KR20070033721A - How to measure algal growth potential - Google Patents

Abstract

The present invention relates to the measurement of algal development mechanisms and more particularly to a method for measuring algal growth potential.

According to the algae growth potential measurement method according to the present invention, by controlling the growth conditions of the corresponding algae algae uniformly can facilitate the growth of algae than conventional centrifugation method, algae growth potential measurement method By providing the optimum measurement conditions for use in algae, algae growth potential can be accurately or easily measured.

Description

Method for Measuring Algal Growth Potential

Figure 1 is a graph showing the results of incubation of C. polykrikoides inoculated in F / 2 medium containing the seawater to be measured by concentration

Figure 2 is a graph showing the change in the concentration of nitrite nitrogen, inorganic phosphorus (inorganic phosphorus) of the medium according to the increase in the number of washing while the medium exposed to artificial seawater for 12 hours using a dialysis membrane

Figure 3 is a graph showing the change in the concentration of nitrite nitrogen, inorganic phosphorus in the medium according to the increase in the number of washing while the medium exposed to artificial seawater for 6 hours using a dialysis membrane

4 is a graph showing changes in the concentrations of nitrite nitrogen and inorganic phosphorus in the medium according to the increase in the number of washings while exposed to artificial seawater for 6 hours in a state containing 1,500 cells / mL of C. polykrikoides in the medium.

5 is a graph showing the proliferation change of C. polykrikoides according to starvation period

The present invention relates to the measurement of algal development mechanisms and more particularly to a method for measuring algal production potential.

In Korea, red tide occurs every year in the sea, causing enormous economic losses. There are no red tide records due to anaerobic bacteria. Among them, the most common species in Korea's coast include Skeletonema, Chaetoceros, and Nitzschia. Diatoms include Gymnodinium and Pro. There are genus Prorocentrum, Heterosigma, and Naticula.

By sea area, red tide was frequently observed in the Naticula genus in the West Sea, Prorocentrum in the South East Sea, and Heterosigma genus in the south sea. In Masan Bay and Haengam Bay, flagella algae, Prorocentrum, Heterosigma, Dangdong Bay and Wonmun Bay, along the Gimnodinium, Gonyaulax, etc. The Cochlodinium genus frequently appears, and in the surrounding waters of Salyang Island, the Cochlodinium genus has caused massive damage to farmed fish since 1989 (Park, Kim, Lee et al., 1993b).

In other words, red tide caused by Cochlodinium polykrikoides occurs every year and causes huge economic losses centering on the coast of South Sea (Kim et al ., 1997; Kim, 1998). To date, there are no fundamental countermeasures to reduce fishery damage caused by the red tide of various algae organisms, including C. polykrikoides , and fishery damage is reduced through the spread of ocher (Bae et al ., 1998).

Therefore, it is necessary to clarify the mechanism of occurrence of the red tide algae in order to reduce the damage to fisheries and to establish fundamental countermeasure. . In other words, since 1965, research on the measurement of algal growth potential has been actively conducted. In Japan, it is widely known as AGP (Algal Growth Potential). Conventional AGP test methods for seawater reported by the Japan National Institute of Pollution include Thalassiosira pseudonana , Dunaliella tertiolecta , and Hornellia after pretreatment of the sample . sp., Heterosigma sp., Skeletonema costatum , and the like, using specific phytoplankton isolated. In addition, after 1980, the limiting nutrient salts were reviewed using the on-site phytoplankton community, and in 1996, the method of dividing the on-site phytoplankton by size in the AGP test was reported.

On the other hand, in Korea, research on the mechanism of C. polykrikoides red tide is being actively conducted to reduce the fishery damage caused by C. polykrikoides red tide and establish fundamental measures (Kim et al ., 1999; Yang et al .). ., 2000) There are still many deficiencies to fully explain the mechanism.

In addition, C. In order to elucidate the mechanism of occurrence of red tide organisms, including birds polykrikoides would have to comply with the AGP test targets C. polykrikoides, it is not always that red tide organisms in the sea waters, even when inevitably some red tide occurred. Therefore, red tide algae should be incubated in the laboratory and used for AGP testing. In the already developed AGP test method, phytoplankton are washed by diluting with artificial seawater by centrifugation. However, if the algae centrifuged according to the conventional method, there is a problem such as poor sedimentation or cells do not proliferate due to injury. In particular, C. polykrikoides. To solve this problem, C. polykrikoides can be washed with artificial seawater using filter paper.In this case, however, C. polykrikoides can pass through the filter paper to obtain C. polykrikoides content. This decreases or takes a long time to filter.

