CA1096228A - Fermentation with thermophilic mixed cultures - Google Patents
Fermentation with thermophilic mixed culturesInfo
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- CA1096228A CA1096228A CA297,516A CA297516A CA1096228A CA 1096228 A CA1096228 A CA 1096228A CA 297516 A CA297516 A CA 297516A CA 1096228 A CA1096228 A CA 1096228A
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- acid
- butanol
- propanol
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Abstract
25,547 FERMENTATION WITH THERMOPHILIC MIXED CULTURES
Abstract of the Disclosure Single cell protein (SCP) and other fermentation products are produced by aerobic fermentation processes at relatively high fermentation temperature conditions employing oxygenated hydrocarbon compounds, such as an alcohol, as carbon and energy source material', and employing a unique thermophilic mixed culture of bacteria NRRL B-8158 as microbial conversion agent.
Abstract of the Disclosure Single cell protein (SCP) and other fermentation products are produced by aerobic fermentation processes at relatively high fermentation temperature conditions employing oxygenated hydrocarbon compounds, such as an alcohol, as carbon and energy source material', and employing a unique thermophilic mixed culture of bacteria NRRL B-8158 as microbial conversion agent.
Description
;228 FERMENTATION WITH THERMOPHILIC MIXED_ CULTURES
Field of the Invention The invention relates to the production of single cell pro-teinO In another aspect, the invention relates to a novel thermo-philic mixed culture.
3e_ Efforts to relieve the impending worldwide shortages of pro-tein have included various biosynthesis processes wherein bio-logically produced single cell protein (SCP) is obtained by the growth of a variety of microorganisms on a variety of carbon-containing substratesO
The carbon and energy sources used as substrates for such processes should be available widely, relatively cheap, uniform and safe in that they do not leave harmful residues in ~he pro-teinaceous product ultimately obtained by the microbial conversions.
Petroleum hydrocarbons have been employed as the carbon and energy source material, but have faced practical difficulties in the lack of water solubility, in the high consumption of oxygerl to assist in the microbial conversion, and allegedly in traces of potentially carcinogenic agents from the petroleum feedstocks entering or ad~
hering to the protein product.
Other proces~es have used oxygenated hydrocarbon derivatives as feedstocks due to the water solubility of such derivatives and hence ease of handling since microbial conversion processes are essentially conducted under aqueous conditions. Such feedstocks are readily available either from petroleum sourcesl natural yas sources, various waste/garbage processing and conversion of methane~ and the like, from fermentation of variou grains and the like, destructlve aiskilLation Qf wood, and so on. Such oxygenated hydrocarbons, whatever their source, are widely available and relatively cheap feedstocks ~or fermentation proce~ses. Advantages accrue in that these feedstocks are partially oxygenated, so that substantially reduced molecular oxygen requirements are involved for the microbial conversion growth process itself.
However, another difficult and limiting factor in the commer-cialization of single cell protein processes has been the necessity to conduct the fermentation at relatively moderate temperatures of about 20 to 50~ C., and preferably not over about 35 C.
Microbial conversions are exothermic oxidation reactions with large quantities of heat being pxoduced. Heat must be removed from the fermentation admixture continuously and consistently, or risk the overheating of the system and either the death of the microorganisms or at least severe limitations of growth encoun-tered as temperatures rise, and hence loss in efficiency.
Many processes have concentrated on the employment Qf one or other of the many available yeasts as the microorganismO
Yeast cells generally are slightly larger than a bacteria cell, and sometimes provide easier separation from the fermentation process media.
However, bacteria offer advantages, since higher crude protein contents of the cell are obtained from bacteria as com-pared to the product obtainable from yeasts in general, sincethe yeasts have higher proportions of nonprotein structural material in their cells. Bacteria usually have a significantly higher true protein content, frequently being higher in the nu~ritionally important sulfur amino acids and lysine.
Discovery of bacteria with the capability o~ rapid growth and high productivity rates at relatively high fermentation process temperatures would be advantageou~. High temperature growth operation means less heat to be removed, less cooling apparatus involved, and ultimately relatively smaller amounts of heat needed for sterilization, coagulation, and separation pro-cesses. Danger of contamination with othex microorganisms is greatly reduced when high temperature ~ermentation can be employed.
Thus, thermophilic or therrnotolerant bacteria are definitely J ~
needed for commarcialization of the single cell protein process.
Summary of_the Invention I have discovered a unique thexmophilic mixed culture of bacteria, containing three separate species of bacteria. These bacteria are individually classified as (1) a large gram-positive curved rod, division bacteria, class ~ , ~rder Eubacteriales, family Bacillaceae, genus ~acillusi (2) a large gram-negative rod, division Bacteria, class ~ , order Eubacteriales, family Bacillaceael genus ~acillus; (3) a short .
gram-negative rod, division Bacteria, class ~ . The mixed thermophilic culture exhibits highly desirable and useful properties. My Mc mixed culture exhibits improved growth at higher temperatures than at conventional temperatures, producing higher cell yields, with reduced foaming tendencies under fer-mentation conditions.
My mixed culture is thermophilic, grows effectively with high productivity on oxygenated hydrocarbon feedstocks, particu-larly lower alcohols, most preferably methanol or ethanol, at temperatures wherein most other kno~m bacteria speci~s either are relatively unproductive, or simply cannot survive/ or are unproductive and intolexant of an oxygenated hydrocarbon feedstock.
This unique mixed culture which I have discovered, and employ in my process, is designated as follows:
Culture Name My Strain Desi~nation .
MC HTB-53 NRRL ~ 8158 The designation NRRL B-8158 reflects the fact that I have deposited my thermophilic mixed cul~ure with the o-f~icial deposi-tory United States Department o~ Agriculture, Agricultural Research Service, North Central Region, Northern Regional Research Center, 1815 North University Street, Peoria, Illinois 61604, by depositing therewith thirty lyophilize~preparations of my mixed culture, prior to filing of this application, and have received rom the depository the NRRL designation B-8158 as indicated.
G2~
My unique mixed culture has been deposited in accordance with the pxocedures of the Department of Agriculture such that progeny of the mixed culture will be available during pendency of this patent application to one determined by the Commissioner of Patents and Trademarks ~o be entitled to access thereto in accord-ance with the Rules of Practice in Patent Cases and 35 U.S.C. 122.
The deposit has been made in accordance with the practices and requirements of the United States Patent and Trademark Office such that all restrictions on availability to the public of pro-geny of the unique mixed culture will be irrevocably removed upongranting of a patent of which this important mixed culture is the subject, so that said culture will be available to provide samples for utilization in accordance with my invention. Thus, any culture samples from this deposit, or from cultures from which the deposit was derived, thus provide mixed culture strains de-rived from the thermophilic mixed culture of my discovery.
Detailed Desc ~
I have discovered a peculiarly and uniquely effective thermophilic mixed culture of bacteria which I have designated as mixe~ strain HTB-53 and which has received U.S.D.A. depository designation NRRL B-8158. This mixed culture, for which I us~ the shorthand designation Mc, is a culture which is highly productive at relatively high fermentation temperatures, producing desirable and valuable single cell protein products with a high protein content of desirable amino acid ~ype and balance.
My unique thermophilic mixed culture is compositionally stabla. The mixed culture employed for lyophilization was iso~
lated from a fermentation run and lyophilized under usual condi-tions which comprise rapidly reezing the microbial cells at a 3~ very low temperaturP followed by rapid deh~dration under high vacuum, and storage at room temperature. To determine viability, some of the lyophilized samples were subsequently reactivated
Field of the Invention The invention relates to the production of single cell pro-teinO In another aspect, the invention relates to a novel thermo-philic mixed culture.
3e_ Efforts to relieve the impending worldwide shortages of pro-tein have included various biosynthesis processes wherein bio-logically produced single cell protein (SCP) is obtained by the growth of a variety of microorganisms on a variety of carbon-containing substratesO
The carbon and energy sources used as substrates for such processes should be available widely, relatively cheap, uniform and safe in that they do not leave harmful residues in ~he pro-teinaceous product ultimately obtained by the microbial conversions.
Petroleum hydrocarbons have been employed as the carbon and energy source material, but have faced practical difficulties in the lack of water solubility, in the high consumption of oxygerl to assist in the microbial conversion, and allegedly in traces of potentially carcinogenic agents from the petroleum feedstocks entering or ad~
hering to the protein product.
Other proces~es have used oxygenated hydrocarbon derivatives as feedstocks due to the water solubility of such derivatives and hence ease of handling since microbial conversion processes are essentially conducted under aqueous conditions. Such feedstocks are readily available either from petroleum sourcesl natural yas sources, various waste/garbage processing and conversion of methane~ and the like, from fermentation of variou grains and the like, destructlve aiskilLation Qf wood, and so on. Such oxygenated hydrocarbons, whatever their source, are widely available and relatively cheap feedstocks ~or fermentation proce~ses. Advantages accrue in that these feedstocks are partially oxygenated, so that substantially reduced molecular oxygen requirements are involved for the microbial conversion growth process itself.
However, another difficult and limiting factor in the commer-cialization of single cell protein processes has been the necessity to conduct the fermentation at relatively moderate temperatures of about 20 to 50~ C., and preferably not over about 35 C.
Microbial conversions are exothermic oxidation reactions with large quantities of heat being pxoduced. Heat must be removed from the fermentation admixture continuously and consistently, or risk the overheating of the system and either the death of the microorganisms or at least severe limitations of growth encoun-tered as temperatures rise, and hence loss in efficiency.
Many processes have concentrated on the employment Qf one or other of the many available yeasts as the microorganismO
Yeast cells generally are slightly larger than a bacteria cell, and sometimes provide easier separation from the fermentation process media.
However, bacteria offer advantages, since higher crude protein contents of the cell are obtained from bacteria as com-pared to the product obtainable from yeasts in general, sincethe yeasts have higher proportions of nonprotein structural material in their cells. Bacteria usually have a significantly higher true protein content, frequently being higher in the nu~ritionally important sulfur amino acids and lysine.
Discovery of bacteria with the capability o~ rapid growth and high productivity rates at relatively high fermentation process temperatures would be advantageou~. High temperature growth operation means less heat to be removed, less cooling apparatus involved, and ultimately relatively smaller amounts of heat needed for sterilization, coagulation, and separation pro-cesses. Danger of contamination with othex microorganisms is greatly reduced when high temperature ~ermentation can be employed.
