Sustainable biomass production

Abstract

The present invention relates to a method for cultivating a microorganism, preferably a Saccharomycetales yeast, capable of utilizing low-grade ethanol as feedstock. 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 PRODUCTIONTechnical FieldThe present invention relates to a method for cultivating a microorganism capable of utilizing low- grade ethanol as feedstock. 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.Background of the InventionThe 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 and fattening 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 huma consumption as well.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, like availability and sustainability of feedstock, like waste-based feedstock. Thus, also feed millers, farmers and food producers still require alternative solutions for protein production to be able to provide low carbon footprint diets while ensuring an environmental-friendly use of the planet's resources.Hence, there is still a need to have at hand an animal feed that comprises protein from an alternative protein source and thus, protein that is neither animal- nor plant-derived. Attempts are being made in industry to produce organic chemicals from waste materials, using renewable energy and renewable feedstocks. It is a challenge, that ideally these renewable, ideally also low-grade, feedstocks can be used to produce protein from an alternative protein source.Description of the FiguresFigure 1 shows yeast single cell protein product from different yeast strains when grown with pure ethanol as carbon source. Shown is the protein on dry matter (%) (w / w) of obtained SCP product (y-axis) per yeast strain (x-axis) of in total 8 genera.Figure 2, left shows the biomass yield (g x gs-1) for Cyberlindnera jadinii CBS621 and Saccharomyces cerevisiae CEN.PK113-7D with pure ethanol as feedstock compared to glucose. Figure 3, right shows the protein content in the biomass (%DM) for Cyberlindnera jadinii CBS621 and Saccharomyces cerevisiae CEN.PK113-7D with pure ethanol as feedstock compared to glucose.Figure 3 shows the protein yield for fermentations with Cyberlindnera jadinii FERM-BP1656 with pure ethanol as feedstock at different temperatures.Figure 4 shows the protein content in the biomass for fermentations with Cyberlindnera jadinii FERM-BP1656 with pure ethanol as feedstock at different pH levels.Figure 5 shows the content of malate synthase in the protein for Cyberlindnera jadinii FERM- BP1656 and Saccharomyces cerevisiae GHP1 with pure ethanol as feedstock compared to glucose.Figure 6 shows the content of isocitrate lyase in the protein for Cyberlindnera jadinii FERM-BP1656 and Saccharomyces cerevisiae GHP1 with pure ethanol as feedstock compared to glucose.Summary of the InventionThus, it is an object of the invention to provide a more sustainable route to prepare a (microbial) biomass or SCP by preparing the (microbial) biomass or SCP from a low-grade ethanol feedstock. In particular, it is an object to provide a method for preparing (microbial) biomass wherein the single cell protein product comprises isocitrate lyase and / OR malate synthase from a low-grade ethanol feedstock. 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.In particular, the present invention relates to a Method for cultivating a microorganism capable of producing at least 40% protein from a low-grade ethanol feedstock, comprising the steps of (i) supplying microorganism to a reactor, (ii) feeding low-grade EtOH as feedstock, (iii) controlling the feed rate, (iv) controlling the temperature, (v) controlling pH and (vi) controlling the growth rate. Preferably, the feed rate is 0.179 - 0.536 gethanoi / gbiomass / h, more preferably 0.179 - 0.469 gethanol / gbiomass / l .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.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.Preferably, the method further comprises the step of (vii) drying the biomass.The present invention also relates to a yeast single cell protein product, wherein the yeast single cell protein product comprising >40% protein on dry cell weight, wherein the protein comprises >0.1%, preferably >0.2% isocitrate lyase and / OR malate synthase.Preferably, the the microorganism is a Saccharomycetales yeast.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.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.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, 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 jadinii CBS841 , 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.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 >40% protein on dry cell weight, wherein the protein comprises >0.1%, preferably >0.2% isocitrate lyase and / OR malate synthase.Preferably, the yeast single cell protein product comprises ethanol fed Saccharomycetales yeast cells.