JPH0446111B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel microorganism and a method for culturing the same. 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 been impractical due to their lack of water solubility and the consumption of significant molecular oxygen to support microbial conversion. Other processes have focused on using oxygenated saturated hydrocarbon derivatives as feedstocks due to their relative water solubility. This is because the aqueous fermentation is easier to handle and the molecular oxygen required for microbial conversion-growth methods is substantially reduced. However, the limiting factor in the commercialization of single-cell protein production is the need to have a moderate yield of dry cells based on the consumed substrate, as well as the need to adjust the fermentation solution to a relatively moderate cell density, its consumption, and its consequences. There was a need to process a large amount of the whole fermentation effluent in order to recover a suitable amount of 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 previous methods have focused on culturing bacteria because the crude protein content typically obtained from yeast is slightly higher than that of bacterial cells.
However, yeast is widely available and relatively easy to cultivate. Because yeast cells are generally slightly larger than bacterial cells, they tend to be more easily separated from the fermentation effluent. The discovery of means and methods to increase cell yield, especially operating at high cell densities, is highly desirable. For example, handling substantially smaller fermentation effluent volumes reduces piping and pump size, reduces water requirements due to reduced sterilization requirements, and reduces equipment size and handling requirements for coagulation and separation steps. That means big savings. The invention described in JP-A No. 57-43682 adds a medium containing a high concentration of inorganic salts to the fermented product in the fermenter, thereby allowing the fermentation solution in the fermenter to have a very high cell density that cannot normally be obtained. A continuous aerobic fermentation process using yeast cultures to manipulate and produce high yields of yeast single cell protein products is disclosed. Cell density means the concentration of cells per volume of fermentation solution based on dry weight. Fermentation liquid refers to the total volume of the aqueous fermentation broth or liquid containing the cells. Cell density is usually expressed as g/. Yield means the weight of cells produced for a given consumption of the carbon energy source or substrate weight supplied to the fermentation liquid. And it is usually expressed in g/g. The high cell densities obtainable by the method of the invention are highly efficient and reduce single cell protein production costs. In many cases, concentrating single cell protein products, such as by centrifugation, can be greatly reduced or even eliminated. If necessary, the cell product is washed with a washing element to remove unconsumed residual salts.
It can then be transferred directly to a drying element such as a spray dryer. The wash solution contains most of the remaining inorganic salts that are not taken up by the cells and can be recycled to the fermenter. Therefore, the water required for the fermentation process is 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. Up until now, continuous methods for producing single-cell protein materials from yeast cultures have generally produced approximately 20
A fermentation effluent with a relatively low yeast cell content, such as ˜25 g of cells, was provided. However, the present invention provides a fermentation method that produces relatively high levels of yeast cells, such as 100 grams 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. The present invention relates to Pichia pastoris NRRL Y-11430,
Yeast cultures of Pichia pastoris NRRL Y-11431 and derivatives thereof are particularly useful for the production of single-cell proteins when provided and cultured aerobically in an aqueous fermentation medium where the carbon source is oxygenated hydrocarbon carbon. According to the invention, the yeast uses effective amounts of molecular enzyme-containing gases, assimilable nitrogen sources, nutritional mineral salts and, if necessary, the addition of additional nutritional organic substances such as vitamins such as biotin and thiamine. and grown under substantially continuous aerobic aqueous fermentation conditions with a suitable carbon energy source as substrate. In this method, inorganic salts can be added at high levels, as described below, resulting in a fermentation process that operates at high cell densities and produces high yields. It is useful to perform fermentations in which high concentrations (relatively) of inorganic salts are added to the fermentation solution and are almost forced onto the cells to obtain high growth rates. The concentration of inorganic salts in the supernatant of the fermentation liquor itself (ie, the fermentation liquor excluding cells) remains at a relatively low level, of course. This is because salt is consumed by cells for growth and reproduction. Therefore, the salt concentration in the cells plus supernatant is very high. Fermentation Conditions The cultivation is carried out 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 inorganic salt can be supplied to the fermentation liquor, and the high concentration is retained in the fermentation liquor. In order to ensure suitable microbial growth, to maximize the assimilation of carbon and energy sources by the cells in microbial conversion methods, and to achieve the highest cell yields at the highest cell densities in fermentation media, Requires appropriate amounts of selected inorganic nutrients in certain proportions. Although the composition of the fermentation broth can vary widely depending in part on the yeast and the substrate used, the mineral content in the fermentation broth according to the invention (i.e., broth plus cells) is relatively high, and is similar to that previously considered suitable or in the prior art. This is at a higher level than that carried out by. Minimum broad and currently preferred concentration ranges for the various elements in the fermentation broth are shown in the table below. Although the concentrations are expressed as those of the elements, it is recognized that all or part of each may be present in soluble ionic form or, in the case of P, in bound form, such as phosphate. 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 the form of sulfate, the minimum concentration of sulfur is usually exceeded. Any or all of the metals indicated can be used or included as sulfates. Mg,
Ca, Fe, Zn, Cu and Mn are preferably used in the form of sulfates or chlorides or in the form of compounds which are converted in situ to sulfates or chlorides. K
are preferably used as sulphates, chlorides or phosphates or in the form of compounds which are converted in situ to sulphates, chlorides or phosphates. P can 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. preferable. Other elements that can be included at least in trace amounts are, for example, halides or sulphates.