Thus, thermophilic or therrnotolerant bacteria are definitely J ~
needed for commarcialization of the single cell protein process.
Summary of_the Invention I have discovered a unique thexmophilic mixed culture of bacteria, containing three separate species of bacteria. These bacteria are individually classified as (1) a large gram-positive curved rod, division bacteria, class ~ , ~rder Eubacteriales, family Bacillaceae, genus ~acillusi (2) a large gram-negative rod, division Bacteria, class ~ , order Eubacteriales, family Bacillaceael genus ~acillus; (3) a short .
gram-negative rod, division Bacteria, class ~ . The mixed thermophilic culture exhibits highly desirable and useful properties. My Mc mixed culture exhibits improved growth at higher temperatures than at conventional temperatures, producing higher cell yields, with reduced foaming tendencies under fer-mentation conditions.
My mixed culture is thermophilic, grows effectively with high productivity on oxygenated hydrocarbon feedstocks, particu-larly lower alcohols, most preferably methanol or ethanol, at temperatures wherein most other kno~m bacteria speci~s either are relatively unproductive, or simply cannot survive/ or are unproductive and intolexant of an oxygenated hydrocarbon feedstock.
This unique mixed culture which I have discovered, and employ in my process, is designated as follows:
Culture Name My Strain Desi~nation .
MC HTB-53 NRRL ~ 8158 The designation NRRL B-8158 reflects the fact that I have deposited my thermophilic mixed cul~ure with the o-f~icial deposi-tory United States Department o~ Agriculture, Agricultural Research Service, North Central Region, Northern Regional Research Center, 1815 North University Street, Peoria, Illinois 61604, by depositing therewith thirty lyophilize~preparations of my mixed culture, prior to filing of this application, and have received rom the depository the NRRL designation B-8158 as indicated.
G2~
My unique mixed culture has been deposited in accordance with the pxocedures of the Department of Agriculture such that progeny of the mixed culture will be available during pendency of this patent application to one determined by the Commissioner of Patents and Trademarks ~o be entitled to access thereto in accord-ance with the Rules of Practice in Patent Cases and 35 U.S.C. 122.
The deposit has been made in accordance with the practices and requirements of the United States Patent and Trademark Office such that all restrictions on availability to the public of pro-geny of the unique mixed culture will be irrevocably removed upongranting of a patent of which this important mixed culture is the subject, so that said culture will be available to provide samples for utilization in accordance with my invention. Thus, any culture samples from this deposit, or from cultures from which the deposit was derived, thus provide mixed culture strains de-rived from the thermophilic mixed culture of my discovery.
Detailed Desc ~
I have discovered a peculiarly and uniquely effective thermophilic mixed culture of bacteria which I have designated as mixe~ strain HTB-53 and which has received U.S.D.A. depository designation NRRL B-8158. This mixed culture, for which I us~ the shorthand designation Mc, is a culture which is highly productive at relatively high fermentation temperatures, producing desirable and valuable single cell protein products with a high protein content of desirable amino acid ~ype and balance.
My unique thermophilic mixed culture is compositionally stabla. The mixed culture employed for lyophilization was iso~
lated from a fermentation run and lyophilized under usual condi-tions which comprise rapidly reezing the microbial cells at a 3~ very low temperaturP followed by rapid deh~dration under high vacuum, and storage at room temperature. To determine viability, some of the lyophilized samples were subsequently reactivated
2~2~
and sub~ected to g~owkh of the lyophilized mixed culture under the same conditions utilized previously. The subsequent fermenta-tions employing the reactivated Mc culture gave essentially the same results in terms of high cell yield and the like, and appar-ent composition of the fermentation culture, as did the earlier runs. Microscopic examination of the reactivated Mc culture indicated ~he same three organisms in the same form and relation-ship existing in the reconstituted culture as in the source fermentation.
This unique hi~h temperature preferring culture provides improved rates of single cell protein production,with reduced cooling requirements, when grown on a carbon and energy substrate of an oxygenated hydrocarbon, preferably a lower alcohol, more preferably methanol or ethanol, and presently preferred is methanol or a substantially methanol-containing substrate.
There are distinct advantages in employing my unique thermo-philic mixed culture Mc in comparison to the utilization of a pure thermophile in the fermentation of methanol or of a similar carbon and energy source material. Firstly, a higher cell yield is obtained using the mixed culture. Cell yield as described herein is defined as the grams of cells produced per 100 grams of carbon and energy source material utili2ed, such as methanol. The higher cell yield is an important practical commercial aconomic advantage.
Another advantage fox my unique thermophilic mixed culture in ~omparison to a pure thermophile, is the fact that foam gen~
eration is substantially less as compared to pure cultures which I have studied. Pure cultures generally tend to produce large amounts of foam during the fermentation process. While foam may be desirable in certain apparatus as a means of assisting in the fermentation process in heat transfer, and in assisting in provid--ing the desired quantities of molecular oxygen for the aerobic fermentation process, nevertheless, many types of fermentation 6~
apparatus means are not designed or equipped to handle excessive or large amounts of foam produced ~y some pure-strain microorgan-isms. Thus, the moderate amounts of foam produced by my unique culture are certainly a distinct advantage.
Another advantaye for my unique mixed culture is the fact that the mixed culture inherently produces a single cell protein product which is a mixture of several varie~ies of cells, and thus the balance of amino acids in the recovered microbial cells from the Mc is expected to have a more desirable balance than exhibited by the product of any single pure culture. A better amino acid balance simply means that there is less likelihood of a deficiency of a particular essential amino acid.
My unique thermophilic mixed culture was discovered by me during work to discover and develop a variety of cultures suitable for microbial conversion processes. A sample of soil was taken two feet below the surface of earth covering a steam line at the Bartlesville, Oklahoma, Research Center of Phillips Petroleum Company. Conventional enrichment techniques in the presence of methanol were employed to isolate several separate or distinct cul~ures. During the course of the work in which several pure thermophilic cultures were isolated, it became apparent that a most unusual compositionally stable mixed culture of thermophilic organisms al90 had been obtained.
The thermophilic mixed culture which I have discovered is composed of three separate microorganisms. The three types of bacteria in my compositionally stable thermophilic mixed culture are described as (1~ a large gram~positive curved rod, (2~ a large gram-negative rod, and (3) a ~hort gram-negative rodO
Repeated attempts at separation were made on this culture
and sub~ected to g~owkh of the lyophilized mixed culture under the same conditions utilized previously. The subsequent fermenta-tions employing the reactivated Mc culture gave essentially the same results in terms of high cell yield and the like, and appar-ent composition of the fermentation culture, as did the earlier runs. Microscopic examination of the reactivated Mc culture indicated ~he same three organisms in the same form and relation-ship existing in the reconstituted culture as in the source fermentation.
This unique hi~h temperature preferring culture provides improved rates of single cell protein production,with reduced cooling requirements, when grown on a carbon and energy substrate of an oxygenated hydrocarbon, preferably a lower alcohol, more preferably methanol or ethanol, and presently preferred is methanol or a substantially methanol-containing substrate.
There are distinct advantages in employing my unique thermo-philic mixed culture Mc in comparison to the utilization of a pure thermophile in the fermentation of methanol or of a similar carbon and energy source material. Firstly, a higher cell yield is obtained using the mixed culture. Cell yield as described herein is defined as the grams of cells produced per 100 grams of carbon and energy source material utili2ed, such as methanol. The higher cell yield is an important practical commercial aconomic advantage.
Another advantage fox my unique thermophilic mixed culture in ~omparison to a pure thermophile, is the fact that foam gen~
eration is substantially less as compared to pure cultures which I have studied. Pure cultures generally tend to produce large amounts of foam during the fermentation process. While foam may be desirable in certain apparatus as a means of assisting in the fermentation process in heat transfer, and in assisting in provid--ing the desired quantities of molecular oxygen for the aerobic fermentation process, nevertheless, many types of fermentation 6~
apparatus means are not designed or equipped to handle excessive or large amounts of foam produced ~y some pure-strain microorgan-isms. Thus, the moderate amounts of foam produced by my unique culture are certainly a distinct advantage.
Another advantaye for my unique mixed culture is the fact that the mixed culture inherently produces a single cell protein product which is a mixture of several varie~ies of cells, and thus the balance of amino acids in the recovered microbial cells from the Mc is expected to have a more desirable balance than exhibited by the product of any single pure culture. A better amino acid balance simply means that there is less likelihood of a deficiency of a particular essential amino acid.
My unique thermophilic mixed culture was discovered by me during work to discover and develop a variety of cultures suitable for microbial conversion processes. A sample of soil was taken two feet below the surface of earth covering a steam line at the Bartlesville, Oklahoma, Research Center of Phillips Petroleum Company. Conventional enrichment techniques in the presence of methanol were employed to isolate several separate or distinct cul~ures. During the course of the work in which several pure thermophilic cultures were isolated, it became apparent that a most unusual compositionally stable mixed culture of thermophilic organisms al90 had been obtained.
The thermophilic mixed culture which I have discovered is composed of three separate microorganisms. The three types of bacteria in my compositionally stable thermophilic mixed culture are described as (1~ a large gram~positive curved rod, (2~ a large gram-negative rod, and (3) a ~hort gram-negative rodO
Repeated attempts at separation were made on this culture
3~ in order to isolate the pure microorganisms. For example, streak plates gave isolated colonies o~ the three organisms on a mineral agar media containing methanol. ~owever, when the isolaked colonies were transfarred to aqueous media for further growth .
- ~ - ' with methanol as the carbon and energy source material, surpris-ingly and unexpectedly no growth took place. Repeated efforts of this type were carried out, but without success.
The cooperative growth by this mixed culture indicates to me a symbiotic relationship between the three microorganisms.
Theorizing, and without intending to be bound by such theorizing, but rather in an effort to help explain the relationship observed, it appears possible ~hat a metabolic product or products of at least one of the microorganisms serves as a necessary substrate for the growth of one or other of the other microorganisms. While the exact nature of such a metabolite is not known at present, it appears possible that such metabolite may be toxic in nature to the microorganism which produces it, thereby explaining the need for ~he presence of the additional microorganism to consume this toxic metabolite in order for the first microorganism ~o continue to grow ade~uately.
