Method and System for Heterotrophic and Mixotrophic Cultivation of Microalgae

Abstract

The invention relates to a method for the simultaneous heterotrophic and mixotrophic cultivation of microalgae, to a system for the simultaneous heterotrophic and mixotrophic cultivation of microalgae and to the use of the method and/or the system for the cultivation of microalgae.

Claims

1. Method for the simultaneous heterotrophic and mixotrophic cultivation of microalgae, comprising the steps of a) providing an inoculum comprising at least one microalgae strain and inoculating a culture medium with the inoculum, b) cultivating the at least one microalgae strain in a first device under heterotrophic culture conditions, c) cultivating the at least one microalgae strain in a second device under mixotrophic culture conditions, wherein at least a portion of the at least one microalgae strain is conveyed from the first device in step b) to the second device in step c) and/or from the second device in step c) to the first device in step b), wherein a process gas is introduced at the lower end of the second device, wherein the process gas is introduced so as to be at least partially compressed or at least partially condensed.

2. Method according to claim 1, characterised in that the process gas in step c) is air or a carbon dioxide-enriched air mixture, preferably an exhaust gas from a heterotrophic cultivation from the first device or produced from technical gases.

3. Method according to claim 1, characterised in that the process gas is introduced in a continuous or clocked manner.

4. Method according to claim 1, characterised in that the amount of process gas introduced is 1 cm.sup.3/s to 100,000 cm.sup.3/s, preferably 1 cm.sup.3/s to 10,000 cm.sup.3/s, particularly preferably 10 cm.sup.3/s to 1,000 cm.sup.3/s.

5. Method according to claim 1, characterised in that at least a portion of the at least one microalgae strain is conveyed in a continuous or clocked manner from the first device in step b) to the second device in step c) and/or from the second device in step c) to the first device in step b), preferably by introducing a process gas in step c) and/or by means of a pump.

6. Method according to claim 1, characterised in that the method comprises at least one further step, the further step being selected from phototrophic cultivation, mixotrophic cultivation, harvesting the biomass and/or drying the harvested biomass.

7. System for the simultaneous heterotrophic and mixotrophic cultivation of microalgae, comprising i. a first device (1), the first device being designed for heterotrophic cultivation, ii. a second device (2) comprising a tube made of translucent material and at least one light source, the second device being designed for mixotrophic cultivation, iii. a first connecting element (3) for connecting the first device (1) to the second device (2), wherein the first connecting element (3) connects the bottom of the first device (1) to the inlet of the second device (2), iv. a second connecting element (4) for connecting the first device (1) to the second device (2), wherein the second connecting element (4) connects the upper part of the first device (1) to the outlet of the second device (2), wherein the second device (2) comprises an injector for introducing a process gas at the lower end or the first connecting element (3).

8. System according to claim 7, characterised in that the tube consisting of translucent material is arranged so as to ascend in a spiral shape around a support frame in the form of a truncated cone or a hollow cylinder, the support frame comprising at least one light source.

9. System according to claim 8, characterised in that the second device (2) comprises at least one further light source for irradiating the tube made of translucent material from the outside, the at least one further light source preferably being a surface spotlight.

10. System according to claim 7, characterised in that the translucent material is selected from silicones, polyvinyl chloride (PVC), polyacrylates, polyethylene or borosilicate glass.

11. System according to claim 10, characterised in that the tube made of translucent material consists of at least one further material selected from UVB- and UVC-permeable materials.

12. System according to claim 7, characterised in that the at least one light source is selected from a sodium vapour lamp or an LED spotlight, preferably having a wavelength in the range of from 400 nm to 520 nm or from 600 nm to 720 nm and a light output in the range of 1 to 150 W/m.sup.2.

13. System according to claim 7, characterised in that the second device (2) comprises at least one further light source, the at least one further light source having a wavelength in the range of from 280 nm to 320 nm.

14. System according to claim 7, characterised in that the volume of the first device and the volume of the tube made of translucent material have a ratio in the range of from 10:1 to 1:5.

15. Use of a method according to claim 1 for the cultivation of microalgae.

16. Use of a system according to claim 7 for the cultivation of microalgae.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0082] The invention will be explained in greater detail in the following with reference to some embodiments and accompanying drawings. The embodiments are intended to describe the invention without having a limiting effect thereon.

