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AbstractUnder the premise of simulating the growth environment of algae in natural conditions, the responses of Microcystis aeruginosa to stress of Rabbosia japonica were studied. The results showed that the maximum specific growth rate of M. aeruginosa inhibited by water extract from R. japonica was -0.083 d-1, and the inhibition effects on the electrical conductivity and pH of M. aeruginosa solution were significant with long duration. When the concentration of water extract from R. japonica was low, the maximum inhibition rate of M.aeruginosa was lower than 20%. When the concentration was 500 or 1 000 mg/L, the inhibition rate was higher than 50% after 144 h. It was proved that the inhibition effect of high concentrations of R. japonica on M. aeruginosa was significant and stable, which can provide reference for the control of water bloom in aquaculture water.
Key wordsMicrocystis aeruginosa; Rabbosia japonica; Water bloom; Inhibition effect
Received: September 15, 2018Accepted: October 31, 2018
Supported by Natural Science Foundation of Tianjin City (17JCYBJC29800); Funding Plan Projects for Various Types of Talent in Tianjin Agricultural University (J01009030702).
Nan YNAG (1996-), female, P. R. China, master, devoted to research about exploitation and utilization of plant resources.
*Corresponding author. Email: njlnie@126.com.
In recent years, with the advancement of science and technology and the development of economy, a large amount of sewage, waste water, excess pesticides and fertilizer have produced and discharged into rivers and lakes, which has made the water eutrophication of main lakes in China increasingly serious and has brought increasing economic loss. Water eutrophication is the main factor causing cyanobacteria bloom. Cyanobacterial bloom will form a layer of bluegreen scented foam on the surface of water, which will hinder the exchange of air and the water. In addition, cyanotoxins will be released during the growth process of cyanobacteria (also called bluegreen algae) and can make water quality to be destroyed, which in turn seriously affects human health［1-3］. There are many species of bluegreen algae leading to water bloom, among which Microystis aeruginosa is the most common, and it is dominant in the bluegreen algae in China and the world. Therefore, research on the inhibition of M. aeruginosa growth has become the focus of researchers at home and abroad［4-6］. Rabbosia japonica is a traditional Chinese herbal medicine of Rabdosia, Labiatae. It is a perennial erect herb with leaves opposite to each other, and the leaf margin is coarsely serrated, while the flowering and fruiting period is from June to September. It is mainly distributed in North China, Northeast China, Japan, North Korea and the former Soviet Union. The overground part of R. japonica can be used as medicine and is bitter and cold. It has effects of invigorating the stomach, invigorating the circulation of blood, clearing heat and detoxicating. It can be used to cure sore throat, a variety of inflammation, cancer, abdominal distension, cold and fever［7］. Moreover, R. japonica has the biological activity of protecting cardiac muscle, diminishing inflammation, anticancer and preventing the liver from being damaged by carbon tetrachloride. Glaucocalyxin A, one of highcontent components of R. japonica, can improve microcirculation and inhibit platelet aggregation［8］.
Different extracts of R. japonica have a certain inhibitory effect on prokaryotes such as Escherichia coli and Staphylococcus aureus. The antibacterial effect mainly comes from the synergistic effect of its own active ingredients such as glaucocalyxin A, diterpenoids, triterpenoids, sterols, etc［9］. Terpenoids can inhibit plant growth and affect cell membrane permeability［10］. In addition, R. japonica has inhibitory effects on algae and various microorganisms including marine fouling bacteria［11］. However, in an artificially simulated environment, the effect of R. japonica on algae has not been reported. In this study, in the artificially simulated natural growth environment of M. aeruginosa, the responses of M. aeruginosa to the inhibition of different concentrations of water extract from R. japonica were studied to analyze the ability of R. japonica to inhibit algae growth and provide scientific reference for the deep research of inhibition of algae growth by R. japonica.
Materials and Methods
Materials
The experimental species of M. aeruginosa was from the Institute of Hydrobiology, Chinese Academy of Sciences. R. japonica used in the experiment was bought from a Chinese medicine shop in Tianjin.
