JPS6240998B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to single cell proteins. In one aspect, the invention relates to the production of single cell proteins. In another aspect, the invention relates to novel yeast strains. Efforts to alleviate the global protein shortage have involved a variety of biosynthetic methods. In that method, single cell proteins (SCPs) are obtained by growing one or other microorganisms on various carbon-containing substrates. Carbon energy substrates should be readily available, relatively inexpensive, uniform and safe. Petroleum hydrocarbons have been used as carbon energy sources, but have encountered practical difficulties with their lack of water solubility and the high consumption of molecular oxygen required to support microbial transformation. Another method is to use it as a feedstock due to the relative water solubility of the oxygen-saturated hydrocarbon derivative and hence its ease of handling in aqueous fermentations and the substantial reduction in molecular oxygen demand for microbial conversion - growth methods. concentrated on. However, the limiting factor in the commercialization of single-cell protein production is the need to have moderate yields of dry cells based on the consumed substrate, as well as the need to adjust the fermentation to relatively moderate cell densities and their consumption, and thus to ensure adequate amounts of dry cells. There was a need to process large amounts of whole fermentation effluent to recover SCP material. Processing of large amounts of aqueous fermentation effluent complicates centrifugation and concentration of single cell protein products as in washing and drying steps. Some existing methods have focused on cell culture, since the crude protein content of cells is generally slightly higher than that obtained from yeast. However, yeast is widely available and relatively easy to cultivate. Yeast cells are generally slightly larger than bacterial cells, and therefore they tend to be more easily separated from the fermentation effluent. The discovery of means and methods to increase cell yield, particularly operating at high cell densities, would be highly desirable. For example, the resulting handling of substantially less fermentation effluent volume reduces piping and pump size, reduces assembly water requirements as well as reduces sterilization requirements, and equipment size and handling for coagulation and separation steps. This would mean significant savings in reduced demand. The present invention adds a medium containing a high concentration of mineral salts to the fermentation solution in the fermenter, making it possible to operate the fermentation solution in the fermenter at an extremely high cell density that is normally unobtainable, thereby producing a high yield of single-cell yeast yeast. A method of operating a continuous aerobic fermentation process using a yeast culture to produce a white product is provided. Cell density is defined as the concentration of cells per volume of fermentation solution on a dry weight basis. Fermentation liquid is defined as the total volume of aqueous fermentation broth or liquid containing cells. Cell density is commonly expressed as g/. Yield is defined as the weight of cells produced for a given consumption of the carbon energy source or weight of substrate supplied to the fermentation liquor. And it is usually expressed in g/g. The high cell density obtainable by the method of the invention is highly efficient and reduces single cell protein production costs. Concentration of single cell protein products, often by centrifugation elements, is greatly reduced or even eliminated. The cell product can, if desired, be washed in a washing element to remove residual unconsumed salts and transferred directly to a drying element, such as a spray dryer. The wash solution contains most of the residual mineral salts that are not taken up by the cells and can be recycled to the fermenter. The water requirements for the fermentation process are thus greatly reduced and, importantly, there is little or no waste water that needs to be disposed of. Alternatively, the entire fermentation liquor, including residual salts, can be dried if desired. To date, continuous processes for producing single-cell protein materials with yeast cultures have typically provided fermentation effluents with relatively low yeast cell content, such as about 20-25 g cells per fermentation liquor. However, the present invention provides a fermentation process that yields relatively high levels of yeast cells, such as 100 g or more per fermentation liquor. Such a high cell density in the fermentation liquor coupled with a high yield of yeast cells means more effective and efficient production, especially in continuous production conditions. According to the invention, the yeast uses effective amounts of molecular oxygen-containing gas, assimilable nitrogen sources, nutritive mineral salts and, if necessary, additional nutritive organic substances such as vitamins such as biotin and/or thiamin. It is grown under substantially continuous aerobic aqueous fermentation conditions on a suitable carbon energy source added as a substrate. In this method mineral salts are added at the high levels described below, resulting in a fermentation process that operates at high cell densities and provides high yields. The invention consists in almost force-feeding the cells by adding to the fermentation liquid a high concentration (relatively) of mineral salts which results in a substantially high growth rate. The concentration of mineral salts in the fermentation supernatant (ie, the fermentation liquor without cells) itself remains, of course, at a relatively low level. This is because salt is consumed by cells for growth and reproduction. The salt concentration in the cells plus supernatant is therefore very high. Cultivation is accomplished in a growth medium consisting of an aqueous mineral salt medium, a carbon energy source material, molecular oxygen, assimilable nitrogen, and, of course, a starting inoculum of the particular species of yeast or yeasts used. In the method of the present invention, a high concentration of Minella salt is supplied to the fermentation liquor, and the high concentration is retained in the fermentation liquor. The supply medium is supplied with selected mineral nutrients in appropriate proportions and amounts to ensure proper microbial growth, maximum assimilation of carbon and energy sources by the cells in microbial conversion methods, and maximum It is necessary to achieve the highest cell yield at a cell density of . Although the composition of the fermentation liquor can vary over a wide range, depending in part on the fermenter and the substrate used, the mineral content in the fermentation liquor according to the invention (i.e., liquor plus cells) is relatively high and has not been previously considered suitable. or at a higher level than that implemented by the prior art. Minimum broad and currently preferred concentration ranges for various elements in the fermentation broth are shown in the table below. Although the concentrations are expressed as those of the elements, all or part of each may be present in the form of soluble ions or, in the case of P, in some type of complex form, such as phosphate. Is recognized. The amount of each element is expressed in g or mg per fermentation solution (aqueous phase containing cells).

[Table] Sulfur is preferably used in the form of sulfate.
Since it is advantageous to add some of the required metals in sulfate form, the minimum concentration of sulfur is usually exceeded. Any or all of the metals listed can be used or included as sulfates. Preferably Mg, Ca, Fe, Zn, Ca and Mn are used in sulphate or chloride form or in the form of compounds which are converted in situ to sulphate or chloride. Preferably, K is used as sulphate, chloride or phosphate or in the form of a compound which is converted in situ to sulphate, chloride or phosphate. P may be used in the phosphoric acid form or in the form of phosphates, such as monohydrogen phosphates or dihydrogen phosphates, such as potassium or ammonium salts, or as compounds that are converted in situ to such salts. is preferred. Other elements which can be included at least in trace amounts are, for example, Na and Co as halides or sulphates, Mo, for example as molybdates, B, for example as borates, for example as selenites or selenium salts. Se, including I as an iodide. In a typical high cell density fermentation, the fermentation solution is approximately
It will consist of 1/2 supernatant medium and 1/2 cells. However, these 1/2 volume cells will contain at least about 2/3 the mineral salt content of the fermentation broth. The method of the invention uses a fermentation culture suitable for growth on a carbon-containing substrate under aqueous fermentation conditions.
