JPH11346760A - Cultivation of microalga - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for cultivating a large quantity of microalgae in high productivity. SOLUTION: This method is conducted by cultivating microalgae by light irradiation in a cultivation reactor while keeping the content of microalgae in a cultivation liquid at a constant level. As the microalga, e.g. Synechocystis PCC6714 PDI (FERM P-14970) may be used, the cultivation is conducted by keeping the microalgal content in the cultivation liquid at a constant value falling within e.g. 100 to 300 mgC/L in terms of the content of the whole organic carbon.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for culturing microalgae, and more particularly, to a method for culturing microalgae with improved productivity in mass cultivation.

[0002]

2. Description of the Related Art Among photosynthetic organisms including terrestrial higher plants and seaweeds, microalgae are said to be the kind of organisms with the highest photosynthetic efficiency per area if culture conditions are suitable. Attention has been focused on the production of industrial raw materials, feed, fine chemical raw materials, etc. by mass culture of algae. However, few of them have been put to practical use, and one of the causes is low productivity. As means for improving productivity, selection and screening of microalgae suitable for culture production and improvement of a reactor for culture have been performed. Culture reactors are broadly divided into two types: one for open pond systems, which is characterized by large-scale applications such as culture ponds, outdoors, and sunlight, and the other for optical fibers and temperature control. It is for a closed system, which is a culture method that skillfully combines these methods.

As an example of selecting microalgae, the present inventors
As previously proposed in Japanese Patent Application No. 7-322966,
Microalgae with a focused light collector size, sunlight or equivalent
Concentration that shows the self-shielding effect of the microalga under strong light conditions
And a method characterized by culturing at a cell concentration of more than
Microalgae Synechocystis with reduced condensing part size
 PCC6714 PD1 (FERM P-1497
0) "to increase productivity in outdoor culture ponds.
Can be improved. What is culture in an outdoor pond?
Outside, for example, a culture tank of 20 cm, 30 cm to several tens cm deep
Drilling (pond) km ^TwoProduce microalgae roughly in the order of size
To be cultured, control of cell concentration, temperature control, microbiology
It was pointed out that it was difficult to control the contamination of objects and small animals.
Have been.

Therefore, in recent years, attention has been paid to improving productivity by culturing in a closed system such as using a transparent pipe or the like for a culturing tank. As a closed system, various types of culturing reactors and improvements have been developed. Proposed. Most of such culture reactors are designed for the purpose of culturing at a relatively high concentration.
The culture conditions can be controlled to a high degree because the scale is much smaller than an outdoor culture pond of about several tens m ³ .
Even in the culture reactor, the same energy source as that of the outdoor culture pond is used as the light energy supply source. Therefore, how to efficiently use the sunlight has been an issue of this system.

[0005] By the way, what is called a batch type (batch type) culture and a continuous culture are known as a culture method. Culture of microalgae is performed at a high concentration due to equipment cost, light utilization efficiency, ease of recovery from the culture solution, etc., but if the microalgae concentration becomes too high during culture, it may be shaded by other microalgae. Since the proportion of microalgae increases and CO ₂ is re-released by dark breathing, culturing at a very high concentration has been avoided. Specifically, when the growth of microalgae has progressed and the ratio in the culture solution has increased, the microalgae is removed from the medium, for example, by collecting the microalgae, and a batch-type culture in which the microalgae is again cultured at a low microalgae concentration is performed. Was. However, there has been no report on quantitative examination of microalgal productivity and microalgal concentration management. In addition, in the case of mass culture of microalgae, there is no report on which of conventional batch culture and continuous culture has higher productivity. The present invention has been made in view of the above situation, and has as an object to provide a culture method which is advantageous for mass culture of microalgae and has improved productivity.

[0006]

Means for Solving the Problems The present invention provides (1) a method for culturing microalgae under light irradiation in a culturing reactor, wherein the culturing is performed while keeping the microalgae concentration in the culture solution constant. (2) The microalgae concentration is set to 100 mg / L or more and 300 mg / L or less (C: organic carbon) in terms of the total amount of organic carbon in the culture solution. The method of culturing the microalgae described, and
(3) The microalga is Synechocystis PCC6714
The method for culturing microalgae according to the above (1) or (2), which is PD1 (FERM P-14970).

