CZ307626B6 - Cultivation process of microalgae strains and use of the microalgae strains cultivated in such a manner - Google Patents

Abstract

SUMMARY OF THE INVENTION In one bioreactor, at least one strain of Sphaeropleales green algae is cultured, after which, after its inoculation, these Sphaeropleales green algae strains are cultured in the bioreactor together with at least one algae strain of the Eustigmatales order.

Description

Technical field

The invention relates to a method of cultivating microalgae strains.

BACKGROUND OF THE INVENTION

Recent methods of cultivating selected species of microalgae with bacteria in order to increase biomass or racial lipid productivity have been described in the literature (see Hong-Wei Yen, PinWen Chen, Li-Juan Chen, The synergistic effects for co-cultivation of oleaginous yeastRhodotorula glutinis and microalgae-Scenedesmus obliquus on biomass and total lipids accumulation, Bioresource Technology, Available online 28 September 2014, and Mauro Do Nascimento, Maria de los Angeles Dublan, Juan César Federico Ortiz-Marquez, Leonardo Curatti, High lipid productivity of Ankistrodesmus-Rhizobium artificial consortium, Bioresource Technology, Volume 146, October 2013, Pages 400-407). There is also a known mixed culture of microalgae with other aquaculture species such as rainbow trout, oysters, or rice roots.

WO 2009/130895 discloses a process for obtaining flucoxanthine (carotenoid, brown dye) alone or flucoxanthine together with polyunsaturated fatty acids.

WO 2012/012671 describes, inter alia, cultures of photosynthesizing microorganisms, i.e. microalgae, which have a shell and can thus provide a substrate on which biofuel producing microorganisms can grow. Photosynthesizing microorganisms - algae produce lipids and non-photosynthesizing microorganisms produce a solvent in which lipids are soluble.

WO 2008/036654 discloses a process for obtaining medium chain fatty acids from a culture of a first algae strain (also including microalgae and cyanobacteria) and hydrocarbons from a culture medium of a second or further algae strain.

The disadvantages of these methods are, in particular, the complicated dilution of the cultures and control of the ratio of the individual strains in the mixture. During cultivation, it is also difficult to optimize the nitrogen dosage to control the formation of the desired metabolites.

SUMMARY OF THE INVENTION

SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for culturing microalgae strains which does not have the disadvantages mentioned above.

This problem is solved by culturing at least one Sphaeropleales green algae strain in one bioreactor, and then, after inoculation, these Sphaeropleales green algae strains are cultured in the bioreactor together with at least one Eustigmatales algae strain.

The advantage of the method according to the invention is, in particular, the possibility of easily adjusting the desired composition of the microalgae strain mixture and easily optimizing the production of the desired metabolites (optimizing the polyunsaturated fatty acid content by limiting the medium irradiation in culture, optimizing the carotenoids and neutral lipids by nitrogen limitation). for cultivation.

In the process according to the invention, they are preferably cultivated as a green algae of the order Sphaeropleales, in particular the green algae Scenedesmus sp., And as a strain of the algae of the order Eustigmatales, in particular

Eustigmatophytic algae Trachydiscus sp, or eustigmatophytic algae Trachydiscus minutus. The green Scenedesmus sp., Containing non-dividing cells inoculated into Trachydiscus minutus, can be cultured.

Microalgae strains cultivated by the method of the invention are used as a nutritional supplement for both animal and human nutrition and for extracting a mixture of polyunsaturated fatty acids and carotenoids.

Clarification of drawings

The invention is further elucidated by way of example with reference to the drawings, in which:

Giant. 1 depletion of nitrates from the culture medium during cultivation of the COBIEM 34 strain,

Giant. 2 co-cultivation of the COBIEM 18 and COBIEM 34 strains, which were cultured separately, at 336 h were diluted and mixed in a cell ratio of 4: 1 (COBIEM 18: COBIEM 34) and cultured in a mixture,

Giant. 3 is a photomicrograph of a mixed culture of COBIEM 18 and COBIEM 34 after 5 days of co-cultivation, the photo on the left shows the autofluorescence of chlorophyll and lipid inclusions in the COBIEM 18 strain, the photo on the right shows the same situation in a light microscope.

