CH629372A5 - Process for the production of biomasses by new monocellular microorganisms of the genus Prototheca - Google Patents

Description

The present invention relates to a process for the production of biomass using new single-cell microorganisms of the genus Prototheca.

Numerous fermentation processes are described in the technical literature and in patents for the production of microorganisms that supply protein substances. For example, processes for the cultivation of yeasts on carbohydrates, contained in molasses, sulphurous lye from paper mills and on fractions of n-alkanes of diesel are described. Similar processes are also described for the production of biomass with bacteria.

During fermentation for the production of yeasts or bacteria, a considerable amount of heat develops due to the oxidation of the substrate, which is greater the lower the degree of oxidation of the same.

The use of partially oxidized substrates limits oxygen consumption and heat production.

Furthermore, the use of microorganisms that grow at high temperatures significantly limits the cooling costs.

In many processes for the production of biomass, an extraction with organic solvents may be necessary to eliminate unwanted or harmful substrate residues such as high molecular weight hydrocarbons or polycyclic hydrocarbons etc.

Very suitable substrates for the production of bioproteins are alcohols. They are soluble in water, are already partially oxidized and are industrially produced with a high degree of purity. Among the aliphatic alcohols, methanol and ethanol are used as first-line substrates.

Economic considerations require that substrates for bioprotein production are low cost.

In comparison with waste products, the cost of ethanol is relatively high. It is therefore essential that the substrate is used completely.

The process according to the invention for the production of biomass using the micro-organism of the genus Prototheca is characterized in claim 1 above.

By choosing ethanol numerous drawbacks of other substrates are avoided. In fact, ethanol is readily available and is used as a food. There are no toxicological problems because it is produced with a high degree of purity; moreover, the ethanol is volatile and any residues are easily eliminated during the drying of the cells. Furthermore, there are no problems of distribution of the substrate in the nutrient medium as with polysaccharides and hydrocarbons.

The strains used according to the present invention also have the great advantage of growing at temperatures up to 40 ° C, preferably at 33-37 ° C, and in a wide pH range (pH 1.0-8.0), preferably at pH 3.0-5.0 which decreases

15

20

TABLE 1 Cultural and physiological characteristics

Cultural characteristics solid medium (24 h): (peptone-glucose)

liquid medium (24 h): (peptone-glucose)

colony diameter 2-3 mm crumbly, rough, opaque without pigments; sediment, thin film.

B. Characteristics of the vegetative cycle:

25 Formation of 2-8 aplanospores (endospores).

After rupture of the mother cell, the aplanospores repeat the cycle, some form hypnospores (resting cells).

C. Characteristics of vegetative cells:

30 Spheroidal or ellipsoidal having the dimensions 15-25 X 20-30 [J ,. Aggregate formation. Core and reserve granules clearly visible.

D. Sexual characteristics:

35 No sex cycle has been observed. .

E. Physiological characteristics:

1) Use of various carbon sources a) Fermentation use

40 acid glucose without gas, slow galactose negative sucrose negative maltose negative lactose negative

45 negative raffinose negative melibiosium negative inulin negative starch negative a-methyl-D-glucoside negative

50

b) assimilative use of carbon sources D-glucose positive D-galactose negative sucrose negative

55 positive fructase maltose negative lactose negative refinery negative melibiosum negative

60 negative inulin soluble starch negative a-methyl-D-glucoside negative

Negative L-sorbose negative salicin

65 arbutin negative cellobiose negative trehalose negative melizitose negative

3

629372

D-xylose

D-arabinose

L-arabinose

D-ribose

L-ramnose methanol ethanol n-propanol iso-propanol n-butanol tert-butanol cyclohexanol glycerol erythritol ribitol dulcitol

D-mannitol sorbitol inositol

DL-lactic acid succinic acid citric acid n-paraffin negative negative negative negative negative negative positive positive negative negative negative positive negative negative negative negative negative negative negative negative negative negative positive positive

2) use of various sources of nitrogen: ammonium, nitrate and urea

3) growth factors requires thiamine

4) growth at different temperatures

4 ° C 15-42 ° C 45 ° C

negative positive negative

Arnold and Ahearn (Mycologia 64, 265, 1972) recently proposed a key for the classification of the species of the genus Prototheca, according to which the strains, which are part of the present invention, belong to the P. zopfii species.

However, the strains marked with the numbers 855 A, B and C differ from the standard description of P. zopfii and from a collection strain (obtained by Prof. Liferri - Pavia) (See table 2).

TABLE 2

growth rates and factors. Thiamine can be supplied as such or as a corn steep liquor or molasses or yeast extract. Ethanol as a carbon source can be added to the culture medium all at once before fermentation or in small doses during the course of fermentation. It is however preferable to add ethanol continuously during fermentation to allow a more complete use.

