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Experimenal Study on the Allelopathy of Two Kind of Submerged Plants


Experimental Study on the Allelopathy of Two Kind of Submerged Plants
WU Shan LI Xiao-hong Yang Long
Civil Engineering College Beijing University of Technology Beijing, China
Abstract—The landscape water body's eutrophication tendency is one of the main issues of recycled water reuses to urban water systems needs to be resolved. The allelopathy of aquatic plants which can inhibit the excessive breeding of algae is a new water quality control technology which is low-carbon and green technology compared with the traditional physical and chemical methods. This process doesn't need dosing potions, and no energy consumption needed. For less damage to the environment, it could get integrated benefits both for water quality environment and socio-economic. This experiment mainly discusses the inhibitory effect of two kinds of Submerged plants ( Ceratophyllum demersum L 、 Hydrilla verticillata ) to Chlorella pyrenoidosa Chick and Scenedesmus oblipuus which are dominant species in the landscape water body of Beijing, in the condition of co-culture. Choosing the content of algal cell’s chlorophyll A, the color of the solution, and the number of algal cells as indicators, the changed situations of algae growth in the process of co-culture are analysis. The results could be used to provide an experimental basis for the technology of allelopathy on the growth of algae control. Keywords-Allelopathy, Co-culture, Ceratophyllum demersum L Hydrilla verticillata,

I.

INTRODUCTION

With the development of economy and the growth of the population, the environmental security and the shortage of water resources is more prominent[1],the recycling of sewage can not only ease the intense contradiction between water supply and demand, but also improve the utilization efficiency of water, in the meantime, it can improve the quality of water environment of the city [2]. However, because the recycled water has unique nutrient levels, it causes the abnormal breeding of algae, occurs eutrophication when it supplements to the urban landscape water body, various phenomena which do harm to the recycling of water will be presented, for example, reduction of the transparency of water, decrease of the dissolved oxygen content, the water ecosystem to be destroyed [3]. Therefore, various technical means and measures must be taken to control the phenomena of eutrophication, maintenance water environmental safety. The research about allelopathy to inhibit the growth of algae began in the late 20th century, but has become a independent research field, it becomes a new way to control eutrophication which is low-carbon and green. Aquatic plants which act as a major producer in water body have a very important role in regulating water functions and maintaining

the ecological balance of water, in particular, during the growth of aquatic plants, they can produce and secrete a chemical substance (allelochemicals) to impact the growth and the metabolism of algae, for example, affecting the enzymatic activity of algae, damaging algae cell membrane, affecting the respiratory metabolism of algae, inhibiting the nutrient absorption of algae, changing the microstructure of algal cells, and so on[4-6].This effect is called the allelopathy. Allelochemicals are mainly distributed in the root, stem, leaf, flower, fruit and seeds of aquatic plants, and the common release ways are the surface volatilization、the stem and leaf leaching 、 the root secretion 、 the plant stump decomposition[7]. These allelochemicals can degrade in the natural condition; will not affect the aquatic ecosystems. Many plants have the allelopathy which inhibits the growth of algal, for example, Phragmitis communis Trin, Acorus calamus, Canna glauca l., Ceratophyllum demersum L., and so on[8-11]. Different aquatic plants will produce different kinds of allelochemicals, which will has the different inhibitory action to the different algae, for example, coumaric acid, sinapic acid, caffeic acid HPLC which are produced by Phragmites communis Trin have the inhibitory action to the Microcystis aeruginosa[6.12-13], N-phenyl-2-naphthalene amine produced by Eichhornia crassipes has the inhibitory action to the Chlamydomonas reinhardtii[6,14], as to the same kind of allelochemical, the allelopathy is different on different kinds of algal, for example, the allelochemicals extracted from Phragmitis communis Trin show significant inhibitory effects on the growth of S.capricornutum, however, no obvious inhibition on the growth of Chlamydomonas reinhardtii can be observed , but the motility of algal cells declined[15]. Therefore, it is very important to seek more aquatic plants which have allelopathy and allelochemicals, and to explore the mechanism of allelopathy, furthermore, the application of allelopathy on the eutrophication control is the focus and key point of current research. This experiment chose two kind of submerged plants (Hydrilla verticillata, Ceratophyllum demersum L.) which are dominant species in landscape water in Beijing, also have the landscape effect, studied the allelopathy of the plants on Chlorella pyrenoidosa Chick and Scenedesmus oblipuus which are the advantage algaes of eutrophic water body. Taking the color of the solution, the content of algal cell’s chlorophyll A, the number of algal cells as indicators, this experiment

