Studies On Growth Of Alexandrium Sp.LC3 And Extinguishing Mechanism By Cupric Glutamate

Recently, the red tides have occurred frequently all over the world, which resulting in the economy loss and the environmental pollution. Red tides are formed usually by the mass gathering of microalgae, protozoan and bacteria that change the color of the seawater or do harm to the other marine organisms, then lead to the ecological abnormality. Alexandrium is one of the most harmful red tide alga distributing in the world, about thirty dinoflagellates in this genera can release natural toxin which caused massive kills of marine animals, and threaten human healthy. Therefore, it is necessary to know about the occurring mechanism of Alexandrium and prevent of the occurrence of this algae.Some environmental factors that induce the occurrence of Alexandrium have been studied and most papers were about the nutrients (nitrogen, phosphorus et al) effect on the proliferation of Alexandrium. Now, the effects of trace heavy metal elements get more attention as research expansion. Spread clay and chemicals are employed widely for eliminating harmful algal blooms (HABs). It was ever a widely accepted method to use cupric sulfate to control the red tide algae worldwide. However, high cupric concentration from cupric sulfate do harm to other organisms and marine ecological environment. Spread clay was becoming a widely accepted method worldwide. Over the past 25 years, clays have been investigated in several countries as a means of removing harmful algae from the water column. Red tide is controlled through the flocculation of clay particle to the red tide organism. Althoughclay is considered as a promising and attractive direct control option, it is not available for some locations with HAB problems, and high transportation cost would quickly render this method uneconomical. It is highly desirable to find alternative options and know about its extinguishing mechanism.Alexandrium sp. LC3 used in this study was provided by Fishery College of China Ocean University, its growth was preliminary studied and the extinguishing mechanism by new algaecide was researched using SEM, TEM, 2D-electrophoresis and MALDI-TOF analysis.(1) An orthogonal designing method was used to study the effects of N, P, Fe and Mn on the growth of Alexandrium sp. LC3 and compare the effect order of these factors. The experiment consisted of sixteen groups, for which we selected four levels for N, P, Fe and Mn, respectively. The variance for cell density, growth rate and generation time were analyzed. The results showed that PO4-P remarkably enhanced the maximum cell density and growth rate of Alexandrium sp. LC3. NO3-N, Fe and Mn enhanced its growth and the algae got the maximum growth rate under 2 646 umol/L NO3-N, 3.5 p.g/L Fe and 18-36 ng/L Mn, respectively. All the four factors had strong enhancing effect on the growth of Alexandrium sp. LC3, the effecting order was PO4-P > Mn > NO3-N > Fe.(2) Removal effects of cpsaicin, sodium percarbonate, cupric sulfate and cupric glutamate on Alexandrium sp. LC3 were studied to find new effective algaecide. Alexandrium sp. LC3 couldn't be removed under low concentration of cpsaicin (0.1, 1.0, 10.0 mg/ml). The amounts of cpsaicin needed to be effective were much. The survival rate of the algae was relative high under sodium percarbonat operation, so it was not fit for these two compounds to control the red tide organisms. Cupric glutamate had an excellent ability to extinguish Alexandrium sp. LC3 and was superior to cupric sulfate, the superiority got more obviously as time lasting, but no enhancement with the improvement of cupric glutamate concentration.A preliminary study on the effect of hexadecyltrimethyleamine bromide (HDTMAB) promotion to the mitigation of Alexandrium sp. LC3 by cupric glutamate was carried out. Effect of different concentration of HDTMAB promotion was studied.The results showed that HDTMAB could increase the extinguishments of harmful algae bloom (HAB) obviously. There was a positive correlation between concentration of HDTMAB and increasing killing effect. Above results indicate the application potential of cupric glutamate and surfactant for red tide prevention as a novel algicide and an accelerant, respectively.(3 ) Changes of cell morphology and ultrastructure of Alexandrium sp. LC3 under cupric sulfate, cupric glutamate, HDTMAB and cupric glutamate-HDTMAB operation were studied with SEM and TEM. Under these stresses, the integrity of cell plasma membrane (cell membrane, chloroplast membrane and mitochondria membrane) was destroyed, which indicated the plasma membrane was the assaulting target.These stress compounds caused structure damage to chloroplast and mitochondria, which affected the respiration and photosynthesis directly and inhibited the growth of algae, even led to the death of cell. Under copper ion stress, the number of starch grain and liposome increased and electron-opaque deposits were observed on the vacuole, nucleus and cell membrane. These materials are probably related with alleviating toxicity of heavy metal.(4) The effect of 0.5xlO'3 mol/L cupric glutamate on the cell membrane permeability, Malonaldehyde (MDA) and SH group content of Alexandrium sp. LC3 after six days incubation was studied. MDA content and cell membrane permeability increased obviously and SH group content decreased under 0.5xlO"3 mol/L cupric glutamate operation, which indicated that cupric glutamate removed the algae cells through making the plasma membrane being seriously damaged and the enhancement of the membrane permeability, then mass extravasation of intracellular components. Study of the effect of different concentration of HDTMAB on the cell physiological level of Alexandrium sp. LC3 showed that the lipid peroxidation level in cells was higher under cupric glutamate-surfactant joint operation than cupric glutamate alone. In addition of the content of sulfhydryl group decreased significantly, plasma membrane permeability of leaked cell solution was increased. These indicated the extinguishing mechanism: the cell membrane was damaged followed by the inhibitionof the cell growth. This verified the conclusion that was described in 4.4.1.