Na and Co; e.g. as molybdates
Mo; B, e.g. as a borate; Se, e.g. as a selenite or selenate; or I, e.g. as an iodide. In a typical high cell density fermentation, the fermentation solution consists of approximately 1/2 supernatant medium and 1/2 cells by volume. However, these 1/2 volume cells contain at least about 2/3 of the inorganic salt content of the fermentation broth. Yeast The method of the invention uses yeast cultures suitable for growth on carbon-containing substrates under aqueous fermentation conditions. The yeast used in the present invention is a new strain of Pichia.
pastoris NRRL Y-114300 and Pichia
pastoris NRRL Y-11431. but,
Candida, Hansenula, Torulopsis,
Saccharomyces, Pichia, Debaryomyces and
It can be used in conjunction with other yeasts, including species from the genus Brettanomyces. The preferred genus is Candida,
Hansenula, Torulopsis, Pichia and
Including Saccharomyces. Examples of suitable species are:

[Table] Includes. 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. For example, Pichia
It has been observed that certain strains of the above species, such as S. pastoris, do not grow in methanol. Pichia pastoris (Culture 21-2) NRRL
Y-11431 and Pichia pastoris (Culture 21
-1) The specific bacterial strain deposited as NRRL Y-11430 or its derivative strain is particularly suitable for use in the production of SCP protein materials with high cell density and high yield. Pichia pastoris (Culture 21-2)
The characteristics of these strains of NRRL Y-11431 and Pichia pastoris (Culture 21-1) NRRL Y-11430 are considered to be particularly unusual for these species.
This is because out of the four Pichia pastoris cultures tested, only two grew on methanol anyway. Pichia pastoris
21-2) NRRL Y-11431 and Pichia
pastoris (Culture 21-1) NRRL Y-11430
is considered new and unique. Carbon energy substrates include oxygenated hydrocarbons, including various carbohydrates suitable as yeast substrates. 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 substrate source. For the purposes of this disclosure, oxygenated hydrocarbons include water-soluble carbides, as well as alcohols, ketones, esters, acids, and aldehydes, and mixtures thereof, which have 1 to 20 carbon atoms per molecule and generally have a significant It is water soluble. More suitable oxygenated hydrocarbons are substantially larger water-soluble ones, typically having up to about 10 carbon atoms per molecule. or generally water-soluble carbohydrates. Examples of carbohydrates include glucose, fructose, galactose, lactose, sucrose,
Contains starch, dextrin, etc. singly or in a mixture. Other types of oxygenated hydrocarbons include 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-
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, dodecanoic acid, eicosanonic acid, methyl formate, methyl acetate, ethyl acetate, propyl butyrate, isopropyl hexanoate,
Contains hexyl 5-methyloctanoate, octyl dodecanoate, etc. 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 paraffins with less than 10 carbon atoms per molecule. Typically these include decane, undecane, dodecane, tridecane, 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 preferred. Water soluble monohydric aliphatic 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. Alcohols of 1 to 4 carbon atoms (other than 2-methyl-1-propanol) are most preferred. Among these, methanol and ethanol are preferred over the others. And methanol is the most preferred of such feedstocks due to its relatively low cost. Petroleum gas can be oxidized. and water-soluble materials such as oxidants such as methane, ethane, etc., used to provide a predominant mixture of the corresponding alcohols along with various aldehydes, ketones, acids, etc., as well as integrated purification and chemical processing complexes, often Suitable hydrocarbon fractions from various petroleum refinery sources produced within the so-called petrocomplexes 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 reproducing cells. Inorganic salts in cells cannot be supplied and applied because some are in bound organic form. Of course, the mineral analysis of the fermentation liquid will reflect the total mineral content. In addition to inorganic 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 biotin and thiamine, or other media components containing these vitamins, such as yeast extract. Therefore, for example
For yeasts such as Hansenula polymorpha, an amount of biotin of about 0.04 to 0.8 mg per 1 aqueous mineral medium and about 4 mg biotin per 1 aqueous mineral medium.
