| Nowadays, along with the development of economy, entrophication is becoming more and more serious and resulting in an increasingly frequent algae bloom, which damages water ecosystem as well as the health of human, animals and plants. Microcystis aeruginosa is the most common species in algal blooms, especially in our country. It produces microcystins (MC), which is one of the most common, dangerous and studied algal toxin, and is found to cause liver disease. Great attentions were paid to cyanobacteria bloom worldwidely in the past decades, and many control methods were found, including biological control method as the most promising strategy. The detection methods for microcystins have also been studied and classified as biological detection, chemical analysis, and biochemical detection.In this study, Lymnaea sp. was selected as the main control species against algae, the changes of various indicators as well as the algal toxin along with the growth of Microcystis aeruginosa controlled by Lamnaea sp. were followed. The control effects were compared with other biological control strategies to find a suitable method to inhibit the growth of Microcystis aeruginosa and to control the algae toxin content simultaneously. Thus method would be applicable and necessary for aquaculture ponds. In detail, growths of four kinds of Microcystis aeruginosa treated by Lamnaea sp. were followed to verify the control effects in this study. At the same time, and the algal toxins were analyzed. Under natural, simulative and laboratory conditions with various nutritiion, the growth and toxin products of Microcystis aeruginosa incubated with and without various biological controlling species were followed. The main results of this thesis are as follows:(1) The analysis for the growth of four types of Microcystis aeruginosa.Fours types of Microcystis aeruginosa (including Microcystis aeruginosa starins of T1343, T905, T942, and T918) were used in this study. The cell densities, the absorbance of the culture in terms of optical density at 600nm(OD600nm), chlorophyll-a, phycocyanin, extracellular MC-LR, total MC, and total toxicity were tracked and compared with each other during no-affected-growth. Results indicated that the cell densities of all four strains showed good exponential relationship with incubation time, and increased 168.08×105 cell/ml,41.70×105 cell/ml,83.25×105 cell/ml, and 33.25×105 cell/ml after 6 days incubations for strains of T1343, T905, T942, and T918, respectively. The increased cell densities were T1343>T942>T905>T918. OD600nm (detected by 96 wells reader), OD600nm (detected by sepectrophotometer), chlorophyll-a, and phycocyanin were found with a linear relationship with cell densities. The greatest slope for OD600nm vs. cell densities were T905=T918 of 0.001 by 96 wells reader and 0.0041 by sepectrophotometer, while the greatest slope for chlorophyll-a or phycocyanin vs. cell densities were T918 of 6.3018 or T905 of 0.1508, respectively. Total toxicities of T905 and T918 were also found with a linear relationship against cell densities. The greatest slope for extracellular MC-LR was 0.1968 for T918, and in the cases of total MC and total toxicity they ware 0.6938 for T918 and 2.0593 for T905, respectively. Total toxicity of T1343 and T942 were found with a logarithmic rather than linear relationship against cell densities. In a word, OD600nm, chlorophyll-a, extracellular MC-LR and total MC of T918 have the greatest growth rate, so we can were focused on T918 for the further study.