constant at this value for 2.5 h. After this, the feed rate was increased exponentially (0.3 IT1) until the end of the fermentation. The feed consisted of 96 %(v / v) ethanol. The maximum oxygen uptake rate (OUR) reached at a feed rate of 123 g / h ethanol (27.1 g / kg / h) was 891 mmol / kg / h. After 11 ,2h, a biomass concentration of 88 g / kg was reached.Table 2 Medium composition of the preculture shake flask, seed fermenter and main fermenter.

[0082] The present invention may also be summarized in the following items:1. Method for cultivating a microorganism capable of producing at least 40% protein, preferably at least 45% protein, more preferably at least 50% on dry cell weight, comprising the steps of:(i) supplying microorganism to a reactor(ii) feeding alcohol as feedstock, and(iii) controlling the oxygen level in the gas flow.2. The method of claim 1 , further comprising the steps of(iv) microsparging with oxygen, and / or(v) increasing the residence time of the gas in the fermentor.3. The method of claim 1 or 2, further comprising the steps of:(vi) control of the feed rate of the alcohol substrate,(vii) controlling the temperature, and / or(vii) controlling pH4. The method of any of claims 1 to 3, wherein the oxygen level in the gas flow is at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%.5. The method of any of claims 1 to 4, wherein the gas flow enters the fermenter through at least two separate gas streams.6. The method of claim 4, wherein at least one gas stream supplies pure oxygen and at least one gas stream supplies air to the fermentation and wherein the oxygen level in the gas flow can be adjusted by adjusting the flow rate of the oxygen.7. The method of claim 5, wherein the oxygen gas stream is microsparged.8. The method of any one of claims 1 to 7, wherein the purity of the oxygen is at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%.9. The method of any one of claims 3 to 8, wherein the feed rate of the alcohol substrate is at least 5 gaicohoi / kg / h, preferably at least 6.2 gaicohoi / kg / h, more preferably at least 7.6 gaicohoi / kg / h, more preferably at least 8.8 gaicohoi / kg / h, more preferably at least 10 gaicohoi / kg / h.10. The method of any of claims 3 to 9, wherein the alcohol substrate is ethanol or methanol.11 . The method of any of claims 3 to 10, wherein the temperature is kept between 30 and 40°C, preferably between 30 and 38°C, more preferably between 30 and 36°C, most preferably between 30 and 34°C.12. The method of any of claims 3 to 11 , wherein the pH is kept between 3.5 and 5.5, preferably between 3.5 and 5.5, more preferably between 3.5 and 4.5.13. The method of any of claims 1 to 12 further comprising the step of recovering the biomass.14. The method of any of claims 1 to 13, further comprising the step of drying the biomass.15 The method of any of claims 1 to 14, wherein the microorganism is a Saccharomycetales yeast.16. The method of any of claims 1 to 15, wherein 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.17. The method of any of claims 1 to 16, wherein the Saccharomycetales yeast is a yeast from Cyberlindnera jadinii, Saccharomyces cerevisiae, Kluyveromyces lactis, Wickerhamomyces anomalus, Pichia anomala, Komagataella pastoris, Ogataea methanolica, Pichia pastoris, or Yarrowia lipolytica, preferably from Cyberlindnera jadinii or Saccharomyces cerevisiae or Kluyveromyces lactis or Wickerhamomyces anomalus.18. The method of any of claims 1 to 16, wherein the Saccharomycetales yeast is a yeast from Cyberlindnera jadinii ATCC 26387, Cyberlindnera jadinii FERM-BP1656, Cyberlindnera jadinii CBS621, Cyberlindnera jadinii CBS841 , Cyberlindnera jadinii AQFM-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, Saccharomyces cerevisiae GHP1, Saccharomyces cerevisiae CEN.PK113-7D, Wickerhamomyces anomalus IFO 569, Wickerhamomyces anomalus CBS 1980, Cyberlindnera jadinii ATCC 9950, Kluyveromyces lactis CBS 2896, Wickerhamomyces anomalus CBS 2576, Komagataella pastoris, Ogataea methanolica CBS8002, Komagataella pastoris CBS704, Ogataea methanolica CBS8002, Candida boidinii CBS5777, Ogataea philodendra CBS6075, Ogataea methanolica CBS6512, Ogataea methanolica CBS6515, Ogataea methanolica CBS8053, Ogataea saltuana CBS10795, Pichia pastoris NRRL-Y11430A, Komagataella pastoris CBS704, Ogataea methanolica CBS6512, Ogataea methanolica CBS8053, Pichia pastoris 86280 or Yarrowia lipolytica CBS 7504, preferably from Cyberlindnera jadinii ATCC 26387, Cyberlindnera jadinii FERM-BP1656, Cyberlindnera jadinii CBS621, Cyberlindnera jadinii CBS841 , Cyberlindnera jac / / n / / AQFM-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, Saccharomyces cerevisiae GHP1, Saccharomyces cerevisiae CEN.PK113-7D, Wickerhamomyces anomalus CBS 1980, Cyberlindnera jadinii ATCC 9950 or Kluyveromyces lactis CBS 2896.19. A yeast single cell protein product, as produced by the methods as claimed in any of the preceding claims.20. An animal feed comprising up to 20% (w / w) of yeast single cell protein product as claimed in claim 19.