A further indication of this symbiotic r&lationship that appears to exist between the three mi~roorgani~ms making up my unique compositionally stable thermophilic mixed culture is the fact that fermentations employing my Mc produce significantly less ~l~antities of foam under typical aerobic fermentation condi-tions as compared to the quantities of foam normally observed under equivalent typical aerobic fermentation conditions but employing pure thermophilic bacteria Bacillu_ ge~us~ This sug-gests to me, though again I do not wish to be bound hy theorizing when I have discovered a unique mixed culture and have demonstrated how to employ it to obtain improved cell yields o~er that obtainable by pure strains, that my Mc does not produce what would otherwise be expected in the way of very large amounts of foam during fermenta~-tion because in an Mc Eermentation an extracellular product,probably of proteinaceous nature~ i5 being consumed by at least one of the symbiotic microorganismæ, thereby continuously deplet-ing such product as a foam-generating material i.n the fermentation - . ~
admixture.
Carbon and Energy Source ~ he carbon and energy source material or substrate for the fermentation process of my inven~ion employing my novel and unique mixed culture is an oxygenated hydrocarbon. The term oxygenated hydrocarbon is a generic term descriptive of ~he compounds employ-able, and not necessarily a limiting term referring to the source of the substrate. The oxygenated hydrocarbons include alcohols, ketones, esters, ethers, acids, and aldehydes, which are carbon-oxygen-hydrogen~containing water-soluble compounds and are sub-stantially water-soluble in character. The oxygenated hydrocar-bons preferably should be of up ~o about 10 carbon atoms per mole-cule for better water solubility, since higher molecular weights tend to reduce water solubility level.
Illustrative examples include: methanol, ethanol, propanol, butanol, pentanol, hexanol, 1,7-heptanediol, 2-heptanol, 2-methyl-
- ~ - ' with methanol as the carbon and energy source material, surpris-ingly and unexpectedly no growth took place. Repeated efforts of this type were carried out, but without success.
The cooperative growth by this mixed culture indicates to me a symbiotic relationship between the three microorganisms.
Theorizing, and without intending to be bound by such theorizing, but rather in an effort to help explain the relationship observed, it appears possible ~hat a metabolic product or products of at least one of the microorganisms serves as a necessary substrate for the growth of one or other of the other microorganisms. While the exact nature of such a metabolite is not known at present, it appears possible that such metabolite may be toxic in nature to the microorganism which produces it, thereby explaining the need for ~he presence of the additional microorganism to consume this toxic metabolite in order for the first microorganism ~o continue to grow ade~uately.
A further indication of this symbiotic r&lationship that appears to exist between the three mi~roorgani~ms making up my unique compositionally stable thermophilic mixed culture is the fact that fermentations employing my Mc produce significantly less ~l~antities of foam under typical aerobic fermentation condi-tions as compared to the quantities of foam normally observed under equivalent typical aerobic fermentation conditions but employing pure thermophilic bacteria Bacillu_ ge~us~ This sug-gests to me, though again I do not wish to be bound hy theorizing when I have discovered a unique mixed culture and have demonstrated how to employ it to obtain improved cell yields o~er that obtainable by pure strains, that my Mc does not produce what would otherwise be expected in the way of very large amounts of foam during fermenta~-tion because in an Mc Eermentation an extracellular product,probably of proteinaceous nature~ i5 being consumed by at least one of the symbiotic microorganismæ, thereby continuously deplet-ing such product as a foam-generating material i.n the fermentation - . ~
admixture.
Carbon and Energy Source ~ he carbon and energy source material or substrate for the fermentation process of my inven~ion employing my novel and unique mixed culture is an oxygenated hydrocarbon. The term oxygenated hydrocarbon is a generic term descriptive of ~he compounds employ-able, and not necessarily a limiting term referring to the source of the substrate. The oxygenated hydrocarbons include alcohols, ketones, esters, ethers, acids, and aldehydes, which are carbon-oxygen-hydrogen~containing water-soluble compounds and are sub-stantially water-soluble in character. The oxygenated hydrocar-bons preferably should be of up ~o about 10 carbon atoms per mole-cule for better water solubility, since higher molecular weights tend to reduce water solubility level.
Illustrative examples include: methanol, ethanol, propanol, butanol, pentanol, hexanol, 1,7-heptanediol, 2-heptanol, 2-methyl-
4-pentanol, pentanoic acid, 2-methylbutanoic acid, 2-pentanolr 2-methyl-4 butanol, 2-methyl-3 butanol, 2-butanol, 2--methyl-1-propanol, 2-methyl-2-propanol, 2-propanol, formic acid, acetic acid, propanoic acid, formaldehyde, acetaldehyde, propanal~ butanal, 2~methylpropanal, butanoic acid, 2-methylpropanoic acid r pentanoic acid, glutaric aaid, hexanoic acid, 2-methylpentanoic acid, hep-tanedioic acid, heptanoic acid, 4-heptanone, 2-heptanone, octanoic acid, 2-ethylhexanoic acid, glycerol, ethylene glycol, propylene glycol, 2-propanone, 2-butanone, diethyl ether, methyl ethyl ethe~r dimethyl ether, di-n-propyl ether, n-propyl isopropyl ether, and the like, including mixtures of any two or more.
Petroleum gases, such as natural gas, such as methane, or other low carbon gases such as ethane and the lik~, can be oxidized ~0 to provide mixtures of predominantly the corresponding alcohols, as well as miscellaneous minor amounts o~ misc~llaneous ketonas, aldehydes, ethers, acids/ and the likeO
~6~
Among the oxygenated hydrocarbons, a presently preferred are the water-soluble alcohols as being suitable carbon and energy source materials for utilization by the thermophilic mixed culture of my discovery. Generally, these will be alcohols of 1 to 7 carbon atoms per molecule. Such alcohols include both linear and branched alcohols, primary, secondary, as well as tertiary.
Such alcohols can be monohydroxy, as well as polyhydroxy.
Exemplary alcohol species include such as methanol, ethanol, propanol, butanol, pentanol, hexanol, 1,7-heptanediol, 2-heptanol, 2-methyl-4-pentanol, 2-p~ntanol, 2-methanol-4-butanol, 2-methyl-3-butanol, 2-butanol, 2-methyl-1-propanol, 2-methyl-2-propanol, 2-propanol, glyceral, ethylene glycol, propylene glycol, and the like, including mixtures of any two or more.
The preferred alcohols are those of 1 to 4 carbon atoms per molecule, and most preferred are the monohydroxy alcohols, because of availability, water solubility, and economics. Methanol presently is an especially preferred alcohol because of its rela-tively low cost, wide availability, wa~er solubili~y, and since governmental regulations on use are less cumbersome than with ethanol. Methanol also is readily obtainable by simplP oxidative conversion of natural gas, which is primarily methane, and thus is or can be made readily available in many areas of the world which presently have surplus stocks of methane and little market or Sanle~ and correspondingly also sometimes have large populations of hungry people in this needlessly protein-deficit world.
~ presently commercially available material sometimes termed "Methyl Fuel'l (C&EN, September 17, 1973, page 23) is exemplary of a commercially available mixture of methanol and controlled per-centages of higher alcohols containing up to about 4 carbon atoms 0 per molecule, and could be employed as a suitable substrate.
Fermentation Condition~
Since the fermentation process employing the thermophilic ~G22~
mixed culture in accordance with my invention is a process of aerobic fermentation, there must also be supplied adequate oxygen for the fermentation admixture. Aerobic fermen~ation processes basically are well known in the art, and means of supplying oxygen to fermentation admixtures are also well known. Generally, the supply of molecular oxygen to the aqueous fermentation reaction admixture can be provi~ed by passing adequate volumes of air of ordinary oxygen content, or oxygen-enriched air if desired, or air and an ~ugmented supply of such as pure oxygen separately, through the fermentation vessel. Offgases can be recovered, recycled if desired, for maximum utiliæation of oxygen, such as by stripping carbon dioxide ~rom the offgases and recycling. In effect, using the oxygenated hydrocarbon as carbon and energy source substrate, a part of the oxygen demand needed for growth of the micro organism is supplied by the oxygen content of the substrate. Nevertheless, additional quantities of molecular oxygen must be supplied for suitable growth, since the assimilation of the substrate and corresponding growth of the microorganism is, in a sense, a combustion process. In general, exemplary is a range o~ about 0.1 and 10, more usually about 0.7 and 2.5, volumes per minute of air of normal oxygen content are supplied to the ermentation admixture per volume of aqueous liquid in the fermentor, ox in terms of oxygen, the respec~ive ranges would be about 0.02 to 2.1, and 0.14 to 0.55.
Pressure employed for my aerobic fermentation process can vary over a wide range. Exemplary would be considered a range of about 0.1 to 100 atmospheres (10.13-10,132 kPa), more usually from about 1 to 30 atmospheres (101.3-3,039 kPa), presently prèferably about 1 to 5 atmospheres (101.3-506.5 kPa), as being suitable and convenient. Pressures greater than atmospheric pressure are advantageous in ~he proces~ since such higher pressures tend to increase the dissolved oxygen content in the aqueous fermen~ation medium, which in turn tends to promote more rapid microbial growth.
.
.
Pressures greater than atmospheric are especially useful employing my thermophilic }nixed culture, since the higher temperatures em-ployed in my thermophilic fermentation tend to decrease oxygen solubility in the aqueous fermentation medium admixture.
The culturing of my unique and novel Mc mixed species of bacteria with the oxygenated hydrocarbon feedstock can be advan-tageously carried out at a temperature in the range of about 45 to 65 C., more preferably for optimum growth rates in accordance with my invention in the range of about 50 to 60 C. It should be noted that the temperature ranges given clearly indicate that the microorganisms utilized in my process are thermophiles in the accepted usage of the term, i.e., ~he microorganisms require such relatively high temperatures for suita~le growth. Lower tempera-tures tend to inhibit (retard) growth rate~.
High concentrations of some of the described carbon and enexgy source substrates, such as methanol, may be inhibitory to satisfactory microbial growth or even toxic to ~he micro-organisms in the fermentati~ns employing the mixed culture, and should be avoided.