Suitable yeasts include Candida, Hansenula, Torulopsis,
Includes species of the genera Saccharomyces, Pichia, Debaryomyces and Brettanomyces. Presently preferred genera include Candida, Hansenula, Torulopsis, Pichia, and Satucharomyces. Examples of suitable species are: Brettanomyces Petrophilium Candida boldinii Candida lipolytica
lipolytica) Candida mycoderma (Candida
Candida utilis (Mycoderma) Candida utilis (Candida utilis)
stellatoidea) Candida robusta Candida clausenii (Candida robusta)
claussnnii) Candida rugosa (Candida rugosa) Candida tropicalis (Candida rugosa)
tropicalis) Hansenula minuta Hansenula minuta
saturnus) Hansenula californica (Hansenula
californica) Hansenula mrakii (Hansenula mrakii) Hansenula silvicola (Hansenula mrakii)
silvicola) Hansenula polymorpha (Hansenula
polymorpha) Hansenula
wickerhamil) Hansenula capsulata (Hansenula
capsulata) Hansenula glucochima (Hansenula
glucozyma) Hansenula henritskii (Hansenula
henricii) Hansenula nonfermentans (Hansenula nonfermentans) Hansenula philodendra (Hansenula
philodendra) Pichia farinosa Pichia polymorpha Pichia membulranaefaciens Pichia pinus Pichia pastoris Pichia trehalophylla
trehalophila) Saccharomyces cervsiae Saccharomyces fragilis
fragilis) Saccharomyces rosei (Saccharomyces
rosei) Saccharomyces acidifaciens Saccharomyces elegans
elegans) Saccharomyces rouxii
rouxii) Saccharomyces lactis
lactis) Torulopsis sonorensis (Torulopsis)
sonorensis) Torulopsis kyanjida (Torulopsis
candida) Torulopsis bormyi (Torulopsis
bolmii) Torulopsis varsatilis (Torulopsis
versatilis) Torulopsis glabrata (Torulopsis
glmbrata) Torulopsis molysiana (Torulopsis
molishiana) Torulopsis nemondendra (Torulopsis
(nemodendra) Torulopsis (Torulopsis)
nitratophila), and Torulopsis pinus. Mixtures of two or more species of yeast can be used if desired. The specific yeast used depends in part on the carbon-containing substrate used. This is because it is well known that different yeasts often require somewhat different substrates for optimal growth. It has been observed that certain strains of the above species, such as Pichia pastoris, do not grow on methanol. Oxygen saturated - those Pichia pastoris and Hansenula polymorpha suitable for growth on hydrocarbon feedstocks, especially lower alcohols such as methanol, are currently preferred. Pichia Pastoris (Culture 21-2) NRRL Y-11431, Pichia Pastoris (Culture 21-1) NRRL Y-
11430, Hansenula polymorpha NRRL Y-
11170, and the strain deposited as Hansenula polymorpha (culture 21-3) NRRL Y-11432, are currently preferred. This is because these strains are particularly suitable for use in the production of SCP protein materials with high cell densities and high yields. Pichia pastoris (Culture 21-2) NRRL Y-11431 and Pichia pastoris (Culture 21-1) NRRL Y-
The characteristics of these strains of 11430 are considered unusual for these species. This is because out of the four Pichia pastoris cultiaria tested, only two grew on methanol anyway. Pichia Pastoris (Culture-21-2) NRRL
11431, Pichia pastoris (Culture-21-
1) NRRL Y-11430 and Hansenula polymorpha (Culture-21-3) NRRL Y-11432 is considered new and unique. The carbon energy substrate can be any carbon energy source suitable as a yeast substrate, such as hydrocarbons, oxygen-saturated hydrocarbons including various carbohydrates, and the like. It is recognized that specific yeasts vary in their preferences for various substrates. Currently preferred substrates for aqueous fermentation conditions are carbon-
It is a highly water-soluble oxygen-hydrogen compound. The term "oxygenated hydrocarbon" is meant as a generic term in this disclosure describing compounds that can be used and is not necessarily a limiting term meaning a source of substrate. For the purposes of this disclosure, oxygen-saturated hydrocarbons include water-soluble carbohydrates, as well as alcohols, ketones, esters, acids and aldehydes, and mixtures thereof, which have from 1 to 20 carbon atoms per molecule and generally have a property of is naturally highly water soluble. More suitable oxygen-saturated hydrocarbons are typically substantially larger water-soluble ones having up to about 10 carbon atoms per molecule, or are generally water-soluble carbohydrates. Exemplary carbohydrates include glucose, fructose, galactose, lactose, sucrose, starch, dextrin, etc. alone or in mixtures. Among other types of oxygen-saturated hydrocarbons, examples are methanol, ethanol, ethylene glycol, propylene glycol, 1-propanol, 2-propanol, glycerol, 1-butanol, 2-butanol, 3-methyl-1-butanol, 1 -Pentanol, 2-hexanol,
1,7-heptanediol, 1-octanol,
2-decanol, 1-hexadecanol, 1-eicosanol, acetone, 2-butanone, 4-methyl-2-pentano, 2-decanone, 3-pentadecanone, 2-eicosanone, formaldehyde, acetaldehyde, propionaldehyde,
Butyraldehyde, hexanal, 7-methyloctanal, tetradecanal, eicosanal, acetic acid,
Propionic acid, butyric acid, glutaric acid, 5-methylhexanoic acid, azelaic acid, dodecanonic acid, eicosanonic acid, methyl formate, methyl acetate, ethyl acetate, propyl butyrate, isopropyl hexanoate, hexyl 5-methyloctanoate, octyl dodecanoate and mixtures thereof. Although less preferred because it is sometimes difficult to remove residual matrix from single-cell protein cells,
It is also possible to use normal paraffins, such as 10 to 20 carbon atoms per molecule, according to the method of the invention. Yeast generally do not assimilate less than 10 carbon atoms per molecule of paraffin. Typically these are decane, undecane, dodecane, tridecane,
Including such as tetradecane, pentadecane, hexadecane, octadecane, eicosane, etc. and mixtures thereof. Water soluble alcohols of 1 to 4 carbon atoms, water soluble acids of 2 to 4 carbon atoms and water soluble carbohydrates are currently preferred. Water-soluble monohydric fatty acid hydrocarbyl alcohols are preferred. 2-methyl-1
-It is noteworthy that propanol is inhibitory to certain yeasts. This alcohol should be avoided in such yeast fermentations. 1~
Alcohols with 4 carbon atoms (other than 2-methyl-1-propanol) are currently also preferred. Of these, methanol and ethanol are currently preferred over the others. Methanol is also preferred among such feedstocks due to its relatively low cost. Petroleum gas can be oxidized. and water-soluble materials such as the oxidation of methane, ethane, etc. used to provide a predominant mixture of the corresponding alcohols along with various aldehydes, ketones, acids, etc., and various purifications produced in centralized rational refineries as well. Appropriate hydrocarbon fractions from sources and chemically processed complexes, often referred to as petroleum complexes, can be used for fermentation purposes. Salts in the supernatant are at relatively low concentrations. This is due to the high uptake by growing and producing cells. Mineral salts in cells cannot be present or applied as a feed because some are in bound organic form. Of course, mineral analysis of the fermentation liquor will reflect the total mineral content. In addition to mineral salts, vitamins (organic growth factors) can be used in the fermentation broth if their presence is desired for the growth of the particular yeast of choice, as known to those skilled in the art. For example, for proper growth many yeasts appear to require the presence of one or both of the vitamins vithione and thiamine, or other media components containing these vitamins, such as yeast extract. Thus, for yeast such as Hansenula polymorpha, an amount of about 0.04 to 0.8 mg biotin per 1 aqueous mineral medium and about 4 to 80 mg per 1 aqueous mineral medium.
