CH631054A5 - Use of the microbial strain of the genus Candida in a process for the production of biomasses - Google Patents

Description

The present invention relates to the use of a new microbial strain of the genus Candida in processes for the production of biomass.

Due to the high reproduction speed of microorganisms and their high protein content, the production of microbial biomass is a very rapid method for the production of proteins.

In the past, waste carbohydrates have been used for the production of biomasses, such as molasses from sugar factories or paper mill leaches.

In more recent times, based on the considerable availability and low price of oil, biomass production processes have been developed, using both crude oil fractions and highly purified n-paraffin mixtures as substrate.

The use of these oil substrates has some technological disadvantages due to their insolubility in water, to the large amount of oxygen necessary for their assimilation by microorganisms, and to the large amount of heat carried out during fermentation. Furthermore, biomass production costs are exacerbated by the need to thoroughly purify the substrate and / or to thoroughly wash the biomass produced to remove potentially dangerous oil components.

These difficulties do not exist if biomass production is carried out using lower alcohols, such as methanol and ethanol, as substrates. In fact their complete solubility in water, the volatility and the fact of being available at a high degree of purity make it possible to obtain a biomass free of unwanted residues, their mix with water avoids the mixing problems that they are found with the oil fractions, while due to the fact that they already contain oxygen in the molecule, they require less for their assimilation: hence the further advantage that the production of biomass is accompanied by the carrying out of a smaller quantity of heat, with consequent reduction of cooling costs.

Ethanol is used by a large number of microorganisms and would be the ideal substrate for biomass production, but its price is somewhat high. Methanol, on the other hand, can be produced cheaply with a high degree of purity. This explains the efforts that have been made to find microorganisms capable of efficiently using methanol. Several bacteria that meet these requirements are described in the scientific and patent literature, while the efficiency of the yeasts described so far is rather limited. The use of the microbial strain of the genus Candida according to the invention is characterized in claim 1 above.

The novelty of the strain will be evident from the description of its characteristics.

The SP M 180 cc strain reproduces by multipolar germination, forming separate ovoid cells or small bows formed by several elongated cells (pseudomycelium).

The SP M 180 cc strain was deposited on January 6, 1977 at the Northern Regional Research Laboratory in Peoria, Illinois, USA, under the filing number NRRL-Y 11062.

In liquid soil it forms a sediment, while in solid soil there are either smooth and shiny colonies or wrinkled and opaque colonies. Subcultarating the strain on solid medium, the strain increasingly assumes the smooth and shiny appearance (which corresponds in liquid to the separate cells) while cultivating the strain in liquid medium under certain conditions, the pseudomice-liar form prevails (which corresponds to the wrinkled colonies and opaque). No spores of any kind have ever been observed. For these morphological characteristics, the strain is believed to belong to the genus Candida, according to the classification proposed by L. Led-der, ed. - The Yeasts; A. Tavernemic Study (1970).

The physiological characteristics of the SP M 180 cc strain are as follows:

1. Fermentative use of some sources of C D-glucose +

D-galactose -

Sucrose -

Maltose -

Trehalose -

Lactose -

2. Growth:

D-glucose +

D-galactose -

L-sorbose -

Sucrose -

Maltose -

Cellobiose +

Trehalose -

Lactose -

Melibiosio -

Raffinosio -

Melzitose -

Inulin -

Starch -

D-xylose +

L-Arabinosio +

D-Arabinosio -

D-ribose + (weak)

L-rhamnose -

Ethanol +

Glycerol -

Erythritol -

5

I

15

20

25

30

35

40

45

50

55

60

65

3

631054

Ribitol

+

MgS02, 7H20

0.2

■ "

galactitol

-

FeS04, 7H20

2.0

mg / 1

D-mannitol

+

CaCl2

2.0

»

D-glucitol

+

ZnS04, 7H20

2.0

»

Lactic acid

-

yeast extract

200

»

Succinic acid

-

Biotin

25

y / i

Citric acid

-

10 Trace Elements

1

ml / 1

Inositol Nitrate without vitamins at 37 °

+ (weak)

15

KH2PO4

NaH2P04, hao (NH4) 2S04

2.0 2.0 5.0

8/1

The trace element solution prepared in diluted HCl (1 ml concentrated HCl in 11 of water) had the following composition:

The comparison of the physiological characteristics of the SP M 180 cc strain with those reported in that Lodder study cited, as well as with those reported in the volume "A new kew to the yeasts" by JA Barnett and RJ Pankhurst (1974) showed that the strain SP M 180 cc differs from all the yeast species described there.

To date, several yeasts capable of using methanol have been described in the scientific and patent literature (see CL Cooney and DW Levine in Single-Cell Protein II - MIT Press 1975), but the characteristics of the SP M 180 cc strain differ from those of all the strains of which we have come to know.