[0007]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present inventors paid attention to the relationship between microalgae concentration during culture and microalgae productivity, and used a closed system that allows easy control of culture conditions. Continuous cultivation (hereinafter abbreviated as "continuous cultivation") and B: batch cultivation without controlling the microalga concentration (hereinafter abbreviated as "batch cultivation") were attempted, and the productivity of both was compared. As a result, it was confirmed that the productivity of microalgae can be increased by continuous culture controlled to a suitable microalgae concentration, and the present invention has been achieved. The implementation of the present invention may be an open pond system or a closed system, but using a closed system is advantageous in that it is easier to control the concentration of microalgae and can highly control various parameters. It is practical. The productivity of the microalgae includes the growth amount of the microalga itself and the production amount of a specific substance produced in the cells. In the present invention, the productivity was compared based on the growth amount of the microalgae.

FIG. 1 is a schematic diagram for schematically explaining the present invention. A culture medium and microalgae are put in a reactor for culture (which may be provided with a ventilation / exhaust means, a heating means and / or a stirring means). Under the irradiation of light from a light source, the concentration of the microalgae in the culture solution is measured while culturing is continued in a predetermined atmosphere, and the culture medium supply amount and the culture solution (microalgae) recovery amount are adjusted based on the measurement result (concentration information), and the culture is performed. Control the concentration of microalgae in the liquid to the set value. That is, when the measured value is higher than the set value, the medium is supplied into the culture reactor, and when the measured value is lower than the set value, the supply of the medium is stopped to wait for the medium to grow to the set concentration.
Thereby, the microalgae concentration in the culture reactor is maintained at the set value, and the culture can be continued at the most preferable microalgae concentration. The concentration measurement is performed online, the microalgae concentration information is input to a culture maintenance device such as a computer, and a concentration control signal (for example, a pump flow rate control signal, etc.) is output to output the pump flow rate of the medium supply means and the culture medium recovery means. By controlling the pump flow rate, a very high degree of concentration control can be realized.

[0009] The concentration of microalgae can be displayed in various ways depending on the measurement method. That is, the number of cells is directly counted using a microscope and a hemocytometer, and is indicated as the number of cells per unit volume (ml) of the culture solution, or expressed as turbidity from turbidity measurement using a commercially available spectrophotometer. Represents the total amount of organic carbon per unit volume (liter) of the culture solution. In the present invention, since the culture may be performed while maintaining the concentration of the microalgae during the culture at a constant level, the measurement method is not particularly limited. In the examples described later, the productivity of microalgae is compared with the growth amount of microalgae per unit time, that is, the increase amount of total organic carbon per unit time. It is shown in the amount (mg), and mgC /
Expressed as liter.

[0010] When the productivity of microalgae is compared with the increase in the amount of total organic carbon as described above, it is ideal to measure the total organic carbon online, but this is not possible with the current commercial equipment. Therefore, the present inventor prototyped a turbidity meter that allows easy online measurement, confirmed that when this turbidity meter showed a constant value, the total organic carbon content also showed a constant value, and managed the cell concentration online. Was performed with the turbidity meter, and the total organic carbon at the time when it was confirmed that the culture was performed stably was measured. The final production rate was calculated from the total organic carbon measurement value. A more specific method will be described in an example described later.

The microalga concentration conditions in the continuous culture of the present invention are slightly different depending on the type of microalga and other conditions. In an example of the present invention, at least 100 mgC / liter or more in a culture reactor having an optical path length of 20 cm.
mg / C or less. When the concentration of microalgae is less than 100 mgC / liter, the disadvantage of reduced productivity due to strong light inhibition (also referred to as photoinhibition) is another 300 mg.
Above C / liter, there is the disadvantage that the consumption of photosynthetic products by dark respiratory volume at night increases.

(Action) FIG. 4 is a graph showing the relationship between the culture time and the concentration of microalgae in the culture solution [Cppm / liter (C means organic carbon)] in an example of batch culture. As shown in FIG. 4, in the batch culture, at a stage where the concentration of microalgae is low, the production rate of microalgae (the amount of increase in microalgae concentration per unit time) temporarily decreases. This is because most of the microalgae that had once grown to a high concentration is recovered, and a part of the microalgae is inoculated on a new medium and the next batch of culture is started. This is considered to cause a bad influence on the growth. The present invention manages the concentration of microalgae and continuously cultures them while maintaining them at a certain concentration range that is optimal for the growth of the microalgae. It is thought that it can be greatly improved.