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 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 , 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 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, Wickerhamomyces anomalus IFO 569, Wickerhamomyces anomalus CBS 1980, Cyberlindnera jadinii ATCC 9950 or Kluyveromyces lactis CBS 2896.Preferably, the Saccharomycetales yeast cells are capable of producing with ethanol as carbon source 34% (w / w) or more protein per gram dry weight of said Saccharomycetales yeast cells, preferably 41 % (w / w) or more protein per gram dry weight of said Saccharomycetales yeast cells, more preferably 42.5% (w / w) or more protein per gram dry weight of said Saccharomycetales yeast cells.Preferably, the yeast SCP product comprises all essential amino acids.Preferably, the Saccharomycetales yeast cells are not genetically engineered.Preferably, the yeast SCP product comprises 34% (w / w) or more protein per gram dry weight of Saccharomycetales yeast cells, preferably 41 % (w / w) or more protein per gram dry weight of Saccharomycetales yeast cells, more preferably 42.5% (w / w) or more protein per gram dry weight of Saccharomycetales yeast cells.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.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. Preferably, the animal feed is a feed for poultry, pigs, horses, camels, cows, sheep or companion animals.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.The present invention also relates to the use of the animal feed according to the present invention for feeding an animal.The present invention also relates to the use of the animal feed according to the present invention for increasing body weight of an animal.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 areselected from catfish, tilapia, seabream, seabass, or carp and / or preferably wherein said cold water fish are selected from cod, salmon or rainbow trout.Detailed description of the InventionThe 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.The method of the invention is an aerobic fermentation forthe 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 is capable of using the low-grade ethanol, which is fed to the aerobic fermentation, as feedstock forthe production of biomass. Preferably, the low-grade ethanol feedstock is at least 40 wt% ethanol, preferablably at least 45 wt% ethanol, preferablably at least 50 wt% ethanol, preferablably at least 55 wt% ethanol, preferablably at least 60 wt% ethanol, preferablably at least 65 wt% ethanol, preferablably at least 70 wt% ethanol, preferablably at least 75 wt% ethanol, preferablably at least 80 wt% ethanol, preferablably at least 85 wt% ethanol, preferablably at least 90 wt% ethanol, preferablably at least 95 wt% ethanol. The term “pure ethanol” or wherever the purity of ethanol is not specified, it refers to ethanol with at least 96 wt% ethanol. In particular, it was surprisingly found that Saccharomycetales yeast cells, when grown with ethanol as carbon source, produce high protein, i.e. 34% (w / w) or more protein per gram dry weight of such Saccharomycetales yeast cells, preferably 41 % (w / w) or more protein per gram dry weight of such Saccharomycetales yeast cells, more preferably 42.5% (w / w) or more protein per gram dry weight of such Saccharomycetales yeast cells (see Figure 1). The Saccharomycetales yeast cells are preferably from the genus Cyberlindnera or Saccharomyces or Kluyveromyces or Wickerhamomyces, and more preferably the Saccharomycetales yeast cells are from the genus Cyberlindnera jadinii or Saccharomyces cerevisiae or Kluyveromyces lactis or Wickerhamomyces anomalus. Further, surprisingly, it was found that Saccharomycetales yeast cells appear to produce more protein, when grown with ethanol as carbon source than when grown on a sugar substrate (see Figure 2). Thus, such Saccharomycetales yeast cells, especially when grown with ethanol feed stock, appear to be suitable as a sustainable single cell protein product, which is a source for, e.g., protein with the aim of at least partially, preferably fully replacing animal-derived protein sources, such as fish meal in animal feed.Interestingly, it was found that the protein content of the biomass can be improved by adjusting the growth rate of the microbial cells at the end of the fermentation process. To improve the protein content in the biomass, 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. The main 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 is subsequently increased exponentially to match the exponential growth rate of the biomass. At a certain point duringthe 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. We found the ethanol feed rate should be defined by the substrate uptake rate of the organism (the qS) calculated by the amount of substrate that is consumed (gethanoi) per amount of biomass in the fermenter (gbiomass) per hour, of 0.179 - 0.536 gethanoi / gbiomass / h, more preferably 0.179 - 0.469 gethanol / gbiomass / l .Interestingly, it was found that the biomass yield decreases with higher temperatures, causing the protein yield (the amount of protein produced per gram of ethanol) to decrease with increasing temperatures (see Figure 3). Preferably, the temperature is kept between 30 and 40°C, preferably between 30 and 38°C, more preferably between 30 and 36°, most preferably between 30 and 34°C. It was further found that protein content does change with the pH level. Protein content is increased at lower pH levels. The biomass yield however drops significantly at pH levels of 3.5 and lower. Preferably, the pH is kept between 3.5 and 5.5, preferably between 3.5 and 5.0, more preferably between 3.5 and 4.5 (see Figure 4).Surprisingly, we found that high levels of the enzymes isocitrate lyase and malate synthase can be found in the biomass, when fed an ethanol feedstock (see Figure 5 and 6).