978-1-4244-5089-3/11/$26.00 ?2011 IEEE

analyzed the inhibition influence due to Hydrilla verticillata and Ceratophyllum demersum L. on the growth of algae in the process of co-culture, and the result will provide an experimental basis for allelopathy inhibition on the growth of algae control. II. A. MATERIAL AND METHOD

12 hours, the cultivation time is seven days, the content of chlorophyll A is measured everyday, the algae counting and the photograph are made separately before and after the experiment. TABLE 1 Composition of Modified Hoagland Solution[16]
Chemical Ca(NO3)2 KNO3 K2HPO4 MgSO4 Solution of ferri-ion (TABLE 2) Solution of microelements (TABLE 3) pH Concentration 945 mg/L 607 mg/L 60.0 mg/L 493 mg/L 2.5 ml/L 5 ml/L 6.0

Materials Hydrilla verticillata is a perennial submerged herb, which is distributed in ponds, lakes and ditches. Ceratophyllum demersum L. is also a perennial submerged herb which is suspended in water, has strong ability to absorb nitrogen, it grows better When high levels of inorganic nitrogen in water. Chlorella pyrenoidosa Chick and Scenedesmus oblipuus are purchased from Institute of Hydrobiology, the Chinese Academy of Sciences. Before the experiment, the algae species mentioned above were pre-cultured in SE culture medium so that it can be in the logarithmic growth phase. B. Methods 20L 1/10 improved Hoagland solution (see TABLE 1) is added into two 50cm×30cm×50cm plexiglass incubators, and Hydrilla verticillata whose wet net weight is 30g are added into a plexiglass incubator, as to the other plexiglass incubator, no Hydrilla verticillata is added; At the same time, the same concentration of algae is added into two incubators, and the initial concentration of Chlorella pyrenoidosa Chick is 4×104 per ml, that of Scenedesmus oblipuus is 6×104 per ml; Uses oxygenizement pump to do aeration, the filled air can not only provide dissolved oxygen, but also play a role of mixture. Coculture system is placed in a room, and the temperature is about 28±3°c, the illumination time is equal to the dark time which is 12 hours, the cultivation time is six days, the content of chlorophyll A is measured everyday, the algae counting and the photograph are made separately before and after the experiment. The co-culture equipment is showed in Figure 1.

TABLE 2 Solution of Ferri-ion[16]
Chemical FeSO4·7H2O Na2EDTA pH Concentration 2.78 g/500mL 3.73 g/500Ml 5.5

TABLE 3 Solution of Microelements[16]
Chemical KI H3BO3 MnSO4 ZnSO4 NaMoO4 CuSO4 CoCl2 Concentration 0.83 mg/L 6.2 mg/L 22.3 mg/L 8.6 mg/L 0.25 mg/L 0.025 mg/L 0.025 mg/L

III.

RESULTS AND DISCUSSION

?

