DK142879B - Process for producing single cell protein by aerobically culturing a culture of thermophilic bacteria. - Google Patents

Description

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The present invention relates to a method for producing single cell protein by aerobically culturing one

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culture of thermophilic bacteria at a temperature between 45 ° C and 65 ° C in an aqueous medium containing one or more alcohols having 1-7 carbon atoms as a carbon and energy source, a nitrogen source and a mineral nutrient.

Attempts to remedy protein deficiency have included biosynthesis ie. biological production of single cell protein (SCP) by culturing microorganisms on carbonaceous substrates.

The carbon and energy sources used as substrates should be widely available, relatively inexpensive, uniform, and be sure not to leave harmful residues in the protein product obtained by microbial conversions. Petroleum hydrocarbons have been used as carbon. -and energy source, but has encountered practical difficulties due to poor water solubility, high oxygen demand and alleged traces of potentially carcinogenic substances from the petroleum raw material entering or adhering to the protein product.

In other methods, oxygenated hydrocarbon derivatives have been used as feedstock as they are water soluble and thus easy to handle because the microbial conversion processes are mainly carried out under aqueous conditions.

Such raw materials are readily available from petroleum, natural gas, various waste conversions and methane conversion, fermentation of various grains and destructive distillation of wood. Such oxygenated hydrocarbons are widely available and constitute relatively inexpensive raw materials for fermentation. It is advantageous that the oxygen demand is thus quite small.

Another difficult and limiting factor in commercial production of single cell protein has been the necessity to carry out the fermentation at low temperature, ie. from 20 to 50 ° C, but preferably not above 35 ° C. Such oxidative microbial conversions are exothermic and provide large amounts of heat to be removed continuously and completely from fermentation.

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2 in the mixture, one should not risk overheating the system so that the microorganisms die or their growth is severely constrained by the rise in temperature with consequent loss in performance.

In particular, many have concentrated on the use of yeast species as a microorganism, especially since yeast cells are generally slightly larger than bacterial cells and easier to separate from the fermentation medium.

However, bacteria have the advantage that their crude protein content in the cell is greater than that of yeast in general, since yeast has a higher content of protein-free structural material in the cell. In addition, bacteria usually have a significantly higher true protein content, particularly of the nutritionally important sulfur-containing amino acids and lysine.

Use of bacteria with the ability of rapid growth and high productivity at relatively high temperature for such fermentation would be clearly advantageous. High growth temperature means that less heat needs to be removed, ie. less cooling apparatus is used and afterwards relatively smaller amounts of heat are required to raise the temperature of the medium for sterilization, coagulation and separation. Furthermore, the risk of infection with other microorganisms will be significantly reduced when high temperature fermentation is used. The use of thermophilic bacteria is thus a necessary prerequisite for commercially viable single cell protein biosynthesis.

25 It has now been found that a novel mixture culture of bacteria can be used with great success for the above purposes.

The process according to the invention is characterized in that the bacterial culture is a mixture culture with deposit designation NRRL B-8158. This mixture culture of thermophilic bacteria exhibits precisely these very desirable properties, viz. improved growth at higher temperatures than the conventionally used temperatures, providing higher cell yields with diminished foaming during fermentation, affecting the rational performance of the process. The blend culture is thermophilic, effectively grows under high production at lower

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Alcohols, especially methanol or ethanol, at temperatures where other known bacterial species are either relatively unproductive or simply unable to survive or tolerate this substrate. 5 This particular blend culture is referred to as:

Culture name Tribe designation Deposit designation

Me HTB-53 NRRL B-8158

The designation NRRL B-8158 shows that the thermophilic blend culture is deposited in official depository at United States Department of Agriculture, Agricultural Research Service, North Central Region, Northern Regional Research Center, 1815 North University Street, Peoria, Illinois 61604, namely in form of 30 lyophilized preparations which received from the depot the NRRL designation B-8158. This mixture culture, with the deposit designation NRRL B-8158, comprises A) an aerobic, gram positive, motile, spore-forming, curved rod-shaped, chain-forming bacterium of the class Schizomycetes, order Eubacteriales, B) an aerobic, gram-negative, motile, spore-forming, long rod-shaped 20 bacterium of the genus Bacillus and C) an aerobic gram-negative, motile, non-spore forming, short rod-shaped, dividing bacterium of the Schizomycetes class.

