TWI487481B - System for culturing nannochloropsis oculata - Google Patents

Description

Cultivating the system of Chlorella

The present invention relates to a system for cultivating Nannochloropsis oculata. In particular, the present invention relates to a Chlorella culture system using a fluorocarbon liquid as an oxygen carrying material. The system can effectively remove the oxygen generated during the growth of Chlorella vulgaris to reduce the growth inhibition effect caused by high dissolved oxygen.

In the prior art, in order to overcome the inhibitory effect of oxygen on the growth of microalgae such as Chlorella, some microalgae photobioreactors use degassing equipment (Degasser) to remove oxygen accumulated in the growing environment; The microalgae photobioreactor itself can violently inject air to remove oxygen accumulated in the growing environment, such as a gas lift reactor. However, the above microalgae photobioreactor has the following disadvantages: (1) high cost; (2) increased access to the culture environment due to a large amount of outside air; (3) dissolved oxygen permeation through the liquid-gas interface The efficiency is not high.

In addition, studies have confirmed that for the microalgae photobioreactor described above, a large amount of air is generated in the culture environment due to the high velocity of air entering the culture environment, the formation of air bubbles continuously at the nozzle of the reactor, and the blasting of air bubbles in the culture medium. Shear stress, but it causes serious damage to the cultured microalgae cells. hurt.

In view of this, the invention is based on the use of four different fluorocarbon liquids as oxygen-carrying materials to improve the oxygen inhibition produced by the culture of Chlorella vulgaris, without causing serious damage to the cultured Chlorella cells. s damage.

An embodiment of the present invention discloses a system for cultivating a chlorella, comprising: placing an oxygen carrier and a cell culture solution in a permeable agitating tank, wherein the oxygen carrier is a fluorocarbon liquid and the cell culture liquid is Walne's medium with a pH of 8; the solutions are immiscible and separate into the lower and upper phases. The permeable agitation tank has a plurality of openings. In use, the temperature of the cell culture solution is controlled to be under 25±2° C., and is continuously irradiated with a light source having an illumination intensity of 80 μE/m ² /s during the culture of the Chlorella vulgaris; The vanes in the tank are mixed with the oxygen carrier and the culture liquid in a continuous stirring manner. The oxygen carrier below can carry the oxygen generated by the organism in the upper culture solution, and a pump is pumped from one of the openings away from the permeable agitation tank into a gas exchange tank, and in the gas exchange tank The inside is contacted with the continuously injected nitrogen gas, and then pumped away from the pump, and again enters the lower portion of the light-permeable stirring tank through the other opening.

20,200‧‧‧Lighting agitation tank (closed three-necked bottle)

60, 600‧‧‧

30, 300‧‧ ‧ gas cylinder

50, 500‧‧‧ light source

10, 100‧‧ ‧ electromagnetic stirrer

40,400‧‧‧Dissolved Oxygen Meter

20a, 30a‧‧‧Perfluorocarbon liquid

20b, 200b‧‧‧Wein medium

200a, 300a‧‧‧hydrofluoroether liquid

70, 700, 90, 900‧‧‧ pipelines

300b‧‧‧ seawater

80, 95, 800, 950 ‧ ‧ openings

1000, 2000‧‧‧ leaves

1500, 2500‧‧‧Oxygen volume flowmeter

Figure 1A is a diagram showing the experimental apparatus of a perfluorocarbon (PFC) photobioreactor.

Figure 1B shows the hydrofluoroether (HFE) light Bioreactor experimental device diagram.

Figure 2 shows the inhibitory effect of oxygen on the growth of Chlorella.

Figure 3 is a graph showing the toxicity test results of four fluorocarbon liquids against Chlorella.

Figure 4 shows the accumulation of oxygen in the four photobioreactor media under continuous perfusion of high oxygenated air (60% O ₂ ), i.e., the four fluorocarbons uptake of dissolved oxygen in the upper medium.

Figure 5 is an analytical curve of the dissolved accumulation rate coefficient of four photobioreactors.

Figure 6 is a graph showing the growth curve of Chlorella in four photobioreactors under continuous perfusion of high oxygen-containing air (60% O ₂ ).