The culture reactor used in the present invention is not particularly limited, and may be any of an open pond system and a closed system, and any of those known in this field can be used. Specific means for measuring the concentration of microalgae in the reactor for culture and controlling the concentration at a constant level will be described in Examples described later, but is not limited thereto. Other culture conditions other than the microalga concentration conditions are appropriately selected according to each microalga.

[0014]

EXAMPLES In accordance with the present invention, a light-harvesting pigment-reducing strain PD-1 (FERM P-14970) of the microalga Synechocystis PCC6714 was continuously cultured using the apparatus shown in FIG. The PD-1 strain was a wild strain Synechocystis PCC obtained from the National Institute of Basic Research, Okazaki Collaborative Research Organization.
It is a mutant strain in which the light-collecting portion size is genetically reduced by artificially mutating 6714, and is deposited with the National Institute of Advanced Industrial Science and Technology under the accession number FERMP-14970.

In FIG. 2, a culture reactor body 2 formed of a transparent material capable of transmitting irradiation light from a light source 1 is provided with a stirrer 3 as a stirring means, and a culture thermostat similarly formed of a transparent material. It is arranged in the tank 4. The culture medium 6 in the culture medium tank 5 is supplied into the culture reactor main body 2 via the culture medium supply line 8 by the culture medium infusion pump 7. Also, the medium 6 in the culture reactor body 2
Microalgae 9 is added (seeded) therein to a predetermined concentration. Reference numeral 10 denotes a culture solution comprising the medium 6 and the microalga 9. An atmosphere gas (air with 1% CO ₂ added) 12 introduced from an atmosphere gas inlet 11 is supplied into the culture solution 10,
Exhaust gas is exhausted from the exhaust port 13. While using an incandescent light bulb as the light source 1 and irradiating it under the condition of a light intensity of 2000 μM photone m ^-2 sec ^-1 (sunlight level at noon level), the culture medium 6 was irradiated.
Using an MDM medium having the composition shown in Tables 1 and 2, the inside of the culture reactor main body 2 was maintained at 25 ° C. by a culture thermostat 4, and cultured with stirring by a stirrer 3. Microalgae concentration setting: high concentration: 300 mgC / liter, low concentration: 10
Two cases of 0 mgC / liter were performed.

[0016]

[Table 1]

[0017]

[Table 2]

While continuing the culture, the culture solution suction pump 14
Is operated to collect the culture solution 10 into the culture solution reservoir 20,
The turbidity of the culture solution 10 is measured by a turbidity meter 16 provided in the middle of the culture solution recovery line 15, and a turbidity data signal 17 obtained from the turbidimeter 16 is input to a computer 18 and set from the input signal 17. The amount of medium to be adjusted to obtain the microalga concentration of the value is calculated, and a pump control data signal 19 for controlling the flow rate of the medium injection pump 7 obtained as a result of the calculation is output to the control system of the medium injection quantitative pump 7. The pump flow rate was controlled to control the concentration of microalgae in the culture solution 10 at a set value. The flow rate of the culture solution suction pump 14 is set so as to always exceed the flow rate of the culture medium injecting constant pump 7. If the measured turbidity is lower than the set value, the injection of the culture medium 6 is stopped and it waits until the set concentration is reached.

As described above, it is impossible to measure the total organic carbon content online as the concentration of microalgae with the current technical level, and in addition, since the commercially available turbidity meter cannot perform appropriate concentration management, On-line concentration control was performed using a prototype turbidimeter that was able to easily measure absorbance by combining a light emitting diode and a photodiode that emit light near the absorption peak of chlorophyll in cells.

It should be noted here that the relationship between the value of the turbidimeter and the value of total organic carbon does not always show a constant value. In other words, the relationship between the turbidimeter and the indicated value of total organic carbon varies depending on the culture conditions (light intensity and cell concentration) or the state of the microalgae (coloring and the like). Therefore, the on-line management of the microalgae concentration was performed by the turbidimeter, and it was confirmed that the culture was performed stably, and the total organic carbon at that time was measured. That is, every time the culture conditions were changed, direct counting was performed using a microscope and a hemocytometer, and the turbidimeter was calibrated. Another purpose of the direct counting is to obtain important information in checking contamination by bacteria or the like and other management of culture conditions.