This thermophilic blend culture is compositively stable and obtained after isolation from a fermentation and lyophilization under usual conditions, scan includes rapid cooling of the microbial cells at very low temperature, followed by rapid dehydration under high vacuum and room temperature storage. To determine viability, some of the lyophilized samples are then reacted and subjected to growth of the resulting blend culture under the same conditions as used previously. Such fermentations using reactivated Mc culture give essentially the same results regarding high cell yield, etc., and apparent composition of the fermentation culture given by the original cultures prior to lyophilization. Microscopic examination of the reactivated Mc ~ culture shows the same three organisms in 142879

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4 the same form and context in the reconstructed culture as in the original culture.

In particular, as lower alcohol is used methanol or ethanol, with methanol or a substantially methanol-containing substrate presently being preferred as the cheapest and most effective.

There are clear advantages in using the thermophilic blend culture M in comparison with the use of a pure culture of thermophilic bacteria in the fermentation of methanol or ethanol. First of all, a higher cell yield is obtained using the blend culture. Cell yield as described herein is defined as grams of cells produced per 100 grams of used carbon and energy source material, e.g. methanol. The higher cell yield is an important practical, commercial, economic benefit.

Another advantage of the blend culture in comparison to a pure culture of a thermophilic bacterium is that foam development is substantially smaller than that of the pure cultures studied, which tend to develop large amounts of foam during the fermentation. While foams may be desirable in certain apparatus as auxiliary in heat transfer and in providing the desired amounts of molecular oxygen for the aerobic fermentation, many types of fermenters are not designed or equipped to accommodate or process the large amounts of foam developed by some pure strain microorganisms. Therefore, the moderate amounts of foam developed by the use of the 25 culture culture in question is a distinct advantage.

Another advantage of the blend culture is that it naturally produces single-cell protein as a blend of several kinds of cells, and therefore the composition of amino acids in these microbial cells derived from the M blend culture will be more stable than the composition obtained by growing each one. as pure culture, in the sense that there is less likelihood of a lack of one or more specific, essential amino acids.

The thermophilic blend culture was found in the work of finding and developing a species of culture suitable for such 35 microbial transformations. A soil sample is sampled 61 cm below the earth's surface over a steam pipe at Bartlesville, Oklahoma,

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Research Center of Phillips Petroleum Company. Conventional enrichment technique in the presence of methanol was used to isolate separate cultures from the many native organisms.

In the course of the work in which several pure thermophilic cultures were isolated, it became clear that one of the cultures constituted an unusually stable mixture culture of thermophilic organisms.

The blend culture is composed of three distinctive thermophilic microorganisms which can be described taxonomically as listed below under A, B and C.

6 142879 NKRL B-8158 q Taxonomic description

Cultural property / test result_A B_C_

Culture Bacillus Bacillus Unknown (probably __pseudomonas) _

Track formation yes yes no 5 Aerobic yes yes yes

Average size (ji) 0.5xl-2.50, 6x1-5 0.5x1

Motility yes yes yes

Optimum temp. 55 ° C 55 ° C 55 ° C

Preferred temp. 50-65 ° C 50-65 ° C 45-60 ° C

10 pH range 5-9 5-9 5-9

Optimum pH 6.2-6.8 6.2-6.5 6-7 Growth factors none none - (Culture C seems to require the presence of the other two organisms when growth is to take place on methanol substrate)

Pigments in IM2 light brown H ^ O educate yellow H ^ O educate pink H_0 educate.

medium + 1.5% CH 2 OH unopl. in Organizational Training. in organic atlm. niche rev.