(5) HDTMAB associated with Ca2+ were used to eliminate the red tide. Attempt was made to study the effect of Ca2+ on the removal of Alexandrium sp. LC3 under HDTMAB stress. The results showed that the eliminate effect of HDTMAB on Alexandrium sp. LC3 was mitigated by Ca2+ obviously, especially under 4 mmol/L of Ca2+. The mitigating mechanism of HDTMAB by Ca2+ was investigated by detecting the SH group, MDA content of the cell membrane and membrane permeability, and indicated that the SH content of cell member increased, the MDA content and membrane permeability decreased when Alexandrium sp. LC3 was treated with Ca2+ and HDTMAB complex, compared to HDTMAB used only. These are suggested that the Ca2+ promotion of HDTMAB stress resistance of Alexandrium sp. LC3 is to reduce the permeability and increase the stability of cell membrane induced by Ca2+.(6) The technology of two-dimensional electrophoresis(2-DE) of protein from Alexandrium sp. LC3 including the preparation of protein sample was studied. The result showed that in SDS-PAGE, the relative molecular weights of proteins were mainly between 14-31 kDa and 43 -66 kDa, and more proteins were detected between 14-31 kDa. The preparation method of protein sample was improved. The two-dimensional electrophoresis patterns of protein from Alexandrium sp. LC3 showed that the relative molecular weights of protein were between 14-100 kDa, and most of them were in the range of 14 -31 kDa, which was consistent with the result of SDS-PAGE. The isoelectric points were found to lie between 3.0-8.0, and most of them were in the range of 3.0-6.0. Better separation effect were acquired with pre-prepared immobilized gradient (IPG) strip (pH 3.0-5.6), about 320 protein spots could be visualized on the 2-DE map by staining. With pH 3.0-10.0 and pH 3.0-5.6 strips, the protein samples of Alexandrium sp. LC3 could be separated well.(7) Study with SDS-PAGE on the effect of different concentration of HDTMAB on the protein expression of algae cell showed that HDTMAB had a negative effect on the content of protein expression. Compared with control group and cupric glutamine operation only, bands of protein decreased. The extinguishing mechanism of Alexandrium sp. LC3 by cupric glutamate was further studied withSDS-PAGE and 2-DE. Results indicated that cupric glutamate not only suppressed the synthesis of high molecular weight proteins (>31 kDa), but also induced the synthesis of some low molecular weight proteins (<31 kDa). This is suggested that the low molecular weight proteins maybe correlate with the auto protection of algae cell under stress. These indicated another extinguishing mechanism by cupric glutamate and promoting mechanism by HDTMAB was that: cupric glutamate or HDTMAB induced denaturation and disintegration of intracellular protein then the cell growth was inhibited.(8 ) Three different protein spots under cupric glutamate stress were excised and identified using peptide mass fingerprint and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. A summary of the identified proteins and their putative functions is presented. Spot no.l was identified as ribulose-bisphosphate carboxylase polyprotein precursor (form II), spot no.2 as RNA polymerase beta1 chain, and spot no.3 was resemble to a hypothetical protein from Guillardia theta. Spots no.l and no.2 were suppressed under cupric glutamate, the reasons for the repression of expression of Rubisco (spot no.l) are: 1) oxidative modification induced by cupric glutamate triggered the catabolism of Rubisco. 2) damage of chloroplast structure promoted the inhibition of synthesis of Rubisco by cupric glutamate. 3) Rubisco degraded as storage protein, which was probably a auto-response to the stress. Oxidative modification and damage of chloroplast structure induce the synthesis inhibition of RNA polymerase (spot no.2). Cupric glutamate induced the synthesis of protein spot no.3, the possible role for this protein may correlate with the auto protection of algae cell under stress.In brief, cupric glutamate had an excellent ability to extinguish Alexandrium sp. LC3 and was superior to cupric sulfate, the superiority got more obviously as time lasting, but no enhancement with the improvement of cupric glutamate concentration. HDTMAB could stimulate the extinguishments by cupric glutamate obviously. There was a positive correlation between concentration of HDTMAB and increasing effect. Remarkable changes of the cell physiological level, morphology and ultrastructure changed obviously under the stress of cupric glutamate or HDTMAB indicated thatthese two compounds inhibited the growth of algae through damaging the cell membrane. Studies on the change of protein under stress using 2-DE and mass spectrometry showed that cupric glutamate induced denaturation and disintegration of intracellular protein, then the cell growth was inhibited. Above results indicated the extinguishing mechanism by cupric glutamate is that: 1) the cell membrane is damaged by cupric glutamate; 2) cupric glutamate induces denaturation and disintegration of intracellular protein, then the cell growth is inhibited. This research indicates the application potential of cupric glutamate and surfactant in controlling harmful algal blooms in the future as a novel algicide and an accelerant respectively due to their efficiency in extinguishing Alexandrium sp. LC3 cells.