Main instruments
Main instruments included DDS307 conductivity meter (Shanghai Precision Scientific Instrument Co., Ltd.), CT6023 pentype acidity meter (Shenzhen Kodida Electronics Co., Ltd.), UH5300 Ultraviolet spectrophotometer (Beijing Puxi General Instrument Co., Ltd.), etc. Methods
Domestication and culture of M. aeruginosa
M. aeruginosa was expanded in BG11 medium［12-13］. It was transferred under sterile conditions on a clean bench. It was cultured at room temperature and under natural light to allow cells of M. aeruginosa to enter the logarithmic phase. After the activation of M. aeruginosa, it was cultured at 30 ℃ in an artificial incubator for 7-10 d, with illumination intensity of 2 000 Lx and illumination ratio of 12∶12.
Preparation of water extract of R. japonica
Firstly, 0.025, 0.05, 0.1, 0.2, 0.5 and 1.0 g of R. japonica powder were weighed accurately and dissolved with water to 1 000 ml. After ultrasonic extraction was conducted for 30 min, they were stood at room temperature for 24 h, filtered by filter paper, and centrifuged for 10 min for use.
Setting of simulation environment
The soil sample that was collected from Jinghai previously and used to simulate the sediment in the growth environment of the algae was aired and sieved with a sieve with 40 meshes. 24 groups of 20 g of the sieved soil sample were put in culture flasks, to which a certain amount of the medium was added to stick the floating soil to the wall of the culture flasks. The culture flasks containing the soil sample were placed in a high temperature sterilization pot for wet sterilization and then were taken out for use after cooling. M. aeruginosa suspension was put in 21 sterilized glass jars (10 cm×10 cm×20 cm), and every 2 000 ml of the medium contained 20 ml of the algae solution and 20 g of the sterilized soil sample. A heating rod was placed in water to control water temperature at around 25 ℃. It was cultured for 5-10 d under natural light.
Test for inhibition of the algae
Firstly, 60 ml of water extract of R. japonica (25, 50, 100, 200, 500 and 1 000 mg/L) was added to the algae solution in the simulation environment. Meanwhile, 60 ml of distilled water was added to the algae solution, which was as the control group (CK). There were three parallel samples in each group. The absorbance, electrical conductivity and pH of the six test groups and the control group were detected after 0, 3, 6, 9, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240 and 264 h respectively. The glass jars were shaken twice a day, and the position was randomly changed. The experiment lasted for 11 d.
Determination methods
Density of algae cells was calculated with a blood counting chamber under a microscope (16×10 time)［13］. Specific growth rate of algae cells was calculated as follows:
μ= ln(Xi /X0)/( i-1)（1）
where μ was specific growth rate of algae cells; Xi was density of algae cells on the ith day after the water extract of R. japonica was added; X0 was initial density of algae cells before the water extract was added.
Inhibition rate of algae cells was calculated as follows:
IR = ( N0-Ns) /N0×100%（2）
where IR was inhibition rate of algae cells; N0 was density of algae cells in the control group; Ns was density of algae cells in the test groups［15］.
In order to detect the electrical conductivity of the algae solution, the algae solution in each glass jar was stirred several times with a glass rod in the upper 1/3 position to make M. aeruginosa distribute evenly. The sensor of a conductivity meter was inserted into the algae solution in the upper 1/3 position, and readings on the sensor were recorded［16］.
To measure the pH of the algae solution, the algae solution in each glass jar was stirred several times with a glass rod in the upper 1/3 position to make M. aeruginosa distribute evenly. Afterwards, the sensor of a pH meter was inserted into the algae solution in the upper 1/3 position, and readings on the sensor were recorded［16］.
Results and Analysis
Inhibition effect of different concentrations of water extract from R. japonica on M. aeruginosa
As shown in Fig. 1, when the concentration of the water extract increased from 25 to 200 mg/L, the cell density of M. aeruginosa reduced and was smaller than the control group, but there was no significant difference between the test groups and the control group. As the concentration of the water extract rose to 500 and 1 000 mg/L respectively, the cell density of M. aeruginosa decreased with the increase of treatment time. When treatment time was up to 264 h, the cell density was 3.646 when the concentration was 500 mg/L, significantly smaller than the control group. It showed that when the concentration of the water extract from R. japonica was higher than 500 mg/L, it would significantly inhibit the reproduction of M. aeruginosa.