Cell appearance large curved rods large rods small rods for long chains no chains

Colon appearance in circular raised circular raised small, needle tip 20 IM2 medium + 1.5% slightly brownish fractured white

CHjOH

o Growth at 55 C pa; nutritional broth - - + broth + 1.5% CH OH - + 25 _ + IM + + 0.5% CH OH + + + IM + 1.5% CH.jOH + + 1 J + IM 2 + 1.5% glucose - - IM2 + 1.5% CH3CH2OH + + - IM2 + 1.5% HCHO + + NR * BiM + 1.5% CH OH +

0.1% NaCl 6 ++ NR

BHM + 5% CH 3 OH + + NR

Plate count + 1.5% CH30H translucent translucently spilled edges shining small 35 IM2 medium see Example 2 BHM medium: KH2PO4: 2.0 g / l, K2HPO4: 3.0 g / l, MgSO4 O: 0.4 g / 1,

CaCl2'2H2O: 0.04 g / l, (NH4) 2SO4: 2 g / l, trace mineral solution: 10 ml / l containing FeSO4 * 7H2O: 0.11 g / l, ZnSO4 * 7H2O: 0.03 g / l, CuSO4'5H2O: 0.02 g / l, MnSO4-H2O: 0.02 g / l, H2SO4 (conc.): 1 ml / l.

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Repeated separation attempts have been made on this blend culture to isolate these microorganisms individually and purify each one. Eg. resulting culture on a mineral agar medium containing methanol resulted in the formation of isolated colonies of all three organisms. However, when the isolated colonies were transferred to aqueous medium for further cultivation with methanol as a carbon and energy source, surprisingly, no growth occurs, even after repeated experiments.

] _q The coexistence of this mixture culture indicates a symbiotic relationship between the three microorganisms. It seems possible that one or more metabolic products of at least one of the microorganisms serve as the necessary substrate for the washing of one or the other microorganisms. Although the exact nature of such a metabolite-L5 is unknown, it seems possible that it may be toxic to the microorganism producing it, thereby providing an explanation that one or both of the other microorganisms must be present to destroy such a toxic metabolite so that the first microorganism can continue to grow sufficiently.

A further hint of this symbiotic relationship is that fermentations using M are produced

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significantly less foam under typical aerobic conditions than normally found under similar typical aerobic conditions 25 using a purified thermophilic bacterium of the genus Bacillus. Thus, since the blend culture Mc does not produce the usual large amount of foam, it is believed that an extracellular product, probably proteinaceous, is consumed by at least one of the symbiotic microorganisms, the amount of 2 .

The carbon and energy source are one or more alcohols with 1-7 carbon atoms per liter. molecule. Such alcohols include both linear and branched alcohols, primary, secondary and tertiary monohydroxy and polyhydroxy.

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Examples of alcohols are methanol, ethanol, propanol, butanol, pentanol, hexanol, 1,7-heptanediol, 2-heptanol, 2-methyl-4-pentanol, 2-pentanol, 2-methanol-4-butanol, 2-methyl 3-butanol, 2-butanol, 2-methyl-1-propanol, 2-methyl-5 -2-propanol, 2-propanol, glyceral, ethylene glycol, propylene glycol, comprising mixtures of two or more.

The most preferred alcohols are those alcohols containing 1-4 carbon atoms per liter. molecule, especially monohydroxy alcohols, due to easy availability, water solubility and low price.

Methanol is particularly preferred due to relatively low cost, ease of access and total water solubility.

A commercially available material termed "Methyl Fuel" (C&EN, September 17, 1973, page 23] is an example of a commercially available mixture of methanol and a few percent higher alcohols containing up to about 4 carbon atoms per molecule that can be used. as an appropriate substrate,

Aerobic fermentation is well known in the art, and 20 means for adding oxygen to fermentation mixtures are also well known. In general, the addition of molecular oxygen to the aqueous fermentation reaction mixture can be accomplished by passing appropriate amounts of air or oxygen-saturated air and optionally additional pure oxygen through the fermentation-2g container. Waste gases can be recycled, if desired, for maximum utilization of oxygen, e.g. by removing carbon dioxide from the waste and recycle gas. Generally, 0.1 to 10 or more generally 0.7 to 2.5, volume of air per unit of air, must be supplied. per minute to the fermentation mixture 20 volume of liquid in the fermentor, or 0.02 to 2.1, and in particular 0.14 to 0.55 volume of oxygen per liter. volume of liquid per minute.