It is desirable to use mg amounts of thiamine hydrochloride. Alternatively, all or part of the biothione and thiamine can be provided through the use of yeast extract or the like. In the production of mineral media used in aqueous aerobic fermentation processes to which growth factors are added, the use of water containing residual chlorine levels, such as those commonly encountered in water from purification processes in some countries, may be accompanied by growth factors, especially If you use aqueous mineral media prepared from water with residual chlorine in such fermentation systems, removing the chlorine before adding the growth factors may tend to deactivate vitamins such as biotin or thiamine. It has long been known that growth factors can be prevented from being lost or inactivated. Therefore, methods have been developed to treat water containing residual chlorine to effectively remove residual trace chlorine and thus avoid vitamin loss. Until now, water containing undesirable amounts of residual chlorine has nevertheless been used without inactivation of the vitamins by chlorine, if the vitamins are added to the fermentation zone as a separate stream from the aqueous nutrient medium. It was found that it can be used in an enzymatic method. Mineral nutrient media can therefore use water containing trace amounts of chlorine. This procedure thus avoids the need for pre-treatment of water containing residual traces of chlorine by expensive and/or time-consuming methods. Separate addition of vitamins to the fermentation zone described above is preferably and advantageously carried out by mixing the vitamins with at least some, preferably all, of the carbon energy substrate stream prior to adding these materials to the fermentation zone. achieved. If an aqueous mixture of vitamin and carbon energy substrates is used, the water used for the initial vitamin dilution should preferably be free of residual chlorine, such as deionized water, and should preferably be free of residual chlorine, such as water-based methanol. Any pre-loss should be avoided before mixing with the substrate. If desired, and preferably the mixture contains water and a water-soluble carbon substrate such as methanol, e.g.
It is produced from a 20% methanol aqueous solution by volume, then the vitamins can be dissolved in the methanol aqueous solution and then fed to the fermenter. In this manner, residual chlorine does not have to be removed first, yet the vitamins are completely preserved. In a more preferred embodiment, further addition of vitamins to the fermentation zone is achieved using a mixture of vitamins, at least some of the carbon energy substrates described above, and further addition of an aqueous trace mineral salt solution. Trace mineral salts are Co, Mo, B, Se, I or Mn,
It consists of those cited above as trace elements such as Cu, Zn and Fe. Use of this more preferred embodiment not only avoids the vitamin inactivation problem caused by traces of chlorine in the water used for aqueous mineral salt media, but also avoids other problems often encountered in fermentation processes. This problem is often the formation of precipitates in heat sterilization zones used to treat aqueous mineral salt media that require cleaning. The presence of trace mineral salts, usually mixed with the initial mineral nutrients, clearly promotes the formation of troublesome precipitates in the heat sterilization zone. Therefore, by not including trace mineral salts in the aqueous mineral salt medium stream, but instead by including trace mineral salts mixed with vitamins and at least some carbon energy substrates, two very troubling problems can be avoided. solve. The water used to produce the mixture of trace mineral salts, at least some carbon energy substrate and vitamins as described above should preferably be free of residual trace amounts of chlorine. The stream consisting of vitamins, some carbon energy substrates and trace minerals can be made sterile by filtration if desired. However, it is preferred and advantageous to combine the stream with the primary carbon energy substrate before entering the fermentation zone and to pass the entire combined stream just before entering the fermentation zone. Fermentations themselves are aerobic processes that require molecular oxygen; they are supplied with a molecular oxygen-containing gas, such as air, oxygen-enriched air, or substantially pure molecular oxygen, in which microorganisms are grown in a growth mode. Maintain an effective partial pressure of oxygen in the fermentation liquor to support growth.
By using an oxygen-saturated hydrocarbon substrate, the total oxygen requirement for microbial growth is less than the requirement when paraffin is used. Even so, an adequate amount of molecular oxygen must be supplied for growth. This is because the assimilation of substrate and the corresponding growth of microorganisms is in part a combustion process. The rate at which molecular oxygen is supplied to the fermentation should be such that yeast growth is not limited by lack of oxygen. Fermentation equipment varies widely in its ability to transport oxygen to the culture. In general, the ventilation rate can be varied over a considerable range;
For fermenters that are very effective at oxygen transfer, aeration is generally carried out to increase the molecular oxygen-containing gas volume per minute per liquid volume in the fermenter (at working pressure and 25°C).
) at a ratio of about 0.5 to 8, preferably about 1 to 6. This amount is based on the normal oxygen content air supplied to the reactor, and in pure molecular oxygen, each range is the volume of molecular oxygen per minute per liquid volume in the fermenter (working pressure and 25
°C) from about 0.1 to 1.7, or preferably from about 0.2 to
Probably 1.3. The pressure used in the microbial fermentation process can vary widely. Typical pressures range from about 0 to
150 psig, currently preferably about 0 to 60 psig, more preferably at least slightly above atmospheric pressure. Pressures above atmospheric pressure are advantageous as such pressures tend to increase dissolved oxygen concentrations in aqueous fermentations. It can then help increase the growth rate of the cells. At the same time, high pressures are offset by the fact that they increase equipment and operating costs. Fermentation temperatures can vary somewhat but will generally be about 25-65°C, generally preferably about 28-50°C. Pichia pastoris culture 21-2 deposited as NRRL Y-11431 and NRRL
Pichia pastoris deposited as Y-1430
The fermentation culture of culture 21-1 is generally about 30
Prefer fermentation temperature of ℃. Hansenula polymorpha
Hansenula polymorpha culture 21-3, deposited as NRRL Y-1170 and NRRL Y-11432, currently appears to prefer fermentation temperatures on the order of about 38-40°C. Yeast require an assimilable nitrogen source. Assimilable nitrogen can be provided by any nitrogen-containing compound or compound capable of liberating nitrogen in a form suitable for vicarious utilization by yeast. Various organic nitrogen source compounds such as protein hydrolysates can be used technically, but typically cheaper nitrogen-containing compounds such as ammonia, ammonium hydroxide, urea and ammonium phosphate, ammonium sulfate, pyrroline Various ammonium salts can be used, such as ammonium acids and ammonium chloride. Ammonia gas itself is advantageous for large-scale operations and can be used in appropriate amounts to foam the aqueous microbial fermentation liquid. At the same time, such ammonia
Also helps with pH adjustment. The pH range of the aqueous microbial fermentation liquid is approximately 3 to 7.