[0083] FIG. 1 shows a schematic view of the system according to the invention for the simultaneous heterotrophic and mixotrophic cultivation of microalgae comprising a first device 1 and a second device 2 comprising a tube made of translucent material.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0084] The first device 1 is designed for heterotrophic cultivation and is preferably a closed stirred tank which can be operated under sterile conditions. The first device 1 preferably comprises an injector for introducing a process gas 5 and an agitator 6, particularly preferably a Rushton turbine agitator. In the bottom region of the first device 1, there is a first connecting element 3 for the leak-tight connection of the first device 1 to the second device 2. The second device 1 preferably comprises a pump 7, particularly preferably a sterilisable rotary lobe pump. The pump 7 is connected to the tube made of translucent material, an injector for introducing a process gas 10 for the mixotrophic cultivation being located between the pump 7 and the tube made of translucent material. The tube made of translucent material is arranged so as to ascend around a support frame (not shown). The first light source 8 enables illumination from the inside and the second light source 9 enables illumination from the outside. The outlet or the upper end of the tube made of translucent material is connected in a pressure-tight manner to the upper part of the first device 1 via a second connecting element 4.

Embodiment 1

[0085] Chlorella sorokiniana is cultivated in an installation according to the invention in accordance with FIG. 1. The first device 1 and the second device 2 each have a working volume of 400 l.

[0086] The system is filled with 600 l of culture medium I and sterilised. The culture medium I is composed as follows: 1.5 g/l KNO.sub.3; 50 mg/l KH.sub.2PO.sub.4; 100 mg/l MgSO.sub.4.7H.sub.2O; 14 mg/l FeSO.sub.4.7H.sub.2O; 1 mg/l MnSO.sub.4.5H.sub.2O; 0.7 mg ZnSO.sub.4.7H.sub.2O; 0.06 mg/l H.sub.3BO.sub.3.sup.; 0.024 mg/l CoSO.sub.4.7H.sub.2O; 0.024 mg/l CuSO.sub.4.5H.sub.2O; 0.01 mg/l (NH.sub.4).sub.6Mo.sub.7O.sub.24.4H.sub.2O and 10 g/l glucose, the glucose being sterilised separately and only being added after the medium has been sterilised. The culture medium is inoculated with 30 l of inoculum containing approx. 20 g/l of Chlorella sorokiniana via the first device 1 and is continuously pumped into the second device 2 with stirring in the first device 1 and from there back into the first device 1. The second device 2 is illuminated with light in the PAR (photosynthetically active radiation) range at an intensity of 80 W/m.sup.2, based on the inner surface of the second device, which is regarded as a truncated cone. Both devices are temperature-controlled to 32 C. and the pH is automatically adjusted to pH 6.8 with sulphuric acid (H.sub.2SO.sub.4). The Chlorella sorokiniana microalgae are cultivated for 4 days, and the required glucose and minerals are supplied discontinuously according to the consumption generated by the algae growth. The amount and composition of the supplied process gases, in particular the carbon dioxide-enriched gas mixture and the oxygen-containing gas mixture, are regulated separately in the first device 1 and the second device 2 in accordance with the algae growth. After 4 days, the biomass in the installation has reached a concentration of 35 g/l. The biomass is harvested, enriched to 100 g/l using a centrifuge and spray-dried. The biomass has a chlorophyll content of 3.0% and contains 3500 ppm lutein based on the dry matter.

Embodiment 2

[0087] Chlorella sorokiniana is cultivated in an installation according to the invention in accordance with FIG. 1. The first device 1 has a working volume of 100 l and the second device 2 has a working volume of 400 l.

[0088] The system is sterilised and filled with 450 l culture medium II. The culture medium II is composed as follows: 0.5 g/l KNO.sub.3; 1 g/l urea; 100 mg/l KH.sub.2PO.sub.3; 100 mg/l MgSO.sub.4.7H.sub.2O; 14 mg/l FeSO.sub.4.7H.sub.2O; 1 mg/l MnSO.sub.4.5H.sub.2O; 0.7 mg ZnSO.sub.4.7H.sub.2O; 0.06 mg/l H.sub.3BO.sub.3.sup.; 0.024 mg/l CoSO.sub.4.7H.sub.2O; 0.024 mg/l CuSO.sub.4.5H.sub.2O; 0.01 mg/l (NH.sub.4).sub.6Mo.sub.7O.sub.24.4H.sub.2O and 0.5 g/l sodium acetate.

[0089] The culture medium is inoculated with 5 l of inoculum containing approx. 100 g/l of Chlorella sorokiniana via the first device 1. Before the inoculation, 5 g of sodium acetate is added to the inoculum, the pH is lowered to pH 3.2 with H.sub.2SO.sub.4 over a period of 30 minutes and then raised to pH 7.0 with a sodium hydroxide (NaOH) solution. With stirring, the microalgae strain and the culture medium from the first device 1 are continuously pumped into the second device 2 and from there back into the first device 1. The second device 2 is illuminated with light in the PAR range with an intensity of 70 W/m.sup.2, based on the inner surface of the second device, which is regarded as a truncated cone. Both devices are temperature-controlled to 30 C. and the pH is automatically adjusted to pH 7.0 with acetic acid. The acetic acid required for the carbon supply is added discontinuously in a pH-controlled manner in accordance with the consumption generated by the algae growth. With the addition of acetic acid, the necessary nitrogen in the form of ammonium acetate and the trace elements iron and manganese are also added. KH.sub.2PO.sub.4 is dosed separately in accordance with the consumption generated by the algae growth. The amount and composition of the supplied process gases, in particular the carbon dioxide-enriched gas mixture and the oxygen-containing gas mixture, are regulated separately in the first device 1 and the second device 2 in accordance with the algae growth. After 8 days, the biomass in the installation has reached a concentration of 12 g/l. The biomass is harvested, enriched to 100 g/l using a centrifuge and spray-dried. The biomass has a chlorophyll content of 3.5% and contains 4000 ppm lutein based on the dry matter.