The pressures used can be widely varied, for example from 0.1 to 100 atmospheres (10.33-10.132 kPa), more commonly from 1 to 30 atmospheres (101.3-3.3039 kPa), but most preferably 1-5 at-35 atmospheres Pressure greater than atmospheric pressure increases the dissolved oxygen content of the aqueous fermentation medium.

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9 142879 medium and thus faster microbial growth. In particular, over-atmospheric pressure is used because the higher temperatures (thermophilic fermentation) decrease the oxygen solubility in the aqueous fermentant.

Cultivation of the M blend culture is carried out at 45-65 ° C, but especially for optimal growth between 50 and 60 ° C. Lower temperatures tend to hinder growth.

High concentrations of some of the described carbon and energy source substrates, e.g. methanol may be inhibitory to satisfactory microbial growth or even toxic to the microorganisms by fermentation of the blend culture and should be avoided.

The fermentation process can be carried out portionwise or continuously. A continuous process is particularly suitable when the carbon and energy source is one of the lower alcohols of methanol or ethanol, which is readily suitable for high efficiency controlled supply. When the fermentor is inoculated with the blend culture, the alcohol is supplied as a separate stream or mixed with water as an aqueous stream for sterilization thereof, or with a mineral agent for sterilization thereof or any of them. Usually, alcohol is added separately for light control of its concentration in the feed stream to the fermentor in the range of 2.5 to 35% by weight, more frequently and conveniently 10-15% by weight.

Cultivation is carried out in an aqueous growth medium comprising an aqueous mineral salt agent, the carbon and energy source material, molecular oxygen and an initial inoculum of the Mc mixture culture.

The fermentation growth rates can be adjusted by the feed material, whose feed rate is controlled so that the amounts fed to the fermentor are substantially the same as the consumption of the organism. This avoids accumulation in the fermentor, especially of toxic materials. A satisfactory control can also be obtained by keeping the feed material content in the fermentor effluent of 0 35 to 0.2% by weight.

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Usually, the residence time of microbial cells in the fermenter is in a continuous process of the order of 2 to 4 hours under such conditions, although this is not critical.

5 The culture Mc requires mineral nutrients and a source of assimilable nitrogen, in addition to molecular oxygen and the described carbon and energy sources. The nitrogen source may be any nitrogen-containing compound capable of releasing nitrogen in a form suitable for metabolic use of the organism. While a variety of organic nitrogen source compounds, e.g. other proteins, urea or the like, may be used, inorganic nitrogen source materials are usually more economical and practical. Such inorganic nitrogen-containing compounds should typically comprise the currently preferred ammonia or ammonium hydroxide, as well as various other ammonium salts, e.g. ammonium carbonate, ammonium nitrate, ammonium phosphate, ammonium sulfate and ammonium pyrophosphate. Ammonia gas is convenient and can be used by simply bubbling it through the aqueous fermentation medium in appropriate amounts.

The pH of the aqueous microbial fermentation mixture should be from 5.5 to 7.5, preferably from 6 to 7. The addition of ammonia helps to maintain the desired pH because otherwise the aqueous medium has an added -25 similarity to becoming slightly acidic. Of course, the preferred pH ranges for microorganisms are generally somewhat dependent on the medium used, and thus the preferred pH range may change at least slightly if the mineral medium changes.

In addition to oxygen, nitrogen and carbon and the energy source, it is necessary to add selected mineral nutrients in necessary amounts and ratios to the feed material to ensure appropriate microorganism growth and to maximize cell assimilation of alcohol by the process.

35 A source of phosphate or other phosphorus, magnesium, calcium, sodium, manganese, molybdenum and copper ions has been found to provide the necessary minerals. One such mineral nutrient medium designated "FM-12" is listed below along with the trace mineral solution which forms part of the FM-12 nutrient medium: 5 Medium FM-12

Component Amount of H3PO4 (85%) 2 ml KC1 1 g

MgSO4-7H2O 1.5 g CaCl2-2H2O 0.2 g

NaCl 0.1 g

Trace mineral solution 5 ml

Distilled water up to 1 liter 15 Trace mineral solution

CuSO 4 * 5H 2 O 0.06 g

At 0.08 g

FeCl-j · 6Η20 4.80 g

MnSO 4 -H 2 O 0.30 g Na 2 Mo 2 O 4 * 2H 2 O 0.20 g

ZnSO4 * 7H2O 2.00 g H3B03 0.02 g H2SO4 (wife) 3 ml

Distilled water up to 1 liter 25 Other mineral medium concentrations can also be used, cf. the examples below.

