Method and system for heterotrophic and mixotrophic cultivation of microalgae
12012581 ยท 2024-06-18
Assignee
Inventors
Cpc classification
C12M43/00
CHEMISTRY; METALLURGY
C12M29/18
CHEMISTRY; METALLURGY
C12M31/10
CHEMISTRY; METALLURGY
International classification
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. A 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 in a culture medium that comprises an organic carbon source, c) cultivating the at least one microalgae strain in a second device under mixotrophic culture conditions in a culture medium that comprises an organic carbon source, 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, wherein 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), by introducing a process gas in step c), and wherein during said method, heterotrophic and mixotrophic cultivation of microalgae are performed simultaneously.
2. The method according to claim 1, wherein the process gas in step c) is air or a carbon dioxide-enriched air mixture.
3. The method according to claim 1, wherein the process gas is introduced in a continuous or clocked manner.
4. The method according to claim 1, wherein the amount of process gas introduced is 1 cm.sup.3/s to 100,000 cm.sup.3/s.
5. The method according to claim 1, wherein 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).
6. The method according to claim 1, wherein 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. The method according to claim 2, wherein the process gas in step c) is an exhaust gas from a heterotrophic cultivation from the first device.
8. The method according to claim 2, wherein the process gas in step c) is produced from technical gases.
9. The method according to claim 4, wherein the amount of process gas introduced is 1 cm.sup.3/s to 10,000 cm.sup.3/s.
10. The method according to claim 4, wherein the amount of process gas introduced is 10 cm.sup.3/s to 1,000 cm.sup.3/s.
11. The method according to claim 1, wherein 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), by means of a pump.
12. The method according to claim 1, wherein introduction of the process gas at the lower end of the second device and the at least partial compression or at least partial condensation of the process gas result in the formation of gas bubbles which flow through the second device, as a result of which thin layers are produced in the microalgae culture between the wall of the second device and gas bubbles.
13. The method according to claim 2, wherein the process gas is introduced in a continuous or clocked manner.
14. The method according to claim 2, wherein the amount of process gas introduced is 1 cm.sup.3/s to 100,000 cm.sup.3/s.
15. The method according to claim 2, wherein the amount of process gas introduced is 10 cm.sup.3/s to 1,000 cm.sup.3/s.
16. The method according to claim 3, wherein the amount of process gas introduced is 1 cm.sup.3/s to 100,000 cm.sup.3/s.
17. The method according to claim 3, wherein the amount of process gas introduced is 10 cm.sup.3/s to 1,000 cm.sup.3/s.
18. The method according to claim 12, wherein: the process gas in step c) is air or a carbon dioxide-enriched air mixture; the process gas is introduced in a continuous or clocked manner; and the amount of process gas introduced is 1 cm.sup.3/s to 100,000 cm.sup.3/s.
19. The method according to claim 18, wherein the amount of process gas introduced is 10 cm.sup.3/s to 1,000 cm.sup.3/s.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) 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.
(2)
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
(3) 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
(4) Chlorella sorokiniana is cultivated in an installation according to the invention in accordance with
(5) 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.Math.7H.sub.2O; 14 mg/l FeSO.sub.4.Math.7H.sub.2O; 1 mg/l MnSO.sub.4.Math.5H.sub.2O; 0.7 mg ZnSO.sub.4.Math.7H.sub.2O; 0.06 mg/l H.sub.3BO.sub.3.sup.?; 0.024 mg/l CoSO.sub.4.Math.7H.sub.2O; 0.024 mg/l CuSO.sub.4.Math.5H.sub.2O; 0.01 mg/l (NH.sub.4).sub.6Mo.sub.7O.sub.24.Math.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
(6) Chlorella sorokiniana is cultivated in an installation according to the invention in accordance with
(7) 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.Math.7H.sub.2O; 14 mg/l FeSO.sub.4.Math.7H.sub.2O; 1 mg/l MnSO.sub.4.Math.5H.sub.2O; 0.7 mg ZnSO.sub.4.Math.7H.sub.2O; 0.06 mg/l H.sub.3BO.sub.3.sup.?; 0.024 mg/l CoSO.sub.4.Math.7H.sub.2O; 0.024 mg/l CuSO.sub.4.Math.5H.sub.2O; 0.01 mg/l (NH.sub.4).sub.6Mo.sub.7O.sub.24.Math.4H.sub.2O and 0.5 g/l sodium acetate.
(8) 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
(9) Chlorella sorokiniana is cultivated in an installation according to the invention in accordance with
(10) 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.
(11) 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
(12) 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.
(13) 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
(14) In an alternative configuration of embodiment 1, Chlorella zofingiensis is cultivated in an installation according to the invention in accordance with
(15) 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
(16) Chlorella vulgaris is cultivated in an installation according to the invention in accordance with
(17) 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.Math.7H.sub.2O; 14 mg/l FeSO.sub.4.Math.7H.sub.2O; 1 mg/l MnSO.sub.4.Math.5H.sub.2O; 0.7 mg ZnSO.sub.4.Math.7H.sub.2O; 0.06 mg/l H.sub.3BO.sub.3.sup.?; 0.024 mg/l CoSO.sub.4.Math.7H.sub.2O; 0.024 mg/l CuSO.sub.4.Math.5H.sub.2O; 0.01 mg/l (NH.sub.4).sub.6Mo.sub.7O.sub.24.Math.4H.sub.2O and 0.5 g/l sodium acetate.
(18) 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
(19) 1 first device, preferably a closed stirred tank which can be operated under sterile conditions 2 second device comprising a tube made of translucent material 3 first connecting element 4 second connecting element 5 injector for introducing a process gas 6 agitator 7 pump 8 first light source 9 second light source 10 injector for introducing a process gas