(2) Controlling effects of Lymnaea sp. against Microcystis aeruginosa and a preliminary analysis of microcystins in the treatmentA comparison analysis of the controlling effects of Lymnaea sp. against four types of Microcystis aeruginosa (including Microcystis aeruginosa starins of T1343, T905, T942, and T918) was performed. The results showed that cell density of strains of T1343, T905, T942, and T918 treated with Lymnaea sp. were 28×105 cell/ml,17.25×105 cell/ml,21.50×105 cell/ml and 4.75 ×105 cell/ml in the end of incubations, which wrere much less than the respective control cell densities of 1172.50×105 cell/ml,127.75×105 cell/ml,245.25×105 cell/ml, and 60.00×105 cell/ml without Lymnaea sp.. It indicates that Lymnaea sp. significantly control the growth of all four types of Microcystis aeruginosa. The numbers of surviving Lymnaea sp. adults were counted at the end of each incubation, and the MC-LR, total MC, and extrecellular total toxicities treated with Lymnaea sp. were monitored in the start and the end of the treatments. The results showed the survival rates of Lymnaea sp. were 33.33% for T1343,60% for T905,26.67% for T942 and 75% for T918. The extrecellular MC-LR treated by Lymnaea sp. were 0 μg/L for T1343,0.106 μg/L for T905, 28.867 μg/L for T918, the values of which were far less than respective controls MC-LR of 0.199 μg/L,25.349 μg/L,34.991 μg/L without Lymnaea sp. addition. Total MC controlled by Lymnaea sp. were 0.856 μg/L for T1343,159.210 μg/L for T905,and 2.090 μg/L for T942, the concentrations of which were also far less than those of 1.785 μg/L,177.622 μg/L,221.080 μg/L in controls of T1343, T905, T942 without Lymnaea sp. additions. Extrecellular total toxicities ofT1343, T905, T942 and T918 in the presence of Lymnaea sp. were equivalent to 4.827 mg/L CuSO4 for T1343,2.448 mg/L CuSO4 for T905,2.046 mg/L CuSO4 for T942,0.796 mg/L CuSO4 for T918, the toxicities were far less than those of 13.602 mg/L CuSO4,586.414 mg/L CuSO4,542.260 mg/L CuSO4,70.755 mg/L CuSO4in the absence of Lymnaea sp., respectively. Therefore, Lymnaea sp. can significantly control extracellular total MC of T942 and total toxicity of T905,T942,and T918.(3) The effects of nutrient conditions against the growth of Microcystis aeruginosaThe growths of Microcystis aeruginosa in different N, P concentrations were studied under the natural condition. The growths, extrecellular MC and total toxicity of Microcystis aeruginosa 918 in different N, P, Fe concentrations were also studied under the laboratory conditions. In the natural condition, A ten folds diluted BG-11 culture medium was used as a basic culture medium. N, P concentration was changed based on the basic medium through the modification of NaNO3 and KH2PO4 concentration, and the basic medium was named as NP(1N1P) while 5 folds increased N and P concentrations were named as 5NP and N5P, respectively. The results showed that the cells densities were 111.375×105cell/ml of N5P>86.750×105cell/ml of NP>43.125×105 cell/ml of 5NP after incubation time of 35 days. The fastest cell growth was observed when NaNO3 is 0.15 g/L and KH2PO4 is 0.02 g/L, and the slowest one was appeared when NaNO3 is 0.75 g/L and KH2PO4 is 0.004 g/L. It indicated that the high P concentration promoted the growth of Microcystis aeruginosa in a certain concentration range, while high N concentration sometimes inhibited Microcystis aeruginosa grows. Surely, there are many factors affecting the growth, the details need further studies.Strain T918 was chosen for the study under laboratory conditions. BG-11 culture medium was used as a basic culture medium. Concentrations of NaNO3, KH2PO4, or Ferric ammonium citrate were 5 folds changed based on BG-11 medium, and the modified media were named as NPFe (BG-11), 1/5NPFe,5NPFe, N1/5PFe, N5PFe, NP1/5Fe, and NP5Fe, respectively. The results showed that the highest cell density was observed as 147.50×105 cell/ml in the culture of 5NPFe while the lowest cell density was 101.50×105 cell/ml in the culture of 1/5NPFe after 8 days incubations. The maximum OD6oonm was 0.211 (96 wells reader) or 0.534 (spectrophotometer) after 8 days incubations. There was no significant difference observed for other treatments. The maximum and minimum chlorophyll-a were 810.92 μg/L and 344.34 μg/L in the culture of N5PFe and Nl/5PFe after 10 days incubations, respectively. The maximum extrecellular MC-LR was 8.830 μg/L in the culture of 1/5NPFe, followed by 5.131 μg/L in theculture of N1/5PFe after 10 days incubations, and there was no significant difference for other treatments. The maximum total MC was 125.112 ug/L in the culture of 5NPFe, followed with 99.346 μg/L in the culture of N5PFe, while the minimum total MC was 53.297 μg/L in the culture of NP1/5Fe