In order to know the culture state of microalgae, color
Element content is also important, and the turbidity meter
Use light-emitting diodes for near-peak light but accurate
Since we do not know the light absorption characteristics,
Using a spectrophotometer [UV3000 manufactured by Shimadzu Corporation]
A₆₈₀-A₇₅₀Of the turbidimeter, and
A₆₈₀-A₇₅₀The relationship between the absorbances of the A₆₈₀-A₇₅₀
Culture system so that the measured value of
Operated the system. Each time the culture conditions change, A₆₈₀-A
₇₅₀Calibration by absorbance measurement
Was. In this embodiment, the microalgae concentration is 300
mgC / liter is turbidity: A₆₈₀-A₇₅₀To 0.45,
Turbidity: A for 100 mgC / liter of low concentration₆₈₀-A ₇₅₀
Was equal to 0.15.

[0022] Thus started several days to several tens days of culture in accordance with the present invention, the medium supply amount, the culture liquid recovery amount, microalga concentration (Prototype turbidimeter direct counting and A ₆₈₀ -A ₇₅₀ absorbance)
When it was confirmed that the system was stable at all points, the total organic carbon content of the microalgae was determined by TOC (Total Or
aganic carbon) analyzer [TOC5, manufactured by Shimadzu Corporation]
00], the flow rate was multiplied by the obtained total organic carbon amount to calculate the growth amount, and the productivity was evaluated as the production rate (growth amount per unit time: mgC / h).

COMPARATIVE EXAMPLE In the continuous apparatus of FIG. 2, a batch culture was performed without controlling the concentration of microalgae. That is, first the same MDM medium 3 liters Example placed in culture reactor body 2, turbidity: condensing portion of such fine algae Synechocystis PCC6714 a 0.15 A ₆₈₀ -A ₇₅₀ dye reduces strain PD -1FERM P-14970 was inoculated, culture was started, culture was continued under the same conditions as in Example 1 except that the culture solution was not collected and the medium was not supplied. Turbidity: A
₆₈₀ -A ₇₅₀ in microalgae until 1.0 the culture liquid 10 is recovered at grown, medium tank 5 so that the turbidity of the initial
Was supplied into the reactor main body 2 for culture.

(Comparison of the results of Examples and Comparative Examples) FIG. 3 shows that the microalgae were prepared at a low concentration (turbidity: A ₆₈₀ -A ₇₅₀ ) according to the present invention.
15) and high concentration (turbidity: the measurement result of the production rate in continuous culture was managed with A ₆₈₀ -A ₇₅₀ 0.45) (Example)
The production rate measurement results when batch cultivation was performed from 0.15 to 1.0 turbidity (Comparative Example) are shown. In FIG. 3, the production rate is indicated by the total organic carbon amount Cppm per unit time (h), but Cppm ≒ mgC / liter (C: total organic carbon). From FIG. 3, the productivity is 1.1 to 1.1 in the case of continuous culture compared to the case of performing batch culture.
It turns out that it becomes 1.3 times higher.

FIG. 4 shows the change in the concentration of microalgae in the culture solution in the batch culture of the comparative example. The horizontal axis represents the elapsed time from the start of the culture, and the vertical axis represents the concentration of microalgae Cppm @ mgC / liter (C: total organic carbon). From this, the initial stage of culture (~ about 4
It can be seen that the production rate of microalgae is temporarily reduced at a low concentration (0 hour). It is considered that the productivity improvement in the continuous culture is because such a temporary decrease in the production rate is eliminated.

[0026]

From the results of the above examples, it can be seen that the microalgae production rate can be clearly improved by culturing while maintaining the microalgae concentration in the culture reactor at a constant level. This is effective as a means of improving economic efficiency in

[Brief description of the drawings]

FIG. 1 is a schematic explanatory view of the present invention.

FIG. 2 is an explanatory diagram of one embodiment of the present invention.

FIG. 3 is a graph showing a comparison of production rates of microalgae in Examples of the present invention and Comparative Examples.

FIG. 4 is a graph showing changes in the concentration of microalgae in a culture solution in a batch culture of a comparative example.

Claims (3)

[Claims] 1. A method for culturing microalgae under light irradiation in a culturing reactor, comprising culturing the microalgae while maintaining a constant concentration of the microalgae in a culture solution. 2. The culture method according to claim 1, wherein the concentration of the microalgae is 100 mgC / L or more and 300 mgC / L or less (C: organic carbon) in total organic carbon in the culture solution. 3. The microalgae is Synechocystis PCC.
The method for culturing a microalga according to claim 1 or 2, wherein the method is 6714 PD1 (FERM P-14970).