In portion or continuous operation, all equipment, reactor or fermentation means, boiler or vessel, pipes, accompanying circulating or cooling means, and the like are usually sterilized using steam having a temperature of approx. 121 ° C for several minutes, e.g. ca.

15 minutes. The sterilized reactor is seeded with a culture of the indicated microorganism in the presence of all the necessary nutrients including oxygen and alcohol.

35 Instrumentation to measure cell density, pH, dissolved oxygen content, alcohol concentration must be maintained.

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12 fermentation mixture, temperature, feed rate and the like. It is preferred that the materials added to the fermentation vessel are first sterilized by e.g. ethanol or methanol is added to the other streams, e.g. the mineral medium, in sterilizing amounts, so that separate heat sterilization is avoided.

Addition of antifoam to the fermentation mixture must be avoided because antifoam agents, e.g. silicones, can be detrimental to the dissolved oxygen content at the recommended high fermentation temperatures, and can cause the organisms to grow slower and even kill. Foam developed by I! The culture is not detrimental to growth and is crucially beneficial in maintaining the organisms in a high dissolved oxygen content system. Foam helps to provide relatively large areas of gas / liquid contact interfaces. Thus, fermentation can be improved and heat transfer can be improved for better control, greater uniformity and avoidance of local superheat.

Of course, if desired, a foaming agent, e.g. with certain detergents, preferably non-ionic, to aid in further foaming, although this is usually not necessary or desirable.

Recovery of microbial cells can be accomplished by conventional techniques, e.g. acidification of fermentation effluent to a pH of approx. 4, and heating the acidified effluent to a temperature capable of killing the microorganisms, e.g. ca. 80 ° C, but is low enough not to damage the protein product. The effluent can then be centrifuged, washed and centrifuged to recover the microbial cells from the fermentation effluent. The cells can be processed for cell solution to recover protein and other materials from the cell. The fermentation also provides a number of desirable by-products that can be recovered from the fermentation effluent. These extracellular products can improve the economy of the process as a whole, as they include valuable substances, e.g. polysaccharides, amino acids, eg, glutamic acid, enzymes, vitamins and the like.

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The single cell protein product is a valuable source of protein for use as animal feed, in particular due to its high nucleic acid content. The present patent does not cover the use of the process in the manufacture of foodstuffs.

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Example 1

A continuous fermentation experiment is carried out using the thermophilic blend culture NRRL B-8158. A 7-liter fermentation vessel equipped with an aerator, stirrer, dissolved oxygen control device and equipment for measuring and controlling the temperature and pH of the fermentation mixture is charged approx. 500 ml of a fermentation reaction mixture from a previous fermentation test using the thermophilic blend culture and 10 ml of methanol on inoculation. The reactor is also charged with 2 liters of fermentation mineral FM-12.

The stirring rate is maintained at 1,000 rpm during the experiment and the pH is kept at 6.2 to 6.35 by the addition of ammonium hydroxide solution. Air is introduced into the fermentation vessel at a rate of 2 liters per liter. per minute during operation of the process. After 6 hours, substantially pure oxygen is also introduced into the fermentation vessel at an addition of 0.5 liters per liter. per minute, then increased to 0.75 liters per minute. per minute after 118 hours, to 1.5 liters per minute. after 174 hours and 2 liters per minute. minute after 190 hours.