Fermentation is carried out at temperatures between 15 and 40 ° C, preferably at 30-38 ° C, and at a pH between 1 and 8, preferably 3.0-5.0.

According to the process described here, which is part of the invention, a yield of 60-70% (g dry biomass / g EtOH) is obtained with a cell concentration in the culture of 15 20-60 g / 1.

An important advantage of this procedure lies in the use of heterotrophic algae of the genus Prototheca. In fact, due to their size, they are easily recovered.

In the case of discontinuous fermentation, the cells 20 are recovered by sedimentation or filtration at the end of the fermentation. Similarly, we proceed to recover biomass during the continuous process.

The biomass thus obtained and dried contains approximately 50-60% of crude protein together with polysaccharides, lipi-25 di and nucleic acids.

According to the invention, it is possible to produce biomass with a high protein content, by growing heterotrophic algae of the genus Prototheca in a simple cultivation medium containing ethanol as the only carbon source.

30 The foregoing and further operating features will be more clearly understandable by examining the examples set out below for the sole purpose of better illustrating the invention, without however limiting its purposes.

35

Example 1

A culture medium was prepared having the following composition:

45

855 A

P. zopfii

P. zopfii (literature data)

50

D-galactose negative positive positive iso-pentanol negative negative positive growth at 42 ° C positive negative -

(NH4) 2S04

5

g / 1

NaH2P04. H20

10

g / 1

KH2PO4

10

g / 1

MgSOé. 7HzO

0.2

g / 1

FeS04. 7h20

2

mg / 1

ZnSO ". 7h20

2

mg / 1

CaCl2

2

mg / 1

Yeast extract

500

mg / 1

sol. of trace elements

1

me

The composition of the trace element solution was:

CuS04. 5h20

5

mg / 1

H3BO3

500

mg / 1

MnSO ,. H20

500

mg / 1

kj

10

mg / 1

CaCl. 6H20

10

mg / 1

Mo03

10

mg / 1

Strain 855 A (wild type) can therefore be considered a stable and defined variant of the species P. zopfii. We propose the name: Prototheca zopfii var. harcourt with the standard description as in table 1. It has been deposited with the Centraalbureau Voor Schimmelcultures Baarn under the number CB5 26S.75.

According to the invention the cells of Prototheca zonfii, var. harcourt 855 A, used for the production of bioproteins, are grown in culture media containing up to 4% ethanol, as a carbon source, nitrogen sources, mineral salts.

After the pH correction to 5.0, the medium was distributed in portions of 100 ml in 500 flasks and after sterilization (30 min at 116 ° C) 1% ethanol (v / v) was added. The flasks were inoculated with a 3-day culture at 30 ° C of the 855 A strain on slant, containing the same medium plus 2% agar and subsequently incubated (orbital agitation-65, 220 rp, m) at 30 ° C . At the 24th hour of incubation, 2% of ethanol was added again.

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

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4

Example 2

Fermentation was carried out at 40 ° C leaving all the other conditions of example 1 unchanged.

The dry cell yield after 48 h of incubation was 18.7 g / l.

Example 2

Fermentation was carried out at 40 ° C leaving all the other conditions of example 1 unchanged.

The dry cell yield after 48 h of incubation was 13.5 g / l.

Example 3

A culture medium was prepared as in example 1, containing 1.5 g / 1 Corn Steep liquor (C.S.L.) instead of the yeast extract.

The dry weight yield after 34 h of fermentation under the conditions of Example 1 at 35 ° C was 17.5 g / l.

Example 4

In small laboratory fermenters with a 4-breakwater turbine with a capacity of 161, 101 of the soil were prepared as in example 1, with the only difference that the concentration of phosphates was 2 g / 1, instead of 10 g / 1. After sterilization for 30 min at 116 ° C, the fermenters were inoculated in the measure of 10% with a culture of 21 h at 35 ° C of the 855 A strain in an Erlenmeyer flask with the same medium as in example 1. The cultivation was carried out at 35 ° C, 1000 rpm and an aeration of 0.5 v / v / min. The pH was controlled at pH 4.0 with soda throughout the fermentation. Ethanol was added to the principle (1% v / v) and to the 24th hour of fermentation (2% v / v).

After 43 hours of fermentation, the cells were collected by filtration on paper and after washing with water they were dried at 90 ° C under vacuum.

The yield of dry cells was 50.5 g / l containing 52.56% of crude protein (N X 6.25) and 4.73% of lipids.

incubation the biomass concentration went from 10.3 g / l to 33.1 g / l.

Example 6

s In a 16 liter fermenter, with 1 turbine and 4 breakwaters, containing 7 liters of soil, a culture of strain 855 A was prepared as in example 4.