A. Co-culture experiment of Hydrilla verticillata The influence of Hydrilla verticillata on chlorophyll A content of two kinds of algae

The co-culture equipment is the same as Hydrilla verticillata, 60L 1/10 improved Hoagland solutionis added into two plexiglass incubators, and Ceratophyllum demersum L. whose wet net weight is 110g is added into a plexiglass incubator, as to the other incubator, no Ceratophyllum demersum L .is added; At the same time, the same concentration of algae is added into two incubators, and the initial concentration of Chlorella pyrenoidosa Chick is 20750 per ml, that of Scenedesmus oblipuus is 2250 per ml; Coculture system is placed in a room, and the temperature is about 30±3°c, the illumination time is equal to the dark time which is

Figure.1 Co-culture Device

Taking the color of the two incubators and the content of algal cell’s chlorophyll A as indicators, the influence of Hydrilla verticillata on the growth of Chlorella pyrenoidosa Chick and Scenedesmus obliquus is analyzed in this experiment. In the beginning of the experiment, the colors which the algae present is almost the same in two incubators; by the end of the experiment, the water color in the incubator where the Hydrilla verticillata is planted is much more transparent than that of the incubator where no Hydrilla verticillata is planted, and this indicates that there is less algae in the first incubator. According to the visual result, the inhibitory action to algae's growth due to the Hydrilla verticillata can be observed. Detail inhibition degree can be seen from the change tendency of content of chlorophyll A of the two kinds of algal. The change tendency of chlorophyll A in two incubators during the coculture process is showed in Figure 2. In this experiment, the volume and the content of nutrient fluid are the same to the incubators, and nutritionally adequate, the illumination degree, the oxygen content (external environment) are also maintain same basically, the incubator

where the Hydrilla verticillata is planted is lower than that where no Hydrilla verticillata is planted, so it shows that the Hydrilla verticillata has inhibited the algae's growth, furthermore, this algae’s chlorophyll A content in the kind of inhibitory action is obvious strengthened with the increase of culture time.
Don't add Hydrilla verticillata Add Hydrilla verticillata
26 24 22

shows that the Hydrilla verticillata can promote the growth of Chlorella pyrenoidosa. B. Co-culture experiment of Ceratophyllum demersum L. ? The influence of Ceratophyllum demersum L. on chlorophyll A content of two kinds of algae Taking the color of the two incubators and the content of algal cell’s chlorophyll A as indicators, the influence of Ceratophyllum demersum L. on the growth of Chlorella pyrenoidosa Chick and Scenedesmus obliquus is analyzed in this experiment. In the beginning of the experiment, the color which the nutrient solution present is almost the same in two incubators; by the end of the experiment, the water color in the incubator where the Ceratophyllum demersum L. is planted is much more transparent than that of the incubator where no Ceratophyllum demersum L. is planted, and this indicates that there is less algae in the first incubator, as to the other incubator, we can see algal blooms. According to the visual result, the inhibitory action to algae's growth due to the Ceratophyllum demersum L. can be observed. Detail inhibition degree can be seen from the change tendency of content of chlorophyll A of the two kinds of algal. The change tendency of chlorophyll A in two incubators during the co-culture process is showed in Figure 3.
Don't add Ceratophyllum demersum L. Add Ceratophyllum demersum L.
34 32 30 28

Chlorophyll A Content( m g/m )

3

20 18 16 14 12 10 8 6 4 2 0 0 1 2 3 4 5 6 7

Incubation Time( d)

Figure 2 The Allelopathy of Hydrilla verticillata on Chlorella pyrenoidosa Chick and Scenedesmus oblipuus in the Co-culture System

?

The different influence of Hydrilla verticillata on two kinds of algae The algae cell’s quantity change by the cytometry is taken as an indicator in the experiment, and the inhibition degree of Hydrilla verticillata on two kinds of algae is analyzed. The change of the quantity of two kinds of algae's cell before and after the experiment is showed in Table 4 and Table 5. TABLE 4 The Change of the Quantity in the Fist Incubator Before and After the Experiment
Chlorella pyrenoidosa (/ml) 4×104 10×104 Scenedesmus oblipuus (/ml) 6×104 4.5×104

Chlorophyll A Content( mg/m )

26 24 22 20 18 16 14 12 10 8 6 4 2 0 0 1 2 3 4 5 6 7

Before the experiment After the experiment

3

Incubation Time( d)

