DE2631047A1 - SINGLE-CELL MICRO-ORGANISMS AND PROCEDURES FOR THEIR BREEDING - Google Patents

Description

Snamprogetti SpA
Corso Venezia 16, Milan / Italy

concerning
Unicellular microorganisms and processes for their cultivation

The invention relates to microorganisms and a method for producing biological proteins by the action of microorganisms, the aliphatic alcohols oxidize with low molecular weight.

The microorganisms used for the process according to the invention belong to the Prototheca group.

The invention also relates to protein products derived from microorganisms which are used as nutritional additives can be used for humans and animals.

There are numerous (fermentation) processes in the literature indicated for the cultivation of microorganisms that provide proteinaceous substances. There are e.g. procedures for the cultivation of yeasts on carbohydrates as indicated they are contained in molasses, sulphite baths from paper mills and n-alkane fractions from light gasoline. Similar Methods are also described for the production of biomass with the help of bacteria.

609883/0957

During the progress of the growth directed towards the formation of yeast and bacteria (fermentation) a considerable heat build-up occurs due to the oxidation of the substrate, which is all the more pronounced is, the lower the degree of oxidation of the substrate itself.

The use of partially oxidized substrates is reduced the oxygen consumption and the heat generation. Furthermore, the cooling costs are significantly reduced by the application of microorganisms that grow at high temperatures.

Many methods of biomass formation may require extraction with organic solvents to avoid unwanted substrate residues or harmful compounds such as high molecular weight hydrocarbons or polycyclic hydrocarbons and the like.

Alcohols are substrates that are very suitable for the formation of bioproteins. They are water soluble, already partially oxidized and produced on an industrial scale in high purity. Of the aliphatic alcohols, methanol and ethanol are primarily used.

Economic considerations make it necessary that the substrates for the formation of bioproteins are cheap.

Compared to waste products, the cost of ethanol is relatively high. It is therefore necessary that the substrate is fully utilized.

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It is the object of the present invention to breed strains of heterotrophic algae of the Prototheca species, utilize ethanol as a substrate. There are numerous disadvantages when using ethanol as a substrate other substrates avoided. Of course, ethanol is readily available and can be used as a nutrient. There are no toxicity problems because it is made with high purity, and besides, it is ethanol Volatile and possibly remaining residues can easily be removed during the drying of the cells will. Furthermore, there is no problem with the distribution of the substrate in the nutrient medium, as with polysaccharides and hydrocarbons.

In addition, the strains used according to the invention have the advantage that they at temperatures up to 40 ° C, preferably at 33 to 37 ⁰ C at a wide pH range (pH 1.0 to 8.0), preferably pH 3 »0 to 5 »0 grow, a fact that reduces the risk of bacterial infection during growth. Many microorganisms do not grow in substrates containing alcohol, others only grow when the concentrations are low. The strains used according to the invention grow in the. In contrast, it is also good at high concentrations and only concentrations above 4 % inhibit growth.

The Prototheca strains according to the invention were isolated and selected from soil samples that were taken have been at Harcourt, Nigeria, and are designated by the numbers 855 A, 855 B and 855 C. The cultural and physiological characteristics are summarized in Table I.

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Table I Breeding and Physiological Characteristics

A. Breeding characteristics

solid medium (24 h): (peptone glucose)

liquid medium (24 h): (Pep ton-GLuko se)

Colonies diameter: 2 ms 3 mm, brittle, coarse, opaque, without pigments

Sediment, thin film

B. Characteristics of the vegetation cycle Formation of 2 to 8 aplanospores (endospores) After the mother cell has been broken up, the aplanospores repeat the cycle and some form hypnospores ("dormant cells")

C. Characteristics of the vegetative cells

Spheroid or ellipsoid, 15 to 25 times in size up to 30 / um. Formation of aggregates. Core and storage granules clearly visible.

D. Gender

No sex cycle was observed.

