Sustainable biomass production

Abstract

The present invention relates to an improved method for cultivating a microorganism capable of utilizing ethanol as feedstock using continuous cultures. The invention is directed to an improved method for the production of biomass using continuous cultures, 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 an improved method for cultivating a microorganism capable of utilizing ethanol as feedstock using continuous cultures. The invention is directed to an improved method for the production of biomass using continuous cultures, 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 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.

[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, 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.

[0004] 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. Attemptsare 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 feedstocks can be used to produce protein from an alternative protein source.III. SUMMARYIV. DETAILED DESCRIPTION

[0033] 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.

[0034] 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 ethanol, which is fed to the aerobic fermentation, as feedstock for the production of biomass.

[0035] It was found that Saccharomycetales yeast cells, when grown with ethanol as carbon source, can produce relatively high protein contents. Further, surprisingly, and against common teachings, it was found that the protein produced from Saccharomycetales yeast cells grown in continuous cultures can be increased by decreasing the growth rate (see Figure 8). As Prior art suggests a positive correlation of growth rate applied on protein content of cellular biomass (Pejin, D. at al. 1993. Continuous Cultivation of the yeast Saccharomyces cerevisiae at Different Dilution Rates and Glucose Concentrations in Nutrient Media. Folia Microbiol. 38 (2), 141-146.).

[0036] 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. 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.

[0037] To adjust the growth rate of the microbial cells in the fermentation process, and thereby 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 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. 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.

[0038] 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.

[0039] 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).

[0040] 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).

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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 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.

[0045] In the context of the present invention, continuous cultures, as opposed to fed-batch cultures, can be achieved using a chemostat. In the context of the present invention, a chemostat is a bioreactorto which fresh medium is continuously added, while culture liquid containing leftover nutrients, metabolic end products and microorganisms is continuously removed at the same rate to keep the culture volume constant. By changing the rate with which medium is added to the bioreactor the specific growth rate of the microorganism can be controlled.

[0046] In the context of the present invention, a fed-batch culture is, an operational technique in biotechnological processes where one or more nutrients (substrates) are fed (supplied) to the bioreactor during cultivation and in which the product(s) remain in the bioreactor until the end of the run. Optionally, a semi-batch culture may be applied, wherein a base medium may support initial cell culture and a feed medium may then be added to prevent nutrient depletion. In some cases, all the nutrients are fed into the bioreactor.

[0047] 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.

[0048] 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.

[0049] In the context of the present invention, the protein content is defined as the amount of protein in the biomass based on dry matter.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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 havebeen 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

[0054] A “yeast cell” is a cell of a yeast, preferably a cell of a yeast as described herein.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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, Cyberiindnera jadinii, Saccharomyces ce evisiae and / or Kluyveromyces lactis. Preferably, the strain is an improved strain.

[0059] 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, Cyberiindnera, Saccharomyces, Kluyveromyces, Yarrowia and / or Pichia, preferably from Cyberiindnera, Saccharomyces, Kluyveromyces and / or Wickerhamomyces. This is advantageous as yeast cells from said genera are capable of growingon a culture medium comprising ethanol as carbon source as also shown herein in the Examples (see, e.g., Figure 1).

[0060] 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., 60% or more protein, preferably from protein, more preferably 70% or more , most preferably 75% or more protein per dry matter and moreover, can fully replace animal-derived protein, such as fish meal, in an animal feed according to the present invention.

[0061] 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, 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 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 jadiniiAQFM-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 and / or Kluyveromyces lactis CBS 2896.

[0062] 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 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.

[0063] 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, version2.2.5, 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] Preferably, the Saccharomycetales yeast cells are not genetically engineered. This is advantageous to ensure that the SCP product comprised in the animal feed comprises only well- defined and / or well characterized yeast cells but no mutated and thus, potentially undefined and / or uncharacterized yeast cells. This is also advantageous for a constant SCP product quality.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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

[0075] Figure 1 shows the protein on dry matter (%) (w / w) in relation to the growth rate (h-1) for improved strain Cyberlindnera jadinii AQFM-009 with ethanol as feedstock grown in a fed-batch manner.