First, it is to be noted that the four different fluorocarbon liquids used in the examples of the present invention are two perfluorocarbons and two hydrofluoroethers, respectively. The two perfluorocarbons are perfluorooctyl bromide (PFOB) and perfluorodecalin (PFD); the two hydrofluoroethers are methoxy-nonafluorobutane (Methoxynonafluorobutane; HFE-7100) and Ethoxynonafluorobutane (HFE-7200). The target cultured organism is a Chlorella sp. which belongs to marine single cell microalgae. The culture device consists of a sealed three-necked bottle, a flow tube device and a gas exchange bottle. The perfluorocarbon and hydrofluoroether systems differ slightly in the setting of the changer bottle. The fluorocarbon liquid in the sealed three-necked bottle is in contact with the cell culture medium below and in the upper layer. The experimental results show that four different materials/devices can successfully overcome the growth inhibition caused by perfusion of high oxygen-containing air (60% O ₂ ), and the algae grows smoothly.

Next, different embodiments of the present algae cultivation system of the present invention will be described based on the drawings.

Figure 1A is a diagram showing the experimental apparatus of a perfluorocarbide photobioreactor. The device is mainly composed of a light-permeable stirring tank (for example, a sealed three-necked bottle) 20, a pump 60, a gas cylinder 30, a magnetic stirrer 10, a dissolved oxygen meter 40, and a light source 50, and the perfluorocarbon liquid 20a is used. Chlorella cultured with oxygen-carrying materials.

The sealed three-necked flask 20 was filled with a perfluorocarbon liquid 20a which was immiscible and divided into lower and upper phases, and a Walne's medium 20b having a pH of 8 as a Nasal cell culture flask. Further, a vane 1000 in the sealed three-necked bottle 20 is continuously stirred with the perfluorocarbon liquid 20a and the Weiin medium 20b.

The gas cylinder 30 is placed in the perfluorocarbon liquid 30a and continuously injected with nitrogen, and the sealed three-port bottle 20 is connected through the pump 60 to form a circulation system. The pump 60 system extracts the oxygen-carrying perfluorocarbon liquid 20a under the sealed three-port bottle 20 through a first opening 80 of the sealed three-port bottle 20 through a first line 70, and sends it to the gas cylinder 30 and the entire gas cylinder 30. The fluorocarbon liquid 30a is mixed, and nitrogen-oxygen gas exchange is performed by the above-described continuous injection of nitrogen. Thereafter, the pump 60 sends the gas-exchanged perfluorocarbon liquid 30a through a second line 90 through a second opening 95 of the sealed three-necked bottle 20 to the underside of the sealed three-necked bottle 20.

In addition, FIG. 1B is a diagram showing the experimental apparatus of the hydrofluoroether photobioreactor. In the embodiment of FIG. 1B, the hydrofluoroether photobioreactor experimental device is mainly composed of a light-permeable stirring tank (for example, a sealed three-necked bottle) 200, a pump 600, a gas cylinder 300, a magnetic stirrer 100, and dissolved oxygen. The meter 400 and the light source 500 are configured to culture the Chlorella vulgaris using the hydrofluoroether liquid 200a as an oxygen-carrying material.

The sealed three-necked bottle 200 was filled with a hydrofluorinated liquid 200a which was immiscible and divided into lower and upper phases, and a Walne's medium 200b having a pH of 8, as a Nasal cell culture flask. Further, a vane 2000 in the sealed three-necked bottle 200 is continuously stirred with the hydrofluoroether liquid 200a and the Weiin medium 200b.

The gas cylinder 3O0 is placed in the hydrofluoroether liquid 300a, the seawater 300b, and nitrogen gas is continuously injected into the hydrofluoroether liquid 300a, and the sealed three-port bottle 200 is connected through the pump 600 to form a circulation system. Among them, the pump 600 system extracts the oxygen-carrying hydrofluoroether liquid 200a under the sealed three-port bottle 200 through the opening 800 of the sealed three-necked bottle 200 through the line 700, and sends it to the gas cylinder 300 and the hydrofluoroether liquid 300a. Mixing and nitrogen-oxygen gas exchange by the above continuously injected nitrogen. Thereafter, the pump 600 passes the gas-exchanged hydrofluoroether liquid 300a through the line 900 to the lower side of the sealed three-necked bottle 200 through the opening 950 of the sealed three-necked bottle 200. In particular, the solution (seawater 300b) used in the upper portion of the gas cylinder 300 is the same as the seawater in the cell culture medium (Wein medium 200b) in the three-neck bottle 200. Further, the nozzle of the line 900 for withdrawing the hydrofluoroether liquid 300a from the gas cylinder 300 shall be placed at the junction of the seawater 300b and the hydrofluoroether liquid 300a.