DETAILED DESCRIPTION OF THE INVENTION

The method is based on the cultivation of two different, systematically distant species of microalgae accumulating different metabolites - Sphaeropleales and Eustigmatales algae, the first of which is characterized by high accumulation of carotenoids in cellular biomass and the second by high accumulation of polyunsaturated fatty acids in its cellular biomass. An example of a polyunsaturated algae accumulating fatty acid is Trachydiscus minutus strain COBIEM 18, and an example of an algae accumulating carotenoid is Scenedesmus sp. strain COBIEM 34, both from the collection kept by the Institute of Botany AS CR. Because the strain COBIEM 34 exhibits a significantly higher specific growth rate than COBIEM 18 (0.126 ± 0.008 h and 0.045 ± 0.008 h ', respectively), the culture of these strains is associated with the risk of unregulated dominance of cells of a faster growing species at the expense of slower growing. Therefore, the process of the invention takes advantage of the fact that the COBIEM 34 strain immediately ceases to divide cells after nitrogen has been depleted from the growth medium. When using 2x diluted medium 1/2 Simmer Setlík (with a concentration of 10 mM NO ₃ -), the nitrogen (contained in the medium in the form of nitrates) is almost completely depleted from the medium already on the first day of cultivation (Fig. 1).

Mixture cultivation itself occurs after inoculation of a COBIEM 34 culture containing non-dividing cells into a COBIEM 18 stationary culture diluted with deionized water to the desired cell concentration ratio of the individual strains. Thus, the resulting medium contains minimal amounts of nitrogen, which prevents cell division and accumulation of specific metabolites may occur (Fig. 2).

Example 1

A culture experiment with delayed inoculation of the second strain was performed in a thin layer photobioreactor with Scenedesnus sp. (carotenoid source) and T. minutus strain Lukavský et Přibyl 2005/1 (source EPA). Total volume of culture medium 145 liters. At the beginning and end of the experiment, samples were taken to determine:

-2GB 307626 B6

1) the ratio of T. minutus to Scenedesmus sp. in suspension which were:

start of experiment: 99.9% T.minus, cell concentration: 2156 cells per ml; dry matter: 0.28 g / L end of experiment: cell ratio: 77.5% T.minus and 22.5% Sc. sp., final biomass: 8.81 g / L of which 2.618 g / L Sc. properties Sp. and 6.192 g / L of T.m. total dry matter of algae mixture: 1,233 kg

2) carotenoids and polyunsaturated fatty acids. Major carotenes were determined by paper chromatography (solvents used were toluene / ethanol 400: 1). Depending on the position of the spots, Scenedesmus sp. mainly beta-carotene (1.4% in dry matter) and lutein (1.3% in dry matter). The total content of selected polyunsaturated fatty acids was determined by gas chromatography and was 3.15 g ARA / kg algae dry matter 16.31 g EPA / kg algae dry matter.

Example 2

The experiment was carried out under laboratory conditions, the algae were cultivated in bubbling cylinders with 1/2 Simmer Šetlík medium, at a constant irradiation of 500 μηιοί m ² with a temperature of 30 ° C and saturated with 2% CO ₂ in air. The slower growing algae Trachydiscus minutus COBIEM 18, which accumulates unsaturated fatty acids, was cultured for 14 days in a batch mode. At 12 days, the cultivation of the rapidly growing Scenedesmus sp. COBIEM 34 producing high amounts of carotenoids and after two days of cultivation, both strains were mixed and diluted at a cell ratio of approximately 4: 1 (ie COBIEM 18: 36.02x10 ⁶ cells / ml, COBIEM 34: 9.49x10 ⁶ cells / ml in the resulting mixtures). The cultivation was then continued under the same conditions for another 5 days.

The mixed culture biomass was 7.41 ± 0.03 g L ¹ DW at the start of the cultivation and reached 10.19 ± 0.15 g L ¹ DW within 5 days. Due to nutrient limitation, especially nitrogen, accumulation of lipid-like metabolites can be expected, as indicated by micrographs of mixed culture at the end of the experiment (Fig. 3). At the beginning and end of the cultivation, samples were taken for fatty acid and carotenoid analyzes.

Industrial applicability

In particular, the invention can be used to obtain biomass for both animal and human nutrition.

PATENT CLAIMS

Claims (3)

Method for culturing microalgae strains for extracting a mixture of polyunsaturated fatty acids and carotenoids, characterized in that at least one Sphaeropleales green algae strain is cultivated in a bioreactor, and after their inoculation these Sphaeropleales green algae strains are cultured in the bioreactor together with at least one strain of algae of the order Eustigmatales. 2. A method for cultivating microalgae strains according to claim 1, characterized in that the green algae Scenedesmus sp. and as eustigmatophytic algae of the order Eustigmatales eustigmatophytic algae Trachydiscus sp. The method of culturing microalgae strains according to claim 2, characterized in that the green algae Scenedesmus sp., Containing non-dividing cells, inoculated into the algae Trachydiscus minutus is cultivated.