[0076] Figure 2 shows the content of malate synthase in the protein for Cyberlindnera jadinii FERM-BP1656 and Saccharomyces cerevisiae GHP1 with ethanol as feedstock compared to glucose.

[0077] Figure 3 shows the content of isocitrate lyase in the protein for Cyberlindnera jadinii FERM-BP1656 and Saccharomyces cerevisiae GHP1 with ethanol as feedstock compared to glucose.

[0078] Figure 4, shows the protein on dry matter (%) (w / w) in relation to the growth rate (h-1) for improved strains Cyberlindnera jadinii AQFM-009, Cyberlindnera jadinii AQFM-035, Cyberlindnera jadinii AQFM-036, Cyberlindnera jadinii AQFM-037 and Cyberlindnera jadinii AQFM-038 with ethanol as feedstock grown in a fed-batch manner.

[0079] Figure 5, shows the protein on dry matter (%) (w / w) in relation to the growth rate (IT1) for improved strains Cyberlindnera jadinii AQFM-039, Cyberlindnera jadinii AQFM-041, Cyberlindnera jadinii AQFM-017, Cyberlindnera jadinii AQFM-042 and Cyberlindnera jadinii AQFM-043 with ethanol as feedstock grown in a fed-batch manner.

[0080] Figure 6, shows the protein on dry matter (%) (w / w) in relation to the growth rate (h-1) for improved strains Cyberlindnera jadinii AQFM-044, Cyberlindnera jadinii AQFM-046, Cyberlindnera jadinii AQFM-048, Cyberlindnera jadinii AQFM-017 and Cyberlindnera jadinii AQFM-049 with ethanol as feedstock in a fed-batch manner.

[0081] Figure 7, shows the protein on dry matter (%) (w / w) in relation to the growth rate (IT1) for improved strains Cyberlindnera jadinii AQFM-050, Cyberlindnera jadinii AQFM-051, Cyberlindnera jadinii AQFM-052, Cyberlindnera jadinii AQFM-053, Cyberlindnera jadinii AQFM- 054, Cyberlindnera jadinii AQFM-055 with ethanol as feedstock in a fed-batch manner.

[0082] Figure 8, shows the protein on dry matter (%) (w / w) in relation to the growth rate (IT1) for Cyberlindnera jadinii CBS 621 (Chemostat cultivation) and Cyberlindnera jadinii (Fed-batch cultivation) with ethanol as feedstock.VI. EXAMPLES

[0083] Example 1 : Production of Cyberlindnera jadinii single cell protein (SCP) in a fed-batch manner

[0084] 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 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 °C. 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 °C. 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 2.1 19 g / h. This feed rate was subsequently exponentially increased with an exponent of 0.2 IT1. 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 qsrange 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.Y1

[0085] 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).