The operating conditions of the photobioreactor experimental apparatus of Figures 1A and 1B are as follows:

(1) The ratio of the fluorocarbon liquid to the cell culture solution (volume ratio) = 1:5.

(2) The stirring rate of the blades in the photobioreactor = 100 rpm.

(3) The flow rate of the fluorocarbon liquid inlet and outlet system (three-necked flask and gas cylinder) = 6 mL/min.

(4) Supply flow rate of mixed air containing 60% oxygen = 5 mL/min (controlled by an external oxygen flow meter (1500, 2500))

(5) Temperature of microalgae culture = 25 ± 2 ° C; luminosity = 80 μE / m ² / s (continuous illumination by light source (50, 500) during culture of Chlorella vulgaris).

Next, the inhibitory effect of oxygen on the growth of microalgae will be described with reference to Fig. 2; and the experimental results of the respective capabilities of the fluorocarbon photobioreactor of Figs. 1A and 1B will be described with reference to Figs.

Figure 2 shows the inhibitory effect of oxygen on the growth of Chlorella. As can be seen from Fig. 2, when the medium was continuously injected into a mixed air containing 60% oxygen (simulated cells were grown in a high oxygen atmosphere), the growth of the Chlorella was almost completely suppressed. Compared to the case where the oxygen concentration is 0% (i.e., without the injection of oxygen-containing air), the growth of the Chlorella is significantly better. The inhibitory effect of high oxygen environment on the growth of Chlorella was demonstrated.

Figure 3 shows the toxicity test results for four fluorocarbon liquids. It can be seen from the growth curve of the comparative control group (the control group (without fluorocarbon) in the figure) that the four fluorocarbon liquids do not have any toxicity to the Chlorella.

Figure 4 shows the accumulation of oxygen in the medium in four photobioreactors under continuous infusion of 60% oxygen mixed air. It can be seen from Fig. 4 that under the same conditions of continuous perfusion of high oxygen gas, whether using perfluorooctyl bromide, perfluorodecalin, methoxy-nonafluorobutane or ethyl nonafluorobutyl ether As a photobioreactor for oxygen-carrying materials, the cumulative amount of oxygen is significantly reduced compared to those who do not use the above oxygen-carrying materials.

Figure 5 is an analytical curve of dissolved oxygen accumulation rate coefficients in four photobioreactors. The results in Figure 5 show the evaluation of the oxygen accumulation rate coefficient ( K _L a ^ac ) for the cell culture medium under the same conditions of continuous perfusion of mixed air containing 60% oxygen, with the perfluorooctyl bromide group having the lowest K _L The a ^ac value indicates that it has the best oxygen mass transfer capability, which provides the highest oxygen elimination efficiency. The oxygen removal efficiency of the four fluorocarbon liquids is from high to low in order: perfluorooctyl bromide > perfluorodecalin > methoxy nonafluorobutane > ethyl perfluorobutyl ether. The K _L a ^ac values of the four systems were significantly lower than those of the oxygen-carrying materials not used above.

Figure 6 is a growth curve of Chlorella vulgaris in four photobioreactors under continuous perfusion of mixed air containing 60% oxygen. According to the results, the high oxygen concentration environment severely inhibited the growth of Chlorella. However, a bioreactor in which perfluorooctyl bromide, perfluorodecalin, methoxy nonafluorobutane, or ethyl perfluorobutyl ether is added during the cultivation of Chlorella is even in a high oxygen atmosphere. All the cells can still grow successfully. As shown in Fig. 6, the order of biomass after 5 days in each group was perfluorobromooctane>perfluorodecalin>methoxy 9-fluorobutane>ethyl perfluorobutyl ether from high to low. This sequence is consistent with the ability of the material to remove oxygen.

Compared with the current microalgae culture bioreactor, the design concept of the device in this case is not complicated, and the oxygen carrying material (PFC or HFE liquid) used can be recycled, so the cost can be effectively controlled in use. Highly economical.

The device of the present invention can set the biological culture environment to be completely sealed in practical operation, and only the fluorocarbon liquid is in and out of the system, and the microorganism is not easy to grow in the fluorocarbon material, so the operation of the device can be greatly improved. Avoid the chance of the system being infected by the outside world.