Embodiment 3

[0090] Chlorella sorokiniana is cultivated in an installation according to the invention in accordance with FIG. 1 in the form of a revolving method. The first device 1 has a working volume of 100 l and the second device 2 has a working volume of 400 l. The system is sterilised and filled with 450 l culture medium II. The culture medium II is composed as follows: 0.5 g/l KNO.sub.3; 1 g/l urea; 100 mg/l KH.sub.2PO.sub.3; 100 mg/l MgSO.sub.4.7H.sub.2O; 14 mg/l FeSO.sub.4.7H.sub.2O; 1 mg/l MnSO.sub.4.5H.sub.2O; 0.7 mg ZnSO.sub.4.7H.sub.2O; 0.06 mg/l H.sub.3BO.sub.3.sup.; 0.024 mg/l CoSO.sub.4.7H.sub.2O; 0.024 mg/l CuSO.sub.4.5H.sub.2O; 0.01 mg/l (NH.sub.4).sub.6Mo.sub.7O.sub.24.4H.sub.2O and 0.5 g/l sodium acetate.

[0091] The culture medium is inoculated with 5 l of inoculum containing approx. 100 g/l of Chlorella sorokiniana via the first device 1. Before the inoculation, 5 g of sodium acetate is added to the inoculum, the pH is lowered to pH 3.2 with H.sub.2SO.sub.4 over a period of 30 minutes and then raised to pH 7.0 with an NaOH solution. With stirring, the microalgae strain and the culture medium from the first device 1 are continuously pumped into the second device 2 and from there back into the first device 1. The second device 2 is illuminated with light in the PAR range with an intensity of 70 W/m.sup.2, based on the inner surface of the second device, which is regarded as a truncated cone. Both devices are temperature-controlled to 30 C. and the pH is automatically adjusted to pH 7.0 with acetic acid. The acetic acid required for the carbon supply is added discontinuously in a pH-controlled manner in accordance with the consumption generated by the algae growth. With the addition of acetic acid, the necessary nitrogen in the form of ammonium acetate and the trace elements iron and manganese are also added. KH.sub.2PO.sub.4 is dosed separately in accordance with the consumption generated by the algae growth. The amount and composition of the supplied process gases, in particular the carbon dioxide-enriched gas mixture and the oxygen-containing gas mixture, are regulated separately in the first device 1 and the second device 2 in accordance with the algae growth. After 8 days, the biomass in the installation has reached a concentration of 12 g/l. The biomass is harvested, enriched to 100 g/l using a centrifuge and spray-dried. The biomass has a chlorophyll content of 3.5% and contains 4000 ppm lutein based on the dry matter.

[0092] Before the inoculation, 5 g of sodium acetate is added to 5 l of the harvested and enriched biomass, the pH is lowered to pH 3.2 with H.sub.2SO.sub.4 over a period of 30 minutes and then raised to pH 7.0 with a sodium hydroxide (NaOH) solution. With this biomass, the system that has been refilled with culture medium II after the total harvest is inoculated without further intermediate disinfection. The cultivation is continued with the above-described method steps after the inoculation. The harvested biomass has a chlorophyll content of 3.5% and contains 4000 ppm lutein based on the dry matter.

Embodiment 4

[0093] 12 l of a 10% Chlorella sorokiniana suspension obtained in accordance with embodiment 1 are used to inoculate a tubular photobioreactor filled with 1200 l of culture medium III. Culture medium III corresponds to culture medium I, but does not contain glucose. The microalgae culture is cultivated in the photobioreactor over a period of 4 weeks. The cultivation takes place in the natural diurnal rhythm, and a minimum light intensity in the PAR range of 80 W/m.sup.2 is set by additional lighting that can be switched on in the day phase, based on the inner surface of the second device, which is regarded as a truncated cone. The pH is automatically regulated by introducing carbon dioxide (CO.sub.2). Mineral nutrients and trace elements are dosed in accordance with the consumption generated by the algae growth. After 5 days, the biomass in the installation has reached a concentration of 3.5 g/l.