[0086] Example 2: Strain improvement; How to generate improved strains, such as Cyberlindnera jadinii AQFM-009Strains Cyberlindnera jadinii FERM-BP 1656 from EP 0299078 A1 and Cyberlindnera jadinii CBS 841 from Westerdijk Fungal Biodiversity Institute, The Netherlands were subjected to classical strain improvement.Strains were grown over night in 100 ml YEPhD comprising 10 g / L yeast extract + 20 g / L BBL Phytone Peptone, 20 g / L glucose, 1 % Penicillin-Streptomycin solution (L0022-100, Biowest) and incubated at 30°C, 280 rpm until 1 ,2>3.0 OD6oo. Cells were washed twice and diluted to 1.0 OD6oo in 0.05 M phosphate buffer pH 6.8. For each mutagenesis condition, 4 ml of the 1x107cells / ml was transferred to 100 ml shake flasks.4-Nitroquinoline 1 -oxide (NQO) and MilliQ water were added up to 5 ml with final NQO concentrations 0 pg / ml, 0.1 pg / ml, 0.2 pg / ml, 0.4 pg / ml, 0.6 pg / ml, 0.8 pg / ml, 1 .0 pg / ml. In parallel, N-methyl-N’-nitro-N-nitrosoguanidine (NTG) and MilliQ water were added up to 5 ml with a finalNTG concentration 0 pg / ml, 10 pg / ml, 20 pg / ml, 30 pg / ml, 40 pg / ml, 50 pg / ml, 60 pg / ml, 70 pg / ml. These mixtures were incubated for 30 min. at 25°C while gently shaken in the water-batch. The mutagenesis was stopped by adding 1 ml 50% (w / v) sodium thiosulfate solution and incubating for 5 minutes. Mutagenized cells were washed twice with YEPhD medium by centrifuging 10 minutes at 4000 g and removing the supernatants. Cells were recovered in 10 ml YEPhD and incubated one hour at 25 °C and 100 rpm. Mutagenized cells were aliquoted in 0.5 ml and mixed with 0.5 ml 20% glycerol and stored in the -80°C. Finally, dilutions of the mutant batches were plated out on YEPhD agar petri plates to determine survival rates.Mutants were plated on YEPhD 2% agar plates and incubated for four days. Single colonies were picked and grown in 96 well plates (Thermo Scientific, Denmark) with 150 pl / well YEPhD agar and incubated for 3 days at 30 °C. Mutants were transferred with a disposable QREP 96 replicator (Molecular Devices, USA) from 96 well YEPhD agar plates to 96 well plates (Thermo Scientific, Denmark) with 150 pl YEPhD and covered with Breathseal (Greiner bio-one, Frickenhausen, Germany). After overnight of cultivation in (30 °C, 750 rpm 550 rpm, 80% humidity) in Microton incubator shaker (Infors AG, Bottmingen, Switzerland), 10 pl of cultivated broth was transferred to half deep well plate (HDWP, 96 wells, 1.2 ml) containing 400 pl SMD-Urea medium (Luttik MA, J Bacteriol 2000;182:7007-7013) with 2% w / w ethanol instead of glucose and cultivated for 24 hours (30 °C, 750 rpm, 80% humidity).Each well was sampled for measuring cell density and protein concentration. One sample of 20 pl was diluted (1 :10) with physiological saline (0.85 % NaCI) to a final volume of 200 pl and cell density was measured in a 96 well, flat bottom, NUNC A / S, (Thermo Scientific) at a wavelength of 600 nm. The second sample (66 pl) was mixed with 134 pl Y-PER (Yeast protein extraction reagent, 78991 , Thermo Scientific) closed with aluminum seal and mixed in Microton incubator shaker at 750 rpm, 30 °C, 80% RH) for 30 minutes. After incubation, the plates were centrifuged (2750 ref) for 5 minutes. Subsequently, 100 pl of supernatant was transferred to a black polystyrene wells 96 well plate with micro-clear flat bottom, (ref 655090, Greiner-bio-one). Finally, the protein (tryptophan) content was measured at a wavelength of 295 - 350 nm (Ex-Em) using a 1.5 g / L BSA solution as standard. The protein on cell density ratio was calculated to select for mutants with increased protein content.Strains selected with the primary screen described above were subsequently cultivated in a shake flask. Pre-cultures were generated in 24 deepwell MTP plate (Axygen Deep well plate) containing 3 ml YEPhD in each well. The precultures were incubated for 3 days in an INFORS shaker set at 30°C, 550 rpm and 80% humidity and sealed with a breath-seal (Greiner Bio-One, 676051). Afterincubation, 30 l of sample material was transferred to fresh 400 pl SMD-Urea medium with 2% w / w ethanol in a 24 deepwell MTP plate for a second pre-cultivation round (3 ml, 30°C, 550 rpm and 80% humidity) and incubated for 24 hours. From this second culture 1 ml broth was transferred to a 500 ml Shake flask containing 100 ml SMD-Urea medium with 2% w / w ethanol, medium and incubated Incubation for 40 hours at 30°C and 250 rpm.The 100 ml culture was divided over two 50 ml Greiner tubes and spun down at 5,000 g for 5 minutes at 5°C. The cell pellets were washed with 25 ml milliQ (8°C) and combined in one 50 ml pre-weighted Greiner tube. After washing, the tubes were again pelletized (5,000 g for 5 minutes at 5°C). Finally, the supernatant was discarded, and the tubes were weighted (wet weight). Cell pellets were stored at -20°C for dry weight and amino acids determination. Dry weight of the strains was determined by measuring the biomass weight after lyophilization of the complete sample (100 ml).Amino acids were determined using method: DBC-SLD-A-02773_AMINO ACIDS AFTER HYDROLYSIS USING ACCQ-TAG METHOD (UPLC)