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.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. 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.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 broilerchickens (referred to herein as broilers), chicks, layer hens (referred to herein 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. 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.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.In the context of the present invention, the protein content is defined as the amount of protein in the biomass based on dry matter.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 aqua-farming 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.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 vegetableprotein, legumes or other plant material. Further, the animal feed is typically fed as a pelleted feed to mono-gastric animals.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.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 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.A “yeast cell” is a cell of a yeast, preferably a cell of a yeast as described herein.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.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.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.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.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). 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 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 have, e.g., 34% (w / w) or more, preferably 41% (w / w) or more, more preferably 42.5% (w / w) or more, protein per dry matter (see Figure 1) and moreover, can fully replace animal-derived protein, such as fish meal, in an animal feed according to the present invention.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 , 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 and / or Yarrowia lipolytica CBS 7504, preferably from Cyberlindnera jadinii ATCC 26387, Cyberlindnera jadinii FERM-BP1656, Cyberlindnera jadinii CBS621 , Cyberlindnera jadinii CBS841 , Wickerhamomyces anomalus IFO 569, Saccharomyces cerevisiae GHP1 , Saccharomyces cerevisiae CEN.PK113-7D, Wickerhamomyces anomalus CBS 1980, Cyberlindnera jadinii ATCC 9950 and / or Kluyveromyces lactis CBS 2896.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 34% (w / w) or more protein per gram dry weight of yeast cells,preferably 41 % (w / w) or more protein per gram dry weight of yeast cells, more preferably 42.5% (w / w) or more protein per gram dry weight of yeast cells. A skilled person can readily test such a capability by culturing yeast cells with 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.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 GCG 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.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 publically 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.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.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.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.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^) 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.Preferably, the Saccharomycetales yeast cells are not genetically engineered. This is also advantageous for a constant SCP product quality.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.In the context of the present invention, the term "essential amino acid" preferably refers to amino acids that cannot be synthesized by an animal from metabolic intermediates. Thus, such amino acids have to be supplied from an exogenous diet as they are required, e.g., for growth. Although variations may be possible, e.g., depending on the metabolic state of an animal, in general the following nine amino acids are considered essential: phenylalanine, valine, tryptophan, threonine, isoleucine, methionine, histidine, leucine, and lysine. Notably, in terms of nutrition, said nine essential amino acids are obtainable by a single complete protein containing all the essential amino acids. Such complete proteins can be derived from animal-based sources of nutrition, whereas plant-based foods represent commonly a source for essential amino acids in the form of incomplete proteins. 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 / or fish. 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.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.