The fermentation process can be carried out in a batch or in a continuous fashion t the presently preerred for economical SCP
production is the growing of microbial cells in large quantities in a continuous process. A continuous process is particularly suitable when a carhon and energy source material is a low~r alcohol, such as methanol or ethanol, which lends itself readily to controllable feed. ~he use o such microorganisms and the use of such feedstocks in a batch fermentation process is considered to be generally uneconomical on a practical basis. However, such substrates are conveniently utilized in a continuous fermentation process with overall high efficiencies and ~conomies. In the fermentation, a~ter the fermentor has been properly inoculated with the mixed culture species, the oxygenat0d hydrocarbon can be Z~
added as a separate stream, or admixed with water as an a~ueous stream to sterilize same, or with mineral media to sterilize same, or any or all of these~ Usually, it is fed separately for ease of control in a concentration in ~he feed stream to the fermentor broadly in the range of about 2.5 to 35 weight percent, more usually and conveniently ahout 10 ~o 15 weight percent~
Culturing is accomplished in an aqueous growth medium compris-ing an aqueous mineral salt medium, the carbon and energy source material, molecular oxygen, and, of course, a starting inoculum o~ the Mc mixed culture.
The fermentation growth rates can be adjusted by controlling the feed of oxygenated hydrocarbon. The feed rate of the carbon and enexgy source material should be adjusted so that the amounts being fed to the fermentor substantially are the same as the rate of consumption by the organism to avoid a significant buildup in the fermentor, particularly of any toxic materials which might inhibit the growth or even kill the microorganisms. A satisfactory condition usually can be exhibited by observation of little or no carbon and every feed material in the effluent being withdrawn from the ferme~tor, though a satisfactory check also can he obtained by watching the feed source material in the fermentor e~luent so as to maintain at a desirable low level of about O to 0.2 weight percent.
Generally the retention time of microbial cells in the fer-mentor means in a continuous process is of the order on the average o~ about ~ to 4 hours under such conditions, though this is not critical and can vary widely.
The unique mixed culture Mc of my discovery requires mineral n~trients and a source of asæimilable nitrogen~ in addition to the molecular oxygen, and the carbon and energy sources as des-cribed. The source of nitrogen can be any nitrogen-containing compound capable of releasing nitrogen in a form suitable for metabolic utilization by the organism. While a variety of organic nitrogen source compounds such as other proteins, urea, or the like, can be employed, usually inorganic nitrogen source materials are more economical and practical. Typicall~, such inorganic nitrogen-containing compounds include the presently preferred ammonia or ammonium hydroxide, as well as various other ammonium salts such as ammonium carbonate, ammonium citrate, ammonium phosphate, ammonium sulfate, ammonium pyrophosphate, and the likeO
Ammonia gas is convenient and can be employed by simply bubbling such through the aqueous fermentation media in suitable amounts.
The pH of the aqueous microbial fermentation admixture should he in the range, in accordance with my investigations, broadly from about 5.5 to 7.5, with a presently preferred range of about 6 to 7. Feed of the ammonia assists in controlling the pH desired, since otherwise the aqueous media tends to be slightly acidic. Of course, pH range preferences for microorganisms generally are dependent to some extent on the media employed, and thus the pH
preference may change at least slightly with a change in mineral media, for example.
In addition to the oxygen, nitrogen, and carbon and energy source, it i5 necessary to supply selected mineral nutrients in necessary amounts and proportions to the feed media in order to assure proper microorganism growth, and to maximize the assimila-tion of the oxygenated hydrocarbon by the cells in th~ microbial conversion processO
A source of phosphate or other phosphorus, magnesium, calcium, sodium, manganese, molybdenum, and copper ion~ appear to provide the essential minerals. The recipe shown below can be used to culture my novel Mc culture of my discovery. A mineral nutrient medium designated by me as FM-12 is useful in the fermentation pro~
cess and is listed below along with the trace mineral solution which forms a part of the FM-12 nutrient medium:
FM-12 Medium Component _ ~mount .
H3PO4 (85%) 2 ml KCl 1 g MgSO4-7H2O 1.5 g CaC12~H2O 0~2 g NaCl 0.1 g Trace Mineral Solution 5 ml Distilled Water To make 1 liter Trace Mineral Solution -CUSO4 ~ SH2O O . 06 g KI 0.08 g Fecl3 6~2o 4.80 g MnSO4 H2O
Na2MOO4 2H2o 0.20 g ZnS04 7H20 2.00 g H3BO3 0.02 g H2SO4 (conc.) 3 ml Distilled Water To make 1 liter Other mineral medium concentrations al~o can be employed, examples of which are provided in this disclosure in the Examples sec~ion.
In either a batch, or in the preferxed continuous operation, all equip~ent, reactor or fermentation means, vessel or other container, piping, attendant circulating or cooling devices, and the like, should be sterilized, such as by the employment o~
steam such as about at least 250 F. ~or at least sevexal minutes, such as about 15 minutes, prior to actual staxtup. The sterilized reactor then IS inoculated with a culture from my mixed culture in the presence o~ all the required nutrients, and including the molecular oxygen and the oxygenated hydrocarbon feed.
In the f0rmentation process, as the culture begins to grow, the introduction of air or other molecular oxygen, nutrient medium, nitrogen source if added separately, and the alcohol or other oxygenated hydrocarbon, are maintained. The addition rate of the feed stream or streams can be varied so as to obtain as rapid a cell growth as possible consistent with the utilization of the carbon and energy source input, so that the objective of a maxi-mized high yield cell weight per weight of feed charged is obtained.
Of course, any of the feed streams can be added either incrementally or continuously as desired ox convenient.
Instrumentation can be maintained to measure cell density, pH, dissolved oxygen content, oxygenated hydrocarbon concentration in the fermentor admixture, temperature, feed rates of input and output streams, and the likeO It presen~ly is preferred ~hat materials fed to the fermen~or be sterilized prior to introduction into the fermentor. When the oxygena~ed hydrocarbon is a material capable of sterilizing other makerials, such as ethanol, or methanol, in some instances it may be convenient to add this component to other streams, such as the mineral media, in steril-izing amounts, and thus accomplish several purposes without the necessity for separate heat sterilization of the min~ral media, for example, thus providing as maximum and economical an operation as possible. Heat added to any stream ultimately generally must be taken out by cooling means in the fermentor, since the aerobic fermentation is one in which heat is being produced.
The type of fermentor employed appears not to be critical in the practice of the fermentation process employing the mixed culture in accordance with my discovery. High productivity of the mixed culture with alcohol appears to be best achieved in a continuous process. Of course, watch must be maintained to con-trol growth rates to avoid foam-out o~ the fermentor which could lower the effective liquid volume and cause some loss of fermentor contents. My unique mixed culture, has a distinct advantage, since while it is a "foamer", it is not an excessive foamer, and liquid levels can be more readily maintained in the fermentor without .
~ 2~
dang~r of foam-outO Particularly, addition of antifoam to the fermentation admixture is to be avoided, if at all possible, since antifoams such as the silicones may be detrimental to the dissolved oxygen content at the reco~nended high fermentation temperatures, and may cause organisms to grow at a slower rate, or even to die.
The foam produced with my Mc mixed culture is not harmful to growth, and definitely is beneficial in maintaining the organisms in a system of high dissolved oxygen content. Foam helps provide relatively large areas of gas/liquid contacting interfaces. ~hus, fermentation can be improved and heat transfer improved as to control, uniformity, and avoidance of hot spots.
Of course, a foam-inducing substance such as some of the detergents, preferably nonionic, could be employed, if desired, to assist or induce additional foaming, though normally this is not necessary, or even desirable, with ~he Mc mixed culture of my discovery.
Product Recovery The recovery of microbial cells from my fermentation process can be accomplished by the usual techniques, such as acidification of fermentation effluent to a pH of such as about 4, and heating the acidified effluent to a temperature suitable to kill the microorganisms, such as about 80 C., though low enough not to damage the protein product. The effluent then can be centrifuged, washed, and recentrifuged to recover the microbial cells from the ermentor effluent. The cells can be treated to cause lysis to expedite recovery of protein and other materials from the cell.
The fermentation also produces a number o~ desirable by-pxoducts which can be recovered from th~ fermentor effluent. These extra-cellular produats can be economically helpful in the overall process, since they include valuable products such as poly-saccharides, amino acids, such as glutamic acid, enzymes, vitamins, and the like.
The single cell protein product in accordance with my process, is a valuable source of protein for humans as well as for animals.
For human consumption, the cells can be trea~ed to reduce the nucleic acid content, if desired, though for animal feed purposes such ~reatment does not appear necessary.
EXAMPLES
Examples following are intended to be descrip~ive of runs employing ~he novel mixed cul-ture of my discovery. Particular amounts and materials, or alcohols employed, should be considered as illustrative, not as limitative of my invention.
XAMPLE I
A continuous fermentation run utilizing the thermophilic mixed culture of the instant invention was carried out. ~ 7 liter fermentor equipped with an aerator, stirrer, dissolved oxygen monitor, and means for measuring and controlling temperature and pH of the fermentation mixture, was charged with about 500 ml of ~ermentation reaction mixture from a previous fermentation run utilizing the thermophilic mixed culture, and 10 ml of methanol as the inoculant. The reactor also was charged with 2 liters of fermentation mineral medium FM-12.
~he stirring rate was maintained at 1,000 rpm throughout the course or the run and the p~ was controlled at from 6.2 to 6.35 by addition as necessary of ammonium hydroxide solution.
~ir wa~ introduced into the fermentor at a rate o~ 2 liters per minute throughout the course o~ the run. At 6 hours into the run essentially pure oxygen also was introduced to the fermentor at a rate of 0.5 liters per minute, then was increased to 0 75 liters per minute at 118 hours, to 1.5 liters per minute at 174 hours and to 2 liters per minute at 190 hour~ duriny the run.
The medium continuously charged initially was the FM-12 medium previously descri~ed. At 22 hours, the mineral medium was changed to an aqueous composition of 7~5% by volume methanol in addition to the FM-12 medium plus 0.75 grams of potassium chloride~ twice the normal trace mineral content, three times the normal manganese component content (all on a per liter basis), and with dele~ion of sodium chloride. A~ 166 hours, tha feed was changed to an aqueous composition of 10% by volume methanol, 2.5 ml phosphoric acid (85%) per liter, 2 grams per liter potassium chloride, 1.75 grams per liter magnesium sulfate 7H20, 0.25 grams per liter of calcium chloride .2H20, 20 ml per liter of a manganese sulfate H2O aqueous solution (0.3 g/l), and 35 ml per liter of the trace mineral solution previously described.