[0087] Example 3: SCP product generation

[0088] Single 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.

[0089] Example 4: Production of Cyberlindnera jadinii single cell protein (SCP) using continuous cultures vs. fed batch

[0090] continuous cultures: Cyberlindnera jadinii CBS 621 was cultivated at 30 °C in 1.5 L bioreactors (Applikon, Delft, The Netherlands) with a working volume of 1.0 L (electric level sensor). Chemically defined, synthetic medium (SM) was used, containing 5 g / L (NH4)2SO4, 3 g / LKH2PO40.5 g / L MgSO47H2O, trace elements (15 mg / L EDTA, 4.5 mg / L ZnSO47H2O, 0.3 mg / L COCI-6H2O, 1 mg / L MnC1 H2O, 0.3 mg / L CuSO4-5H2O, 4.5 mg / L CaC12-2H2O, 3 mg / L FeSO47H2O, 0.4 mg / L Na2MoO42H2O, 1 mg / L H3BO3, 0.1 mg / L KI) and vitamins (0.05 g / L D-(+)- biotin, 1 .0 g / L D-calcium pantothenate, 1 .0 g / L nicotinic acid, 25 g / L myo-inositol, 1 .0 g / L thiamine hydrochloride, 1.0 g / L pyridoxal hydrochloride, 0.2 g / L 4-aminobenzoic acid) (Verduyn et al., 1992), unless otherwise stated. The pH was adjusted to 6.0 (KOH) and medium was subsequently autoclaved at 120 °C for 20 min. Vitamins were filter-sterilised and added to medium prior to inoculation. SM was supplemented with 0.2 g / L Pluronic PE 6100 (anti-foam) and ethanol. The pH was maintained at 5.0 by the automatic addition of 2 M KOH. Stirrer speed was kept at 800 rpm and air was supplied at a flow of 0.5 L / min. After the substrate was exhausted during the batch phase, a continuous cultivation mode started at specific dilution rates.

[0091] At least two pre-steady-state samples were collected from the effluent line within 5 volume changes prior to final sampling. When values of dry weight and CO2did not differ more than 5% for two additional volume changes, steady-state samples were collected directly from the culture. Assuming a biomass to Cmol conversion of 26.4 g-Cmol’1(Lange & Heijnen, 2001), carbon recovery was calculated for all experiments and values between 95-105% were obtained.

[0092] Fed-batch: Fed-batch cultivations with C. jadinii CBS 5947 (vitamin prototroph) were conducted at 30 °C in 20 L Bio Bench reactors (Applikon, Delft, The Netherlands) with an initial working volume of 8.0 L. Stirrer speed was kept at 800 rpm and air (21% O2, 0.05% CO2) was supplied at a flow of 7 L / min. In both the batch and fed-batch phases, an overpressure of 1 .0 bar was applied to enhance oxygen transfer (2.0 bar total pressure). A polarographic oxygen electrode (model 32 275 6800; Ingold) was used to continuously monitor the dissolved oxygen concentration, ensuring it remained above 20% of air saturation (van Hoek et al., 2000).