after 10 days incubations. The maximum extrecellular total toxicity represented by CUSO4 concentrations was 14.813 mg/L of 5NPFe, followed with 7.834 mg/L in the culture of of NPFe, while the minimum was 1.676 mg/L in the culture of Nl/5PFe. Therefore, P concentration was the factor most significantly affecting OD600m and chlorophyll-a of strain T918. Both OD600nm and chlorophyll-a increased constantly when KH2PO4 concentration increased from 0.008 g/L to 0.2 g/L without the changes of N, Fe concentrations. N concentration was the factor most significantly affecting cell density, extracellular MC-LR, total MC, and total toxicity. Within 0.3-7.5 g/L of NaNO3, the cell density, extracellular total MC, and total toxicity increased along with ascent of NaNO3 concentration from 0.3 to 7.5 g/L without changes of P and Fe concentrations.(4) Effect of different biological methods against Microcystis aeruginosa and a preliminary analysis of microcystins in the treatmentsUnder laboratory conditions, the controlling effects of treatments of Lymnaea sp., effective microorganisms (EM, probiotics), duckweed, Lymnaea sp. plus duckweed, Lymnaea sp. plus EM, and Chlorella sp. against strain T918 were studied, and intracellular and extracellular MC as well as MC in the controlling species of Lymnaea sp. were analyzed. In addition, the samiliar process for simulating conditions of nature ecosystem was also followed. The results under the laboratory were as follows:After 8 days culture, cell densities of strain T918 were decreased significantly to a level near to zero in the treatments of Lymnaea sp., duckweed, Lymnaea sp. plus duckweed, and Lymnaea sp. plus EM while the control was 105.50×105 cell/ml. In the case of OD600nm (96 wells reader), those in the treatment of Lymnaea sp., duckweed, and Lymnaea sp. plus duckweed were 0.034,0.035, and 0.038, respectively, and were far less than that of 0.203 in controls. OD600nm (spectrophotometer) of 0.051 for Lymnaea sp.,0.054 for duckweed, and 0.053 for Lymnaea sp. plus duckweed were observed, and were also far less than 0.473 in controls. Chlorophyll-a contents were significantly decreased to 31.24 μg/L,41.98 μg/L,52.14 μg/L, and 67.25 μg/L in the treatments of Lymnaea sp., duckweed, Lymnaea sp. plus duckweed, and Lymnaea sp. plus EM for strain T918 after 8 days culture, respectively. They were far less than that of 370.02 μg/L in controls. Intracellular and extracellular MC-LR were as low as nondetectable for the treatments of Lymnaea sp., duckweed,Lymnaea sp. plus duckweed, and Lymnaea sp. plus EM while intracellular MC-LR of 153.018 μg/L and extracellular MC-LR of 7.403 ∴g/L were observed in the controls after 8 days culture. After 8 days culture, intracellular total MC of strain T918 in the treatment of Lymnaea sp., duckweed, Lymnaea sp. plus duckweed, and Lymnaea sp. plus EM were 0.061 μg/L,0.019μug/L, 0.016 μg/L,2.218 μg/L, respectively, while it was 108.903 μg/L for control. In the cases of extracellular total MC, they were 8.134 μg/L for Lymnaea sp.,7.067 μg/L for duckweed,6.598 μg/L for Lymnaea sp. plus duckweed,11.629 μg/L for Lymnaea sp. plus EM. They were also far less than 116.006 μg/L in controls. The extracellular total toxicities (represented by CuSO4 concentrations) of strain T918 were significantly decreased by the treatments of Lymnaea sp., duckweed, and Lymnaea sp. plus duckweed to 10.578 mg/L,8.699 mg/L,5.892 mg/L after 3 days culture, respectively, and were far less than that of 635.118 mg/L in controls.The results from simulating conditions of nature ecosystem were as follows. In the absence of carp fishes, the lowest cell density of 1.25 ×105 cell/ml at 4 day was observed in the treatment of Lymnaea sp. plus duckweed without carps, while it was 13.25×105 cell/ml for controls. In the presence of carp fishes, the lowest cell density of 2.25×105 cell/ml at 4 day was observed in the treatment of Lymnaea sp. plus duckweed, while it was 5.75×105 cell/ml for controls. In the absence of carp fishes, the lowest OD600nm (spectrophotometer) of 0.047 after 5 days culture was observed in the treatment of Lymnaea sp. plus duckweed, which was far less