The media feed rate during the run ranged from 700 ml per hour at 22 hours to 817 ml per hour at 118 hours, 781 ml per hour at 166 hours, and 763 ml per hour at 382 hours.
Samples of the fermentation efluent were with~rawn from time to ~ime to recover the cells therefrom. Values obtained for cell content in terms of dry weight of cells in grams per liter and ~he yields calculated are presented below in Table I.
Also shown in the table are values calculated for productivity in terms of grams of cells per liter per hour for the fermentation process.
At about 126 hours into the run, samples of the fermentation admixture were withdrawn and prepared for lyophilization o~ the microbial cells according to procedures known in the art. These lyophilized samples then were stored for subsequent use in a later fermentation run and for supplying samples of HTB-53 to a deposi~ory for microorganims operated by the United States Department of Agriculture, Northern ~egional Research Laboratory at Peoria, Illinois.
Periodic samples also were tak.en from the fermentation reac-tion mixture for microscopic examination o~ the microbial cells in terms of their gross morphology. Such microscopic examination showed that the Mc culture was composed of a large Gram positive curved rod, a large Gram negative rod, and a small Gram negative rod. Occasionally, a large Gram positive rod (not curved) also was observed, but this was believed to be a transitory variant of the large curved Gram positive rod.
Table I
Time, Hours46 118 166 190 Z86 358 Retention Time, Hours2.36 2.27 2.38 2.83 2.33 2.43 Cells(a) g/l 2~.86 27.13 27.04 35.21 35.53 35.57 Solids(b) g/l 26.82 27.66 27.87 35.5 36.66 36.76 10 Cell Yieldtc) ~ 44.7 46.1 46.47 44.4 45.8 45.9 Productivity(d) g/l/hr 1104 12.2 11.7 12.5 15.7 15.1 (a) Value obtained by evaporating a 10 ml sample of fermentex effluent overnight at 100 C and subtractlng weight of mineral solids contained in 10 ml of medium.
(b) Value obtained by centrifuging 100 ml sample of fermentor effluent, resuspending solids in distilled water and centrifuging again to recover solids which are dried overnlght at 100 C.
(c) Value obtained by dividing recovered solids (g/l) by methanol charged (g/l)x 100.
(d) Value obtained by dividing recovered solids (g/l) by retention time (hr).
After about one month, a single tube of the lyophilized HTB-53 microbial culture from the fermentation run of Example I a~ove was opened aseptically by conventional procedures and added to 100 ml of a fermentation medium designated IM2 which also contained 0.5~ methanol. The composition of medium IM2 is ~hown below.
IM2 ~edium C9 ~S~Y~ Amou_t, g XH2PO4 2.0 K2HPO4 3.0 MgS4 7H20 0.4 CaC12 2H20 Narl 0. 1 (NH4)2so4 2.0 Trace Mineral Solution 0.5 ml Distilled Water To make 1 liter . . ..
- ... . : . : .
; .. :" ' ' ~6~
The flask charged with the revived lyophilized culture was incu-bated with shaking a~ 55 C. After 24 hours, the shake flask showed good growth of the culture and 5 ml of the mixture was transferred to 100 ml of medium IM2 also containing 1.5% by volume methanol. Good growth developed within 24 hours and a third tran~-fer was made to the same medium in two flasks, each containing 500 ml of IM2 medium plus 1.5% by volume methanol. This third transfer involved 100 ml of the culture being added ~o each of the two flasks. The culture was allowed to grow for 32 hours and then was utilized as the inoculant for a continuous fermentation run in the apparatus described above in Example I. The fermentor was charged with 1,000 ml of FM-12 mediuml and 1,000 ml of the inoculant to which was added 10 ml of methanol. The temperature was maintained a~ 55 C, and the pH was controlled at from 6.25 to 6.4 by the continuous addition of ammonium hydroxide solution as before. Initially the stirrer was operated at 300 rpm, and air was introduced at a rate of 0.5 liters per minutel while the culture was permitted to establish itsel in the fermentor. After 7 hours, a continuous introduction of feed media having the same composition as the last named feed media shown in Example I above was introduced. In addition, the air rate was increased to 2 liters per minute while the rpm was set at 1,000 for the stirrex~
After 30 hours, the air rate was reduced to 1.75 liters per minute, while oxygen was introduced at 0.75 liters per minute, later increased to 1 liter per minute at 54 hours, and to 1.5 liters per minute at 198 hours. Again the fermentation effluent was sampled from time to time to provide data on the cell content in terms of grams per liter based on a dry weight and in terms of the yield and productivity of the fermentation. The data obtained during the run are presented below in Table II.
.
Table II(a) Time, Hours 70 166 190 214 Retention Time, Hours 2.60 2.70 2~63 2.64 Cells g/l 3~.33 33.56 33.87 34.8 Solids g/l 34.44 35.4 34.55 35~52 Cell Yield ~ 43.6 44.2 43.2 44.2 Productivity g/l/hr 13.2 13.1 13.1 13.5 (a) See footnotes for Table I above.
Samples of the mi.crobial culture were also obtained during the fermentation run for microscopic examina~ion as described in Example I. In this instance, there was also obser~ed the large Gram positive curved rods and the large and small Gram negative staining rods. At 238 hours into the run, the culture was lost while attempting to change ~he feed to a higher alcohol concentra~
tion.
Example III
Presented below in Table III are analytical data character~
izing ~ha microbial ~ulture obtained in the run of Example I in terms o a chemical analysis of the microbial cells recovered. In Table IV there is also presented an amino acid content analysis of microbial cells recovered from another fermentation run util-izing the mixed thermophilic culture of my invention. For purposes of comparison, an amino acid content analysis of a pure thermo-philic microorganism obtained during the course o the isolation ; 30 of the thermophiles from the initial soil sample previously described is also presented in Tahle IV.
. .
6~
Table III
Chemical Analysis of Microbial Cells Obtained in A Run With HTB-53 Crude Protein(a), wt. % 85.63 Ash, wt. % 9.19 Amino nitrogen, wt. % 13.4 Carbon, wt. ~ 44.7 Hydxogen, wt. % 6.79 Nitrogen, wk. % 13.7 Phosphorous, wt. % 1.71 Potassium, wt. % 0.91 Magnesium, wt. ~ O.26 Calcium, wt. % 0.1 Sodium, wt. ~ < 0.01 Iron, ppm 1300 Zinc, ppm 55.8 Manganese, ppm 126 Coppex, ppm 20 (a) Nitrogen content (13.7) x 6.25.
TabIe IY
Amino Acid Content of Thermo~hilic Cultures Grown on MethanoI- Grams .
Per 100 Grams Product _ _ _ .
Chem ~1)( Score(l) Essential Amino Acids Pure ValuesMixed Values (HTB-7) leucine 5.37 75 5.76 104 isoleucine 4.56 83 4.90 118 30 lysine 5.54 96 5.67 141 methionine 1.50 f 1.22 34 ~36 cystine * * t ~hreonine 2.95 90 2.79 88 phenylal~nine 2.68 ( 2.78 : 80 88 tyrosine 2.55 2.72 tryptophan 0.60 43 0.89 90 valine 5.13 91 5.46 121 (*~= not detected) `
,, :- , ~
, &~
Table IV (continued) Amino Acld Content of Thermophilic Cultures Grown on Methanol _ Grams Per lO0 Grams Product . _ Chem.Chem.
Score(1)(2) score(l) Essential Amlno Acids Pure _ Values~-HTB-~2) -alanine 5.65 6.19 lO arginine 3.39 3.01 aspartic acids 6.50 6.26 glycine 3.71 4.19 glutamic acid 10.47 10.69 histidine 1.26 1.22 proline 2.32 2.38 serine 2.09 1.75 Total Essential amino acids 30.88 32.19 Total ~mino Acids66.27 67.88 Crude Protein 85.63 84.4 (l) Chemical Score Values: based on essential amino acid content of egg as 100 for same weight of protein.
(2) Based on averages from five pure thermophiLe runs.
It can be noted that the percentage of total amino acids which are essential amino acids is slightly higher for the mixed culture product (47%) than for the pure culture product (46%). Furthermore, if the essential sulfur-containing amino acids are supplied by addition of synthetic methionine, which is very likely since essentially all SCP's have been ~ound to 30 ba low in these amino acids, the Chemical Score values show that the mixed culture product is twice as good from a nutritional standpoint as the pure culture product based on the next lowest essential amino acid Chemical Score value.
The disclosure, including data, illustrate the value and effectiveness of my invention. The examples, knowledge and ~3 background of the field of the invention, general principles of microbiology, chemistry, and other applicable sciences, have formed the bases from which the broad descriptions of my inven-tion, including the ranges of conditions and generic groups of operant componen~s have been developed, which have formed the bases for my claims here appended.
,' .~' , ' ' . - '
Petroleum gases, such as natural gas, such as methane, or other low carbon gases such as ethane and the lik~, can be oxidized ~0 to provide mixtures of predominantly the corresponding alcohols, as well as miscellaneous minor amounts o~ misc~llaneous ketonas, aldehydes, ethers, acids/ and the likeO
~6~
Among the oxygenated hydrocarbons, a presently preferred are the water-soluble alcohols as being suitable carbon and energy source materials for utilization by the thermophilic mixed culture of my discovery. Generally, these will be alcohols of 1 to 7 carbon atoms per molecule. Such alcohols include both linear and branched alcohols, primary, secondary, as well as tertiary.
Such alcohols can be monohydroxy, as well as polyhydroxy.
Exemplary alcohol species include such as methanol, ethanol, propanol, butanol, pentanol, hexanol, 1,7-heptanediol, 2-heptanol, 2-methyl-4-pentanol, 2-p~ntanol, 2-methanol-4-butanol, 2-methyl-3-butanol, 2-butanol, 2-methyl-1-propanol, 2-methyl-2-propanol, 2-propanol, glyceral, ethylene glycol, propylene glycol, and the like, including mixtures of any two or more.