[0093] The cultivation medium contained 2 g / L (NH4)2SO4, 20 g / L KH2PO46 g / L MgSO47H2O, and trace elements (150 mg / L EDTA, 45 mg / L ZnSO47H2O, 3 mg / L CoCI 6H2O, 10 mg / L MnC1 H2O, 3 mg / L CuSO4-5H2O, 45 mg / L CaC12-2H2O, 30 mg / L FeSO47H2O, 4 mg / L Na2MoO42H2O, 10 mg / L H3BO3, 1 mg / L KI) and 0.2 g / L Pluronic PE 6100. Ammonium hydroxide (25% solution in water) was used as the titrant (pH set at 5.0) and nitrogen source. For the initial batch phase, 12 g / L ethanol was used as carbon source and the reactor was inoculated at initial OD 0.2. At the end of the batch phase (decrease in CO2production and increase in DO signal), the fed-batch phase was initiated. Absolute ethanol was used as carbon source, which was pumped into the reactor with a controllable Masterflex model 7518-00 pump (Cole-ParmerInstrument Company, Chicago, IL) with flow rates between 0.006 and 0.050 kgEtoH-h’1. The exponential feed was calculated and controlled by an online control system (BIODACS Applikon, Delft, The Netherlands) according to equation 2 below.

[0095] Where FHOH is the flow rate of ethanol in kgEtoHTr1, Cxo is the initial biomass concentration (kgDw kgbroth'1), Wois the initial culture weight (kgbroth) and t is the time (h) after starting the feed. is the maximum biomass yield on substrate (kgDwgEtoH-1) and msis the maintenance coefficient (kgEtoH kgDw'1h_1), both calculated from chemostat experiments. The specific growth rate was kept at 0.10 h-1until the DO decreased below 15% of air saturation, when the feed was changed from exponential to constant.

[0096] Analytical methods: Dry weight measurements were conducted as previously described (Postma et al., 1989). Briefly, nitrocellulose filters (pore size 0.45 pm, Gelman Laboratory, Ann Arbor, Ml) were kept at 80 °C, subsequently weighed and used to filter the medium of 10 mL culture samples. Biomass-containing filters were washed with demineralized water (demiH2O), dried for 20 min at 350 W in a microwave (Bosch, Stuttgart, Germany) and weighed. Protein content was determined according to (Verduyn et al., 1991 b). Briefly, cell suspension (20 mL) was centrifuged at 10,000 g for 5 min and the pellet was washed with demiH2O. Subsequently, the pellet was resuspended in 10 mL H2O (total volume) and kept at -20 °C. For protein measurements, thawed samples were mixed with 3 M NaOH (2:1), boiled at 100 °C for 10 min and cooled on ice. CuSO45H2O (2.5% w / v) was added to samples (1 :3), which were incubated at room temperature for 5 min and centrifuged at 13,000 rpm for 5 minutes. The absorbance of the clear supernatant was measured at 510 nm. A stock solution of 5 g / L BSA (Merck-Sigma A9647) was taken in parallel and used for a standard curve.***