Fig. 1Effects of different concentrations of water extract from R. japonica on the cell density of M. aeruginosa
Seen from Fig. 2, as the concentration of the water extract was 25-200 mg/L, the inhibition rate of algae cells changed slightly and was always smaller than 20%. After treatment time exceeded 216 h, the inhibition rate of algae cells began to decrease when the concentration of the water extract was 25, 100 and 200 mg/L. As the concentration of the water extract was 500 and 1 000 mg/L, the inhibition rate of algae cells rose with the increase of treatment time. That is, it increased from 50% to 60% with the increase of treatment time from 144 to 168 h, and it was above 60% when treatment time was 192, 216, 240 and 264 h. The results indicated that low concentrations of the water extract inhibited the reproduction of M. aeruginosa cells slightly, but the inhibition effect was obvious when the concentration was higher than 500 mg/L. The inhibition effect of water extract from R. japonica on M. aeruginosa showed clear timeeffect and dosageeffect relationship. According to Table 1, the total degree of freedom was 20, and the degree of freedom among groups and in a group was 6 and 14 respectively. The calculation results showed that F=17.310, P=0.000<0.01, that is, there was 99% confidence that there was a significant difference between different concentrations of water extract from R. japonica in the inhibition effect of M. aeruginosa, thereby rejecting H0 and accepting H1. The inhibition effect of different concentrations of water extract from R. japonica on M. aeruginosa was significant at 0.01 level.
Fig. 2Inhibition rate of different concentrations of water extract from R. japonica on M. aeruginosa
Table1Analysis of variance of the inhibition effect of M. aeruginosa by different concentrations of R. japonica
Source of variationSum of squares (SS)Degree of freedom (df)Mean square (MS)FP value
Among groups1.53550.30719.0080
In a group0.194120.016
Total1.72917
Effects of different concentrations of water extract from R. japonica on the specific growth rate of M. aeruginosa in various treatment periods
Seen from Table 2, the specific growth rate of M. aeruginosa cells decreased with the increase of treatment time in the control group. The specific growth rate in the test groups was smaller than that of the control group, showing that the growth of M. aeruginosa was inhibited by different concentrations of water extract from R. japonica. When the concentration of the water extract was 25-200 mg/L, the specific growth rate was positive, indicating that the effect was not significant. As the concentration of the water extract was 500 mg/L, the specific growth rate became negative after 96 h, that is, the growth of M. aeruginosa cells was inhibited after 96 h. When the concentration of the water extract was 1 000 mg/L, the growth of algae cells was completely inhibited from the beginning. Seen from the changes of the specific growth rate, in the same concentration, the inhibition effect of the water extract increased with the increase of treatment time. In the simulation environment, with the increase of treatment time, the higher the concentration of water extract from R. japonica was, the lower the specific growth rate of M. aeruginosa, and the better the inhibition effect was.
Agricultural Biotechnology2019
Table 2Specific growth rate of M. aeruginosa inhibited by different concentrations of water extract from R. japonica Time∥h
Specific growth rate∥d-1
0 mg/L 25 mg/L50 mg/L100 mg/L 200 mg/L500 mg/L1 000 mg/L
240.1880.1100.1180.1190.1070.069-0.047
480.1250.0980.0670.0710.0910.028-0.028
720.1010.0760.0540.0650.0760.004-0.026
960.0960.0680.0520.0620.061-0.011-0.029
1200.0870.0700.0570.0560.056-0.034-0.044
1440.0770.0620.0500.0550.057-0.05-0.046
1680.0700.0560.0440.0490.051-0.069-0.060
1920.0570.0450.0330.0390.041-0.065-0.064
2160.0520.0370.0320.0350.041-0.061-0.065
2400.0520.0420.0290.0430.047-0.054-0.074
2640.0490.0380.0280.0400.044-0.051-0.083
Effects of different concentrations of water extract from R. japonica on the electrical conductivity of M. aeruginosa solution in various treatment periods
As shown in Fig. 3, in the simulation environment, the electrical conductivity of M. aeruginosa solution in the control group fluctuated from 881 to 979 μs/cm within 216 h, but the change was not significant. With the increases in the concentration of water extract from R. japonica (25-200 mg/L) and treatment time, the electrical conductivity of M. aeruginosa solution rose, but there was no significant difference between the test groups and the control group. When the concentration of the water extract was 1 000 mg/L, the electrical conductivity of M. aeruginosa solution increased to 1 014 μs/cm after 120 h, significantly higher than that of the control group. It revealed that after the addition of high concentration of the water extract from R. japonica, the permeability of cell membranes of M. aeruginosa changed significantly, and their damage became more and more serious, so that the electrical conductivity of M. aeruginosa solution increased.