More preferably and usually it should be in the range of about 3.5 to 5.5. The preference of certain microorganisms for pH ranges depends to some extent on the medium used, as well as on the particular microorganism. Therefore, some variation can be made by changing the culture medium, as can be readily determined by those skilled in the art. The average retention time of the fermentation liquor in the enzyme vessel can vary considerably, depending in part on the fermentation temperature and the yeast culture used. Generally the hold time will be about 2 to 30 hours based on average hold, preferably about 4 to 14 hours at present. High concentrations of some of the carbon and energy substrates mentioned, especially methanol or formaldehyde, can be inhibiting to satisfactory microbial growth or even toxic to fermentation microorganisms. Therefore, relatively high substrate concentrations should be avoided, and it is generally desirable to maintain fermentation substrate concentrations at the maximum acceptable level. For some lower alcohols, this level in the fermentate is generally between about 0.001 and 5% by volume, preferably between about 0.01 and 5% by volume.
0.05% by volume, whereas for aldehydes, due to the toxicity of aldehydes, these
Should be 1/10. When carbon and energy source materials contain aldehydes in amounts potentially harmful to microorganisms, harmful aldehyde effects can be avoided by subtracting the substrate with a suitable amount of a nitrogen compound, preferably ammonia, ammonium hydroxide, or other active ammonium compound per mole of aldehyde. Mitigation can be achieved by initial treatment with a ratio of about 0.01 to 10 molar equivalents of such nitrogen-containing compounds. Such treated substrates then contain not only carbon energy depletion but also at least some of the necessary assimilable nitrogen. The carbon-containing substrate can be adjusted as a limiting factor, thereby advantageously fermenting in a manner that ensures sufficient conversion of the carbon-containing substrate into the yeast cells and avoids possible contamination of the yeast cells with substantial amounts of unconverted substrate. It is done. The latter is not a problem due to water-soluble substrates. This is because any residual traces are easily washed out. However, there may be problems with non-aqueous substrates such as higher n-paraffins which require additional product treatment steps such as removal of residual hydrocarbons by a suitable solvent wash step. Continuous operation is highly preferred because of the ease of control, production of uniform product in uniform quantities, and the most economical use of all equipment. In a continuous process, carbon and energy source materials as substrates, an aqueous mineral medium, an assimilable nitrogen source and a molecular enzyme-containing gas are continuously added to the fermentation liquor in the fermenter in combination with continuous withdrawal of the fermentation liquor. The volume ratio of added carbon energy substrate:added aqueous mineral medium can vary over a wide range, depending in part on the nature of the carbon-containing substrate, but generally ranges from about 1:9 to 6:4, presently and preferably. will range from about 2:8 to 5:5. If desired, all parts of the carbon energy source material and/or part of the assimilable nitrogen source, such as ammonia, can be added to the aqueous mineral medium before passing it through the fermenter.
It was also advantageous to use a feed ratio of approximately 40% alcohol to 60% mineral salts medium by volume in this method at high cell densities. Each stream introduced into the reactor has a predetermined proportion or carbon and energy substrate concentration;
Adjustments are made according to requirements that can be determined by monitoring such as pH, lytic enzymes, oxygen or carbon dioxide in the gases exiting the fermenter, cell density as measured by light transmission, etc. The feeding ratios of the various materials can be varied to obtain as high a yield of yeast cells on added substrate as possible, consistent with efficient utilization of carbon and energy sources, and to obtain the fastest possible cell growth rate. By the method of the invention, yeast cells can be obtained with a yield of about 30 to 110 g per 100 g of added substrate, depending in part on the particular substrate used. All equipment, reactors or fermentation elements, vessels, piping, attached circulation or cooling equipment, etc.
It is also preferred to sterilize by using steam, typically at about 250°C (121°C) for at least about 15 minutes. The sterilizing reactor is inoculated with a culture of specific microbial powder in the presence of all necessary nutrients, including molecular oxygen and carbon-containing substrates. The type of fermenter used is not critical in carrying out the fermentation process of the invention, although operation in a foam-filled fermenter is currently preferred. Fermenters designed to promote and maintain foam production are necessary to maintain the desired high cell densities and rapid growth rates, and are advantageous in the present method of achieving increased oxygen transfer. At the start of the fermentation, an aqueous mineral medium, an appropriate concentration of carbon source, assimilable nitrogen, trace ingredients if desired and starting inoculum of yeast are placed in a sterile fermenter, and a suitable flow of oxygen and various feeds is gradually introduced. will be started on. If desired, the initial fermentation substrate can be gradually changed to methanol or the like as cell density increases, such as glucose. In aqueous fermentations, one can start with low mineral salt levels and build up to high mineral salt levels by feeding the fermentation liquor with an aqueous mineral medium having a high concentration of mineral salts. However, usually the high salt medium is simply added to the fermenter at the beginning and operation is immediately stopped. Those skilled in the art will appreciate that there will typically be a short lag time after departure before the inoculum has formed sufficient cells for complete input of salt and substrate used. Yeast cells produced by the high cell density method of the invention can be recovered from the fermentation mixture effluent by conventional methods such as centrifugation or filtration. If desired, extracellular products can be recovered from the substantially cell-free residual supernatant by conventional methods. The substantially cell-free effluent can be treated with, for example, acetone or a lower alcohol such as methanol or ethanol to precipitate any extracellularly produced polymeric material. The cell-free effluent can be processed by solvent extraction and/or base extraction to recover other extracellular products such as pigments, vitamins, or organic acids produced during the culturing process, if desired. . The cell-free effluent, with or without such ancillary treatment, is returned to the fermenter as part of the aqueous component, or as substantially or almost all of the aqueous component, to avoid waste problems as much as possible. can. Microbial cells are typically killed by heat or chemical means, which can occur before or after separation of the cells from the fermentation effluent. Yeast cells are a valuable source of protein for humans as well as animals. For human consumption, processing can be performed to reduce the nucleic acids when necessary, but for animal feed purposes such processing does not currently appear to be necessary. High yields can be obtained with the present invention using manipulation of cell densities, for example within the range of dry standard yeast cells per fermentation mixture. If desired, cells can be recovered from the fermentation mixture by centrifugation or other separation methods. If desired, the concentrated cells can then be washed, such as by mixing with water, separated, such as by recentrifugation, or substantially free of cells by adding water before or during centrifugation. It can be washed with mineral medium. The wash solution containing the separated mineral medium can then be returned to the fermenter as water and mineral medium assembly components, thus substantially reducing or avoiding waste problems. The harvested cells can then simply be dried to produce a dry product for future use. If desired, the high cell density fermenter effluent is dried in its entirety to produce a total dry product of dry cells and residual water-soluble materials containing salts, and this total dry product is high in protein and high in salt content. can be used as extremely useful animal feed. Tests using the method according to the invention are described below. The particular amounts of materials, the particular types of feedstock used, the particular species or strains of yeast are to be considered as examples and not as limitations on the invention. Example: In a test conducted under continuous aerobic fermentation method conditions:
Yeast Pichia pastoris culture 21 deposited as NRRL Y-11431 in methanol and aqueous mineral salt media in a volume ratio of 30.15 to 69.85, respectively.