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.The present invention may also be summarized in the following items:1 . Method for cultivating a microorganism capable of producing at least 40% protein, comprising the steps of:(i) supplying microorganism to a reactor(ii) feeding low-grade ethanol as feedstock(iii) controlling the feed rate(iv) controlling the temperature(v) controlling pH(vi) controlling the growth rate2. The method of claim 1 , wherein the low-grade ethanol feedstock is at least 40 wt% ethanol, preferablably at least 45 wt% ethanol, preferablably at least 50 wt% ethanol, preferablably at least 55 wt% ethanol, preferablably at least 60 wt% ethanol, preferablably at least 65 wt% ethanol, preferablably at least 70 wt% ethanol, preferablably at least 75 wt% ethanol, preferablably at least 80 wt% ethanol, preferablably at least 85 wt% ethanol, preferablably at least 90 wt% ethanol, preferablably at least 95 wt% ethanol.3. The method of claim 1 or 2, wherein the feed rate is 0.179 - 0.536 gethanoi / gbiomass / h, more preferably 0.179 - 0.469 gethanoi / gbiomass / h.4. The method of any of claims 1 to 3, wherein the temperature is kept between 30 and 40°C, preferably between 30 and 38°C, more preferably between 30 and 36°, most preferably between 30 and 34°C.5. The method of any of claims 1 to 4, wherein the pH is kept between 3.5 and 5.5, preferably between 3.5 and 5.5, more preferably between 3.5 and 4.5.6. The method of any of claims 1 to 5 further comprising the step of recovering the biomass.7. The method of any of claims 1 to 6, further comprising the step of drying the biomass.8. The method of any of claims 1 to 7, wherein the microorganism is a Saccharomycetales yeast.9. The method of any of claims 1 to 8, 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.10. The method of any of claims 1 to 9, wherein the Saccharomycetales yeast is a yeast from Cyberlindnera jadinii, Saccharomyces cerevisiae, Kluyveromyces lactis, Wickerhamomycesanomalus, Pichia anomala or Yarrowia lipolytica, preferably from Cyberlindnera jadinii or Saccharomyces cerevisiae or Kluyveromyces lactis or Wickerhamomyces anomalus.11 . The method of any of claims 1 to 10, wherein 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, 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 jadinii CBS841 , Wickerhamomyces anomalus IFO 569, Saccharomyces cerevisiae GHP1 , Saccharomyces cerevisiae CEN.PK113-7D, Wickerhamomyces anomalus CBS 1980, Cyberlindnera jadinii ATCC 9950 or Kluyveromyces lactis CBS 2896.12. A yeast single cell protein product, wherein the yeast single cell protein product comprising >40% protein on dry cell weight, wherein the protein comprises >0.1%, preferably >0.2% isocitrate lyase and / OR malate synthase.13. The yeast single cell protein product of claim 12, wherein the yeast is a Saccharomycetales yeast.14. The yeast single cell protein product of claim 13, 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.15. The yeast single cell protein product of any of claim 13 or 14, wherein the Saccharomycetales yeast is a yeast from Cyberlindnera jadinii, Saccharomyces cerevisiae, Kluyveromyces lactis, Wickerhamomyces anomalus, Pichia anomala or Yarrowia lipolytica, preferably from Cyberlindnera jadinii or Saccharomyces cerevisiae or Kluyveromyces lactis or Wickerhamomyces anomalus.16. The yeast single cell protein product of any of claims 13 to 15, wherein the Saccharomycetales yeast is a yeast from Cyberlindnera jadinii ATCC 26387, Cyberlindnera jadinii FERM-BP1656, Cyberlindnera jadinii CBS621 , Cyberlindnera jadinii CBS841 , Wickerhamomyces anomalus IFO 569, Saccharomyces cerevisiae GHP1 , Saccharomyces cerevisiae CEN.PK113-7D, 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 jadinii CBS841 , Saccharomyces cerevisiae GHP1 , Saccharomyces cerevisiae CEN.PK113-7D, Wickerhamomyces anomalus IFO 569, Wickerhamomyces anomalus CBS 1980, Cyberlindnera jadinii ATCC 9950 or Kluyveromyces lactis CBS 2896.17. An animal feed comprising up to 20% (w / w) of yeast single cell protein product as claimed in any of the preceding claims.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.Unless otherwise stated, the following terms used in this document, including the description and claims, have the definitions given below.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.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.Example 1Production of Cyberlindnera jadinii single cell protein (SCP)Yeast strain Cyberlindnera jadinii FERM-BP1656 was cultivated in a shake flask (100 ml) for 24h at 32 oC 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 250 ml seed fermenter was inoculated with 10 ml material, resulting in a starting weight of 100 g. The medium composition of this fermenter is described in Table 1 . During the fermentation, pH was controlled at 5.0 by addition of 10% (w / w) ammonia. Temperature was controlled at 30 OC. Airflow was controlled at 0.25 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 0.615 g / h. This feed rate was subsequently exponentially increased with an exponent of 0.1 h-1 . After a feed rate of 3.0 g / h was reached, the feed rate was kept constant at this value. The feed consisted of 10 % (w / w) ethanol. This cultivation was run for 72h, after which biomass was harvested for inoculation of the main fermenter. At the end of this fermentation, a biomass concentration of 32.35 g dry weight / kg was obtained.The 250 ml main fermenter was inoculated with 0.6 g dry matter or approximately 19 ml culture, resulting in a starting weight of 150 g. The medium composition of this fermenter is described in Table 1 . During the fermentation, pH was controlled at 5.0 by addition of 10% (w / w) ammonia. Temperature was