TABLE 5 The Change of the Quantity in the Second Incubator Before and After the Experiment
Chlorella pyrenoidosa (/ml) 4×104 6×104 Scenedesmus oblipuus (/ml) 6×104 21×104

Figure 3 The Allelopathy of Ceratophyllum demersum L. on Chlorella pyrenoidosa Chick and Scenedesmus oblipuus in the Co-culture System

Before the experiment After the experiment

Compared to the beginning of the experiment, in the incubator which plants the Hydrilla verticillata , there is a decrease of 1.5×104 per ml in the number of Scenedesmus oblipuus at the end of the experiment, however, in the other incubator which has not planted the Hydrilla verticillata, there is an increase of 15×104 per ml in the number of Scenedesmus oblipuus at the end of the experiment, this experiment shows that the Hydrilla verticillata has a very strong inhibitory action to the growth of Scenedesmus oblipuus. At the same time, there is an increase of 6×104 per ml in the number of Chlorella pyrenoidosa at the end of the experiment, however, in the other incubator which has not planted the Hydrilla verticillata, there is an increase of 2×104 per ml in the number of Chlorella pyrenoidosa at the end of the experiment, this experiment

The algae’s chlorophyll A content in the incubator where the Ceratophyllum demersum L. is planted is lower than that where no Ceratophyllum demersum L. is planted in the case of the same culture environment, and this gap is becoming increasingly obvious, it shows that the Ceratophyllum demersum L. has inhibited the algae's growth control. ? The different influence of Ceratophyllum demersum L. on two kinds of algae The algae cell’s quantity change by the cytometry is taken as an indicator in the experiment, and the inhibition degree of Ceratophyllum demersum L. on two kinds of algae is analyzed. The change of the quantity of two kinds of algae's cell before and after the experiment is showed in Table 6 and Table 7. The number of Scenedesmus oblipuus is 0 per ml in the incubator which plants the Ceratophyllum demersum L. at the end of the experiment, however, in the other incubator which has not planted the Ceratophyllum demersum L., the number is 3.5×105per ml, it shows that the Ceratophyllum demersum L. has a very strong inhibitory action to the growth of Scenedesmus oblipuus. At the same time, the number of

Chlorella pyrenoidosa Chick in the incubator which plants the Ceratophyllum demersum L. compared to has not planted the Ceratophyllum demersum L. increased 0.75×105 per ml at the end of the experiment, it shows that the Ceratophyllum demersum L. can promote the growth of Chlorella pyrenoidosa Chick. TABLE 6 The Change of the Quantity in the Fist Incubator Before and After the Experiment
Chlorella pyrenoidosa (/ml) 2.075×104 3×105 Scenedesmus oblipuus (/ml) 2250 0