It is desirable to use an amount of thiamine hydrochloride of ~80 mg. Alternatively, all or part of the biotin and thiamine can be provided through the use of yeast extract or the like. Use in aqueous aerobic fermentation processes to which growth factors are added In the production of mineral media, 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 biotin or thiamine. and that if such fermentation systems use aqueous mineral media prepared from water containing residual chlorine, removing the chlorine before adding the growth factors will destroy the growth factors. It has long been known to avoid loss or inactivation. 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 a fermentation method. Therefore, mineral nutrient media can 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. The separate addition of vitamins to the fermentation zone is preferably and advantageously achieved 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. be done. If an aqueous mixture of vitamins and carbon energy substrates is used, the water used for the initial vitamin dilution should preferably be free of traces of residual chlorine, such as deionized water, and should preferably be free of traces of residual chlorine, such as deionized water. Any prior loss should be avoided before mixing with the aqueous carbon 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. With this embodiment, residual chlorine does not have to be removed first, but the vitamins are still fully retained. In a more preferred embodiment, further addition of vitamins to the fermentation zone is accomplished 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 inorganic salts include Co, Mo, B, Se, I, Mn, Cu, Zn and
It consists of the trace elements mentioned above, such as Fe. Use of this more preferred embodiment not only avoids the vitamin inactivation problem caused by trace chlorine in the water used in 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 process aqueous mineral salt media that require cleaning. The presence of trace mineral salts, which usually mix 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 rather by including trace mineral salts in a mixture with vitamins and at least some carbon energy substrates, two very troubling problems can be solved. 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 traces of chlorine. The stream consisting of vitamins, some carbon energy substrates and trace minerals can be made sterile by filtration if necessary. However, it is preferred and advantageous to combine the stream with the primary carbon energy substrate before entering the fermentation zone and to filter the entire combined stream immediately before entering the fermentation zone. Fermentation itself is an aerobic process that requires molecular oxygen, which is supplied by air, a molecular oxygen-containing gas such as oxygenated air, or substantially pure molecular oxygen, and which supports the growth of microorganisms in a multiplicative manner. The fermentation liquor is maintained by an effective partial pressure of oxygen to help.
By using an oxygenated hydrocarbon substrate, the total oxygen demand for microbial growth is lower than when using paraffin. Even so, adequate amounts of molecular oxygen must be provided for growth. This is because the assimilation of the substrate and the growth that microorganisms are responsible for are in part a combustion process. The rate at which molecular oxygen is supplied to the fermentation liquid should be such that yeast growth is not limited by lack of oxygen. Fermenter design varies widely in its ability to transport oxygen to the culture. The overall aeration rate can vary over a considerable range, but in fermenters that are very effective at oxygen transfer, the aeration is generally at a rate of molecular oxygen-containing gas volume per minute of liquid volume in the fermenter (working pressure and (at 25℃) about 0.5 to 8,
Preferably, the ratio is about 1 to 6. This amount is based on the normal oxygen content air and pure molecular oxygen supplied to the reactor, each range being the volume of molecular oxygen per minute per liquid volume in the fermenter at working pressure and 25°C. ) will be about 0.1 to 1.7, or preferably about 0.2 to 1.3. The pressure used in the microbial fermentation process can vary widely. Typical pressures range from about 0 to
150 psig, preferably about 0 to 60 psig, more preferably at least slightly above atmospheric pressure. Pressures higher than atmospheric pressure are advantageous as they tend to increase the dissolved oxygen concentration 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 are generally about 25-65°C, preferably about 28-50°C.