The preferred alcohols are those of 1 to 4 carbon atoms per molecule, and most preferred are the monohydroxy alcohols, because of availability, water solubility, and economics. Methanol presently is an especially preferred alcohol because of its rela-tively low cost, wide availability, wa~er solubili~y, and since governmental regulations on use are less cumbersome than with ethanol. Methanol also is readily obtainable by simplP oxidative conversion of natural gas, which is primarily methane, and thus is or can be made readily available in many areas of the world which presently have surplus stocks of methane and little market or Sanle~ and correspondingly also sometimes have large populations of hungry people in this needlessly protein-deficit world.
~ presently commercially available material sometimes termed "Methyl Fuel'l (C&EN, September 17, 1973, page 23) is exemplary of a commercially available mixture of methanol and controlled per-centages of higher alcohols containing up to about 4 carbon atoms 0 per molecule, and could be employed as a suitable substrate.
Fermentation Condition~
Since the fermentation process employing the thermophilic ~G22~
mixed culture in accordance with my invention is a process of aerobic fermentation, there must also be supplied adequate oxygen for the fermentation admixture. Aerobic fermen~ation processes basically are well known in the art, and means of supplying oxygen to fermentation admixtures are also well known. Generally, the supply of molecular oxygen to the aqueous fermentation reaction admixture can be provi~ed by passing adequate volumes of air of ordinary oxygen content, or oxygen-enriched air if desired, or air and an ~ugmented supply of such as pure oxygen separately, through the fermentation vessel. Offgases can be recovered, recycled if desired, for maximum utiliæation of oxygen, such as by stripping carbon dioxide ~rom the offgases and recycling. In effect, using the oxygenated hydrocarbon as carbon and energy source substrate, a part of the oxygen demand needed for growth of the micro organism is supplied by the oxygen content of the substrate. Nevertheless, additional quantities of molecular oxygen must be supplied for suitable growth, since the assimilation of the substrate and corresponding growth of the microorganism is, in a sense, a combustion process. In general, exemplary is a range o~ about 0.1 and 10, more usually about 0.7 and 2.5, volumes per minute of air of normal oxygen content are supplied to the ermentation admixture per volume of aqueous liquid in the fermentor, ox in terms of oxygen, the respec~ive ranges would be about 0.02 to 2.1, and 0.14 to 0.55.
Pressure employed for my aerobic fermentation process can vary over a wide range. Exemplary would be considered a range of about 0.1 to 100 atmospheres (10.13-10,132 kPa), more usually from about 1 to 30 atmospheres (101.3-3,039 kPa), presently prèferably about 1 to 5 atmospheres (101.3-506.5 kPa), as being suitable and convenient. Pressures greater than atmospheric pressure are advantageous in ~he proces~ since such higher pressures tend to increase the dissolved oxygen content in the aqueous fermen~ation medium, which in turn tends to promote more rapid microbial growth.
.
.
Pressures greater than atmospheric are especially useful employing my thermophilic }nixed culture, since the higher temperatures em-ployed in my thermophilic fermentation tend to decrease oxygen solubility in the aqueous fermentation medium admixture.
The culturing of my unique and novel Mc mixed species of bacteria with the oxygenated hydrocarbon feedstock can be advan-tageously carried out at a temperature in the range of about 45 to 65 C., more preferably for optimum growth rates in accordance with my invention in the range of about 50 to 60 C. It should be noted that the temperature ranges given clearly indicate that the microorganisms utilized in my process are thermophiles in the accepted usage of the term, i.e., ~he microorganisms require such relatively high temperatures for suita~le growth. Lower tempera-tures tend to inhibit (retard) growth rate~.
High concentrations of some of the described carbon and enexgy source substrates, such as methanol, may be inhibitory to satisfactory microbial growth or even toxic to ~he micro-organisms in the fermentati~ns employing the mixed culture, and should be avoided.
The fermentation process can be carried out in a batch or in a continuous fashion t the presently preerred for economical SCP
production is the growing of microbial cells in large quantities in a continuous process. A continuous process is particularly suitable when a carhon and energy source material is a low~r alcohol, such as methanol or ethanol, which lends itself readily to controllable feed. ~he use o such microorganisms and the use of such feedstocks in a batch fermentation process is considered to be generally uneconomical on a practical basis. However, such substrates are conveniently utilized in a continuous fermentation process with overall high efficiencies and ~conomies. In the fermentation, a~ter the fermentor has been properly inoculated with the mixed culture species, the oxygenat0d hydrocarbon can be Z~
added as a separate stream, or admixed with water as an a~ueous stream to sterilize same, or with mineral media to sterilize same, or any or all of these~ Usually, it is fed separately for ease of control in a concentration in ~he feed stream to the fermentor broadly in the range of about 2.5 to 35 weight percent, more usually and conveniently ahout 10 ~o 15 weight percent~
Culturing is accomplished in an aqueous growth medium compris-ing an aqueous mineral salt medium, the carbon and energy source material, molecular oxygen, and, of course, a starting inoculum o~ the Mc mixed culture.
The fermentation growth rates can be adjusted by controlling the feed of oxygenated hydrocarbon. The feed rate of the carbon and enexgy source material should be adjusted so that the amounts being fed to the fermentor substantially are the same as the rate of consumption by the organism to avoid a significant buildup in the fermentor, particularly of any toxic materials which might inhibit the growth or even kill the microorganisms. A satisfactory condition usually can be exhibited by observation of little or no carbon and every feed material in the effluent being withdrawn from the ferme~tor, though a satisfactory check also can he obtained by watching the feed source material in the fermentor e~luent so as to maintain at a desirable low level of about O to 0.2 weight percent.
Generally the retention time of microbial cells in the fer-mentor means in a continuous process is of the order on the average o~ about ~ to 4 hours under such conditions, though this is not critical and can vary widely.
The unique mixed culture Mc of my discovery requires mineral n~trients and a source of asæimilable nitrogen~ in addition to the molecular oxygen, and the carbon and energy sources as des-cribed. The source of nitrogen can be any nitrogen-containing compound capable of releasing nitrogen in a form suitable for metabolic utilization by the organism. While a variety of organic nitrogen source compounds such as other proteins, urea, or the like, can be employed, usually inorganic nitrogen source materials are more economical and practical. Typicall~, such inorganic nitrogen-containing compounds include the presently preferred ammonia or ammonium hydroxide, as well as various other ammonium salts such as ammonium carbonate, ammonium citrate, ammonium phosphate, ammonium sulfate, ammonium pyrophosphate, and the likeO
Ammonia gas is convenient and can be employed by simply bubbling such through the aqueous fermentation media in suitable amounts.
The pH of the aqueous microbial fermentation admixture should he in the range, in accordance with my investigations, broadly from about 5.5 to 7.5, with a presently preferred range of about 6 to 7. Feed of the ammonia assists in controlling the pH desired, since otherwise the aqueous media tends to be slightly acidic. Of course, pH range preferences for microorganisms generally are dependent to some extent on the media employed, and thus the pH
preference may change at least slightly with a change in mineral media, for example.
In addition to the oxygen, nitrogen, and carbon and energy source, it i5 necessary to supply selected mineral nutrients in necessary amounts and proportions to the feed media in order to assure proper microorganism growth, and to maximize the assimila-tion of the oxygenated hydrocarbon by the cells in th~ microbial conversion processO
A source of phosphate or other phosphorus, magnesium, calcium, sodium, manganese, molybdenum, and copper ion~ appear to provide the essential minerals. The recipe shown below can be used to culture my novel Mc culture of my discovery. A mineral nutrient medium designated by me as FM-12 is useful in the fermentation pro~
cess and is listed below along with the trace mineral solution which forms a part of the FM-12 nutrient medium:
FM-12 Medium Component _ ~mount .
H3PO4 (85%) 2 ml KCl 1 g MgSO4-7H2O 1.5 g CaC12~H2O 0~2 g NaCl 0.1 g Trace Mineral Solution 5 ml Distilled Water To make 1 liter Trace Mineral Solution -CUSO4 ~ SH2O O . 06 g KI 0.08 g Fecl3 6~2o 4.80 g MnSO4 H2O
Na2MOO4 2H2o 0.20 g ZnS04 7H20 2.00 g H3BO3 0.02 g H2SO4 (conc.) 3 ml Distilled Water To make 1 liter Other mineral medium concentrations al~o can be employed, examples of which are provided in this disclosure in the Examples sec~ion.
In either a batch, or in the preferxed continuous operation, all equip~ent, reactor or fermentation means, vessel or other container, piping, attendant circulating or cooling devices, and the like, should be sterilized, such as by the employment o~
steam such as about at least 250 F. ~or at least sevexal minutes, such as about 15 minutes, prior to actual staxtup. The sterilized reactor then IS inoculated with a culture from my mixed culture in the presence o~ all the required nutrients, and including the molecular oxygen and the oxygenated hydrocarbon feed.
In the f0rmentation process, as the culture begins to grow, the introduction of air or other molecular oxygen, nutrient medium, nitrogen source if added separately, and the alcohol or other oxygenated hydrocarbon, are maintained. The addition rate of the feed stream or streams can be varied so as to obtain as rapid a cell growth as possible consistent with the utilization of the carbon and energy source input, so that the objective of a maxi-mized high yield cell weight per weight of feed charged is obtained.
Of course, any of the feed streams can be added either incrementally or continuously as desired ox convenient.
Instrumentation can be maintained to measure cell density, pH, dissolved oxygen content, oxygenated hydrocarbon concentration in the fermentor admixture, temperature, feed rates of input and output streams, and the likeO It presen~ly is preferred ~hat materials fed to the fermen~or be sterilized prior to introduction into the fermentor. When the oxygena~ed hydrocarbon is a material capable of sterilizing other makerials, such as ethanol, or methanol, in some instances it may be convenient to add this component to other streams, such as the mineral media, in steril-izing amounts, and thus accomplish several purposes without the necessity for separate heat sterilization of the min~ral media, for example, thus providing as maximum and economical an operation as possible. Heat added to any stream ultimately generally must be taken out by cooling means in the fermentor, since the aerobic fermentation is one in which heat is being produced.