E. Physiological property

1) Utilization of various carbon sources a. fermentative utilization (for growth)

Glucose acidic, without gas, slowly

Galactose negative

Sucrose negative

Maltose negative

Lactose negative

Raffinose negative

Melibiosis negative

Inulin negative

Strength negative

0 ( -Methyl-D-glucoside negative

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b. assimilative utilization D-glucose D-galactose Sucrose fructose maltose lactose raffinose melibiose Inulin soluble starch # -methyl-D-glucoside L-Sorbose Salicin Arbutin Cellobiose Trealose Melicitis D-xylose D-arabinose L-arabinose D-ribose L-rhamnose methanol Ethanol n-propanol Iso-propanol n-butanol tert-butanol Cyclohexanol Glycerin Erythritol Ribit Dulcit D-mannitol sorbitol

from carbon sources positive negative negative positive negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative negative positive positive negative positive negative negative positive negative negative negative negative negative

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Inositol. negative

DL-lactic acid negative

Succinic acid negative

Citric acid negative

η-paraffin positive

2) Utilization of various nitrogen sources Ammonium, nitrate and urea

3) growth factors

requires thiamine

4) Growth at different temperatures, 4 ° C Ms negative 15 42 ° C positive 54 ⁰ C negative

Arnold and Ahearn (Mycologia, Vol. 64, p. 265, 1972) some time ago had a key to classification proposed by Prototheca, according to which the strains according to the invention belong to the group of P. Zopfii species belong.

However, the strains designated with the symbols 855 A, B and C differ from the standard description from P. Zopfii and towards the strains present in the collections (as from Prof. Liferri, Pavia preserved, see tables).

Table II

855 A P. Zopfii P. Zopfii

(Literature)

D-galactose negative positive positive Jso-pentanol negative negative positive growth at 42 ⁰ C positive negative -

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The strain 855 A (wild type) can thus be regarded as a stable and defined variant of the species P. Zopfii. It becomes the name Prototheca Zopfii, Var. Harcourt, "suggested in the standard description given in Table I. The strain was deposited with the Centraalbureau voor Schimmelcultures Baarn and received the number CB5 26S.75.

The cells of Prototheca Zopfii, var. Harcourt 855A, as used according to the invention for the formation of bioproteins, are grown in culture media which contain up to 4% ethanol as a carbon source, nitrogen sources, mineral salts and growth factors. Thiamine can be added as such or in the form of corn water or molasses or yeast extract. The ethanol as a carbon source can be added all at once before fermentation or in small portions during fermentation. However, it is preferred to continuously add ethanol as the fermentation proceeds to enable more complete utilization.

The fermentation is carried out at temperatures in the range of 15 to 40 ° C, preferably 30 to 38 ⁰ C at a pH in the range of 1 to 8, preferably from 3.0 to 5.0.

According to the method described above, a yield of 60 to 70 % (g / biomass / g ethanol) is obtained with a cell concentration in the culture of 20 to 60 g / l.

An exceptional advantage of this procedure is the use of heterotrophic algae of the species Prototheca. Because of their size, they are of course easy to win.

In the case of batch work, the cells are obtained by sedimentation or filtration after complete Fermentation. The same process is used to obtain the biomass in continuous processes.

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The biomass obtained and dried in this way contains approximately 50 to 60 % crude proteins together with polysaccharides, lipids and nucleic acids.

According to the invention, it is possible to produce biomasses with high protein contents by growing heterotrophic ones Prototheca algae in a simple culture medium containing ethanol as the only carbon source.