Pichia deposited as NRRL Y-11431
pastoris culture 21-2 and NRRL Y-
Yeast cultures of Pichia pastoris culture 21-1, deposited as 11430, generally prefer fermentation liquor temperatures of about 30°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 metabolic utilization by the yeast. Although various organic nitrogen source compounds such as protein hydrolysates can be used technically, inexpensive nitrogen-containing compounds such as ammonia, ammonium hydroxide, urea and ammonium phosphate, ammonium sulfate, pyrroline are commonly used. 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
Useful for pH adjustment. The PH range of the aqueous microbial fermentation liquid should be in the range of about 3 to 7, preferably and usually about 3.5 to 5.5. The preference of certain microorganisms for PH ranges depends to some extent on the medium used and on the particular microorganism. Accordingly, some variation may be made by changing the medium and can be readily determined by one skilled in the art. The average retention time of the fermentation liquid in the fermenter can vary considerably, depending in part on the fermentation temperature and the yeast culture used. Generally, the retention time is about 2 to 30 hours, preferably about 4 to 14 hours, based on average retention. High concentrations of some of the carbon and energy substrates mentioned, in particular methanol or formaldehyde, are 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 the substrate concentration in the fermentation liquor at the maximum acceptable level. For some lower alcohols, this level in the fermentation liquor is generally around 0.001
~5% by volume, preferably about 0.01-0.05% by volume, while for aldehydes, so as not to starve or inhibit the growth rate of the selected microorganisms.
It should be 1/10 of these in terms of aldehyde toxicity. If the carbon and energy source materials contain aldehydes in amounts that can be harmful to microorganisms, harmful aldehyde effects can be avoided by treating the substrate with an appropriate amount of a nitrogen-containing compound, preferably ammonia, ammonium hydroxide, or other active ammonium compound, for one mole of aldehyde. Approximately 0.01 to 10 such nitrogen-containing compounds per
Mitigation can be achieved by initially treating in molar equivalent proportions. Such treated substrates then contain not only a carbon energy source, but also at least some of the necessary assimilable nitrogen. The carbon-containing substrate can be adjusted as a limiting factor, thereby conducting the fermentation in such a way that the carbon-containing substrate is fully converted into the yeast cells and avoids possible contamination of the yeast cells with substantial amounts of unconverted substrate. It is better. The latter is not a problem due to water-soluble substrates. This is because any residual traces are easily washed out. However, problems arise with water-insoluble substrates such as higher n-paraffins, which require additional product treatment steps such as removal of residual hydrocarbons by appropriate solvent washing steps. Continuous operation is highly preferred for ease of regulation, production of uniform product in uniform quantities, and most economical use of all equipment. Continuous methods use carbon and energy source materials as substrates, an aqueous mineral medium,
Assimilable nitrogen sources and molecular enzyme-containing gases are continuously added to the fermentation liquor in the fermenter in combination with continuous recovery of the fermentation liquor. Added carbon energy substrate:
The volume ratio of the added aqueous mineral medium can vary widely depending, in part, on the nature of the carbon-containing substrate, but generally ranges from about 1:9 to 6:4, preferably about 2:
The ratio is in the range of 8 to 5:5. If necessary, a portion of all carbon energy source materials and/or a portion of an assimilable nitrogen source such as ammonia can be added to the aqueous mineral medium before passing it through the fermentor. In this method of high cell density fermentation, approximately 40
It was also advantageous to use a feed ratio of % alcohol by volume to 60% by volume mineral salt medium. Each stream introduced into the reactor is monitored at a predetermined rate or for carbon and energy substrate concentrations, pH, dissolved oxygen, oxygen or carbon dioxide in the gas exiting the fermenter, cell density as measured by optical transmission, etc. It can be measured and adjusted according to the requirements. 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 most preferred to sterilize using steam, typically at about 250°C (121°C) for at least about 15 minutes. The sterilized reactor is inoculated with a culture of specific microorganisms 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 foam-filled fermenters is preferred. Fermentors designed to promote and maintain foam production are advantageous for methods that increase the oxygen transfer necessary to maintain the necessary high cell densities and rapid growth rates. At the start of the fermentation, an aqueous mineral medium, an appropriate concentration of carbon source, assimilable nitrogen, trace ingredients if necessary and a starting yeast inoculum are placed in a sterile fermenter, with an appropriate flow of oxygen and various feeds. Start gradually. If necessary, the initial fermentation substrate is glucose or the like, which can be gradually changed to methanol or the like as the cell density increases. In aqueous fermentations, one can start with low inorganic salt levels and build up to high inorganic salt levels by feeding the fermentation with an aqueous mineral medium having a high concentration of inorganic salts. However, it is usual to simply first add a high-salt medium to the fermenter and immediately start the operation. Those skilled in the art will appreciate that there is typically a short lag time after starting before the inoculum has formed sufficient cells for complete input of salt and substrate used. Product Recovery 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 lower alcohols (methanol or ethanol) to precipitate any polymeric substances produced extracellularly. The cell-free effluent is treated by solvent extraction and/or base extraction to remove, if desired, the dye produced during the culturing process.