REFERENCES
[1] Huang Weiwei.Research on Characteristics of Eutrophication in Urban Landscape Water Body Supplemented by Reclaimed Water[D].Tangshan:Hebei Polytechnic University.2008(in Chinese) Zhao Lejun,Wang Xiuduo,Liu Chunguang , et al. Research on trend of eutrophication in Landscape Water Body Supplemented by Reclaimed Water[J].Water & Wastewater Engineering. 2008,34(12):13~16(in Chinese) Zhao Budiao,Liu Baizhu,Lu Xiaofang,et al.Formation,Hazard and Prevention of Eutrophication in Water Body[J].Anhui Agricultural Science Bulletin,2007,13(17):51-53(in Chinese) Wu Xiaohui.Studies on the allelopathy of several Submerged Potamogetonaceae species on Planktonic algae[D]. Wuhan: Chinese Academy of Sciences .2005 (in Chinese) Li Fengmin,Hu Hongying,Zhong Yunxiao,et al. Effects of Allelochemical EMA Isolated from Phragmites communis on Algal Cell Membrane Lipid and Ultrastructure[J]. Environmental Science,2007,28(7):1534-8.(in Chinese) Hong Yu,Hu Hongying. Research and application of inhibitory allelopathy from aquatic plants on algae[J].Chinese Science Bulletin,2009,54(03): 287-293. (in Chinese) Hu Hongying,Men Yujie,Li Fengmin. Research progress on phytoallelopathic algae control[J]. Ecology and Environment, 2006,15(1):153157(in Chinese) Li Fengmin,Hu Hongying. Allelopathy and Inhibitory Effect of Extracts from Macrophytes on Algae Growth[J]. China Water & Wastewater, ,2004, 20(11) :18-21(in Chinese) Ding Huijun,Zhang Weihao,Wang Chao , et al. Allelopathic Effect of Acorus calamus on Some Species of Algae[J]. Environmental Science and Technology, 2007,30(6):20-22(in Chinese) Li Ruihua,Guan Yuntao,He Miao,et al. Treating polluted river water withCanna glauca l.,Vetiveria zizanioides and Scirpus yagara ohw zones[J]. Journal of Tsinghua University(Science and Technology), 2006,46(3):366-370(in Chinese) Gross E M,Erhard D,Enik? I.Allelopathic activity of Ceratophyllum demersum L.and Najas marina ssp.Intermedia(Wolfgang)Casper[J].Hydrobiologia,2003,506-509:583589 Li F M,Hu H Y.Isolation and characterization of a novel antialgal allelochemical from Phragmites communis[J].Appl Environ Microbiol,2005,71:6545-6553 Zhou S,Nakai S,Hosomi M,etal.Allelopathic growth inhibition of cyanobacteria by reed[J].Allelopathy J,2006,18(2):277-285 Sun Wenhao,Yu Shuwen,Yang Shanyuan,et al. Allelochemicals from Root Exudates of Water Hyacinth(Eichhornis crassipes) [J].Acta phytophysiologica Sinica,1993,19(1):92-96 (in Chinese) Men Yujie,Li Fengmin,Hu Hongying.Effects of an allelopathic fraction in Phragmitis commuis Trin on the growth characteristics of Selenastrum capricornutum and Chlamydomonas reinhardtii[J].Journal of Lake Sciences,2007,19(4):473-478(in Chinese) Zhang Binzhi.Studies on the Allelopathy of Elodea nuttallii on Microcystis aeruginosa [D].Wuhan: Chinese Academy of Sciences.2007(in Chinese)

[2]

[3]

[4]

Before the experiment After the experiment

[5]

[6]

TABLE 7 The Change of the Quantity in the Second Incubator Before and After the Experiment
Chlorella pyrenoidosa (/ml) 2.075×104 2.25×105 Scenedesmus oblipuus (/ml) 2250 3.5×105

[7]

Before the experiment After the experiment

[8]

[9]

IV.

CONCLUSIONS

[10]

Through the co-culture experiments in the same environmental condition, the allelopathy of two kinds of submerged plants (Hydrilla verticillata, Ceratophyllum demersum L.) on Chlorella pyrenoidosa Chick and Scenedesmus oblipuus was discussed. Taking the color of the solution, the content of algal cell’s chlorophyll A and the number of algal cells as indicators, the results show that the Hydrilla verticillata and Ceratophyllum demersum L. can hinder the growth of two kinds of algae as a whole and can reduce the concentration of algae in the solution. However, the allelopathy of Hydrilla verticillata and Ceratophyllum demersum L. affected on Chlorella pyrenoidosa Chick and Scenedesmus oblipuus are not the same. Both submerged plants have an inhibitory effect on the growth of Scenedesmus oblipuus and the inhibitory action is obvious strengthened with the increase of culture time; but both of the two submerged plants have no simulative effect on the growth of Chlorella pyrenoidosa Chick. By the way, because the experiment is carried out under the co-culture system, two kinds of algae also exist, the influence of competition between Chlorella pyrenoidosa Chick and Scenedesmus oblipuus cannot be excluded. The application of the technology of allelopathy, has also maintained the environment security during control the eutrophication of reclaimed water.

[11]

[12]

[13] [14]

[15]

[16]


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