Therefore, there is a lot of demand in the art for the algae growth potential measurement method that can measure the growth potential of algae causing red tide including C. polykrikoides without the same problems as the conventional AGP test method.

Therefore, the present inventors have made a thorough study to solve the problems of the conventional AGP test method, it is easy to remove the material required for the growth of algae in the medium absorbed during subculture when washing algae with artificial seawater using a dialysis membrane. The present invention has been focused on.

Accordingly, it is an object of the present invention to provide an optimal measurement method for measuring algal growth potential.

Another object of the present invention is to provide an optimal measurement condition used in the algae growth potential measurement method.

In order to achieve the above object, the present invention comprises the steps of separating the algae; Culturing the isolated algae; Uniformly adjusting the growth conditions of the cultured algae; It provides an algae growth potential measurement method comprising the step of inoculating and propagating in a medium containing the seawater to be adjusted to the controlled algae.

The step of uniformly adjusting the growth conditions is characterized in that it comprises the step of washing the cultured algae with artificial seawater using a dialysis membrane.

Washing the cultured algae with artificial seawater using a dialysis membrane is characterized in that the cultured algae is made by washing at least five times by exposing the cultured algae to artificial seawater for 3-9 hours.

Inoculating and propagating in the medium containing the seawater to be measured the growth conditions controlled algae, characterized in that it comprises the step of inoculating more than 100 cells of the algal organisms with the growth conditions controlled per 1ml of seawater to be measured It is done.

Inoculating the cultured algae in a medium containing artificial seawater, characterized in that it further comprises the step of propagating.

Hereinafter, the present invention will be described in detail with reference to Examples.

Example

Algae growth potential measurement of Cochlodinium polykrikoides ( hereinafter “ C. polykrikoides ”)

1. C. Separation of polykrikoides

C. polykrikoides was isolated from the South Sea in summer 2002.

2. C. Culture of polykrikoides

The isolated C. polykrikoides were passaged in F / 2 medium except Na ₂ SiO ₃ using seawater around Jeju Island, Korea.

3. Control of growth conditions of the cultured C. polykrikoides

In order to remove all the materials necessary for the growth of C. polykrikoides in the cultured medium, the medium was exposed to dialysis membrane for 6 hours and washed 6 times with artificial seawater.

At this time, the dialysis membrane was Spectrum Spectra / Por ^® 7 Membrane (MWCO 50,000). C. polykrikoides Single cell size varies depending on the culture, but is generally 28-48 ?? m long and 17–31 ?? m wide. Considering this size, we chose the maximum MWCO that C. polykrikoides would not pass through.

4. Inoculation and propagation of C. polykrikoides with uniform growth conditions

C. polykrikoides washed 6 times with artificial seawater and washed six times using dialysis membrane as described above were inoculated and incubated in F / 2 medium containing seawater to be measured by concentration, and the results are shown in FIG. 1. C. From the 11th day after the inoculation of polykrikoides at 20 and 50cells / mL, from the 4th day after incubation at the initial concentration of 100cells / mL, from the 2nd at 200cells / mL, and from the beginning at 500cells / mL. It started to multiply distinctly. In other words, although the growth rate was different, C. polykrikoides proliferated even if C. polykrikoides were washed with artificial seawater 6 times at 6 hour intervals.

Here, the algae growth potential when to increase the initial concentration of the phytoplankton in the measurement method and requires a lot of plankton, because it takes a long time to determine if the result is less important inoculation concentration, an initial concentration of C. polykrikoides 50cells / mL or less It took more than 16 days to confirm the proliferation, and it took about 11 days at the concentration above 100 cells / mL. Therefore, initial C. polykrikoides concentrations of at least 100 cells / mL are required.

It does not specifically described luggage nodi nium (Gymnodinium) in the car stuffing ticks (kinetics) experiment, compared to the initial inoculation concentration in the load nodi Stadium (Gymnodinium) in using the dialysis membrane without any effect on proliferation may be washed C. polykrikoides It can be seen that.