[0083] 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.

[0084] Unless otherwise stated, the following terms used in this document, including the description and claims, have the definitions given below.

[0085] 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.

[0086] 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 using no 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.

[0087] 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".

[0088] 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.

[0089] 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”.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] Other embodiments are within the following claims. In addition, where features or aspects of the present invention are described in terms of Markush groups, those skilled in the art will recognize that the present invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0094] 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. Method for cultivating a microorganism capable of producing at least 40% protein, preferably at least 45% protein, more preferably at least 50% on dry cell weight, comprising the steps of:(i) supplying microorganism to a reactor(ii) feeding alcohol as feedstock, and(iii) controlling the oxygen level in the gas flow.

2. The method of claim 1 , further comprising the steps of(iv) microsparging with oxygen, and / or(v) increasing the residence time of the gas in the fermentor.

3. The method of claim 1 or 2, further comprising the steps of:(vi) control of the feed rate of the alcohol substrate,(vii) controlling the temperature, and / or(vii) controlling pH4. The method of any of claims 1 to 3, wherein the oxygen level in the gas flow is at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%.

5. The method of any of claims 1 to 4, wherein the gas flow enters the fermenter through at least two separate gas streams.

6. The method of claim 4, wherein at least one gas stream supplies pure oxygen and at least one gas stream supplies air to the fermentation and wherein the oxygen level in the gas flow can be adjusted by adjusting the flow rate of the oxygen.

7. The method of claim 5, wherein the oxygen gas stream is microsparged.

8. The method of any one of claims 1 to 7, wherein the purity of the oxygen is at least 30%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%.

9. The method of any one of claims 3 to 8, wherein the feed rate of the alcohol substrate is at least 5 gaicohoi / kg / h, preferably at least 6.2 gaicohoi / kg / h, more preferably at least 7.6 gaicohoi / kg / h, more preferably at least 8.8 gaicohoi / kg / h, more preferably at least 10 gaicohoi / kg / h.

10. The method of any of claims 3 to 9, wherein the alcohol substrate is ethanol or methanol.11 . The method of any of claims 3 to 10, wherein the temperature is kept between 30 and 40°C, preferably between 30 and 38°C, more preferably between 30 and 36°C, most preferably between 30 and 34°C.

12. The method of any of claims 3 to 11 , wherein the pH is kept between 3.5 and 5.5, preferably between 3.5 and 5.5, more preferably between 3.5 and 4.5.

13. The method of any of claims 1 to 12, wherein the microorganism is a Saccharomycetales yeast.

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

15. An animal feed comprising up to 20% (w / w) of yeast single cell protein product as claimed in claim 14.

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