Preliminary Study Of The Biological Control On The Anabaena Flos-aquae

The eutrophication of water bodies is a serious environmental problem in today's society,which normally leads to cyanobacterial blooms and brings great harm to the water dody.It not only reduced the water landscape function,but also releases large amount secondary metabolites,which destroys the water environments and human health.These toxicants are mainly cyanotoxins,which causing serious damage to aquatic ecosystem,changing the water bodies to be black and niffy,even depriving the water of basic functions.China is one of the countries which are contaminated with the most eutrophic water and are affected deeply by the cyanobacterial blooms.Especially,the cyanobacterial blooms are a extremely serious problem in the potable water area and aquaculture ponds.In this study,we selected a representative species—Anabaena flos-aquae as target.An aquatic animal Lymnaea sp.and an aquatic microoganism of a marine bacterium were employed as biological controlling tools to inhibit A.flos-aquae in laborary condition.Based on the preliminary quantitative studies between the inhibitory strength of Lymnaea sp.or the marine bacterium and A.flos-aquae growth,the combination effects of these two biological tools were examined.Based on the data of the inhibitory effects obtained in laboratory experiments,the biological controlling effect as well as the water quality were studied in the presence of A.flos-aquae,Lymnaea sp.and the marine bacterium.The main results of this study are as follows.(1)In the experiments of Lymnaea sp.against A.flos-aquae,Lymnaea sp.was found efficiently controlling the growth of A.flos-aquae.Taking 5 adults of Lymnaea sp.in 250 ml water as an example,althrough the initial biomass of A.flos-aquae in the control groups and the treatment group were same as 7.4×106?m·mL-1,the biomass of A.flos-aquae increased to 12.6×100?m·mL-1 after 5 days in the control but decreased to 1.1×100?m·mL-1 in the treatment.The inhibition rate achieved 83.7%.Meanwhile,the concentration of chlorophyll-a,chycocyanin,soluble protein in the treatments were decreased by 76.4%,80.7%and 99.5%in comparison with those in the controls,respectively.The extracellular soluble polysaccharide,however,was increased in the treatments with a percentage of 21.9%.By analysis on the data with various ratio between Lymnaea sp.and initial biomass of A.flos-aquae,it was found that the best biological controlling effects was observed with more Lymnaea sp.added and less initial biomass of A.flos-aquae.The applicability of using Lymnaea sp.to control algae bloom were then verified.(2)A bacterial strain of HY339 was selected as the bioclogical controlling tool based on its strong inhibitory effects against A.flos-aquae in the preliminary screening and among 6 potential marine bacteria strains.The inhibitory effects by various adding dose,incubation time,the initial biomass of A.flos-aquae,or the desalination of bacterial medium were examined.The results indicated that the better inhibitions were observed with the more addition of bacrerial culture.Taking both the inhibitory effect and reacting time into account,3%(volume of bacterial culture:algal culture)was the opitimal ratio.Differente incubation time of the bacterium led various inhibitory effect against A.flos-aquae.among which incubation time of 5 days was better than others.The lower the initial algal biomass resulted the better inhibition effect.Importantly,the desalination of bacterial medium did not cause significant difference in the inhibitory effects against A.flos-aquae.Therefore,we draw a conclusion that the salinity in culture medium do not influence the inhibitory effect of bacterial strain of HY339 against A.flos-aquae.(3)The biomass of A.flos-aquae decreased from 6.4×106 ?m·mL-1 to 2.7×106?m·mL-1 during 5 days with addition of 3%(v:v)bacterial culture of HY339,which had been incubated for 5 days.The inhibitory rate(5d)was 57.5%.Meanwhile,the concentration of chlorophyll-a,chycocyanin,soluble protein,extracellular soluble polysaccharide,and soluble protein in the treatments were decreased by 18.8%,39.7%,15.3%and 7.7%in comparison with those in the controls,respectively.The study on the inhibition of strain HY339 showed that the secondary metabolites from the strain of HY339 were the active component and resistant to high temperature.The extract by ethyl acetate(mixed with 5%acetone)of the strain possessed 55.5%of the inhibitory effect of the culture.By the determination of NMT(non-invasive micro-test technique),the stream of extracellular H+ and Na+ of A.flos-aquae cells treated for 1 day by bacterial metabolites were detected,and the results of which show that H+ were flowing into the cell while Na+was discharging from the cells.The microscopic observation for thus treated A.flos-aquae cells showed that cell structure was destroyed.Therefore,the treatment with the active substances destroyed the structure of algal cell and caused cell membranet not being able to maintain the osmotic pressure,which then led abnormal ion flows.A 16S rDNA genetic sequence analysis for strain HY339 was performed,the obtained sequence from which was compared with those of other strains in gene bank.The strain HY339 was identified as Bacillus cereus based on phylogenetic tree builded.(4)After the biological controlling experiment by single organisms was conducted,the joint treatments with different strength of Lymnaea sp.and strain HY339 against A.flos-aquae were studied.The results showed that the joint treatment possess better activites than single ones.And the more addition of Lymnaea sp.adults or strain HY339 cultutr brought the higher biological controlling activity.The replacement of bacterial cultire with those extracted by ethyl acetate(mixed with 5%acetone)in the joint treatments led less inhibitory activitiers.(5)The joint treatments with Lymnaea sp.and strain HY339 against A.flos-aquae were than employed in a larger and open water tank to simulate natural water conditions.Lymnaea sp.and strain HY339 were added in different growth phase of Anabaena flos-aquae.The results from low initial biomass treatment indicated that less number of snails achieved higher inhibitory effect against the growth of the A.flos-aquae in comparison with those in laboratory conditions.In logarithm growth phase,the certain number of Lymnaea sp.also inhibited the rapid growth in biomass.In the case of high initial biomass,larger number of Lymnaea sp.was needed to reduce the algal biomass rapidly,and the small number only slowed down the growth of A.flos-aquae.(6)The concentration changes of total nitrogen and total phosphorus in the joint treatment were determinated,and the results of which showed that the concentrations of total nitrogen and total phosphorusc were reducing in both controls and treatments.But the reduction in the treatments was lower than those in the controls.The survival rates of Lymnaea sp.in treatment groups in this open water were analyzed,from which not only the effective inhibition against the growth of A.flos-aquae but also the higher survival rates of Lymnaea sp.than those in lab conditions were observed.The freshly released spawns were also observed in the treatment tanks,which indicated that the adults of Lymnaea sp.were at healthy conditions and highly tolerant to toxins released by A.flos-aquae.