-2 were inoculated into fermenters individually. No preconditioned media or substrates were used. The fermenter was a 4-fermenter with automatic adjustment of 2-liquid volume, PH, temperature and level. Stirring is 1000~
It was carried out using two blades rotating at 1200 rpm. The aeration rate is 1 to 1.5 volumes per fermentation volume per minute (approximately atmospheric pressure and
at 25 °C) and about 20% of which will dissolve in the fermentation mixture saturated with air at atmospheric pressure and about 30 °C.
An amount of dissolved oxygen equal to the amount of dissolved oxygen was maintained in the fermentation mixture. Aqueous ammonium hydroxide (2 volumes concentrated ammonium hydroxide and 1 volume deionized water) was added at a rate to maintain the pH of the fermentation mixture at about 3.5. The aqueous mineral salt medium used is 1 solution:
12.5ml 85% H ₃ PO ₄ , 2.5g 85% KOH, 8.5g
KCl, _7.0gMgSO4・_7H2O , _1.5gCaCl2・
2H ₂ O, 25ml trace mineral solution A, 25ml trace mineral solution B, 10ml bithione-thiamine hydrochloride solution, about 0.08ml antifoam agent (Mazu DF-37C),
and sufficient deionized water were mixed to prepare a solution of 1. Trace mineral solution A is 4.8g per solution.
of FeCl ₃ ·6H ₂ O, 2.0 g ZnSO ₄ ·7H ₂ O, 0.02 g
H ₃ BO ₃ , 0.20 g Na ₂ MoO ₄ 2H ₂ O, 0.30 g
_{MnSO4.H2O ,} 0.08g KI, 0.06g _CuSO4 .
A solution of 1 was prepared by mixing each of 5H ₂ O, 3 ml of concentrated H ₂ SO ₄ and enough deionized water. Trace mineral solution B is 2.0g per solution.
FeCl ₃ ·6H ₂ O, 2.0 g ZnSO ₄ ·7H ₂ O, 0.3 g
MnSO ₄ .H ₂ O, 0.6 g CuSO ₄ .5H ₂ O, 2 ml conc.
Mix H ₂ SO ₄ and enough deionized water to
A solution was prepared. A biotin-thiamine hydrochloride solution was prepared by mixing 2 mg of biotin, 200 mg of thiamine hydrochloride, and 50 ml of deionized water. Fermentation was carried out at approximately 30° C. and approximately atmospheric pressure with a hold time of 7.0 hours. Yeast cells were separated from the fermentation effluent by centrifugation, washed in a water suspension, recentrifuged, dried overnight at 100°C, and weighed. On a dry basis, yeast cells per 100 g of methanol supplied
A yield of 42.3g was produced. Cell density is effluent 1
The level was as high as 100.7 g of cells per cell. Example Another fermentation test was carried out with some changes in the composition of the aqueous mineral salt medium, the volume ratio of methanol to aqueous mineral salt medium was 40.8 to 59.2, and the aqueous ammonium hydroxide for pH adjustment was 3 volumes of concentrated ammonium hydroxide and 1 volume of concentrated ammonium hydroxide. The procedure was substantially as described in the example except that the fermentation hold time was 8.35 hours. The aqueous mineral salt medium used in this test was
For the solution, 20.0 ml 85% H ₃ PO ₄ , 4.0 g 85%
KOH, 12.0 g KCl, 10.4 g _{MgSO4.7H2O ,}
2.4 g of CaCl ₂ .2H ₂ O, 40 ml of the trace mineral solution A as described in the example, 40 ml of the trace mineral solution B as described in the example, 16 ml of the biotin-thiamine hydrochloride solution as described in the example, approximately 0.08 ml of antifoaming agent, and enough deionized water to form a solution of 1. Yeast cells were separated from the fermentation effluent, washed and dried as in the example. On a dry basis, the yeast cells produced a yield of 41.4 g per 100 g of methanol fed. Cell density is 133.3g per effluent
This was an extremely desirable high level. EXAMPLE Continuous aerobic fermentation was carried out in a fermenter as described in the example, this time with yeast deposited as NRRL Y-11432 inoculated with Hansenula polymorpha culture 21-3. 300 per total solution in the fermenter
A methanol and aqueous mineral salt medium mixture containing ml methanol was fed. The stirred fermentation mixture is
Supply air with and containing sufficient oxygen to 2 volumes per fermentation volume per minute (approximately atmospheric pressure and approximately 25°C).
aeration) by passing it through the fermenter to maintain an amount of dissolved oxygen in the fermentation mixture equal to about 20% of that which would dissolve in an air-saturated fermentation mixture at atmospheric pressure and about 38°C. Ta. Aqueous ammonium hydroxide (2 volumes of concentrated ammonium hydroxide and 1 volume of deionized water) was added at a rate to maintain the pH of the fermentation mixture between 3.7 and 4.1. The mixture of methanol and aqueous mineral salt medium is 1 part solution: 300 ml methanol, 6 ml
85% H ₃ PO ₄ , 3 g KCl, 4.5 g MgSO ₄ .
_7H2O , 0.6g _{CaCl2.2H2O ,} 0.3g NaCl, 10
ml of trace mineral solution A as described in the example, 10 ml of trace mineral solution B as described in the example, 4 ml of biotin-thiamine hydrochloride solution as described in the example, 4 drops of antifoam and enough deionized water to make one solution. It was produced by mixing. The fermentation was carried out at about 38° C. and about atmospheric pressure with a hold time of 5.66 hours. Yeast cells were separated from the fermentation effluent, washed and dried as in the example. On a dry basis, the yeast cells produced a yield of 31.0 g per 100 g of methanol fed. Cell density was 73.3 g cells per effluent. Example: In a continuous aerobic fermentation method, 36.9 pairs each
Methanol and aqueous mineral medium in a volume ratio of 63.1 were individually fed to a fermenter inoculated with the yeast Pichia pastoris culture 21-1 deposited as NRRL Y-11430. The fermenter was a 1500 bubble filled fermenter with a liquid capacity of approximately 600, automatic adjustment of pH, temperature, level and ventilation pipes.