The four oxygen carrying materials (PFOB, PFD, HFE-7100, HFE-7200) used in the device of this case have excellent dissolved oxygen properties, and the oxygen transfer is between the same phase (liquid-liquid interface). The experiment proves that the device of this case is excellent in oxygen removal ability with any oxygen-carrying material.

According to the design of the device of the present invention, the fluorocarbon liquid is located under the cell culture liquid and enters and exits the system at the bottom of the photobioreactor, and does not generate shear stress in the biological culture environment, thereby avoiding occurrence on the current device. Shortcomings. In the future, if there is a need to supplement the gas (such as CO ₂ ) required for respiration of the Phytophthora in the culture solution, it can be set in a timed quantitative low speed manner to reduce damage to the cells.

The PFOB and PFD used in the device of this case are two perfluorocarbons which are most widely used in medicine and biotechnology. Therefore, they all have excellent biocompatibility. In the future, this system has the potential to expand its application to animal cell culture.

The HFE used in the photobioreactor of this case has been confirmed to be a substance that is harmless to the environment, ecology and human health. As used HFE-7100 and HFE-7200 have better dissolved oxygen than PFC, and the price is much cheaper than PFC.

In addition, the photobioreactor device of the present invention is small in size, and can achieve a small space and high density microalgae cell culture requirement.

20‧‧‧Lighting agitation tank (closed three-necked bottle)

60‧‧‧ pump

30‧‧‧Ventilator

50‧‧‧Light source

10‧‧‧Electromagnetic stirrer

40‧‧‧Dissolved Oxygen Meter

20a, 30a‧‧‧Perfluorocarbon liquid

20b‧‧‧Wein medium

70, 90‧‧‧ pipeline

80, 95‧‧‧ openings

1000‧‧‧ leaves

1500‧‧‧Oxygen volume flowmeter

Claims (6)

A system for cultivating a chlorella, comprising: a light-permeable stirring tank having at least three openings and an oxygen carrier and a cell culture solution through a first opening, wherein the oxygen carrier is fluorocarbon The compound liquid is not miscible with the cell culture solution and is separated into the lower and upper phases. Wherein the cell culture medium is a Walne's medium having a pH of 8 and the temperature is controlled at 25±2° C., and wherein the vanes in the permeable agitation tank continuously stir the oxygen carrier and a cell culture solution; a light source, continuously irradiating the cell culture medium during the cultivation of the Chlorella algae at an illumination intensity of 80 μE/m ² /s; and a pump for the oxygen carrier under the light-permeable stirring tank Oxygen generated by the organism in the culture medium above is taken away from a second opening of the permeable agitation tank into a gas cylinder, and after contact with the continuously injected nitrogen gas in the gas cylinder The pump is evacuated and re-enters the lower portion of the light-permeable stirring tank through a third opening of the light-permeable stirring tank. The system for cultivating a marine algae according to claim 1, wherein the leaf is located at an interface between the cell culture fluid and the oxygen carrier, and the stirring rate is 100 rpm. The system for cultivating Chlorella vulgaris according to claim 1, wherein the oxygen carrier is perfluorooctyl bromide (PFOB), perfluorodecalin (Perfluorodecalin; PFD), methoxy-nonafluorobutane (HFE-7100), or Eethoxynonafluorobutane (HFE-7200). The system for cultivating Chlorella vulgaris according to claim 1, wherein the volume ratio of the cell culture fluid to the oxygen carrier is 5:1. The system for cultivating a chlorella, as described in claim 1, wherein the oxygen carrier circulates between the permeable agitating tank and the ventilating cylinder according to the dissolved oxygen content and the cell culture above. Set the oxygen accumulation rate in the liquid. The system for cultivating Chlorella sp., wherein the oxygen carrier is methoxy-nonafluorobutane (HFE-7100) or ethyl hexafluorobutyl ether (Ethoxynonafluorobutane; HFE). -7200), a seawater is to be placed in the gas cylinder, and the seawater is the same as the seawater as the component of the Weiner medium, and the oxygen carrier is removed from the mouth of the gas cylinder and the seawater is placed in the seawater. At the junction with methoxy-nonafluorobutane or ethyl nonafluorobutyl ether.