Experimental Example 1

Frequency and exposure time of washing with artificial seawater when controlling the growth conditions of the cultured C. polykrikoides

1. Experimental method

First, the medium was prepared by adding inorganic phosphorus and nitrite nitrogen to seawater used for subculture to determine the dilution of the C. polykrikoides medium using a dialysis membrane.

The bottom of the dialysis membrane was closed with a closure, and then 100 mL of medium containing inorganic phosphorus and nitrite nitrogen was added thereto, and the top was closed with a closure. Thereafter, the dialysis membrane was placed in a 1L measuring cylinder, and 900 mL of sterilized artificial seawater was added thereto, and the measuring cylinder was blocked with Parafilm. The measuring cylinder was placed in the culture chamber for a certain time. After a certain time, the medium in the dialysis membrane was taken and at the same time, new artificial seawater was added to the measuring cylinder. The volume ratio of the medium and artificial seawater was set to 1: 9. Nitrogen and phosphorus in the collected media were analyzed by A Manual for Oceanographical Observation. To investigate the effect of C. polykrikoides on the nitrogen and phosphorus washing of the medium, C. polykrikoides was cultured in large quantities, and inorganic phosphorus and nitrite nitrogen were added to the dialysis membrane.

Next, the C. polykrikoides were cultured to see if they proliferated after washing C. polykrikoides with artificial seawater using a dialysis membrane. Washing was performed 6 times at 6 hour intervals with artificial seawater using a dialysis membrane. The culture was inoculated with washed C. polykrikoides in 5 mL F / 2 medium in a 15 mL test tube (Φ 15 mm × 125 mm). Inoculation concentrations of C. polykrikoides were 20, 50, 100, 200, 500 cells / mL. The culture conditions were temperature 23 ± 2 ℃, light 7,000 ± 500 lx, 12:12 h contrast cycle, and the chlorophyll was measured by Phyto-pam at 1 ~ 3 days interval.

In addition, it was cultured again after washing to confirm the need for starvation. C. polykrikoides washed 6 times and 6 times with artificial seawater 5mL was put in a 15mL test tube (Φ15mm × 125mm) and incubated for 0, 3, 6 days under the same conditions as above. After starvation, C / 2 polykrikoides was added to the F / 2 medium except for seawater and Na ₂ SiO ₃ to confirm the presence of excess absorbed material.

2 experimental results

The concentration of nitrite nitrogen and inorganic phosphorus in the medium according to the increase in the number of washings was shown in FIG. 2 while the medium was exposed to artificial seawater for 12 hours using a dialysis membrane. The initial concentrations of nitrite nitrogen and inorganic phosphorus were 59.02 and 41.81 μM, respectively. After washing once every 12 hours with artificial seawater, the concentrations of nitrite nitrogen and inorganic phosphorus were 7.45 and 11.37μM, respectively. As the number of washings increased, the concentration drastically decreased. At the fifth time, nitrite nitrogen and inorganic phosphorus decreased to 0.23 and 0.08 μM, respectively. Figure 3 is a change in the concentration of nitrite nitrogen, inorganic phosphorus in the medium according to the increase in the number of washing while the medium exposed to artificial seawater for 6 hours using a dialysis membrane. Initial concentrations of nitrite nitrogen and inorganic phosphorus were 39.20 and 28.73 μM, respectively. As the number of washings increased, the concentration sharply decreased. At the fifth time, the concentrations of nitrite nitrogen and inorganic phosphorus decreased to 0.26 and 0.09 μM, respectively. In Figure 4, C. polykrikoides is a case containing 1,500 cells / mL. Nitrite nitrite, inorganic phosphorus concentration decreases rapidly as the number of washing increases as shown in FIGS.

From the above results, irrespective of the population of C. polykrikoides, the concentrations of nitrite nitrogen and inorganic phosphorus were lowered to 0.26 and 0.09 μM or less when the medium was washed 5 times or more in artificial seawater. On the other hand, the concentrations of nitrite nitrogen and inorganic phosphorus in artificial seawater were 0.3 and 0.1 µM, respectively. Therefore, the concentrations of nitrite nitrogen and inorganic phosphorus in artificial seawater do not seem to be lowered any more. In other words, the concentration of substances not contained in artificial seawater is likely to have been lowered. From the above results, it can be seen that in order to remove all the substances necessary for the growth of C. polykrikoides in the medium, most of the substances will be removed by washing 5 times or more with artificial seawater at the exposure time of 6 hours.