After 22 hours, the mineral agent is changed to an aqueous mixture containing 7.5% by volume of methanol in addition to medium FM-12 plus 0.75 grams of potassium chloride, twice the normal trace mineral content, three times the normal manganese component content (all per liter basis) and excluding sodium chloride. After 166 hours, the feed material is changed to an aqueous mixture containing 10% by volume of methanol, 2.5 ml of phosphoric acid (85%) per ml. liter, 2 grams per liter. liter of potassium chloride, 1.75 grams per liter. 0.25 grams per liter of magnesium sulfate * 7H 2 O liter of calcium chloride · 20 µl, 20 ml. liters of an aqueous solution of manganese sulphate · H2 O (0.3 g / -35 liters), and 35 ml per liter. liter of trace mineral solution as previously described.

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The rate of addition of the medium ranges from 700 ml per ml. to 817 ml per hour after 22 hours. hour after 118 hours, 781 ml per hour, after 166 hours and 763 ml per hour. hour after 382 hours.

5 Samples from the fermentation effluent are taken from hour to ten minutes to recover cells therefrom. Values obtained for the cell content denoted as dry weight cells in grams per gram. Table I and the calculated yields are given in Table I. The table also lists values calculated for productivity designated as grams of cell -] 0 clay per liter. liter per liter. hour.

At about. 126 hours, samples are taken from the fermentation mixture and prepared for lyophilization of the microbial cells according to known methods. These lyophilized samples are then stored for subsequent use in subsequent fermentation and to provide samples of HTB-53 to a microorganism repository operated by the United States Department of Agriculture, Northern Regional Research Laboratory in Peoria, Illinois.

Periodic samples are also taken from the fermentation reaction mixture for microscopic examination of microbial cells for their gross morphology. Such a microscopic study shows that the M culture is composed of a large gram-positive curved rod, a large gram-negative rod, and a small gram-negative rod. Sometimes a large 25 gram-positive rod (not curved) is also found, but this is assumed to be a short-term variant of the large curved gram-positive rod, cf. the taxonomic description mentioned above.

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Table I

Time, hours 46 118 166 190 286 358

Residence time, hours 2.36 2.27 2.38 2.83 2.33 2.43 5 Cells (a) g / liter 26.86 27.13 27.04 35.21 35.53 35.57

Solids ^ g / liter 26.82 27.66 27.87 35.5 35.66 36.76

Cell yield (c)% 44.7 46.1 46.47 44.4 45.8 45.9

Productivity (d> g / liter / h 11.4 12.2 11.7 12.5 15.7 15.1 (all Value determined by evaporating a 10 ml sample of fermentation effluent overnight at 100 ° C and subtracting the weight of mineral solids contained in 10 ml of medium.

(bl Value determined by centrifuging 100 ml sample of fermentation effluent, resuspending solids in distilled water, and again centrifuging to recover solids which are dried overnight at 100 ° C.

(cl Value determined as recovered solids (g / liter} divided by methanol added (g / liter) x 100).

(dl Value determined as recovered solids (g / liter) divided by residence time (hours).

Example 2

After about 1 month, a single preparation glass with lyophilized HTB-53 microbial culture is opened from the fermentation experiment in 3q Example 1 aseptically in the usual manner and added to 100 ml of fermentation medium designated scan IM2, scan also contains 5% methanol. The composition of medium IM2 is shown below.

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Medium IM2

Component Amount, g KH2PO4 2.0 K2HPO4 3.0 5 MgSO4 * 7H20 0.4

CaCl 2 * 2H 2 O 0.04

NaCl 0.1 (NH 4) 2 SO 4 2.0

Trace mineral solution 0.5 ml 10 Distilled water up to 1 liter

The flask with the reactivated culture is incubated with shaking at 55 ° C. After 24 hours, the culture shows good washing, and 5 ml of the mixture is transferred to 100 ml of medium IM2, which also contains 1.5% by volume of methanol. Good growth is developed over 24 hours and a third transfer is made to the same medium in two flasks, each containing 500 ml of medium IM2 plus 1.5% by volume of methanol. This third transfer causes 100 ml of the culture to be added to each of the two flasks. The culture is allowed to grow for 32 hours and then used as a graft for continuous fermentation in the apparatus described above in Example 1. The fermentation vessel is charged with 1,000 ml of medium FM-12 and 1,000 ml of graft to which 10 ml of methanol is added. The temperature is maintained at 55 ° C and the pH is adjusted to 6.25 to 6.4 by continuous addition of ammonium hydroxide solution as previously mentioned. Initially, the stirrer operates at 300 rpm and air is supplied at a rate of 0.5 liters per minute. per minute, while the culture is allowed to take root in the fermentation vessel. After 7 hours, continuous feed with 30 compositions together as the latter feed material is introduced in Example 1.