A peculiar characteristic of this strain is its ability to assimilate methanol with greater efficiency than ethanol. The strain can be grown in discontinuous cultures and contains, but its properties are best exploited in continuous culture.

Furthermore, due to its ability to form pseudo-mi-celio, a culture can be obtained which sediments with great ease: this property can be exploited to conduct continuous culture with partial biomass recycling (feedback) which allows to obtain a higher hourly productivity. The ease of sedimentation also simplifies the collection of biomass.

In practice, the process consists in inoculating a culture medium with the SP M 180 cc strain, containing the essential elements (N, P, K, Mg, Fe, Ca), the growth factors (yeast extract and biotin), mineral trace elements and, as a source of C and energy, methanol. The broth is incubated under stirring at a temperature between 20-35 ° C, preferably between 30 and 33 ° C, keeping the pH between 2.5 and 6.5, preferably between 4 and 4.5 ensuring a constant supply of a mixture of gas containing oxygen, for example, air.

Yeast cells that multiply at the expense of supplied nutrients are collected by sedimentation or filtration, washed with water and dried by heating.

The biomass obtained can be used as such as a protein supplement in food and feed, or noble products such as proteins, amino acids, nucleic acids can be extracted from it.

The invention is illustrated but not limited by the following examples.

Example 1

500 ml flasks were prepared, containing 50 ml of a medium of the following composition:

20

CuS04, 5H20

H3BO3

Mn SO ,. H, 0

kj

CoCl ,. 6H, 0

25

MoO,

200 500 500 10 10 10

mg / 1

30

35

40

The pH of the medium was brought to 5.0 and the flasks were then sterilized at 116 ° C for 20 minutes. 0.75 ml (1.9% v / v) of methanol was added to two flasks and the flasks were then inoculated by a slant of the SP M 180 cc strain.

The flasks were incubated for 72 hours on a rotary shaker (220 rpm), with a displacement diameter of 3.5 cm, thermostat at 32.5 ° C.

With 5 ml of this preculture other flasks were inoculated containing the same medium and added with 1% v / v of methanol, 2% v / v of ethanol and 2% v / v of glucose; after 24 hours of incubation, 1% v / v methanol was added to the flasks containing methanol. After 48 total incubation hours the biomass content of the flasks was determined, with the following results:

50

Substrate

Optical density (1:10) at 660 nm

Dry biomass g / 1 Glucose protein

0,340

3.99

54.5

methanol

0,270

3.68

51.0

ethanol

0,095

1.18

54.1

Proteins were determined by the biuret method.

Example 2

55 A fermenter having a useful capacity of about 8 1, containing the same medium as in Example 1, was inoculated with a suspension of the SP M 180 cc strain. Methanol was gradually added to the fermenter thermostated at 32.5 ° C, ensuring that the residual concentration of the broth never exceeded 1% v / v.

When the culture had grown well, continuous ag-guinta was started at the sterile broth fermenter, containing 29.6 g / l of methanol, while at the same time a quantity of broth-culture identical to that of the added soil was taken. . The amount of soil added and of broth-culture removed were increased until the dilution rate (D = inlet flow / volume of the cultures) of 0.166 h-1 was reached. Under these conditions the outgoing flow contained

631054

4

10.28 g / 1 of dry biomass at 146 ppm of residual methanol, with a yield of 35% and an hourly productivity of 1.72 g / 1 of biomass. The biomass obtained contained 55.6% protein (biuret).

Example 3

To a fermenter like the one described in example 2 a sedimenter was applied on the outlet flow and a part of the biomass enriched culture was regularly sent back to the fermenter. Adding to the fermenter fresh soil, containing 24 g / 1 of methanol, at a flow such as to reach a dilution speed of 0.267 h_1, a broth-culture containing 7.80 g was obtained in the non-recycled part leaving the sedimenter / 1 of biomass and 120 ppm of residual methanol, with a yield of 32.5% and an hourly productivity of 2.08 g / 1.

The biomass obtained contained 53.0% of protein (biuret).

v

Claims (3)

631054 1. Use of the microbial strain of the genus Candida, deposited on January 6, 1977 at the Northern Regional Research Laboratory of Peoria, Illinois, USA, under the deposit number NRRL-Y 11062, for the production of high protein biomass by cultivation of said strain of the genus Candida, characterized in that the reaction is carried out on a growth medium consisting of a saline solution containing exclusively methanol as a source of energy and carbon. Use according to claim 1, characterized in that the fermentation is carried out at temperatures between 20 ° and 35 °. 2 Use according to one of claims 1 or 2, characterized in that the fermentation is carried out at a pH between 2.5 and 6.5.