Fig. 3Effects of different concentrations of water extract from R. japonica on the electrical conductivity of M. aeruginosa solution
Effects of different concentrations of water extract from R. japonica on the pH of M. aeruginosa solution in various treatment periods
Seen from Fig. 4, in the simulation environment, the pH of M. aeruginosa solution in the control group was 8.3-10.2, showing an increasing trend. Meanwhile, the density of M. aeruginosa cells also increased gradually, showing that the higher the pH was, the higher the reproductive efficiency of M. aeruginosa. The pH of M. aeruginosa solution in the test groups (25-200 mg/L) was lower than that of the control group, but there was no significant change. When the concentration of water extract from R. japonica increased to 500 and 1 000 mg/L, the pH of M. aeruginosa solution was significantly lower than that of the control group, and decreased with the increase of treatment time after 192 h. When the concentration was 1 000 mg/L, the pH was lower than 8 after 24 h. The results were consistent with the result of effects of different concentrations of water extract from R. japonica on the specific growth rate of M. aeruginosa, which further proved that M. aeruginosa had a stronger response to the inhibition of high concentrations of water extract from R. japonica.
Fig. 4Effects of different concentrations of water extract from R. japonica on the pH of M. aeruginosa solution
Discussion
M. aeruginosa, which is the most common algae causing algal bloom, produces toxins and leads to absence of oxygen in water during the process of decomposition to destroy the normal food web and directly threaten human health and the survival of other animals and plants. Therefore, it is important to develop an environmentally friendly natural reagent used to inhibit algae growth. Studies have shown that the current natural environmental factors have more significant effects on the community structure of algae than human factors［17］. Environmental factors have synergistic effects on algae growth, and experimental conditions should be close to the environment during the process of research to obtain more reliable conclusions［18］. Therefore, temperature, light and soil conditions close to the environment were set in this experiment. It was found that in the artificial simulated natural environment, high concentrations of water extract from R. japonica could significantly inhibit the proliferation of M. aeruginosa cells.
M. aeruginosa is a prokaryote, and its structure is similar to that of E. coli and other bacteria. The inhibition effect of R. japonica on M. aeruginosa may be the result of pharmacological synergy of different types of chemical components［11］. The experimental results showed that when the concentration of the water extract exceeded 500 mg/L, the electrical conductivity of M. aeruginosa solution increased significantly. The reason is that the terpenoids in R. japonica might destroy the structure of M. aeruginosa cell membranes［10］, thereby inhibiting the proliferation of M. aeruginosa cells, which is consistent with the result of effect of different concentrations of water extract on the cell density of M. aeruginosa.
Most of the lakes in China are alkaline and the pH is high. Studies have shown that in the process of water bloom, the pH of water environment increases significantly with the growth of algae, and the pH of the water environment suitable for the growth of M. aeruginosa is 8-9 on average. Other studies real that high pH (about 11.0) can promote the growth and proliferation of algae cells［19-21］. As the concentration of the water extract was 1 000 mg/L, the pH of M. aeruginosa solution declined significantly after 24 h, lower than 8, while the pH of M. aeruginosa solution in the control group was always higher than 9. It showed that the environment with high pH was more suitable for the growth of M. aeruginosa, while the pH of water environment in the treatments containing water extract from R. japonica would be decreased to inhibit the growth of M. aeruginosa. The inhibition effect of water extract from R. japonica on M. aeruginosa showed clear timeeffect and dosageeffect relationship. Conclusions
In the simulated natural growth environment, the responses of M. aeruginosa to the inhibition of different concentrations of water extract from R. japonica were studied. The results showed that low concentrations of water extract from R. japonica (25-200 mg/L) had no significant effect on the reproduction of M. aeruginosa. When the concentrations of water extract from R. japonica was higher than 500 mg/L, the cell density, specific growth rate and pH of M. aeruginosa decreased, while the inhibition rate and electrical conductivity of M. aeruginosa rose with the increase of treatment time and water extract concentration. According to the results of variance analysis, there was 99% confidence that the inhibition effect of different concentrations of water extract from R. japonica on M. aeruginosa was extremely significant. It provides reference for the application of R. japonica in water protection and the development of a new reagent used to inhibit algae growth in aquaculture water.
References
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［7］ REN XX, ZHOU HC, HE S, et al. Chemical constituents and antitumor activity of Isodon japonica var. glaucocalyx［J］. Journal of Xinxiang Medical University,2016,33(4): 216-266. (in Chinese).