Other extracellular products such as vitamins or organic acids can be recovered. With or without such ancillary treatment, the cell-free effluent can be returned to the fermentor as part of the aqueous component or as substantially or almost all of the aqueous component, minimizing waste problems as much as possible. can be avoided. Microbial cells are typically killed by heat or chemical means, and this can be done before or after separating the cells from the fermentation effluent. Yeast cells are a valuable source of protein for humans and animals. For human consumption, the cells can be optionally treated to reduce nucleic acids, but for animal feed purposes such treatment does not currently appear to be necessary. According to the invention, high cell densities, e.g. about 60 to 160 g of yeast cells on a dry basis per fermentation mixture,
High yields can be obtained using manipulation of cell densities, preferably within the range of about 70-150 g. If desired, cells can be recovered from the fermentation mixture by centrifugation or other separation methods. Alternatively, if desired, the concentrated cells can then be washed, for example by mixing with water, and separated, such as by recentrifugation, or water can be added before or during centrifugation to substantially liberate the cells in the mineral medium. The wash liquor containing the separated mineral medium can then be returned to the fermentor as water and mineral medium, 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 fermentation 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 a high-protein, high-salt product. It can be used as a very 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:
Methanol and aqueous mineral salt media in a volume ratio of 30.15 to 69.85, respectively, were fed individually to a fermenter inoculated with the yeast Pichia pstoris culture 21-2, deposited as NRRL Y-11431. No preconditioned media or substrates were used. The fermenter was a 2-liquid 4 fermenter with automatic adjustment of pH, temperature and level. Stirring was performed with two blades rotating at 1000-1200 rpm. The aeration rate is 1 to 1.5 volumes per fermentation volume per minute (at approximately atmospheric pressure and 25 °C) of the supply air with sufficient oxygen to dissolve the air into the saturated fermentation mixture at atmospheric pressure and approximately 30 °C. An amount of dissolved oxygen was maintained in the fermentation mixture equal to approximately 20% of the amount that would occur. Aqueous ammonium hydroxide (2 parts concentrated ammonium hydroxide and 1 part deionized water) was added at a rate to maintain the pH of the fermentation mixture at about 3.5. The aqueous inorganic salt base used per solution was
12.5ml 85% H ₃ PO ₄ , 2.5g 85% KOH, 8.5g
KCl, _7.0gMgSO4・_7H2O , _1.5gCaCl2・_2H2O ,
25ml trace mineral solution A, 25ml trace mineral solution B, 10ml biotin-thiamine hydrochloride solution,
A solution of 1 was prepared by mixing about 0.08 ml of antifoam (Mazu DF - 37°C) and enough deionized water. 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 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
A solution of 1 was prepared by mixing MnSO ₄ .H ₂ O, 0.6 g CuSO ₄ .5H ₂ O, 2 ml concentrated H ₂ SO ₄ , and enough deionized water. A biotin-thiamine hydrochloride solution was prepared by mixing 2 mg of biotin, 200 mg of thiamine hydrochloride, and 50 ml of deionized water. Fermentations were conducted at approximately 30° C. and approximately atmospheric pressure with a holding time of 7.0 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, yeast cells weigh 42.3g per 100g of methanol supplied.
produced in a yield of . Cell density was at a high level of 100.7 g cells per effluent. Example Another fermentation test was carried out with some changes in the composition of the aqueous inorganic salt base, with methanol to aqueous inorganic salt base capacitances of 40.8 to 59.2, and aqueous ammonium hydroxide for pH adjustment to 3 parts concentrated ammonium hydroxide and ion. Prepared with 1 part removed water and fermentation retention time
The procedure was carried out using substantially the same method as described in the example, except that the time period was 8.35 hours. The aqueous inorganic salt medium used in this test was 20.0 ml of 85% H ₃ PO ₄ and 4.0 g of 85% H 3 PO 4 per solution.
