Studies On The Allelopathic Effects Of Macroalgae On Red Tide Microalgae

Severe outbreaks of harmful algal blooms (hereafter, HABs) cause serious problems with regard to effective utilization of water resources such as fisheries and water-supply reservoirs, moreover, about 2000 cases of human poisoning resulting from algal toxin are reported every year. Because of the severe economic and public health problems caused by harmful microalgae, many studies about the blooms of harmful microalgae have been conducted. Some promising methods have been developed including the use of yellow losses to sediment red tide organisms, chemical agents such as copper sulfate, hydrogen peroxide, and some biological controls in the form of viruses or bacteria. Although these methods seem effective in some short-term experiments, they may have potentially dangerous environmental consequences; therefore there are very limited investigations of a direct and specific control of marine harmful algal blooms with few environmental side-effects.To look for HABs control agents that are efficient and benign to the environment, many scientists show a growing interest for growth inhibition of HAB species by allelopathic substances released by other aquatic organisms. Macroalgae and microalgae were known to have an antagonistic relationship in both natural and experimental aquatic ecosystems.The present studies deal with the allelopathic effects of three macroalgae Ulva pertusa (Chlorophyta), Corallina pilulifera (Rhodophyta) and Sargassum thunbergii (Phaeophyta) on three red tide microalgae Heterosigama akashiwo, Alexandrium tamarense and Skeletonema costatum, and isolation and characterization of these allelochemicals. The three main objectives of these studies were to provide proof of the allelopathic effects of three macroalgae on these three microalgae, to gain an insight into the chemical interactions between macroalgae and microalgae in coastal areas, and to present evidence for the feasibility of using many macroalgae to control the growth of red tide microalgae. Results show that:1. Comparative studies on the allelopathic effects of three macroalgae on three microalgaeWe conducted laboratory experiments under stable environmental conditions, precluding the nutrient, pH, light conditions and effects of bacteria in order to investigate the allelopathic effects of fresh tissue, dry powder and aqueous extracts of three macroalga U. pertusa, C. pilulifera and S. thunbergii on the growth of microalgae H. akashiwo, A. tamarense and S. costatum were evaluated using coexistence culture systems in which concentrations of three macroalga were varied. The effects of macroalga culture medium filtrate on three microalgae were also investigated. The results coexistence assay showed that the growth of the three microalgae was strongly inhibited by using fresh tissues and dry powder; this indicated that there are the allelopathic effects of three macroalgae on three microalgae. Aqueous extracts of various concentrations of three macroalga can inhibit the growth of three microalgae and indicated that there are allelochemicals in three macroalgae; The effects of the macroalga culture medium filtrate exhibited no apparent inhibitory effect under initial filtrate addition but show apparent inhibitory effect under semi-continuous addition, which suggested that continuous release of small quantities of rapidly degradable allelochemicals from the fresh tissue of macroalga were essential to effectively inhibit the growth of three microalgae. In our study, we also concluded that the allelopathic effects of macroalgae on red tide microalgae show universality and species-specific. 2. Growth inhibition of H. akashiwo and A. tamarense by the extracts of C. pilulifera and S. thunbergiiGrowth inhibitory effects of different concentrations of distilled water extracts and 4 organic solvent (methanol, acetone, ether and chloroform) extracts of C. pilulifera and S. thunbergii on the two microalgae H. akashiwo and A. tamarense were investigated. Methanol extracts of the highest growth inhibitory activity on two microalgae, and they killed all the cells of two microalgae at relatively higher concentrations. Growth of the two microalgae was significantly inhibited by the distilled water extracts of two macroalgae at relatively higher concentrations while cells of the two microalgae did not die completely even at the highest concentration. The other 3 organic solvent extracts of C. pilulifera and S. thunbergii had no apparent effect on the two microalgae, which suggested that the growth inhibitory substances from the extracts of two macroalgae had relatively high polarities. The methanol extracts of the two macroalgae were partitioned to petroleum ether phase, ethyl acetate phase, butanol phase and distilled phase by liquid-liquid fractionation. The bioassays of the activity of every fraction were carried out on two microalgae, indicated that petroleum ether phase and ethyl acetate phase had strong algicidal effect on the microalgae, fatty acids are probably main allelochemicals in the tissue of macroalgae.3. Isolation and characterization of allelochemicals in the tissues of C. pilulifera and S. thunbergiiThe petroleum ether phase and ethyl acetate phase of the methanol extracts of C. pilulifera and S. thunbergii were analyzed by GC/MS. The results of GC/MS revealed that there were 12 fatty acids in the 2 phase, and most of them were unfatty acids. The results of bioassays demonstrated that 5 unsaturated fatty acids in the tissue of C. pilulifera and S. thunbergii had strong algicidal effect on H. akashiwo and A. tamarense, and the effective concentrations were below 7.1 mg l^-1. The results this study demonstrated that unsaturated fatty acids in the tissue of C. pilulifera and S. thunbergii might be one of the components of the allelochemicals in the macroalgae. Taken together, it is speculated that unsaturated fatty acids in the tissue of C. pilulifera and S. thunbergii has a potential to be considered as allelochemicals for the control of several harmful algae. However, it is not clear yet whether this compound can practically be applied to open waters. So further studies on the algicidal should focus on efficacy and effects on the algal control spectrum and mode of action are needed.4. Influence of temperature, salinity, irradiance and pH on the effectiveness of allelopathic effects of C.pilulifera on H. akashiwo and S.thunbergii on A. tamarense and two microalgae growthTwo-factor combinations of different levels of temperature (10, 15, 25 and 30), salinity (10, 20, 30 and 40), irradiance (20,100,200 and 400μmol m^-2s^-1) and pH (5.5, 7, 8.5 and 10) on the effectiveness of allelopathic effect of C. pilulifera on H. akashiwo or S. thunbergii on A. tamarense and growth of the two microalgae were investigated. Two-factor combinations of different levels of temperature, salinity, irradiance and pH had significant (p<0.01) influence on the mean growth rates of two microalgae, and on the microalgal growth inhibition rates by C. pilulifera or S. thunbergii. The optical growth conditions for H. akashiwo was: 25°C, 30, 100μmol m^-2s^-1 and pH=8.5, and for A. tamarense was: 20²5°C, 30, 100μmol m^-2s^-1 and pH=7-8.5. The effectiveness of allelopathic effects of C. pilulifera on H. akashiwo or S. thunbergii on A. tamarense was increased by low temperature (15°C), low salinity (20), high irradiance (400μmol m^-2s^-1) and high pH (10).