［8］ WEN HY, ZHENG S, LANG YY, et al. Quality standard of Plectranthus japonicas (Burm.f.) Koidz. var. Glaucocalyx (Maxim.) Koidz.［J］.Central South Pharmacy, 2018,16(2): 233-237. (in Chinese). ［9］ JIN ZM, SHA W, HU XY. Study on bacteriostasis of the extracts of Isodon japonica ( Burm. f.) Hara var. glaucocalyx (Maxim.) Hara［J］. Guangxi Sciences,2007(2): 160-162. (in Chinese).
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［11］ LIU HC, XIANG ZB, WANG Q, et al. Monomeric and dimeric entkauranoidtype diterpenoids from rabdosia japonica and their cytotoxicity and antiHBV activities［J］. Fitoterapia, 2017,118: 94-100. (in Chinese).
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［15］ NIE JL, WANG T, SHI C, et al. Inhibitory effect of extract from Galla chinensis on the growth of Microcystis aeruginosa［J］. Bulletin of Botanical Research,2011,31(2):231-234. (in Chinese).
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Key wordsMicrocystis aeruginosa; Rabbosia japonica; Water bloom; Inhibition effect
Received: September 15, 2018Accepted: October 31, 2018
Supported by Natural Science Foundation of Tianjin City (17JCYBJC29800); Funding Plan Projects for Various Types of Talent in Tianjin Agricultural University (J01009030702).
Nan YNAG (1996-), female, P. R. China, master, devoted to research about exploitation and utilization of plant resources.
*Corresponding author. Email: njlnie@126.com.
In recent years, with the advancement of science and technology and the development of economy, a large amount of sewage, waste water, excess pesticides and fertilizer have produced and discharged into rivers and lakes, which has made the water eutrophication of main lakes in China increasingly serious and has brought increasing economic loss. Water eutrophication is the main factor causing cyanobacteria bloom. Cyanobacterial bloom will form a layer of bluegreen scented foam on the surface of water, which will hinder the exchange of air and the water. In addition, cyanotoxins will be released during the growth process of cyanobacteria (also called bluegreen algae) and can make water quality to be destroyed, which in turn seriously affects human health［1-3］. There are many species of bluegreen algae leading to water bloom, among which Microystis aeruginosa is the most common, and it is dominant in the bluegreen algae in China and the world. Therefore, research on the inhibition of M. aeruginosa growth has become the focus of researchers at home and abroad［4-6］. Rabbosia japonica is a traditional Chinese herbal medicine of Rabdosia, Labiatae. It is a perennial erect herb with leaves opposite to each other, and the leaf margin is coarsely serrated, while the flowering and fruiting period is from June to September. It is mainly distributed in North China, Northeast China, Japan, North Korea and the former Soviet Union. The overground part of R. japonica can be used as medicine and is bitter and cold. It has effects of invigorating the stomach, invigorating the circulation of blood, clearing heat and detoxicating. It can be used to cure sore throat, a variety of inflammation, cancer, abdominal distension, cold and fever［7］. Moreover, R. japonica has the biological activity of protecting cardiac muscle, diminishing inflammation, anticancer and preventing the liver from being damaged by carbon tetrachloride. Glaucocalyxin A, one of highcontent components of R. japonica, can improve microcirculation and inhibit platelet aggregation［8］.
Different extracts of R. japonica have a certain inhibitory effect on prokaryotes such as Escherichia coli and Staphylococcus aureus. The antibacterial effect mainly comes from the synergistic effect of its own active ingredients such as glaucocalyxin A, diterpenoids, triterpenoids, sterols, etc［9］. Terpenoids can inhibit plant growth and affect cell membrane permeability［10］. In addition, R. japonica has inhibitory effects on algae and various microorganisms including marine fouling bacteria［11］. However, in an artificially simulated environment, the effect of R. japonica on algae has not been reported. In this study, in the artificially simulated natural growth environment of M. aeruginosa, the responses of M. aeruginosa to the inhibition of different concentrations of water extract from R. japonica were studied to analyze the ability of R. japonica to inhibit algae growth and provide scientific reference for the deep research of inhibition of algae growth by R. japonica.
Materials and Methods
Materials
The experimental species of M. aeruginosa was from the Institute of Hydrobiology, Chinese Academy of Sciences. R. japonica used in the experiment was bought from a Chinese medicine shop in Tianjin.