KOH, 12.0 g KCl, 10.4 g _{MgSO4.7H2O ,}
2.4 g of CaCl ₂ 2H ₂ O, 40 ml of trace mineral solution A as described in the example, trace mineral solution B 40 as described in the example
ml, biotin-thiamine hydrochloride solution 16 as described in the example
ml, about 0.08 ml of antifoam, and enough deionized water to make a solution of 1 part. 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 methanol fed. Cell density is 133.3g per effluent
This was an extremely desirable high level. Example (Reference) Continuous aerobic fermentation, this time using NRRL Y-11432
The yeast Hansenula polymorpha culture 21-3, deposited as Y. 300 ml of total solution per fermenter
A methanol and aqueous inorganic salt medium mixture containing methanol was fed. The stirred fermentation mixture has sufficient oxygen and is supplied with air at a rate of 2 per fermentation volume per minute.
(at about atmospheric pressure and about 25°C) is aerated by passing it through a fermentation tank, and into the fermentation mixture about 20% of the air at about atmospheric pressure and about 38°C will be dissolved in the fermentation mixture. % of dissolved oxygen was maintained. Aqueous ammonium hydroxide (2 times concentrated ammonium hydroxide and 1 part 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 inorganic salt base is
300ml methanol to 1 part solution, 6ml 85%
H ₃ PO ₄ , 3 g KCl, 4.5 g MgSO ₄ 7H ₂ O,
0.6 g of _CaCl2.2H2O , 0.3 g of NaCl, 10 ml of trace mineral solution A as described in the example, 10 ml of trace mineral solution B as described in _example , 4 ml of biotin-thiamine hydrochloride solution as described in example, 4 drops of antifoaming agent and 1
It was prepared by mixing enough deionized water to form a solution. Fermentation was carried out at about 38° C. and about atmospheric pressure with a holding 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
Yeast deposited as NRRL Y-11430 with methanol and aqueous mineral medium at a volume ratio of 63.1
They were individually fed into fermenters inoculated with Pichia pastoris culture 21-1. The fermenter was a 1500 bubble filled fermenter with approximately 600 liquid volume, 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 to 1 solution.
ml 75% H ₃ PO ₄ , 6.0 g KCl, 6.0 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 (drinking water should first be treated with enough sodium thiosulfate to remove the free (reacted with 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 biotin-thiamine hydrochloride solution was prepared by mixing the ingredients in a ratio of 0.4 g biotin to 40 g thiamine hydrochloride in 1 part deionized water. Fermentations were 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 salt medium in a volume ratio of
Yeast pichia deposited as NRRL Y-11430
pastoris culture 21-1 inoculated into fermenters. The fermenter has a liquid capacity of about 610,
1500 with automatic adjustment of PH, temperature and level
It was a fermentation tank filled with bubbles. The stirring was carried out by two conventional paddle-type turbines driven at 1000 rpm. The aeration rate is approximately 4 parts air per part of fermentation liquid in the fermenter per minute (approximately 38 psig and approximately
(at 25℃). Anhydrous ammonia was added at a rate to maintain the pH of the fermentation mixture at approximately 3.5. The aqueous inorganic salt medium contains 15.86 ml of 75% H ₃ PO ₄ , 9.53 g of K ₂ SO ₄ , and 7.8 g of drinking water.
of MgSO ₄ .7H ₂ O, 0.6 g CaCl ₂ .2H ₂ O, and 2.6 g 85% KOH. The trace mineral solution plus biotin was fed separately via the methanol stream at a rate of 10 ml per methanol. 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 ml of biotin. Trace mineral solution D is 65g for solution 1.