The invention is illustrated in more detail by the following, non-limiting examples:

example 1

A culture medium of the following composition was prepared:

(NH ₄ ) ₂ S0 ₄ 5 g / l NaH ₂ PO ₄ .H ₂ O 10 g / l KH ₂ PO ₄ . . 10 g / l MgSO ₄ .7H ₂ O 0.2 g / l FeSO ₄ .7H ₂ O 2 mg / 1 ZnSO ₄ .7H ₂ O 2 mg / 1 CaCl ₂ 2 m.g / 1 Yeast extract 500 mg / 1 Solution of trace 1 ml

The trace element solution consisted of: CuSO ₄ .5H ₂ O 5 mg / l

H ₃ BO ₃ 500 mg / l

MnSO ₄ .H ₂ O 500 mg / 1

KJ 10 mg / 1

CaCl ₂ .6H ₂ O 10 mg / 1

MoO ₃ 10 mg / 1

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After adjusting the pH to 5.0, the medium in amounts of in each case was 100 ml in 500 ml flask and after 30 minutes a long time sterilization at 116 ⁰ C of 1% ethanol (vol./vol.) Was added. The flasks were then inoculated with a 3 day old culture (30 ⁰ C) of the stem 855A of a slant containing the same medium and 2% agar, and then with stirring (orbital stirrer) at 220 rpm at 30 ⁰ C incubated. After incubation for 24 hours, a further 2 % ethanol was added.

The dry cell yield after 33 hours of incubation was 18.7 g / l.

Example 2

A fermentation was carried out at 40 ° C., the other conditions of Example 1 not being changed became. The yield of dry cells after incubation for 48 hours was 13.5 g / l.

Example 5

A culture medium was prepared as in Example 1, but instead of yeast extract 1.5 g / l Contained corn water (C.S.L.). The ... yield, as dry weight, after fermentation for 34 hours under the conditions given in Example 1 at 35 ° C. was 17.5 g / l. ■

Example 4

In small laboratory fermenters equipped with a turbine stirrer and 4 (antisloshing) baffle plates and had a volume of 16 1, 10 1 of the medium were given as in Example 1, with the only difference that the concentration of phosphate salts was 2 g / l instead of 10 g / l. After 30 minutes of sterilization

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Sieren at 116 ⁰ C the fermenter with 10% of a 21 hour old culture of the strain 855A in a flask containing the same medium were inoculated as in Example 1, of 35 ° C. The cultivation was performed at 35 ⁰ C under stirring at 1,000 rpm and an aeration of 0.5 vol./vol. χ min. carried out. The pH was kept at 4.0 by adding soda throughout the fermentation period. Ethanol was added at the beginning (1% by volume) and after 24 hours (2% by volume).

After fermentation for 43 hours, the cells were filtered off on paper and, after washing with water, ^{dried at 90 °} C. in vacuo. The yield of dry cells was 50.5 g / l, containing 52.56 % crude proteins (N χ 6.25) and 4.73% lipids.

Example 5

A laboratory fermenter with a volume of 20 1 containing 12 1 of the KuIturmediums used in Example 4 _f, was as indicated in this example, and inoculated 24 hours under stirring (800 rpm) at 35 ° C incubated with introduction of 0.5 vol ./Vol. χ min.air.

Two laboratory fermenters of the type described in Example 4, each containing 10 l of the same culture medium and 1 % ethanol, were inoculated with 1.5 l of this preculture each. The fermenters were stirred at 1000 rpm and 0.5 v / v. χ min. ventilated. The fermentation temperature was 30 ⁰ C of a fermenter, the other of 35 ° C. To adjust the pH value to the desired value (about 4) to _- hold a mixture was used, consisting of 32 F and 95% aqueous ammonia 'Mnanol in the ratio 20: 80 was prepared by adding this mixture not only of the pH kept at an optimal value for growth, but also the carbon source is automatically added.

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After incubation for 20 hours, the concentration of biomass was 20.6 g / l (30 ^° C.) or 21.3 g / l (35 ^° C.).

The agitation speed was then increased to 1500 rpm and 0.75 v / v. χ min. ventilated. After further For 25 hours, the biomass concentrations were 53.2 g / l (30 ° C) and 58.1 g / l (35 ° C).