Main instruments
Main instruments included DDS307 conductivity meter (Shanghai Precision Scientific Instrument Co., Ltd.), CT6023 pentype acidity meter (Shenzhen Kodida Electronics Co., Ltd.), UH5300 Ultraviolet spectrophotometer (Beijing Puxi General Instrument Co., Ltd.), etc. Methods
Domestication and culture of M. aeruginosa
M. aeruginosa was expanded in BG11 medium［12-13］. It was transferred under sterile conditions on a clean bench. It was cultured at room temperature and under natural light to allow cells of M. aeruginosa to enter the logarithmic phase. After the activation of M. aeruginosa, it was cultured at 30 ℃ in an artificial incubator for 7-10 d, with illumination intensity of 2 000 Lx and illumination ratio of 12∶12.
Preparation of water extract of R. japonica
Firstly, 0.025, 0.05, 0.1, 0.2, 0.5 and 1.0 g of R. japonica powder were weighed accurately and dissolved with water to 1 000 ml. After ultrasonic extraction was conducted for 30 min, they were stood at room temperature for 24 h, filtered by filter paper, and centrifuged for 10 min for use.
Setting of simulation environment
The soil sample that was collected from Jinghai previously and used to simulate the sediment in the growth environment of the algae was aired and sieved with a sieve with 40 meshes. 24 groups of 20 g of the sieved soil sample were put in culture flasks, to which a certain amount of the medium was added to stick the floating soil to the wall of the culture flasks. The culture flasks containing the soil sample were placed in a high temperature sterilization pot for wet sterilization and then were taken out for use after cooling. M. aeruginosa suspension was put in 21 sterilized glass jars (10 cm×10 cm×20 cm), and every 2 000 ml of the medium contained 20 ml of the algae solution and 20 g of the sterilized soil sample. A heating rod was placed in water to control water temperature at around 25 ℃. It was cultured for 5-10 d under natural light.
Test for inhibition of the algae
Firstly, 60 ml of water extract of R. japonica (25, 50, 100, 200, 500 and 1 000 mg/L) was added to the algae solution in the simulation environment. Meanwhile, 60 ml of distilled water was added to the algae solution, which was as the control group (CK). There were three parallel samples in each group. The absorbance, electrical conductivity and pH of the six test groups and the control group were detected after 0, 3, 6, 9, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240 and 264 h respectively. The glass jars were shaken twice a day, and the position was randomly changed. The experiment lasted for 11 d.
Determination methods
Density of algae cells was calculated with a blood counting chamber under a microscope (16×10 time)［13］. Specific growth rate of algae cells was calculated as follows:
μ= ln(Xi /X0)/( i-1)（1）
where μ was specific growth rate of algae cells; Xi was density of algae cells on the ith day after the water extract of R. japonica was added; X0 was initial density of algae cells before the water extract was added.
Inhibition rate of algae cells was calculated as follows:
IR = ( N0-Ns) /N0×100%（2）
where IR was inhibition rate of algae cells; N0 was density of algae cells in the control group; Ns was density of algae cells in the test groups［15］.
In order to detect the electrical conductivity of the algae solution, the algae solution in each glass jar was stirred several times with a glass rod in the upper 1/3 position to make M. aeruginosa distribute evenly. The sensor of a conductivity meter was inserted into the algae solution in the upper 1/3 position, and readings on the sensor were recorded［16］.
To measure the pH of the algae solution, the algae solution in each glass jar was stirred several times with a glass rod in the upper 1/3 position to make M. aeruginosa distribute evenly. Afterwards, the sensor of a pH meter was inserted into the algae solution in the upper 1/3 position, and readings on the sensor were recorded［16］.
Results and Analysis
Inhibition effect of different concentrations of water extract from R. japonica on M. aeruginosa
As shown in Fig. 1, when the concentration of the water extract increased from 25 to 200 mg/L, the cell density of M. aeruginosa reduced and was smaller than the control group, but there was no significant difference between the test groups and the control group. As the concentration of the water extract rose to 500 and 1 000 mg/L respectively, the cell density of M. aeruginosa decreased with the increase of treatment time. When treatment time was up to 264 h, the cell density was 3.646 when the concentration was 500 mg/L, significantly smaller than the control group. It showed that when the concentration of the water extract from R. japonica was higher than 500 mg/L, it would significantly inhibit the reproduction of M. aeruginosa.