[0094] 400 l of culture suspension is harvested and replaced with fresh culture medium III. This process is repeated until the end of week 4, the last harvest cycle is designed to be a total harvest.

Embodiment 5

[0095] In an alternative configuration of embodiment 1, Chlorella zofingiensis is cultivated in an installation according to the invention in accordance with FIG. 1, with both devices being temperature-controlled to 28 C.

[0096] 12 l of the accordingly obtained 10% Chlorella zofingiensis suspension are used to inoculate a tubular photobioreactor filled with 1200 l of culture medium III. Culture medium III corresponds to culture medium I, but does not contain glucose. The microalgae culture is cultivated in the photobioreactor over a period of 4 weeks at 28 C. daytime temperature and 20 C. night temperature. The cultivation takes place in the natural diurnal rhythm, and a minimum light intensity in the PAR range of 80 W/m.sup.2 is set by additional lighting that can be switched on in the day phase, based on the inner surface of the second device, which is regarded as a truncated cone. The pH is automatically regulated by introducing carbon dioxide (CO.sub.2). Mineral nutrients and trace elements are dosed in accordance with the consumption generated by the algae growth. After 5 days, the biomass in the installation has reached a concentration of 3.0 g/l. 400 l of culture suspension is harvested and replaced with fresh culture medium III. This process is repeated until the end of week 4, the last harvest cycle is designed to be a total harvest. The biomass harvested in each case contains, in addition to other carotenoids, 1300 ppm astaxanthin, based on the dry matter.

Embodiment 6

[0097] Chlorella vulgaris is cultivated in an installation according to the invention in accordance with FIG. 1, with an area of 50 cm of the tube made of translucent material in the second device 2 having been replaced with a separately illuminated tube made of quartz glass. The first device 1 has a working volume of 100 l and the second device 2 has a working volume of 400 l.

[0098] The system is sterilised and filled with 450 l culture medium II. The culture medium II is composed as follows: 0.5 g/l KNO.sub.3; 1 g/l urea; 100 mg/l KH.sub.2PO.sub.3; 100 mg/l MgSO.sub.4.7H.sub.2O; 14 mg/l FeSO.sub.4.7H.sub.2O; 1 mg/l MnSO.sub.4.5H.sub.2O; 0.7 mg ZnSO.sub.4.7H.sub.2O; 0.06 mg/l H.sub.3BO.sub.3.sup.; 0.024 mg/l CoSO.sub.4.7H.sub.2O; 0.024 mg/l CuSO.sub.4.5H.sub.2O; 0.01 mg/l (NH.sub.4).sub.6Mo.sub.7O.sub.24.4H.sub.2O and 0.5 g/l sodium acetate.

[0099] The culture medium is inoculated with 5 l of inoculum containing approx. 100 g/l of Chlorella vulgaris via the first device 1. Before the inoculation, 5 g of sodium acetate is added to the inoculum, the pH is lowered to pH 3.2 with H.sub.2SO.sub.4 over a period of 30 minutes and then raised to pH 7.0 with an NaOH solution. With stirring, the microalgae strain and the culture medium from the first device 1 are continuously pumped into the second device 2 and from there back into the first device 1. The second device 2 is illuminated with light in the PAR range with an intensity of 70 W/m.sup.2, based on the inner surface of the second device, which is regarded as a truncated cone. The portion of the tube made of quartz glass is exposed to UVB radiation. Both devices are temperature-controlled to 27 C. and the pH is automatically adjusted to pH 7.0 with acetic acid. The acetic acid required for the carbon supply is added discontinuously in a pH-controlled manner in accordance with the consumption generated by the algae growth. With the addition of acetic acid, the necessary nitrogen in the form of ammonium acetate and the trace elements iron and manganese are also added. KH.sub.2PO.sub.4 is dosed separately in accordance with the consumption generated by the algae growth. The amount and composition of the supplied process gases, in particular the carbon dioxide-enriched gas mixture and the oxygen-containing gas mixture, are regulated separately in the first device 1 and the second device 2 in accordance with the algae growth. After 8 days, the biomass in the installation has reached a concentration of 8 g/l. The biomass is harvested, enriched to 100 g/l using a centrifuge and spray-dried. The biomass has a chlorophyll content of 3.5% and contains, in addition to carotenoids, 0.05 ppm vitamin D2 based on the dry matter.

LIST OF REFERENCE NUMERALS

[0100] 1 first device, preferably a closed stirred tank which can be operated under sterile conditions [0101] 2 second device comprising a tube made of translucent material [0102] 3 first connecting element [0103] 4 second connecting element [0104] 5 injector for introducing a process gas [0105] 6 agitator [0106] 7 pump [0107] 8 first light source [0108] 9 second light source [0109] 10 injector for introducing a process gas