Allelopathic activity and nutrients competition between Ceratophyllum demersum and Microcystis aeruginosa
State Key Laboratory of Pollution Control and Resource Reuse, School of Environment, Nanjing University, Nanjing 210093, China. E-Mail: xianqm@hotmail.com
This paper compared the depletion of NH4-N and PO4_P in separated and coexistent culture of Ceratophyllum demersum and Microcystis aeruginosa. The results indicated the depletion of NH4-N in the coexistent medium was more than in the separated, while the depletion of PO4_P was nearly the same. The amount of algae decreased in the coexistence more than in the separation by counting the number of algae. Competition of N and P nutrients was not the primary power leading to the decrease of algae but allelopathic activity of C. demersum against M. aeruginosa. Allelochemicals in the culture solution were analyzed with Solid phase extraction – high performance liquid chromatography (SPE-HPLC). Content of chemicals in the coexistence was different from the separation at wavelength of 254 nm. In the coexistence experiment the presence of algae might stimulate the release of some potential allelochemicals from macrophytes to inhibit the growth of algae.
Not nutrients competition but allelopathic chemicals inhibited the growth of algae in co-culture water of Ceratophyllum demersum and Microcystis aeruginosa.
Keywords
Ceratophyllum demersum L., Microcystis aeruginosa Kütz, coexistence, competition, allelopathic activity.
Introduction
Shallow eutrophic lakes tend to be either in a turbid state dominated by phytoplankton or in a clearwater state dominated by submerged macrophytes (Scheffer et al. 1993). Ceratophyllum demersum L. played an important role in stabilizing and maintaining a Clearwater state at high P concentrations (Mjelde and Faafeng, 1997), and had low epiphyte densities under N, but not under P limitation (Fitzgerald 1969). This alternative nutrient source may enable macrophytes to grow even when nutrient concentrations in the water are very low, thus, providing them with a competitive advantage over the phytoplankton (Barko and James, 1998). A few studies also showed allelopathic effects of C. demersum on the growth of blue-green algae (Gross et al. 2003; Xian et al. 2005). However, dominating role of either nutrients competition or allelopathic effect was seldom separated.
In the present study, we performed a series of laboratory experiments to compare the effects of nutrients competition and allelopathic interactions between macrophytes and algae, and analyze allelopathic chemicals in co-culture with SPE-HPLC.
Material and methods
Macrophytes
C. demersum L. plants were collected from local ponds, washed thoroughly and maintained in multiple sets of aquaria at ambient temperature in diffuse light. 600-gram of the plants were cultivated with 1/10 Hoagland’s solution. The plants were acclimatized to laboratory conditions for more than one month prior to experiments.
Algae
Microcystis aeruginosa Kütz, were obtained from the Environmental Biological Laboratory, Nanjing University. Before experiment the algae were cultivated in 1/10 Hoagland’s solution at 25±1 °C with a 14 h light/10 h dark photoperiod and a light intensity of 4000-6000 lux till the concentration of algae reaching about 107 cells/ml.
Co-culture of algae and macrophytes
In a 500 mL Erlenmeyer flask with sterile silica gel-stoppered, macrophytes (6 g-wet) were cultured in 200 mL 1/10 Hoagland’s solution where algae were inoculated. Co-culture was cultivated in the same condition of algal growth, control groups were prepared with separated culture of algae and macrophytes, and all the experiments were repeated twice. Concentrations of NH4-N and PO4-P were measured with photometric methods (Wei 1989; Liu 1999) and densities of algal cell were counted with a hemocytometer per 72 h, macrophytes were weighed at the end of experiment.
Extraction of allelochemicals by solid phase extraction and analysis by HPLC
1 L incubation water after 72 h cultivation (Fraction 1) was filtered through 0.22 µm membrane filter and passed over a SPE-C18 filter (AccuBond SPE ODS-C18 Cartridges, 1g, 6 mL) preconditioned with 10 mL dichloromethane and methanol in turn to adsorb exuded compounds. The filtrate (Fraction 2) and Fraction 1 were bioassayed. The SPE-C18 filter was then dried in stream of nitrogen gas for 5 min, and then the adsorbed compounds were washed with 20 mL methanol. The eluate was concentrated in a rotation evaporator and re-dissolved in 2 mL with methanol for the analyses in HPLC (Hewlett Packard Series 1100, including a diode array detector at the wavelengths of 254 nm, software: HP-Chemstation), equipped with a C18 reversed-phase column (ODS, 5 µm, 15 cm × 4.5 mm ID) at 30 °C. Mobile phase composition is methanol-water (40:60, v/v) at a flow rate of 1.0 ml min-1. A 10 µL injection of each sample was done in duplicate using an autosampler.