Fig. 1Effects of different concentrations of water extract from R. japonica on the cell density of M. aeruginosa
Seen from Fig. 2, as the concentration of the water extract was 25-200 mg/L, the inhibition rate of algae cells changed slightly and was always smaller than 20%. After treatment time exceeded 216 h, the inhibition rate of algae cells began to decrease when the concentration of the water extract was 25, 100 and 200 mg/L. As the concentration of the water extract was 500 and 1 000 mg/L, the inhibition rate of algae cells rose with the increase of treatment time. That is, it increased from 50% to 60% with the increase of treatment time from 144 to 168 h, and it was above 60% when treatment time was 192, 216, 240 and 264 h. The results indicated that low concentrations of the water extract inhibited the reproduction of M. aeruginosa cells slightly, but the inhibition effect was obvious when the concentration was higher than 500 mg/L. The inhibition effect of water extract from R. japonica on M. aeruginosa showed clear timeeffect and dosageeffect relationship. According to Table 1, the total degree of freedom was 20, and the degree of freedom among groups and in a group was 6 and 14 respectively. The calculation results showed that F=17.310, P=0.000<0.01, that is, there was 99% confidence that there was a significant difference between different concentrations of water extract from R. japonica in the inhibition effect of M. aeruginosa, thereby rejecting H0 and accepting H1. The inhibition effect of different concentrations of water extract from R. japonica on M. aeruginosa was significant at 0.01 level.
Fig. 2Inhibition rate of different concentrations of water extract from R. japonica on M. aeruginosa
Table1Analysis of variance of the inhibition effect of M. aeruginosa by different concentrations of R. japonica
Source of variationSum of squares (SS)Degree of freedom (df)Mean square (MS)FP value
Among groups1.53550.30719.0080
In a group0.194120.016
Total1.72917
Effects of different concentrations of water extract from R. japonica on the specific growth rate of M. aeruginosa in various treatment periods
Seen from Table 2, the specific growth rate of M. aeruginosa cells decreased with the increase of treatment time in the control group. The specific growth rate in the test groups was smaller than that of the control group, showing that the growth of M. aeruginosa was inhibited by different concentrations of water extract from R. japonica. When the concentration of the water extract was 25-200 mg/L, the specific growth rate was positive, indicating that the effect was not significant. As the concentration of the water extract was 500 mg/L, the specific growth rate became negative after 96 h, that is, the growth of M. aeruginosa cells was inhibited after 96 h. When the concentration of the water extract was 1 000 mg/L, the growth of algae cells was completely inhibited from the beginning. Seen from the changes of the specific growth rate, in the same concentration, the inhibition effect of the water extract increased with the increase of treatment time. In the simulation environment, with the increase of treatment time, the higher the concentration of water extract from R. japonica was, the lower the specific growth rate of M. aeruginosa, and the better the inhibition effect was.
Agricultural Biotechnology2019
Table 2Specific growth rate of M. aeruginosa inhibited by different concentrations of water extract from R. japonica Time∥h
Specific growth rate∥d-1
0 mg/L 25 mg/L50 mg/L100 mg/L 200 mg/L500 mg/L1 000 mg/L
240.1880.1100.1180.1190.1070.069-0.047
480.1250.0980.0670.0710.0910.028-0.028
720.1010.0760.0540.0650.0760.004-0.026
960.0960.0680.0520.0620.061-0.011-0.029
1200.0870.0700.0570.0560.056-0.034-0.044
1440.0770.0620.0500.0550.057-0.05-0.046
1680.0700.0560.0440.0490.051-0.069-0.060
1920.0570.0450.0330.0390.041-0.065-0.064
2160.0520.0370.0320.0350.041-0.061-0.065
2400.0520.0420.0290.0430.047-0.054-0.074
2640.0490.0380.0280.0400.044-0.051-0.083
Effects of different concentrations of water extract from R. japonica on the electrical conductivity of M. aeruginosa solution in various treatment periods
As shown in Fig. 3, in the simulation environment, the electrical conductivity of M. aeruginosa solution in the control group fluctuated from 881 to 979 μs/cm within 216 h, but the change was not significant. With the increases in the concentration of water extract from R. japonica (25-200 mg/L) and treatment time, the electrical conductivity of M. aeruginosa solution rose, but there was no significant difference between the test groups and the control group. When the concentration of the water extract was 1 000 mg/L, the electrical conductivity of M. aeruginosa solution increased to 1 014 μs/cm after 120 h, significantly higher than that of the control group. It revealed that after the addition of high concentration of the water extract from R. japonica, the permeability of cell membranes of M. aeruginosa changed significantly, and their damage became more and more serious, so that the electrical conductivity of M. aeruginosa solution increased.