[0097] The present invention may also be summarized in the following items:1. Method for cultivating a microorganism capable of producing at least 60% protein, preferably at least 65% protein, more preferably at least 70%, most preferably at least 75% protein, comprising the steps of:(i) supplying microorganism to a reactor,(ii) continuously feeding EtOH as feedstock and(iii) continuously removing culture liquid comprising microorganisms.2. The method of claim 1 , further comprising the step of (iv) controlling the growth rate.3. The method of claim 1 or 2, further comprising one or more of the steps of:(v) controlling the feed rate,(vi) controlling the temperature, and(v / 7) controlling pH.4. The method of any of claims 1 to 3, further comprising the step of (viii) drying the biomass5. The method of any of claims 2 to 4, wherein the growth rate is below 0.2 h’1, preferably below 0. 15 h-1, more preferably below 0. 12 h’1, more preferably below 0. 1 h’1.6. The method of any of claims 2 to 4, wherein the growth rate is between 0.01 h-1and 0.2 h’1, preferably between 0.015 h’1and 0.2 h-1preferably between 0.02 h-1and 0.2 h’1, preferably between 0.015 h’1and 0.2 h’1, preferably between 0.015 h-1and 0.15 h’1, preferably between 0.015 h’1and 0.12 h-1, preferably between 0.015 h-1and 0.1 h-1.7. The method of any of claims 1 to 6, wherein the feed rate is 0.179 - 0.536 gethanoi / gbiomass / h, more preferably 0.179 - 0.469 gethanoi / gbiomaSS / h.8. The method of any of claims 1 to 7, 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.9. The method of any of claims 1 to 8, wherein the pH is kept between 3.5 and 5.5, preferably between 3.5 and 5.5, more preferably between 3.5 and 4.5.10. The method of any of claims 1 to 9 further comprising the step of recovering the biomass.11 . The method of any of claims 1 to 10, further comprising the step of drying the biomass.12. The method of any of claims 1 to 11 , wherein the microorganism is a Saccharomycetales yeast.13. The method of any of claims 1 to 12, wherein the Saccharomycetales yeast is a yeast from the genus Cyberiindnera, Saccharomyces, Kluyveromyces, Wickerhamomyces, Pichia orYarrowia, preferably from the genus Cyberlindnera or Saccharomyces or Kluyveromyces or Wickerhamomyces.14. The method of any of claims 1 to 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. The method of any of claims 1 to 13, 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 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 cerevisiae GHP1, Saccharomyces cerevisiae CEN.PK113-7D, Wickerhamomyces anomalus CBS 1980, Cyberlindnera jadinii ATCC 9950 or Kluyveromyces lactis CBS 2896.16. A yeast single cell protein product, wherein the yeast single cell protein product comprising at least 60% protein, preferably at least 65% protein, more preferably at least 70%, most preferablyat least 75% protein on dry cell weight, wherein the protein comprises >0.1 %, preferably >0.2% isocitrate lyase and / OR malate synthase.17. The yeast single cell protein product of claim 16, wherein the yeast is a Saccharomycetales yeast.18. The yeast single cell protein product of claim 17, 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.19. An animal feed comprising up to 20% (w / w) of yeast single cell protein product as claimed in any of the preceding claims.

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

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

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

[0101] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain 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.

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

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

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

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

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

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

[0108] 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.

[0109] 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 60% protein, preferably at least 65% protein, more preferably at least 70%, most preferably at least 75% protein, comprising the steps of:(i) supplying microorganism to a reactor,(ii) continuously feeding EtOH as feedstock and(iii) continuously removing culture liquid comprising microorganisms.

2. The method of claim 1 , further comprising the step of (iv) controlling the growth rate.

3. The method of claim 1 or 2, further comprising one or more of the steps of:(v) controlling the feed rate,(vi) controlling the temperature, and(v / 7) controlling pH.

4. The method of any of claims 1 to 3, further comprising the step of (viii) drying the biomass5. The method of any of claims 2 to 4, wherein the growth rate is below 0.2 h’1, preferably below 0. 15 h-1, more preferably below 0. 12 h’1, more preferably below 0. 1 h’1.

6. The method of any of claims 2 to 4, wherein the growth rate is between 0.01 h-1and 0.2 h’1, preferably between 0.015 h-1and 0.2 h-1preferably between 0.02 h-1and 0.2 h-1, preferably between 0.015 h-1and 0.2 h-1, preferably between 0.015 h-1and 0.15 h-1, preferably between 0.015 h’1and 0.12 h-1, preferably between 0.015 h-1and 0.1 h-1.

7. The method of any of claims 1 to 6, 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.

8. The method of any of claims 1 to 7, 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.

9. The method of any of claims 1 to 8 further comprising the step of recovering the biomass.

10. The method of any of claims 1 to 9, further comprising the step of drying the biomass.

11. The method of any of claims 1 to 10, wherein the microorganism is a Saccharomycetales yeast.

12. A yeast single cell protein product, wherein the yeast single cell protein product comprising at least 60% protein, preferably at least 65% protein, more preferably at least 70%, most preferably at least 75% 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 orKluyveromyces or Wickerhamomyces.

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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