controlled at 30 OC. Airflow was controlled at 0.25 nL / min. Dissolved oxygen concentration was controlled at 20% using the agitation rate. The ethanol feed rate was started upon inoculation at 2.119 g / h. This feed rate was subsequently exponentially increased with an exponent of 0.2 h-1 . After a feed rate of 7.86 g / h was reached, the feed rate was kept constant at this value. The feed consisted of 10 % (w / w) ethanol. The ethanol feed rate range can also be expressed as a qS range of 0.035 gS / gX / h to 0.450 gethanoi / gbiomass / h. The oxygen uptake rate (OUR) reached at this feed rate was 190 mmol / kg / h. This cultivation was run for 48h, at which point a biomass concentration of 38.46 g dry weight / kg was obtained, with a sum of hydrolysed amino acids (excluding cysteine and tryptophan) of g / gbiomass and a Kjeldahl protein content (N*6.25) of 57.4 %. During the fermentation, samples were taken at t = 6 h, t = 23 h, t = 30 h and t = 48 h, which were subsequently analysed for dry matter content, Kjeldahl protein content, RNA content, amino acid content, polysaccharide content and residual ethanol concentration.Table 1 . Medium composition of the preculture shake flask, seed fermenter and main fermenter.Proteomics method description:Prior to Proteomics analysis the samples were normalized for their biomass concentration, based on DM measurements, and subsequently lysed by adding lysis buffer (PreOmics) and incubation at 95 °C for 20 minutes. Cell lysates were processed further by reduction, alkylation, and digestion using trypsin. Samples were analyzed in technical triplicates by liquid chromatography tandem mass spectrometry (LC-MS / MS) using a Vanquish UHPLC coupled to an Orbitrap Exploris 480 MS (Thermo Fisher Scientific). Peptides were separated using reverse-phase chromatography on a ACQUITY UPLC CSH C18 Column, 130A, 1 .7 pm, 2.1 mm X 100 mm analytical column (Waters) using a gradient of water with 0.1 % formic acid (solvent A) and 20% water and 0.1 % formic acid in acetonitrile (solvent B) from 5% B to 40% B in 20 min. Data- independent acquisition (DIA) was performed with a full MS scan resolution setting of 60,000 within the 350 to 1 ,200 m / z range, followed by high-energy collision-induced dissociation activated (HCD) MS / MS with 17 m / z isolation width covering 400 m / z to 1000 m / z in a resolution setting of 15,000. Raw files were analyzed with Spectronaut (Biognosys), version 17, against the proteins of a C.jadinii or S.cerevisiae database. Label-free quantification was performed using the top three peptides measured for each protein. Retention time realignment was done based on non-linear regression and normalization was set to total peptide amount.High levels, above 0.1%, of the enzymes isocitrate lyase and malate synthase can be found in the biomass, when fed an ethanol feedstock (see Figure 5 and 6).Example 2SCP product generationSingle cell protein products were generated using Saccharomycetales yeast cells from different yeast genera using ethanol as carbon source. More specifically, strains of the genera Cyberlindnera, Kluyveromyces, Wickerhamomyces, Yarrowia, Saccharomyces, Pichia, Ogateae and Blastobotrys were investigated. Protein (g) per dry matter (%) (w / w) of SCP product obtained in the Example is given in Figure 1 for SCP products obtained using Saccharomycetales yeast cells from Yarrowia lipolytica, Kluyveromyces marxianus, Blastobotrys adeninivorans, Ogateae polymorpha, Pichia anomala, Pichia pastoris, Saccharomyces cerevisiae, Wickerhamomyces anomalus, Cyberlindnera jadinii and Kluyveromyces lactis. As shown, comparatively well performing yeast cells were Saccharomycetales yeast cells from Wickerhamomyces anomalus, Kluyveromyces lactis, and Cyberlindnera jadinii in view of protein (g) per dry matter (g) given as protein on dry matter (%) with (up to) over 41% (w / w) observed.Exam le 3Production of Cyberlindnera jadinii single cell protein (SCP) using different ethanol feedstocksYeast strain Cyberlindnera jadinii FERM-BP1656 was cultivated in a shake flask (100 ml) for 24h at 32 oC 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 fermenter was inoculated with 15 ml material, resulting in a starting weight of 150 g. The medium composition of this fermenter is5 described in Table 2. During the fermentation, pH was controlled at 5.0 by addition of 10% (w / w) ammonia. Temperature was controlled at 32 OC. Airflow was controlled at 15 nL / h. Dissolved oxygen concentration was controlled at 20% using the agitation rate. After all glucose in the fermenter was consumed, the ethanol feed rate was started at 1 .017 g / h. This feed rate was subsequently exponentially increased with an exponent of 0.1 h-1 . After a feed rate of 7.86 g / h w was reached, the feed rate was kept constant at this value. The feed consisted of one of 5 ethanol mixtures, described in table 1 . This cultivation was run for 72h.Table 2 Overview of the different ethanol feed mixtures evaluated in this fermentation experiment.Table 3 Medium composition of the preculture shake flask, seed fermenter and main fermenter.Table 4 Overview of protein content of SCP produced by the different ethanol feed mixtures evaluated in this fermentation experiment.