The type of fermentor employed appears not to be critical in the practice of the fermentation process employing the mixed culture in accordance with my discovery. High productivity of the mixed culture with alcohol appears to be best achieved in a continuous process. Of course, watch must be maintained to con-trol growth rates to avoid foam-out o~ the fermentor which could lower the effective liquid volume and cause some loss of fermentor contents. My unique mixed culture, has a distinct advantage, since while it is a "foamer", it is not an excessive foamer, and liquid levels can be more readily maintained in the fermentor without .
~ 2~
dang~r of foam-outO Particularly, addition of antifoam to the fermentation admixture is to be avoided, if at all possible, since antifoams such as the silicones may be detrimental to the dissolved oxygen content at the reco~nended high fermentation temperatures, and may cause organisms to grow at a slower rate, or even to die.
The foam produced with my Mc mixed culture is not harmful to growth, and definitely is beneficial in maintaining the organisms in a system of high dissolved oxygen content. Foam helps provide relatively large areas of gas/liquid contacting interfaces. ~hus, fermentation can be improved and heat transfer improved as to control, uniformity, and avoidance of hot spots.
Of course, a foam-inducing substance such as some of the detergents, preferably nonionic, could be employed, if desired, to assist or induce additional foaming, though normally this is not necessary, or even desirable, with ~he Mc mixed culture of my discovery.
Product Recovery The recovery of microbial cells from my fermentation process can be accomplished by the usual techniques, such as acidification of fermentation effluent to a pH of such as about 4, and heating the acidified effluent to a temperature suitable to kill the microorganisms, such as about 80 C., though low enough not to damage the protein product. The effluent then can be centrifuged, washed, and recentrifuged to recover the microbial cells from the ermentor effluent. The cells can be treated to cause lysis to expedite recovery of protein and other materials from the cell.
The fermentation also produces a number o~ desirable by-pxoducts which can be recovered from th~ fermentor effluent. These extra-cellular produats can be economically helpful in the overall process, since they include valuable products such as poly-saccharides, amino acids, such as glutamic acid, enzymes, vitamins, and the like.
The single cell protein product in accordance with my process, is a valuable source of protein for humans as well as for animals.
For human consumption, the cells can be trea~ed to reduce the nucleic acid content, if desired, though for animal feed purposes such ~reatment does not appear necessary.
EXAMPLES
Examples following are intended to be descrip~ive of runs employing ~he novel mixed cul-ture of my discovery. Particular amounts and materials, or alcohols employed, should be considered as illustrative, not as limitative of my invention.
XAMPLE I
A continuous fermentation run utilizing the thermophilic mixed culture of the instant invention was carried out. ~ 7 liter fermentor equipped with an aerator, stirrer, dissolved oxygen monitor, and means for measuring and controlling temperature and pH of the fermentation mixture, was charged with about 500 ml of ~ermentation reaction mixture from a previous fermentation run utilizing the thermophilic mixed culture, and 10 ml of methanol as the inoculant. The reactor also was charged with 2 liters of fermentation mineral medium FM-12.
~he stirring rate was maintained at 1,000 rpm throughout the course or the run and the p~ was controlled at from 6.2 to 6.35 by addition as necessary of ammonium hydroxide solution.
~ir wa~ introduced into the fermentor at a rate o~ 2 liters per minute throughout the course o~ the run. At 6 hours into the run essentially pure oxygen also was introduced to the fermentor at a rate of 0.5 liters per minute, then was increased to 0 75 liters per minute at 118 hours, to 1.5 liters per minute at 174 hours and to 2 liters per minute at 190 hour~ duriny the run.
The medium continuously charged initially was the FM-12 medium previously descri~ed. At 22 hours, the mineral medium was changed to an aqueous composition of 7~5% by volume methanol in addition to the FM-12 medium plus 0.75 grams of potassium chloride~ twice the normal trace mineral content, three times the normal manganese component content (all on a per liter basis), and with dele~ion of sodium chloride. A~ 166 hours, tha feed was changed to an aqueous composition of 10% by volume methanol, 2.5 ml phosphoric acid (85%) per liter, 2 grams per liter potassium chloride, 1.75 grams per liter magnesium sulfate 7H20, 0.25 grams per liter of calcium chloride .2H20, 20 ml per liter of a manganese sulfate H2O aqueous solution (0.3 g/l), and 35 ml per liter of the trace mineral solution previously described.
The media feed rate during the run ranged from 700 ml per hour at 22 hours to 817 ml per hour at 118 hours, 781 ml per hour at 166 hours, and 763 ml per hour at 382 hours.
Samples of the fermentation efluent were with~rawn from time to ~ime to recover the cells therefrom. Values obtained for cell content in terms of dry weight of cells in grams per liter and ~he yields calculated are presented below in Table I.
Also shown in the table are values calculated for productivity in terms of grams of cells per liter per hour for the fermentation process.
At about 126 hours into the run, samples of the fermentation admixture were withdrawn and prepared for lyophilization o~ the microbial cells according to procedures known in the art. These lyophilized samples then were stored for subsequent use in a later fermentation run and for supplying samples of HTB-53 to a deposi~ory for microorganims operated by the United States Department of Agriculture, Northern ~egional Research Laboratory at Peoria, Illinois.
Periodic samples also were tak.en from the fermentation reac-tion mixture for microscopic examination o~ the microbial cells in terms of their gross morphology. Such microscopic examination showed that the Mc culture was composed of a large Gram positive curved rod, a large Gram negative rod, and a small Gram negative rod. Occasionally, a large Gram positive rod (not curved) also was observed, but this was believed to be a transitory variant of the large curved Gram positive rod.
Table I
Time, Hours46 118 166 190 Z86 358 Retention Time, Hours2.36 2.27 2.38 2.83 2.33 2.43 Cells(a) g/l 2~.86 27.13 27.04 35.21 35.53 35.57 Solids(b) g/l 26.82 27.66 27.87 35.5 36.66 36.76 10 Cell Yieldtc) ~ 44.7 46.1 46.47 44.4 45.8 45.9 Productivity(d) g/l/hr 1104 12.2 11.7 12.5 15.7 15.1 (a) Value obtained by evaporating a 10 ml sample of fermentex effluent overnight at 100 C and subtractlng weight of mineral solids contained in 10 ml of medium.
(b) Value obtained by centrifuging 100 ml sample of fermentor effluent, resuspending solids in distilled water and centrifuging again to recover solids which are dried overnlght at 100 C.
(c) Value obtained by dividing recovered solids (g/l) by methanol charged (g/l)x 100.
(d) Value obtained by dividing recovered solids (g/l) by retention time (hr).
After about one month, a single tube of the lyophilized HTB-53 microbial culture from the fermentation run of Example I a~ove was opened aseptically by conventional procedures and added to 100 ml of a fermentation medium designated IM2 which also contained 0.5~ methanol. The composition of medium IM2 is ~hown below.
IM2 ~edium C9 ~S~Y~ Amou_t, g XH2PO4 2.0 K2HPO4 3.0 MgS4 7H20 0.4 CaC12 2H20 Narl 0. 1 (NH4)2so4 2.0 Trace Mineral Solution 0.5 ml Distilled Water To make 1 liter . . ..
- ... . : . : .
; .. :" ' ' ~6~
The flask charged with the revived lyophilized culture was incu-bated with shaking a~ 55 C. After 24 hours, the shake flask showed good growth of the culture and 5 ml of the mixture was transferred to 100 ml of medium IM2 also containing 1.5% by volume methanol. Good growth developed within 24 hours and a third tran~-fer was made to the same medium in two flasks, each containing 500 ml of IM2 medium plus 1.5% by volume methanol. This third transfer involved 100 ml of the culture being added ~o each of the two flasks. The culture was allowed to grow for 32 hours and then was utilized as the inoculant for a continuous fermentation run in the apparatus described above in Example I. The fermentor was charged with 1,000 ml of FM-12 mediuml and 1,000 ml of the inoculant to which was added 10 ml of methanol. The temperature was maintained a~ 55 C, and the pH was controlled at from 6.25 to 6.4 by the continuous addition of ammonium hydroxide solution as before. Initially the stirrer was operated at 300 rpm, and air was introduced at a rate of 0.5 liters per minutel while the culture was permitted to establish itsel in the fermentor. After 7 hours, a continuous introduction of feed media having the same composition as the last named feed media shown in Example I above was introduced. In addition, the air rate was increased to 2 liters per minute while the rpm was set at 1,000 for the stirrex~
After 30 hours, the air rate was reduced to 1.75 liters per minute, while oxygen was introduced at 0.75 liters per minute, later increased to 1 liter per minute at 54 hours, and to 1.5 liters per minute at 198 hours. Again the fermentation effluent was sampled from time to time to provide data on the cell content in terms of grams per liter based on a dry weight and in terms of the yield and productivity of the fermentation. The data obtained during the run are presented below in Table II.
.
Table II(a) Time, Hours 70 166 190 214 Retention Time, Hours 2.60 2.70 2~63 2.64 Cells g/l 3~.33 33.56 33.87 34.8 Solids g/l 34.44 35.4 34.55 35~52 Cell Yield ~ 43.6 44.2 43.2 44.2 Productivity g/l/hr 13.2 13.1 13.1 13.5 (a) See footnotes for Table I above.
Samples of the mi.crobial culture were also obtained during the fermentation run for microscopic examina~ion as described in Example I. In this instance, there was also obser~ed the large Gram positive curved rods and the large and small Gram negative staining rods. At 238 hours into the run, the culture was lost while attempting to change ~he feed to a higher alcohol concentra~
tion.
Example III
Presented below in Table III are analytical data character~
izing ~ha microbial ~ulture obtained in the run of Example I in terms o a chemical analysis of the microbial cells recovered. In Table IV there is also presented an amino acid content analysis of microbial cells recovered from another fermentation run util-izing the mixed thermophilic culture of my invention. For purposes of comparison, an amino acid content analysis of a pure thermo-philic microorganism obtained during the course o the isolation ; 30 of the thermophiles from the initial soil sample previously described is also presented in Tahle IV.
. .
6~
Table III
Chemical Analysis of Microbial Cells Obtained in A Run With HTB-53 Crude Protein(a), wt. % 85.63 Ash, wt. % 9.19 Amino nitrogen, wt. % 13.4 Carbon, wt. ~ 44.7 Hydxogen, wt. % 6.79 Nitrogen, wk. % 13.7 Phosphorous, wt. % 1.71 Potassium, wt. % 0.91 Magnesium, wt. ~ O.26 Calcium, wt. % 0.1 Sodium, wt. ~ < 0.01 Iron, ppm 1300 Zinc, ppm 55.8 Manganese, ppm 126 Coppex, ppm 20 (a) Nitrogen content (13.7) x 6.25.