Agitation was provided by a turbine operating at 750 rpm at the bottom of the draft tube. The aeration rate is approximately 1.6 volumes of air (approximately 38 psig) per volume of fermentation liquid in the fermenter per minute.
and at about 25°C). Anhydrous ammonia was added at a rate to maintain the pH of the fermentation mixture at approximately 3.5. The initial aqueous mineral salt medium is
12.2 ml 75% H ₃ PO ₄ , 6.0 g KCl, 6.0 g
MgSO ₄ 7H ₂ O, 0.8 g CaCl ₂ 2H ₂ O, 2.0 g
85% KOH, 2.0 ml of trace mineral solution C, 0.8 ml of biotin-thiamine hydrochloride solution and enough drinking water to make solution 1 (potable water should first be treated with enough sodium thiosulfate to contain (reacted with free chlorine). Trace mineral solution C is 65g per solution 1.
FeCl ₃ ·6H ₂ O, 18 g ZnSO ₄ ·7H ₂ O, 5.0 g
It was prepared by mixing MnSO ₄ .H ₂ O, 6.0 g CuSO ₄ .5H ₂ O, 2.0 ml concentrated H ₂ SO ₄ and enough deionized water to make solution 1. A bithione-thiamine hydrochloride solution was prepared by mixing the ingredients in a ratio of 0.4 g bithione to 40 g thiamine hydrochloride in 1 part deionized water. Fermentation was carried out at 30° C. and 38 psig pressure with a hold time of 12.7 hours. Yeast cells were separated from the fermentation effluent by centrifugation, washed in a water suspension, recentrifuged, and
Dry overnight at 100°C and weigh. On a dry basis, the yeast cells produced a yield of 37 g per 100 g of methanol fed. The cell density was a highly desirable 110.3 g cells per effluent. Example: 40 vs 60 respectively in continuous aerobic fermentation method
Methanol and aqueous mineral salts medium in a volume ratio of 100 ml were individually fed to a fermenter inoculated with the yeast Pichia pastoris culture 21-1 deposited as NRRL Y-11430. The fermenter has a liquid capacity of about 610, PH, temperature and level automatic adjustment of 1500
It was a fermenter filled with bubbles. The stirring is
This was done with two conventional paddle-type turbines driven at 1000 rpm. The aeration rate is approximately 4 volumes of air per volume of fermented product in the fermenter per minute (approx.
at 38 psig and about 25°C). Anhydrous ammonia was added at a rate to maintain the pH of the fermentation mixture at approximately 3.5. The aqueous mineral salt medium is 1 part drinking water,
15.86 ml of 75% H ₃ PO ₄ , 9.53 K ₂ SO ₄ , 7.8 g
MgSO ₄ 7H ₂ O, 0.6 g CaCl ₂ 2H ₂ O, and
It was prepared by mixing 2.6g of 85% KOH. Trace mineral solution plus biotin per 1 methanol
A portion of 10 ml was fed separately via the methanol stream. Trace mineral solution plus biotin was prepared by mixing 780 ml of trace mineral solution D, 20 ml of water, 200 ml of methanol and 0.032 g of biotin. Trace mineral solution D is 65g for solution 1.
of FeSO ₄ ·7H ₂ O, 20 g of ZnSO ₄ ·7H ₂ O, 3.0 g of
It was prepared by mixing MnSO ₄ .H ₂ O, 6.0 g CuSO ₄ .5H ₂ O, 5.0 ml concentrated sulfuric acid, and enough deionized water to make solution 1. Aqueous mineral salt medium was fed at a rate of 31.5 per hour and methanol was fed at a rate of 21 per hour. Fermentation was carried out at 30° C. and approximately 38 peig pressure with a hold time of 11.6 hours. For analytical purposes, yeast cells were separated from the fermentation effluent by centrifugation, washed with aqueous suspension, recentrifuged, dried overnight at 100°C and weighed. Yeast cells are fed methanol on a dry basis
A yield of 40.6 g per 100 g was produced. The cell density was a highly desirable 128.4 g cells per effluent. The total solids content of the fermentation liquor (effluent from the fermenter) was 134.7 g/l (cells plus dissolved solids). The effluent from the fermenter was passed through a sterilizer to kill yeast and fed directly to the spray dryer without further concentration or treatment. Example: 29 vs 71 respectively in continuous aerobic fermentation method
methanol and aqueous mineral salts medium in a volume ratio of
Fermenters inoculated with 11170 were fed individually. The fermenter was a 1500 bubble filled fermenter with approximately 560 liquid capacity, automatic adjustment of PH, temperature and level, and vent tubes. Stirring is done at the bottom of the ventilation pipe.
This was done by a turbine driven at 810 rpm. The aeration rate was approximately 5 volumes of air per volume of fermentation liquid in the fermenter per minute (approximately 38 psig and approximately 25°C).
Anhydrous ammonia was added at a rate to maintain the pH of the fermentation mixture at approximately 3.5. The aqueous inorganic salt solution is 10.38 ml of 75% H ₃ PO ₄ , 4.29
g KCl, _6.44 g _MgSO4.7H2O , 0.86 g
CaCl ₂ 2H ₂ O, 0.43 g NaCl, 3.0 ml trace mineral solution C and in 1000 ml of drinking water (the drinking water is first treated with enough sodium sulfate to react with the free chlorine contained therein). It was prepared by mixing 0.64 ml of biotin-thiamine hydrochloride solution. Fermentation was carried out at 39-40°C and 38 psig pressure with a hold time of 7.6 hours. For analysis, yeast cells were separated from the fermentation effluent by centrifugation, washed in a water suspension, recentrifuged, dried overnight at 100°C, and weighed. On a dry basis, yeast cells are fed methanol 100%
produced with a yield of 33.3 g/g. Cell density was the desired 76.2 g cells per effluent. EXAMPLE Aerobic growth of Candida utilis (Torula) was carried out in a bench top fermenter in a continuous aseptic manner. After the initial sterilization of the fermenter containing the medium of 2, ethanol was added to a final concentration of 0.5% (v/v) and the pH was adjusted to 3.5-4.0 with ammonia.
Adjusted to. The inoculum grown in flasks was added to the fermenter and grown through a number of split cycles as in a batch test. As soon as all the alcohol was consumed, sterile ethanol was added at a rate that allowed complete consumption of the culture. Usually this is monitored by dissolved oxygen response. After adding a total of 300 ml of ethanol to the growth container,
Both sterile mineral feed and ethanol were added to the fermenter. Fermenter capacity during continuous method is 1.8
Maintained at ~2.0. Oxygenated air was dispersed and the dissolved oxygen concentration was maintained at approximately 10% saturation. Stable conditions were obtained by adjusting the dilution rate of the medium. The substrate ethanol has always been the limiting nutrient.
Fermentation was maintained at a temperature of 32±1°C. The cell concentration depends on the ethanol concentration of the feed solution and the yield of cell aggregates.
It was in the range of 120-140g/. After reaching steady-state conditions, the fermentation broth samples were collected, centrifuged, washed, and then dried, and the cell mass yield was determined. The results are shown in the table below. The fastest growth rate obtained was with a holding time of 3 to 4 hours. Yield (78~
82%) is the usual low cell density method (Table Y-1084 ^a )
The productivity of our method using a high cell density method at laboratory scale (33 g/
/hour) is more than three times higher than the low density method (10g/hour).