Fig. 3Effects of different concentrations of water extract from R. japonica on the electrical conductivity of M. aeruginosa solution
Effects of different concentrations of water extract from R. japonica on the pH of M. aeruginosa solution in various treatment periods
Seen from Fig. 4, in the simulation environment, the pH of M. aeruginosa solution in the control group was 8.3-10.2, showing an increasing trend. Meanwhile, the density of M. aeruginosa cells also increased gradually, showing that the higher the pH was, the higher the reproductive efficiency of M. aeruginosa. The pH of M. aeruginosa solution in the test groups (25-200 mg/L) was lower than that of the control group, but there was no significant change. When the concentration of water extract from R. japonica increased to 500 and 1 000 mg/L, the pH of M. aeruginosa solution was significantly lower than that of the control group, and decreased with the increase of treatment time after 192 h. When the concentration was 1 000 mg/L, the pH was lower than 8 after 24 h. The results were consistent with the result of effects of different concentrations of water extract from R. japonica on the specific growth rate of M. aeruginosa, which further proved that M. aeruginosa had a stronger response to the inhibition of high concentrations of water extract from R. japonica.
Fig. 4Effects of different concentrations of water extract from R. japonica on the pH of M. aeruginosa solution
Discussion
M. aeruginosa, which is the most common algae causing algal bloom, produces toxins and leads to absence of oxygen in water during the process of decomposition to destroy the normal food web and directly threaten human health and the survival of other animals and plants. Therefore, it is important to develop an environmentally friendly natural reagent used to inhibit algae growth. Studies have shown that the current natural environmental factors have more significant effects on the community structure of algae than human factors［17］. Environmental factors have synergistic effects on algae growth, and experimental conditions should be close to the environment during the process of research to obtain more reliable conclusions［18］. Therefore, temperature, light and soil conditions close to the environment were set in this experiment. It was found that in the artificial simulated natural environment, high concentrations of water extract from R. japonica could significantly inhibit the proliferation of M. aeruginosa cells.
M. aeruginosa is a prokaryote, and its structure is similar to that of E. coli and other bacteria. The inhibition effect of R. japonica on M. aeruginosa may be the result of pharmacological synergy of different types of chemical components［11］. The experimental results showed that when the concentration of the water extract exceeded 500 mg/L, the electrical conductivity of M. aeruginosa solution increased significantly. The reason is that the terpenoids in R. japonica might destroy the structure of M. aeruginosa cell membranes［10］, thereby inhibiting the proliferation of M. aeruginosa cells, which is consistent with the result of effect of different concentrations of water extract on the cell density of M. aeruginosa.
Most of the lakes in China are alkaline and the pH is high. Studies have shown that in the process of water bloom, the pH of water environment increases significantly with the growth of algae, and the pH of the water environment suitable for the growth of M. aeruginosa is 8-9 on average. Other studies real that high pH (about 11.0) can promote the growth and proliferation of algae cells［19-21］. As the concentration of the water extract was 1 000 mg/L, the pH of M. aeruginosa solution declined significantly after 24 h, lower than 8, while the pH of M. aeruginosa solution in the control group was always higher than 9. It showed that the environment with high pH was more suitable for the growth of M. aeruginosa, while the pH of water environment in the treatments containing water extract from R. japonica would be decreased to inhibit the growth of M. aeruginosa. The inhibition effect of water extract from R. japonica on M. aeruginosa showed clear timeeffect and dosageeffect relationship. Conclusions
In the simulated natural growth environment, the responses of M. aeruginosa to the inhibition of different concentrations of water extract from R. japonica were studied. The results showed that low concentrations of water extract from R. japonica (25-200 mg/L) had no significant effect on the reproduction of M. aeruginosa. When the concentrations of water extract from R. japonica was higher than 500 mg/L, the cell density, specific growth rate and pH of M. aeruginosa decreased, while the inhibition rate and electrical conductivity of M. aeruginosa rose with the increase of treatment time and water extract concentration. According to the results of variance analysis, there was 99% confidence that the inhibition effect of different concentrations of water extract from R. japonica on M. aeruginosa was extremely significant. It provides reference for the application of R. japonica in water protection and the development of a new reagent used to inhibit algae growth in aquaculture water.
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