Mt 2 1 of the 35 ° C culture was a third fermentor of the same type, containing 10 1 of the same inoculated culture medium, and also at 35 ⁰ C with stirring at 1500 rpm and an air feed of 0.75 vol./vol. χ incubated for min. After 8 hours of incubation, the concentration of biomass increased from 10.3 g / l to 33.1 g / l.

Example 6

In a 16-1 fermenter with a turbine pipe and 4 (antisloshing) baffles and contained 7 1 medium, a culture of the strain 855A was accordingly Example 4 produced.

For the continuous fermentation, a nutrient medium (feed solution) was prepared with the following composition:

Na ₂ HPO ₄ .H _£ 0 2.0 g / l KH ₂ PO ₄ 2.0 g / l (NH ₄ ) ₂ S0 ₄ 5.0 g / l MgSO ₄ .7H ₂ O
FeSO ₄ .7H ₂ O 0.2 g / l
22 mg / 1 ZnSO ₄ .7H ₂ O 20 mg / 1 CaCl ₂ 20 mg / 1 Solution of traces
elements
Yeast extract
Ethanol (S ₀ ) 2 ml / 1
0.5 g / l
37.6 g / l

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The medium (without ethanol) was adjusted to a pH of 4.0 and ^{sterilized at 116 °} C. for 30 minutes.

With a dilution rate (D = / u) of 0.190 h ~ ¹ , 2000 rpm and an aeration of 1.5 v / v χ niin. the following results were obtained:

S = concentration of ethanol in

the culture 0.067 g / l

X = concentration of biomass

in the culture 25.12 g / l

Y = yield (g biomass / g substrate) 0.66

P = productivity 4.8 g / lxh

The biomass was centrifuged off and after washing with water it was dried in a vacuum oven. The composition the biomass is given in Table III.

Table III

Analysis data of the biomass from Prototheca Zopfii, Var. Harcourt 855A

Humidity 0.60 %

Ashes 7.04 %

N (Kjeldahl) 8.45 %

raw proteins (Nx6.25) 52.62 %

Protein (biuret) 45.70 %

raw lipids 4.82 %

Fatty acids 3.08 %

soluble carbohydrates 11.96 %

raw fibers 2.50%

RNS 15J7 %

DNS 0.42 %

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Elemental analysis:
C = 46.10 %
H = 6.71 %
N = 8.32 %

percentage composition of fatty acids:

nC _i4 1.38 %

nC _i6 29.12 %

nC _i6 : 1 *) 0.90 %

nC ₁₈ 4.44 %

nC ₁₈ : 1 28.46 %

nC ₁₈ : 2 35.70 %

percentage composition of amino acids (g / 100 g dry biomass)

Lysine 1.88

Histidine 0.779

Arginine. . 5.11

Aspartic acid 3.03

Threonine 1.73

Serine 1.56

Glutamic acid 4.85

Proline. 3.13

Glycine 1.98

Alanine 2.26

Valine 2.11

Methionine 0.83

Isoleucine 1.12

Leucine 2.95

Tyrosine 1.32

Phenylalanine 1.55

Tryptophan 0.23

*) Double bonds

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Claims (4)

Claims nj Single-cell microorganisms of the Prototheca species, characterized in that they have grown in a culture medium which contains ethanol as the only carbon source. 2.0 Microorganisms according to claim 1, characterized in that they are grown in a culture medium in the ethanol in an amount up to 4 wt -% is ent-.Hold.. 3. A method for growing the microorganisms according to claim 1 or 2, characterized in that the fermentation is carried out at a temperature of 15 to 40 ⁰ C, preferably 30 to 38 ° C. 4. The method according to claim 3 »characterized in that the fermentation is carried out at a pH value between 1 and 8, preferably between 3 and 5. 5, use of the microorganisms according to claim 1 or for the production of bioproteins. 6280 .- * ■■ " 609883/095 7