Studies On The Ecophysiological Effects Of Microcystins Against Microbes

Toxic cyanobacterial blooms in fresh water bodies have been becoming a kind of ecological disaster worldwide. One of the most harmful effects of cyanobacterial blooms is typically from the toxins produced by the algae,which represent a major threat to human, livestock and wildlife health. Of all the algal toxins, microcystins are among the most abundant and diverse. Most investigations into the toxicity of the microcystins are focused on animals and higher plants. In this paper the ecophysiological effects of microcystins on bacteria and its possible mechanism were studied. The main results are as follows:1. Microcystins from Microcystis bloom in Lake Dianchi were extracted and purified with high performance liquid chromatography (HPLC). MC-RRwith a purity over 95% was finally obtained by preparative HPLC.2. The growth and biochemical response of Escherichia coli (E.coli) and Bacillus subtilis (B. subtilis) to MC-RR were studied. The results showed that the growth and cell viability of bacteria were inhibited for a short period compared with that of the control when exposed to MC-RR. But the dose of MC-RR did not have a lethal effect. E.coli and B. subtilis only showed growth inhibition at the initial growth phase when cells were treated with MC-RR. Indeed the normal rate of growth was gradually re-established and the growth curves of the toxin-treated and untreated bacteria became parallel. The cell viability of E.coli and B. subtilis were also inhibited for a short period compared with that of the control which was similar to the growth of bacteria. The contents of protein and soluble sugar in cells increased compared with that of the control at the beginning of MC-RR exposure and then gradually decreased. The results showed the stress of MC-RR on E.coli and B. subtlis. The change of protein and soluble sugar make cells suit for the stress of MC-RR gradually.3. The permeabilising ability of MC-RR under different concentrations to the cell outer membrane of E. coli and B. subtilis was demonstrated by a rapid and sustained reduction in the OD675 values of lysozyme-treated cells. And the decrease of the absorbance values significantly showed a time- and dose-effect. The extravasations of protein and soluble sugar increased with the increment of the treated-concentration of MC-RR and the prolonged of the treated-time. The results showed that MC-RR could increase the permeability of cell outer membrane of E.coli and B.subtilis. Though the permeability effect of microcystins on cell outer membranes is still controversial and inconclusive our results once again showed that MC-RR could increase the permeability of cell outer membrane of E.coli and B.subtilis. And the synergistic effects of MC-RR and lysozyme on bacteria might indicate that MC-RR might play the ecological role on bacteria by combining with other substance in some aquatic environments.4. In the present paper, E.coli was undertaken to determine the effect of microcystin-RR on microbes. For this purpose, six biochemical parameters including reactive oxygen species (ROS), thiobarbituric acid reactive substances (TBARS), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione (GSH) were investigated in E.coli when exposed to 0.1, 1, 5, 10 mg/L MC-RR. With high concentrations MC-RR exposure, the results showed that the ROS level and TBARS contents of E.coli were obviously increased after 1 h exposure to MC-RR. At the same time, the activities of antioxidant enzymes including SOD and CAT as well as GR activity and GSH contents in bacterial cells were also increased when exposed to MC-RR for 1 h, subsequently decreased. These results suggested that MC-RR could cause the accumulation of ROS in E.coli and induce the oxidant stress for a short period. The antioxidant system protects E.coli from oxidative damage.5. B. subtilis was undertaken to determine the dose- and time- effect of MC-RR, and the results showed that the activity of antioxidant enzymes including SOD and CAT was significantly increased than that of the control when exposed to 5 or 10 mg/L MC-RR for 1 h. The contents of TBARS and GSH as well as GR activity were obviously increased only when exposed to 10 mg/L MC-RR. For the time-effect of MC-RR on B. subtilis, the activities of antioxidant enzymes including SOD and CAT as well as GR activity and TBARS, GSH content in B. subtilis were first significantly increased and than subsequently decreased. These results suggested that MC-RR could induce the oxidative stress of B. subtilis for a short period. The antioxidant system protects B. subtilis from oxidative damage.6. Two strains denitrifying bacteria and Organic phosphorus bacteria were isolated to further study the effect of MC-RR on the population of functional bacteria. From the morphological characteristics and results of 16S rDNA sequence analysis, the denitrifying bacteria were identified as Bacillus gibsonii and Oceanobacillus iheyensis, the organic phosphorus bacteria were identified as Serratia marcescens.7. The growth and denitrification function of denitrifying bacteria were prolonged by MC-RR. It showed a time- and dose-effect. The decrease of Nitrate and the increase of nitrite were both inhibited as a result of the growth and NR activity inhibition. Indeed with the toxin-treated time increased, the normal rate of growth was gradually re-established and the growth curves of the toxin-treated and untreated bacteria became parallel. The effect of MC-RR on organic phosphorus bacteria was similar to that on denitrifying bacteria. During all the experiment the enzyme activity of ACP and ALP were lower than that of the control and finally the content of phosphate in liquid medium decreased with the exposure of MC-RR.