Claims

Claims1. Method for cultivating a microorganism capable of producing at least 40% protein, comprising the steps of:(i) supplying microorganism to a reactor(ii) feeding low-grade ethanol as feedstock(iii) controlling the feed rate(iv) controlling the temperature(v) controlling pH(vi) controlling the growth rate2. The method of claim 1 , wherein the low-grade ethanol feedstock is at least 40 wt% ethanol, preferablably at least 45 wt% ethanol, preferablably at least 50 wt% ethanol, preferablably at least 55 wt% ethanol, preferablably at least 60 wt% ethanol preferablably at least 65 wt% ethanol, preferablably at least 70 wt% ethanol preferablably at least 75 wt% ethanol, preferablably at least 80 wt% ethanol preferablably at least 85 wt% ethanol, preferablably at least 90 wt% ethanol preferablably at least 95 wt% ethanol.

3. The method of claim 1 or 2, wherein the feed rate is 0.179 - 0.536 gethanoi / gbiomass / h, more preferably 0.179 - 0.469 gethanoi / gbiomass / h.

4. The method of any of claims 1 to 3, wherein the temperature is kept between 30 and 40°C, preferably between 30 and 38°C, more preferably between 30 and 36°, most preferably between 30 and 34°C.

5. The method of any of claims 1 to 4, 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.

6. The method of any of claims 1 to 5 further comprising the step of recovering the biomass.

7. The method of any of claims 1 to 6, further comprising the step of drying the biomass.

8. The method of any of claims 1 to 7, wherein the microorganism is a Saccharomycetales yeast.

9. The method of any of claims 1 to 8, 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.

10. The method of any of claims 1 to 9, wherein the Saccharomycetales yeast is a yeast from Cyberlindnera jadinii, Saccharomyces cerevisiae, Kluyveromyces lactis, Wickerhamomyces anomalus, Pichia anomala or Yarrowia lipolytica, preferably from Cyberlindnera jadinii or Saccharomyces cerevisiae or Kluyveromyces lactis or Wickerhamomyces anomalus.

11. A yeast single cell protein product, wherein the yeast single cell protein product comprising >40% protein on dry cell weight, wherein the protein comprises >0.1%, preferably >0.2% isocitrate lyase and / OR malate synthase.

12. The yeast single cell protein product of claim 11 , wherein the yeast is a Saccharomycetales yeast.

13. The yeast single cell protein product of claim 12, 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.

14. The yeast single cell protein product of any of claim 12 or 13, wherein the Saccharomycetales yeast is a yeast from Cyberlindnera jadinii, Saccharomyces cerevisiae, Kluyveromyces lactis, Wickerhamomyces anomalus, Pichia anomala or Yarrowia lipolytica, preferably from Cyberlindnera jadinii or Saccharomyces cerevisiae or Kluyveromyces lactis or Wickerhamomyces anomalus.

15. An animal feed comprising up to 20% (w / w) of yeast single cell protein product as claimed in any of the preceding claims.

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