of Fe ₂ SO ₄ 7H ₂ O, 20 g ZnSO ₄ 7H ₂ O, 3.0 g
of _MnSO4.H2O , 6.0g _{of CuSO4.5H2O ,} 5.0ml
of 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. Fermentations were conducted at 30° C. and approximately 38 psig 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 (Reference) In continuous aerobic fermentation method, 29 vs. 71, respectively.
menatol and aqueous mineral salts medium in a volume ratio of
Yeast Hansenula polymorpha NRRL Y-11170
were individually fed to the inoculated fermenters. The fermenter is approximately
It was a 1500 foam-filled fermenter with a liquid capacity of 560, automatic adjustment of pH, temperature and level, and venting. Stirring is done at the bottom of the ventilation pipe.
This was done by a turbine driven at 810 rpm. The aeration rate was about 5 parts air per part of fermentation liquor in the fermentor per minute (at about 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 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 thiosulfate to react with the free chlorine contained therein). 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, the yeast cells produced a yield of 33.3 g per 100 g of methanol fed. Cell density was the desired 76.2 g cells per effluent. The discovery of yeast with the capacity for rapid growth and high productivity is clearly advantageous. The present invention presents three very unique yeast cultures that have highly desirable and useful properties: NRRLY
Pichia pastoris (culture 21-2) deposited as -11431 and Pichia pastoris (culture 21-1) deposited as NRRLY-11430 are provided. 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 a new Pichia
Of particular note is pastoris. This is because some cultures of the species Pichia pastors do not grow easily in methanol. These unique species are named as follows:

[Table] NRRL Y-11431, NRRL Y-11431 and
NRRLY−11430 is the official custodian of the new yeast culture, United States Department of
Agriculture, Agricultural Research Service,
Northern Regional Research Laboratory
Deposited by Applicants by depositing two slanted agar cultures in Peoria, Illinois 61604;
We then received individual NRRL strain numbers as shown from the custodian. Therefore, culture samples from these deposits or from applicants' cultures that have made deposits 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 as:

[Table] New and unique microorganisms can be further characterized by properties as shown in the table below:

[Table] Growth factor Biotin Biotin

【table】

Table: Of course, as with all microorganisms, some of the characteristics can 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 on the high salt media described herein, but also under 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) Add immediately before use Amount of trace metal solution components CuSO ₄・5H ₂ O 0.06g Kl 0.08g MnSO ₄・H ₂ O 0.3g Na ₂ MoO ₄・2H ₂ O 0.2g H ₃ BO ₃ 0.02g ZnSO ₄・7H ₂ O 2.0g FeCl ₃・6H ₂ O 4.8g Distilled water 1000ml H ₂ SO ₄ (concentrated) 3ml This disclosure including materials is the value of the present invention and exemplify the effect.

Claims (1)

[Claims] 1. Pichia that has the ability to grow under methanol
pastoris NRRL Y-11430 (FRI 5682) or
Pichia pastoris NRRL Y-11431 (FRI 5683). 2. Pichia according to claim 1, which has no long-term logarithmic growth phase, has the ability to metabolize methanol, and has the ability to grow in methanol at high cell density without loss of yield.
pastoris. 3. The cellulose according to claim 1, which requires biotin in order to grow in methanol at high cell density.
Pichia pastoris. 4. Pichia pastoris according to claim 1, in the form of a biologically pure culture. 5 Pichia pastoris NRRL Y-11430 (FRI
5682) or Pichia pastoris NRRL Y-11431
Pichia pastiris Y-, named or derived from a yeast strain named (FRI 5683), characterized by culturing it under aerobic fermentation conditions in an aqueous medium using enzymatic hydrocarbons as carbon and energy substrates.
11430 (FRI 5682) and NRRL Y-11431 (FRI
5683) cultivation method. 6. The method according to claim 5, wherein the produced microbial cells are decomposed and recovered as a single-cell protein material. 7 Enzymed hydrocarbon carbon and energy substrates include one or more substantially water-soluble alcohols, ketones,
7. The method of claim 5 or 6, comprising an ester, ether, acid or aldehyde of up to about 10 carbon atoms per molecule. 8. The method of claim 5, wherein the carbonic acid and the energy substrate comprise a water-soluble lipophilic monohydric hydrocarbon alcohol. 9. The method according to claim 8, wherein the alcohol is methanol.