Bioassay
Algal growth inhibition of aqueous solution was performed with M. aeruginosa as follows. In sterile foam-stoppered 250 mL Erlenmeyer flask, 150 mL aqueous solution was added and then 3 mL algae inoculated into them. The algae were cultivated for 72 h under the same condition as above. Algal growth was monitored with a microscope and a hemocytometer by counting cell number at the stationary phase in 0 h, 72 h. Control groups were prepared with 1/10 Hoagland’s solution substituting culture water. Each experiment included triplicate treatments and the experiments were repeated twice.
Result and discussion
Competition for nutrients
Biomass of macrophytes and algae increased rapidly in separated cultivation, however, biomass of macrophytes increased slowly and algae died out after 72 h (Figure 1) in the co-culture experiment. Weight of macrophytes increased by 40% in the separated experiment but increased by 20% in the co-culture during 2-weeks cultivation (Figure 2). The growth of macrophytes was affected by algae due to the competition of nutrients. Depletion of N and P nutrients was significant during the periods of growth (Figure 3), and was faster in group 2 (macrophytes) and group 3 (algae and macrophytes) than in group 1 (algae). Uptake rates of NH4-N were higher than of PO4-P, more than 70% of NH4-N, but less than 50% of PO4-P was depleted in two weeks cultivation. Uptake of NH4-N was more different between macrophytes and algae, especially during early growth periods. However, uptake of PO4-P was almost the same. After 6-day growth, nutrients (NH4-N and PO4-P) maintained stable level in the groups containing macrophytes.
Competition for nutrients between macrophytes and algae was noticed in co-culture, which was coincident with previous research results (Ozimek et al. 1990; van Donk et al. 1993). And after 2-week cultivation nutrients levels showed no difference among three groups, but exponential growth of M. aeruginosa in group1 and disappearance in group 3 were found. Uptake potential of ammonium N by C. demersum was stronger than that of phosphate P. However, Lombardo et al (2003) found that C. demersum could absorb P at a high rate in higher trophic state. Nutrients were not the primary factors causing algae to die away in the co-culture. In addition, light was also not a restrained condition in our experiment. Allelopathic activity between macrophytes and algae may play a significant role to inhibit algal growth.
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Figure 1. Algal growth inhibition in co-culture. |
Figure 2. Macrophytes growth in co-culture. |
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Figure 3. Uptake of nutrients (NH4-N and PO4-P). 1, algae; 2, macrophytes; 3, algae and macrophytes | |
SPE-HPLC analysis
Growth inhibition of Fraction 1 and 2 on M. aeruginosa showed that inhibitory effect of Fraction 2 was weaker than that of Fraction 1 (Figure 4). Allelopathic chemicals in culture water were effectively adsorbed in SPE-C18 filter. The chemical compositions and contents of the three culture analyzed in HPLC measurement in 254 nm wavelength showed different analyte signals, especially in second half part of chromatogram. Content of compounds with the retention time within 0.5-2 min in diagram a were more than in diagram b, which showed these compounds released were inhibited in co-culture water.
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Figure 4. Allelopathic effect of co-culture water through SPE-C18 filter. |
Figure 5. SPE-HPLC diagram of culture water (a, macrophytes; b, macrophytes and algae; c, algae). |
A compound with retention time at 7 min should be paid attention since its peak area in diagram b was much more than in diagram a, but the compound was not found in diagram c. The compound increased insignificantly its content in co-culture water, and macrophytes were stimulated to release the compound into water in the stress of algae. Neighboring competition stress could induce occurrence of allelopathic activity of organism, and further enhance organism’s competitive ability. There might be chemical signals transmitted between macrophytes and algae. In addition, strong polar chemicals passed through column faster than weak polar chemicals due to the application of reversed-phase column, so weak polar compounds may be the main allelochemicals to inhibit the growth of M. aeruginosa. It needs further structural identification of the mixture and confirmation of its allelopathic effect. Hyphenated techniques such as LC/UV, LC/MS and more recently LC/NMR, quickly provides plenty of structural information, leading to a partial or a complete on-line structure determination of the secondary metabolite of plants (Wolfender et al. 2003).
Conclusion
In laboratory condition, competition for nutrients between C. demersum and M. aeruginosa and algal death were found in co-culture water. They adsorbed NH4-N more than PO4-P in the earlier period of their growth. Allelopahtic activity was the primary reason causing algae to die off, but not the competition for nutrients. Allelochemicals might be concentrated in the SPE-C18 filter, and the analysis of HPLC indicated that concentrations of allelochemicals was different under the stress of algae. Weak polar compounds may contribute more allelopathic effect on algae.
Acknowledgement
This work was supported by The National Basic Research Program of China (No: 2002CB412307).
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