Experimental Example 2

Necessity experiment of hunger culture

As described above, the maximum growth rate was higher as the initial inoculation concentration was higher, despite the same medium washed 6 times with artificial seawater and washed six times using the dialysis membrane as described above. Since phytoplankton have been reported to absorb excessive amounts of abundant substances in the medium, C. polykrikoides may also be ingested in excess of the substances necessary for growth. Therefore, the higher the concentration of initial C. polykrikoides, the more C. polykrikoides is absorbed by the excess material into the culture vessel. Therefore, because C. polykrikoides proliferated more by using the excess C. polykrikoides absorbed by the addition of F / 2 medium, starvation was performed.

In other words, C. polykrikoides were hung incubated 6 hours and 6 times with artificial seawater using dialysis membrane. And culturing C. polykrikoides cultured again, the results can be seen from Figure 5, a graph showing the proliferation change of C. polykrikoides according to the hunger culture period, the F / 2 medium in hunger culture on day 0 There was no change in the concentration of C. polykrikoides in the control group without the addition of the substance. However, the addition of F / 2 medium increased up to 2 times the initial concentration in the culture for 6 days. The 3 and 6 day hunger cultures also increased as did the 0 day hunger cultures. As mentioned above, phytoplankton consume a lot of the substances in the medium. Theoretically, even if hunger culture is limited to a certain amount of proliferation, phytoplankton is more difficult to proliferate due to the restriction substance, and the substance absorbed by phytoplankton may remain in the cell. In general, starvation is carried out to minimize the effect of the culture. In this experiment, however, the proliferation of F / 2 medium after starvation indicates that the excess absorbed material was not removed by starvation. Therefore, short-term hunger culture of C. polykrikoides does not seem to be meaningful in measuring algal growth potential. Rather, control experiments using artificial seawater are necessary.

From the above examples and experiments, the algae growth potential measurement method of washing C. polykrikoides with artificial seawater using the dialysis membrane according to the present invention, the exposure time is 3-9 in order to eliminate all the substances necessary for the growth of C. polykrikoides in the medium. Time, more preferably 6 hours, the number of years of washing with artificial seawater is found to be about 5 times or more. In addition, even by using a dialysis membrane washing C. polykrikoides in artificial sea water 6 times 6 hours is C. polykrikoides were both proliferation and inoculation concentration of the initial C. polykrikoides is preferably a 100cells / mL or more, for algae growth potential measured C The starvation of polykrikoides seems to be meaningless, indicating that control experiments using artificial seawater are necessary.

Therefore, those skilled in the art will be able to make various modifications, changes, additions, and the like within the spirit and scope of the present invention, and such modifications should be regarded as belonging to the following claims.

As described above, the method for measuring the algae growth potential according to the present invention has the following effects.

According to the algae growth potential measurement method according to the present invention, it is possible to facilitate the growth of algae than the conventional centrifugal method by uniformly adjusting the growth conditions of the red tide algae.

In addition, according to the algae growth potential measurement method according to the present invention, it is possible to easily measure the algae growth potential by providing the optimum measurement conditions used in the algae growth potential measurement method.

Claims (5)

Separating the algae; Culturing the isolated algae; Uniformly adjusting the growth conditions of the cultured algae; Algae growth potential measurement method comprising the step of inoculating and propagating in a medium containing the seawater to be adjusted to the controlled algae The method of claim 1, wherein the controlling of the growth conditions uniformly comprises washing the cultured algae with artificial seawater using a dialysis membrane. The algae of claim 2, wherein the washing of the cultured algae with artificial seawater using a dialysis membrane is performed by exposing the cultured algae to artificial seawater for 3-9 hours in a dialysis membrane to wash at least five times. How to measure growth potential. The method of claim 1, wherein the step of inoculating and propagating in the medium containing the seawater to be measured the algae, the growth conditions are adjusted to 100 cells of the algae organisms with the growth conditions controlled per 1ml of the medium containing the seawater to be measured Algae growth potential measurement method comprising the step of inoculating abnormalities. The method according to any one of claims 1 to 4, further comprising the step of inoculating and propagating the regulated algae in a medium containing artificial seawater.

Images