than that of 0.091 in controls. In the presence of carps fishes, OD600nm (spectrophotometer) was significantly reduced by the addition of carps fishes, and resulted in 0.052 after 5 days culture when that of the blank group was 0.091. The values were not significant among other treatments. The similar situation was observed in the case of OD6000nm (96 wells reader) as that in OD600nm (spectrophotometer). In the absence of carp fishes, the lowest chlorophyll-a contend of 36.14 μg/L after 4 days culture was observed in the treatment of Lymnaea sp. plus duckweed, which was far less than 74.16 μg/L of controls. In the presence of carp fishes, chlorophyll-a contend after 4 days was most efficiently reduced to 58.07 μg/L in the treatment of Lymnaea sp. plus duckweed, which was far less than 77.06 μg/L in controls.The results of MC and toxicity in Lymnaea sp. in the end of experiments was as follows. Under the laboratory condition, the survival rates of Lymnaea sp. were 40% in the treatment of Lymnaea sp.,77% in the treatment Lymnaea sp. plus duckweed, and 0% in the treatment of Lymnaea sp. plus EM. Thrse results indicated that a certain amount of duckweed was beneficial to Lymnaea sp. survives, while EM inhibited the survivals of Lymnaea sp. In the end of experiment, Lymnaea sp. in the treatment of Lymnaea sp. had Oug/kg MC-LR,15.551 μg/kg total MC, and 2332.982 mg/kg (CuSO4) total toxicity. Lymnaea sp. in the treatment of Lymnaea sp. plus duckweed had 0 μg/kg for MC-LR,6.749 μg/kg for total MC, and 1789.718 mg/kg (CuSO4) for total toxicity. Above results indicated that a certain amount of duckweed was not only beneficial to Lymnaea sp. survivals, but also able to reduce the MC and total toxicity in Lymnaea sp. Under the simulating conditions of nature ecosystem, the survival rates of carp fishes in the treatment of control, Lymnaea sp. plus duckweed, and Lymnaea sp. were 80%,80%, and 60%, respectively. It indicated that a certain amount of duckweed was beneficial to carp fishes survives. The survival rate of Lymnaea sp. in the treatment of Lymnaea sp. with and without carp fishes was 48% and 52%, respectively. The survival rate of Lymnaea sp. in the treatment of Lymnaea sp. plus duckweed with and without carp fishes were both same as that in the treatment of Lymnaea sp with carp fishes. in the presence of carp fishes. So, a certain amount of duckweed was not harmful to Lymnaea sp.. In the end of the experiments, MC-LR in Lymnaea sp. was all Oug/kg in the treatment of Lymnaea sp. and Lymnaea sp. plus duckweed with or without carps fishes. In the treatments of Lymnaea sp. with and without carps fishes, Lymnaea sp. plus duckweed with or without carps, total MC in Lymnaea sp. were 17.453 μg/kg,22.515 μg/kg, 6.535 μg/kg, and 10.694 μg/kg, respectively, while total toxicities (represented by CuSO4 concentration) in Lymnaea sp. were 2534.472 mg/kg,2526.969 mg/kg,1732.377 mg/kg, and 1743.984 mg/kg in sequence. Total MC of Lymnaea sp. in the presence of carp fishes was higher than those in the absence of carp fishes, while total MC and toxicity in Lymnaea sp. treated with duckweed were lower than those without duckweed. Therefore, a certain amount of duckweed can reduce the MC and toxicity in Lymnaea sp..The results of this study showed that the growth of Microcystis aeruginosa was significantly inhibited by Lymnaea sp.. At the same time, the high cell density of Microcystis aeruginosa also caused Lymnaea sp. death, and the color of water becoming yellow, residual MC and toxicity in water remaining high. Therefore, other species were also involved in the biological methods and compared with that only using Lymnaea sp. The results led the conclusion that Lymnaea sp. plus duckweed not only can inhibit Microcystis aeruginosa growth, improve the survival rate of Lymnaea sp. and carp fishes, but also is able to reduce intracellular and extracellular MC, toxicity of algae and their residue in Lymnaea sp. bodies. Present method was a promising method to inhibit Microcystis aeruginosa growth after the quantitative relationship being further investgated. |
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