Further, the air speed is increased to 2 liters per hour. while stirring is set to 1,000 rpm. After 30 minutes, the airspeed is reduced to 1.75 liters per minute. per minute, while oxygen is introduced at 0.75 liters per minute. per minute, later at 1 liter per minute. per minute at 54 hours, and 1.5 liters per minute. minute at 198 hours. Ferments are tested from time to time for cell contents' 142879

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17 expressed in grams per gram. liters based on dry watch and for fermentation yield and productivity. The data obtained during the trial are given in Table II below.

5 Table II 1

Time, hours 70 166 190 214

Stay time, hours 2.60 2.70 2.63 2.64

Cells 10 g / liter 34.33 33.56 33.87 34.84

Solids g / liter 34.44 35.4 34.55 35.52

Cell yield,% 43.6 44.2 43.2 44.2

Productivity 15 g / liter / hour 13.2 13.1 13.1 13.5 (a) see footnotes to Table I.

Samples of the culture are taken during the fermentation for microscopic examination as described in Example 1. In this case, too, large gram-positive granular sticks and large and small gram-negative staining rods are found. After running the test for 238 hours, the culture is destroyed by attempting to raise the feed's alcohol concentration.

Below Table III provides analytical data, scan is characteristic of the microbial culture used in Example 1, and shows chemical analysis of the microbial cells. Table IV also lists an analysis of the amino acid content of microbial cells extracted from another fermentation experiment using the thermophilic blend culture. For comparison, Table IV also gives an amino acid analysis of a ther-3q mofil microorganism culture generated by isolation from the same soil samples as described above, *

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Table III

Chemical Analysis of Microbial Cells Obtained in an HTB-53 Experiment Ingredient Weight Percent 5 Crude Protein ^ 83.63

Ash 9,19

Aminonitrogen 13.4

Carbon 44.7

Hydrogen 6.79 10 Nitrogen 13.7

Phosphorus 1.71

Potassium 0.91

Magnesium 0.26

Calcium 0.1 Sodium \ 0.01

Trace element ppm

Iron 1300

Zinc 55.8 20 Manganese 126

Copper 20 1

Nitrogen content (13.7) x 6.25

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Table IV

Amino acid content of thermophilic cultures grown on methanol: grams per 100 grams of product: 5 Kem. Kem.

measure.-W <2> measure- ^

Essential amino acids Pure values Mixed values (HTB-7) (HTB-42) leucine 5.37 75 5.76 104 10 isoleucine 4.56 83 4.90 118 lysine 5.54 96 5.67 141

methionine 1.50 to 1.22 C

cystine x "x34 x (_, 36 threonine 2.95 90 2.79 88 phenylalanine 2.68 J80 2.78 Jg tyrosine 2.55 L 2.72 tryptophan 0.60 43 0.89 90 valine 5, 13 91 5.46 121 20 (»= not detected) 0 142879 20

Table IV (cont.)

Amino acid content of thermophilic cultures grown on methanol: grams per 100 grams of product_ 5 Non-essential amino acids_ Pure Mixed (HTB-7) (HTB-42) alanine 5.65 6.19 arginine 3.39 3.01 10 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 amino acids 66.27 67.88 20 Crude protein 85.63 84.4 (11 Chemical measurement values: based on essential amino acid content of eggs as 100 for the same weight of protein.

(2) Based on the average of five experiments with thermo-25 file pure cultures.

It should be noted that the percentage of total amino acids which are essential is slightly higher for the blend culture (47%) than for the pure culture (46%). If the essential sulfur-containing amino acids are determined by the addition of synthetic methionine, the chemical measurement values show that the mixture culture grown here is twice as good from a nutritional standpoint as the cultured pure culture based on the second lowest chemical measurement value for essential amino acids.