TabIe IY
Amino Acid Content of Thermo~hilic Cultures Grown on MethanoI- Grams .
Per 100 Grams Product _ _ _ .
Chem ~1)( Score(l) Essential Amino Acids Pure ValuesMixed Values (HTB-7) leucine 5.37 75 5.76 104 isoleucine 4.56 83 4.90 118 30 lysine 5.54 96 5.67 141 methionine 1.50 f 1.22 34 ~36 cystine * * t ~hreonine 2.95 90 2.79 88 phenylal~nine 2.68 ( 2.78 : 80 88 tyrosine 2.55 2.72 tryptophan 0.60 43 0.89 90 valine 5.13 91 5.46 121 (*~= not detected) `
,, :- , ~
, &~
Table IV (continued) Amino Acld Content of Thermophilic Cultures Grown on Methanol _ Grams Per lO0 Grams Product . _ Chem.Chem.
Score(1)(2) score(l) Essential Amlno Acids Pure _ Values~-HTB-~2) -alanine 5.65 6.19 lO arginine 3.39 3.01 aspartic acids 6.50 6.26 glycine 3.71 4.19 glutamic acid 10.47 10.69 histidine 1.26 1.22 proline 2.32 2.38 serine 2.09 1.75 Total Essential amino acids 30.88 32.19 Total ~mino Acids66.27 67.88 Crude Protein 85.63 84.4 (l) Chemical Score Values: based on essential amino acid content of egg as 100 for same weight of protein.
(2) Based on averages from five pure thermophiLe runs.
It can be noted that the percentage of total amino acids which are essential amino acids is slightly higher for the mixed culture product (47%) than for the pure culture product (46%). Furthermore, if the essential sulfur-containing amino acids are supplied by addition of synthetic methionine, which is very likely since essentially all SCP's have been ~ound to 30 ba low in these amino acids, the Chemical Score values show that the mixed culture product is twice as good from a nutritional standpoint as the pure culture product based on the next lowest essential amino acid Chemical Score value.
The disclosure, including data, illustrate the value and effectiveness of my invention. The examples, knowledge and ~3 background of the field of the invention, general principles of microbiology, chemistry, and other applicable sciences, have formed the bases from which the broad descriptions of my inven-tion, including the ranges of conditions and generic groups of operant componen~s have been developed, which have formed the bases for my claims here appended.
,' .~' , ' ' . - '
Claims (27)
1. A method of producing a single cell protein material which comprises culturing a mixed culture of thermophilic bacteria species microorganisms NRRL B-8158 in aqueous medium employing an oxygenated hydrocarbon as carbon and energy source under aerobic fermentation conditions at a fermentation temperature of at least about 45° C., and recovering the resulting microorganisms as a single cell protein material.
2. A process for the production of microbial cells which comprises aerobically culturing under thermophilic fermentation conditions at a fermentation temperature in the range of about 45 to 65° C. a strain of mixed thermophilic bacteria derived from a mixed culture deposited as NRRL B-8158 in a culture medium containing at least one oxygenated hydrocarbon as a primary carbon and energy source, nutrients, and a nitrogen source.
3. The process according to claim 2 comprising the further step of separating and recovering said microbial cells so produced from said culture medium.
4. The process according to claim 3 wherein said oxygenated hydrocarbon is characterized as a water-soluble alcohol, ketone, ester, ether, acid, aldehyde, or mixture, containing up to about 10 carbon atoms per molecule.
5. The process according to claim 4 wherein said oxygenated hydrocarbon is methanol, ethanol, propanol, butanol, pentanol, hexanol, 1,7-heptanediol, 2-heptanol, 2-methyl-4-pentanol, pen-tanoic acid, 2-methyl-butanoic acid, 2-pentanol, 2-methyl-4 butanol, 2-methyl-3-butanol, 2-butanol, 2-methyl-1-propanol, 2-methyl-2-propanol, 2-propanol, formic acid, acetic acid, propan-oic acid, formaldehyde, acetaldehyde, propanal, butanal, 2-methylpropanol, butanoic acid, 2-methylpropanoic acid, pentanoic acid, glutaric acid, hexanoic acid, 2-methylpentanoic acid, heptanedioic acid, heptanoic acid, 4-heptanone, 2-heptanone, octanoic acid, 2-ethylhexanoic acid, glycerol, ethylene glycol, propylene glycol, 2-propanone, 2-butanone, diethyl ether, methyl ethyl ether, dimethyl ether, di-n-propyl ether, n-propyl isopropyl ether, or mixture of any two or more.
6. The process according to claim 4 wherein said oxygenated hydrocarbon comprises a monohydric or polyhydric alcohol of 1 to 7 carbon atoms per molecule.
7. The process according to claim 6 wherein said alcohol is methanol, ethanol, propanol, butanol, pentanol, hexanol, 1,7-heptanediol, 2-heptanol, 2-methyl-4-pentanol, 2-pentanol, 2-methyl-4-butanol, 2-methyl-3-butanol, 2-butanol, 2-methyl-1-propanol, 2-methyl-2-propanol, 2-propanol, glycerol, ethylene glycol, propylene glycol, or mixture of any of these.
8. The process according to claim 6 wherein said alcohol contains 1 to 4 carbon atoms per molecule, and is methanol, ethanol, a propanol, or a butanol.
9. The process according to claim 8 wherein said alcohol comprises predominantly methanol or ethanol.
10. The process according to claim 9 wherein said culturing is conducted at a fermentation temperature in the range of about 50° C. to 60° C., and said alcohol comprises methanol.
11. The process according to claim 10 wherein the culture medium is maintained at a pH in the range of about 5.5 to 7.5.
12. The process according to claim 11 wherein said pH is maintained in the range of about 6 to 7.
13. The process according to claim 12 wherein said fermenta-tion conditions are maintained so that the amount of methanol in the fermentor means effluent is in the range of 0 to 0.2 weight percent.
14. The process according to claim 13 wherein said aerobic culturing of said mixed culture includes fermentation conditions employing about 0.02 to 2.1 volumes of oxygen per minute per volume of liquid in said culture medium, and said culture medium is maintained under pressure of about 0.1 to 100 atmospheres.
15. The process according to claim 14 wherein said culturing is conducted under foam culture fermentation conditions.
16. The process according to claim 14 wherein said microbial cells are subjected to lysis.
17. A nonviable single cell protein material prepared by the process which comprises culturing a mixed culture of bacteria microorganisms species NRRL B-8158 in an aqueous medium employing an oxygenated hydrocarbon as carbon and energy source under aerobic fermentation conditions at a fermentation temperature of at least about 45° C. and recovering from the resulting microorganisms said single cell protein material.
18. A nonviable single cell protein material prepared by the process which comprises aerobically culturing, under thermo-philic aerobic fermentation conditions in fermentation means at a fermentation temperature in the range of about 45° CO to 65° C., a strain of mixed bacteria derived from NRRL B-8158 in an aqueous culture medium containing at least one oxygenated hydrocarbon as a primary carbon and energy source, mineral nutrients, and an assimi-lable nitrogen source, thereby preparing microbial cells, and recovering therefrom said nonviable single cell protein material.
19. The SCP according to claim 18 wherein said oxygenated hydrocarbon contains up to about 10 carbon atoms per molecule.
20. The SCP according to claim 19. wherein said alcohol is selected from the group consisting of methanol, ethanol, propanol, butanol, pentanol, hexanol, 1,7-heptanediol, 2-heptanol, 2-methyl-4-pentanol, 2-pentanol, 2-methyl-4-butanol, 2-methyl-3-butanol, 2-butanol, 2-methyl-l-propanol, 2-methyl-2-propanol, 2-propanol, glycerin, ethylene glycol, propylene glycol, and mixture of any two or more of these.
21. The SCP according to claim 19 wherein said oxygenated hydrocarbon comprises a monohydric or polyhydric alcohol of 1 to 7 carbon atoms per molecule.
22. The SCP prepared according to claim 21 wherein said alcohol contains 1 to 4 carbon atoms per molecule, and is methanol, ethanol, a propanol, or a butanol, wherein said culturing is con-ducted at a fermentation temperature in the range of about 50° C.
to 60° C., and wherein the culture medium is maintained at a pH
in the range of about 5.5 to 7.5.
to 60° C., and wherein the culture medium is maintained at a pH
in the range of about 5.5 to 7.5.
23. The SCP prepared according to claim 22 wherein said alcohol comprises predominantly methanol, and wherein said pH is maintained in the range of about 6 to 7.
24. The SCP prepared according to claim 23 wherein said fermentation conditions are maintained so that the amount of methanol in the fermentor means effluent is in the range of 0 to 0.2 weight percent.
25. The SCP prepared according to claim 24 wherein aerobic culturing of said mixed culture includes fermentation conditions employing about 0.02 to 2.1 volumes of oxygen per minute per volume of liquid in said culture medium, and said culture medium is maintained under pressure of about 0.1 to 100 atmospheres and a temperature of about 50° C. to 60° C.
26. The SCP prepared according to claim 18 wherein said recovering of said microorganisms so produced from said culture medium comprises a treating step effective to kill the micro-organisms without substantial harm to the protein thereof, and a separation step separating microorganisms from said culture medium.
27. The SCP prepared according to claim 26 wherein said recovering comprises the steps of acidifying the cultured media containing the microbial cells to a temperature effective to kill the cells without substantial harm to the protein thereof, centri-fuging. washing, and effectuating lysis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA297,516A CA1096228A (en) | 1978-02-21 | 1978-02-21 | Fermentation with thermophilic mixed cultures |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA297,516A CA1096228A (en) | 1978-02-21 | 1978-02-21 | Fermentation with thermophilic mixed cultures |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1096228A true CA1096228A (en) | 1981-02-24 |
Family
ID=4110842
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA297,516A Expired CA1096228A (en) | 1978-02-21 | 1978-02-21 | Fermentation with thermophilic mixed cultures |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1096228A (en) |
-
1978
- 1978-02-21 CA CA297,516A patent/CA1096228A/en not_active Expired
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