For continuous fermentation a nutrient medium (= feed) was prepared having the following composition:

I

Na ^ PO ,. HjO 2.0 g / 1

KH2P04 2.0 g / 1

(NH4) 2S04 5.0 g / 1

MgS04. 7HzO 0.2 g / 1

15 FeS04. 7H20 22 mg / 1

20 mg / 1

ZnS04. 7H20 CaCl,

sol. of trace elements yeast extract 20 ethanol (Sc)

20 mg / 1 2 ml / 1 0.5 g / 1 37.6 g / 1

The medium (without ethanol) was brought to pH 4.0 and sterilized at 116aC for 30 min.

The following results were obtained at a dilution rate (D = jx) of 0.190 hr1, 2000 rpm / 25 min and an aeration of 1.5 v / v / min:

S = concentration of ethanol in the culture 0.067 g / 1

30 X = concentration of biomass in the culture 25.12 g / 1

YSUb = yield (g biomass / g substrate) 0.66

P = productivity 4.8 g / l.h

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The biomass was collected by centrifugation and after washing with water, dried in a stove under vacuum. The composition of the biomass is shown in table 3.

Example 5

A laboratory fermenter with a capacity of 20 liters and containing 12 liters of the culture medium used in example 4, was inoculated as described therein and incubated for 24 hours at 35 ° C, under stirring (800 rpm) and with the introduction of 0.5 v / v / min of air.

With 1.5 liters each of this preculture two laboratory fermenters of the type described in Example 4 were inoculated and containing 101 of the same culture medium, with 1% of ethanol. The fermenters were stirred at 1000 rpm with an aeration of 0.5 v / v / minute: the incubation temperature of one of the fermenters was maintained at 30 ° C, while that of the other was 35 ° C. To maintain the pH at the desired value (about pH 4) a mixture consisting of 32% aqueous ammonia and 95% ethanol in the ratio 20: 80 was used. In this way not only the pH was kept around the optimal value for growth, but the source of C. was automatically provided

After 20 hours of incubation the biomass concentrations were 20.6 g / 1 (30 ° C) and 21.3 g / 1 (35 ° C) respectively. The stirring speed was then raised to 1500 rpm and aeration to 0.75 v / v / minute. After another 25 hours of incubation, the biomass concentrations were 53.2 g / 1 (30 ° C) and 58.1 g / 1 (35 ° C) respectively.

With two liters of the culture at 35 ° C a third fermenter of the same type was inoculated containing 10 liters of the same culture medium which was also incubated at 35 ° C, 1500 rpm and 0.75 v / v / min of air. In eight hours of

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TABLE 3

Analytical data of the biomass of Prototheca zopfii var. harcourt 855 A

45 humidity 0.60%

ashes 7.04%

N (Kjeldahl) 8.45%

crude protein (N X 6.25) 52.62%

protein (biuret) 45.70%

50 raw lipids 4.82%

fatty acids 3.08%

soluble carbohydrates 11.96%

raw fiber 2,50%

RNA 15.17%

55 DNA 0.42%

60

65

Elementary analysis C H N

46.10% 6.71% 8.32%

Percentage composition of fatty acids:

n-C14

1.38%

n-C16

29.12%

n-C16:

: 1

0.90%

n-CI8

4.44%

n ~ C18:

1

28.46%

n-C18:

2

35.70%

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Percentage composition of amino acids: (g / 100 g of 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

5 methionine 0.83

isoleucine 1.12

leucine 2.95

tyrosine 1.32

phenylalanine 1.55

I tryptophan 0.23

v

Claims (4)

629372 1. Procedure for the production of bioproteins consisting of cultivating the single-cell microorganism of the genus Prototheca, deposited with the Centraalbureau Voor Schimmelcultures Baarn with the number CB5 265.75 in a culture medium containing ethanol as a carbon source, thus obtaining a biomass containing bioproteins. 2. Process according to claim 1, characterized in that ethanol is added to the culture medium up to 4% by weight. 2 3. Process according to claim 1, characterized in that the fermentation is carried out at temperatures between 15 and 40 ° C, preferably between 30 and 38 ° C. 4. Process according to claim 3, characterized in that the fermentation is carried out at a pH between 1 and 8, preferably between 3 and 5. the danger of bacterial infection during fermentation diminishes. Many microorganisms do not grow in substrates containing • alcohols, others grow only if concentrations are low. Our strains, on the other hand, grow well even at high concentrations and only concentrations higher than 4% are inhibitory. The Prototheca strains, object of the present invention, have been isolated and selected from soil samples taken from Harcourt in Nigeria and are marked with the numbers 855 A, 855 B and 855 C. The cultural and physiological characteristics are summarized in table 1 .