[Table] Example: Kluyveromyces fragilis was first developed by USDA
Obtained from Northern Regional Research Laboratories (Peoria, IL) and the American Type Culture Collection (Rotskville Maryland). Yeast were cultured on YM agar slants or plates. One colony isolated from YM plate
1.2% in a 250ml Erlenmeyer flask
100 ml of minimal medium supplemented with whey permeate solids (Express Foods, Louisville, Kentucky) was inoculated. The medium contains the following components (g/):
KH ₂ PO ₄ , 5.0; (NH ₄ ) ₂ SO ₄ , 3.0; MgSO ₄・
7H ₂ O, 0.5; CaCl ₂ 2H ₂ O, 0.1; FeSO _4
7H2O , 0.15; _CuSO4・_5H2O , 0.0125; _ZnSO4・
7H ₂ O, 0.095; MnSO ₄ H ₂ O, 0.0063;
Na ₂ MoO ₄ 2H ₂ O, 0.0038 and CaCl ₂ 6H ₂ O,
Contained 0.0005. The flasks were incubated at 37°C in a New Brunswick G-25 shaker. The overnight culture (16-24 hours) was then used as inoculum for benchtop fermentation. The benchtop fermenter is a 4-fermenter that I ordered.
For high cell density methods, initially 75%
H ₃ PO ₄ , 2.1 ml/: H ₂ SO ₄ , 1.2 ml/; Niacin, 3.1 mg/; D-pantothenic acid, 1.0 mg/
Whey permeate solids, 100 g/; and trace mineral solution, 1.5 ml/; were placed in a sterile medium. The trace mineral solution contains the following minerals (g/
): FeSO ₄・7H ₂ O, 60; CuSO ₄・5H ₂ O, 5;
ZnSO ₄・7H ₂ O, 25: MnSO ₄・H ₂ O, 2:
Contains _NaMoO4.2H2O , ₁ and _{CaCl2.6H2O ,} 0.2. After inoculating the overnight culture, the fermenter is heated at PH4.6 for 13-16 hours until all the lactose is consumed.
and operated aerobically at 37°C. A sterile feed solution was then added continuously at a rate that allowed the culture to completely consume the lactose contained in the feed solution. This was usually monitored by dissolved oxygen response. Lactose content was determined by ion chromatography. The continuous feed liquid is 75% H ₃ PO ₄ (6.2 ml/), H ₂ SO ₄ (3.7 ml/
), D-pantothenic acid (3.1 mg/), niacin (9.3 mg/) and trace mineral solution (4.4
ml/) of 30% whey permeate solids (permeate dry powder or concentrated permeate). Food grade antifoam agent FG-10 (Dow Chemical, Midland, Michigan) was used for foam control. The gas-free liquid capacity of the fermenter was maintained at 1.8-2.0 by continuous withdrawal of the fermenter broth. Oxygenated air was sparged to maintain the dissolved oxygen concentration at 20-30% saturation. Stable state was achieved when there was no change in the cell mass at a constant dilution rate. Gaseous ammonia was used not only as a nitrogen source but also for pH adjustment. Lactose, the sugar in whey, has always been a limiting nutrient. By using concentrated whey permeate as a substrate, food grade yeast (Kluyveromyces fragilis) can be grown in fermenters at very high cell densities (140 g/
(total broth dry weight) was continuously grown. It was found that the high pressure content of the whey permeate was not inhibitory to cell growth. Cell mass yield was constant across the board with pH ranging from 3.9 to 5.1, temperature from 31 to 37°C, and 6 to 30% whey permeate solids in the feed solution. It was found that the dilution rate (0.1-0.3 h ^-1 ) had little effect on cell mass yield. Yield decreased at temperatures higher than 38℃. The high cell density fermentation broth was directly dried without centrifugation and washing, and the composition of the dried product was determined directly. Analysis showed that the product had acceptable protein and mineral content. The amino acid profile, vitamin content and fatty acid composition of the product suggest that it is an excellent protein product. The discovery of yeasts capable of rapid growth and high productivity is a clear advantage. The present invention describes three very unique yeast cultures that are highly desirable and useful:
Pichia pastoris (Culture 21-2) deposited as NRRL Y-11431, Pichia pastoris (Culture 21-2) deposited as NRRL Y-11430
-1), and Hansenula polymorpha (Culture 21-3) deposited as NRRL Y-11432.
provide. These unique cultures grow particularly well at high mineral salt levels and cell densities. These three unique yeast cultures grow effectively and with high productivity on oxygen-saturated hydrocarbons, especially lower alcohols, most preferably methanol or ethanol. This is particularly noteworthy with respect to the new Pichia pastoris. This is because some cultures of the species Pichia pastoris do not grow easily on methanol. These unique species are named as follows:

[Table] Hua names NRRL Y-11431, NRRL Y-11430 and NRRL Y-11432 are new yeast cultures officially custodian, United States Department of Agriculture,
Agricultural Research Service, Northern
Regional Research Laboratory, Peoria;
Applicants' deposit of two slanted agar cultures in Illinois 61604 (deposit under the Budapest Treaty) reflects the fact that they received individual NRRL strain designations as indicated by the custodian.
Culture samples from these deposits or from applicants' cultures that have made the deposits therefore provide strains derived from applicants' discovered species. The present invention provides a method for culturing oxygen-saturated hydrocarbon-assimilating microbial cells belonging to three novel cultures or strains of microorganisms under aqueous aerobic culture conditions. These strains were classified;

【table】

【table】

【table】

Table: Of course, as with all microorganisms, some of the characteristics may be subject to some variation depending on the culture medium and particular conditions. The medium formulation given in the example can be used to cultivate the novel yeast species of the invention. However, they will grow on other than methanol-containing substrates. These new yeast cultures can be used not only with the high salt media described herein, but also with customary fermentation conditions using media such as: Media 1M - 1 Ingredients KH ₂ PO ₄ 5.0g MgSO ₄・7H ₂ O 0.5g CaCl ₂・2H ₂ O 0.1g KCl 0.5g (NH ₄ ) ₂ SO ₄ 3.0g Biotin 0.04mg Thiamine 4.0mg Trace mineral solution ^(a) 2.5ml Water 1000ml Sterile methanol ^{( b)} 0.5 to 1.0% by volume (a) Refer to the recipe below (b) Amount of trace metal solution components added just before use CuSO ₄・5H ₂ O 0.06g Kl 0.08g MnSO ₄・H ₂ O 0.3g Na ₂ MoO ₄・2H ₂ O 0.2 g H ₃ BO ₃ 0.02 g ZnSO ₄・7H ₂ O 2.0 g FeCl ₃・6H ₂ O 4.8 g Distilled water 1000 ml H ₂ SO ₄ (concentrated) 3 ml Illustrate the effect.

Claims (1)

[Scope of Claims] 1. At least one yeast species is cultivated under aqueous aerobic fermentation conditions in an aqueous fermentation broth using effective amounts of carbon and energy substrates and assimilable nitrogen, and an aqueous mineral salt medium is added to the fermentation broth. In a method for producing a single-cell protein material, the aqueous mineral salt medium contains salts of the following elements, and at least the specified weight per fermentation liquid: The following elements: P-1.9g, K-1g, Mg-0.15gCa-0.06
g, S-0.1g, Fe-6mg, Zn-2mg, Cu-0.6
mg, and Mn - 0.6 mg, are added to the fermentation liquor in proportions to maintain it in the fermentation liquor, and the fermentation is carried out under conditions to maintain a cell density of at least about 60 to 160 g per fermentation liquor on a dry basis. The above-mentioned manufacturing method is characterized in that the manufacturing method is carried out. 2. The method according to claim 1, wherein the yeast is one of the genera Candida, Hansenula, Torulopsis, Satucharomyces, Pichia, Debaryomyces, and Brettanomyces. 3. The yeasts are Brettanomyces petrophyllium candida boijinii candida lipolytica candida mycoderma candida utilis candida stellatoidea candida robusta candida clausenii candida rugosa candida tropicalis deviomyces hansenii hansenula minuta hansenula satour Eggplant Hansenula Californica Hansenula Murakii Hansenula Silvicola Hansenula Polymorpha Hansenula Vitskelamii Hansenula Capsulata Hansenula Glucochima Hansenula Henritskii Hansenula Nonhuamen Chest of Hansenula Phylodendra Pichia Huarinosa Pichia Polymorpha Pichia Membranefuasciens Pichia Pinus Pichia pastoris Pichia Trehalophylla Satucalomyces cerevisiae Satucalomyces Fragilis Satucalomyces rosei Satucalomyces acideiphaciens Satucalomyces elegans Satucalomyces roxii Satucharomyces lactis torulopsis sonorensis torulopsis candida torulopsis bormyi torulopsis voursatilis torulopsis glabrata torulopsis molysiana torulopsis nemonendra The method according to claim 2, which is Torulopsis nitratophylla or Torulopsis pinus. 4 Carbon and energy substrates are any one or more carbohydrates; alcohols, ketones, aldehydes, acids or esters having 1 to 20 carbon atoms per molecule;
4. A method according to any one of claims 1 to 3, wherein the paraffin is a normal paraffin having 10 to 20 carbon atoms. 5. The method of claim 4, wherein the carbon and energy substrate is an alcohol having 1 to 4 carbon atoms. 6. The method according to claim 5, wherein the alcohol is methanol or ethanol. 7. The method of claim 4, wherein the carbon and energy substrate is one or more carbohydrates: glucose, fructose, galactose, lactose, sucrose, starch and dextrin. 8. The method of claim 4, wherein the carbon and energy substrate is any one or more of normal paraffin, decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, octadecane and eicosane. 9 Carbon and energy substrates can be any one or more alcohols, ketones, aldehydes, acids and esters, ethylene glycol, propylene glycol, 1-propanol, 2-propanol, glycerol, 1-butanol, 2
-Butanol, 3-methyl-1-butanol, 1
-Pentanol, 2-hexanol, 1,7-heptanediol, 1-octanol, 2-decanol, 1-hexadecanol, 1-eicosanol, acetone, 2-butanone, 4-methyl-2-
Pentanone, 2-decanone, 3-pentadecanone, 2-eicosanone, formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, hexanal, 7-methyloctanal,
Tetradecanal, eicosanal, acetic acid, propionic acid, butyric acid, glutaric acid, 5-methylhexanoic acid, azelaic acid, dodecanonic acid, eicosanonic acid, methyl formate, methyl acetate, ethyl acetate, propyl butyrate, isopropyl hexanoate, hexyl 5-methylocta 5. The method according to claim 4, which is noate and octyl dodecanoate. 10 Each fermentation liquid 1 is P-1.9~20g, K-1~
20g, Mg-0.15~3g, Ca-0.06~1.6g, S-
0.1~8g, Fe-6~140mg, Zn-2~100mg, Cu
-0.6 to 16 mg, and Mn -0.6 to 20 mg, the method according to any one of claims 1 to 9. 11 Each fermentation liquid 1 is approximately P-2.2~10g, K-1.5
~10g, Mg-0.3~1.2g, Ca-0.08~0.8g, S
-0.2~5g, Fe-9~80mg, Zn-3~40mg, Cu
- Contains in the range of 1 to 10 mg and Mn - 0.9 to 8 mg,
The method according to claim 10. 12 The aqueous fermentation liquid further contains sodium, cobalt,
12. The method according to any one of claims 1 to 11, comprising at least one of molybdenum, boron and selenium. 13. The method according to any one of claims 1 to 12, wherein at least one vitamin is added to the fermentation liquid. 14. The method according to claim 13, wherein the vitamin is biothione and/or thiamine. 15 Vitamins are mixed with aqueous diluted methanol or ethanol and nutrients Fe, Zn, Mn and
Add thereto a trace mineral medium containing compounds that supply Cu, and the resulting mixture contains nutrients P,
15. A method according to claim 13 or claim 14, wherein the fermentation zone is fed separately from the initial mineral medium containing compounds supplying K, Mg, S and Ca. 16 Patent using fermentation temperatures ranging from 25 to 65°C, pH ranging from about 3 to 7, pressures ranging from about 0 to 150 psig, and fermentation times ranging from about 2 to 30 hours based on average retention. Claims 1 to 15
The method described in any one of paragraphs. 17 Approximately 70~ per fermentation mixture on dry basis
17. A method according to any one of claims 1 to 16, wherein a cell density of 150 g is maintained. 18. Claims 1 to 17 maintain a cell yield of about 30 to 110 g per 100 g of added substrate.
The method described in any one of paragraphs. 19 Pichia pastoris NRRL Y as yeast
-11430, Pichia pastoris NRRL Y-11431
Or Hansenula Polymorpha NRRL Y-
19. A method according to any one of claims 1 to 18, using a strain designated as or derived from 11432. 20 The fermentation effluent containing cells is subjected to a water washing process,
The residual aqueous mineral medium is thereby substantially separated from the cells, leaving a wet cell mass, the wet cells are dried to produce dry cells, and the washed and separated mineral medium is recycled to remove the water and minerals. 20. A method according to any one of claims 1 to 19, wherein at least a portion of the culture medium assembly is provided. 21. Any one of claims 1 to 19, wherein the entire fermentation effluent containing cells and residual salts is dried, thereby producing a dried cell product containing residual water-soluble substances, including salts. The method described in section. 22.Centrifuging a fermentation effluent containing cells, thereby producing a concentrated cell stream and a thin recirculating liquid, washing the concentrated cell material with water, and drying the washed cells. The method according to any one of paragraphs 19 to 19. 23. Claim 22, wherein at least a portion of the dilute recirculating liquid and/or the wash water from the washing step is used as at least part of the assembly medium.
The method described in section.