Physiological Responses to Ferrate (VI) Stress in Microcystis aeruginosa Liming Liu1, 2, 3 , Lin Li1, Zhongxing Wu1, Lirong Song1, 1
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, 430072, China Engineering Research Center of Eco-environment in Three Gorges Reservoir Region, Ministry of Education, China Three
Beijing, 100049, China First author Email address: [email protected]
Abstract—Potassium ferrate (VI) has been considered to be an environmentally friendly oxidant. In this paper, a research on the physiological and biochemical changes of the ferrate (VI)’s acting on Microcystis aeruginosa FACHB-905, a common kind in algal bloom, has been performed. Under the action of ferrate (VI), chlorophyll-a content, the photosynthetic oxygen evolution rate, maximum electron transport rate (ETRm) and maximum photochemical efficiency (Fv/Fm) of Microcystis aeruginosa were significantly decreased, while MC-LR, an extracellular toxin of Microcystis aeruginosa increased. Malonaldehyde (MDA) is oxidatively stressed by low concentration of ferrate (VI). With the increasing concentration of ferrate (VI), MDA content increases, and the activity of SOD and CAT also shows a significant rise. After the treatment of low concentration ferrate (VI), GST activity decreased rapidly, while with the further increasing of ferrate (VI), GST activity increased gradually. This shows that ferrate (VI) produces oxidative stress on Microcystis aeruginosa. The mechanism is that ferrate (VI) inhibits the activity of microcystin photosynthetic PS II system, which leads to the damage to photosynthesis. The accumulation of excessive intracellular free radicals can further lead to the lipid peroxidation of membrane, increasing the membrane permeability, finally causes the death of Microcystis aeruginosa. Keywords- Microcystis aeruginosa; Potassium Ferrate (VI); Physiological Responses
I. Introduction With the rapid development of industry and agriculture and the expansion of the cities, a large amount of industrial wastewater, domestic sewage and farmland surface runoff discharge into the lake, increasing eutrophication of the lake. Eutrophication process often leads to the blooms induced by the algae, especially algae mass-breeding. On the one hand the algae bloom leads to the decline of water clarity, the decreased of dissolved oxygen and the increase of suspended solids and COD. On the other hand, algae produce large quantities of secondary metabolites, such as toxins, odor compounds. These secondary metabolites not only affect the quality of drinking water and aquatic products, but also cause the toxicity of aquatic organisms and some of land animals, and even endanger human health. Potassium ferrate is a strong oxide either in acidic or alkaline conditions for its standard electrode potential in acidic conditions and alkaline conditions is 2.20V and 0.72V, respectively. Therefore, its strong oxidation can effectively
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Gorges University, Yichang, Hubei, 443002, China Graduate School of the Chinese Academy of Sciences,
inactivate microorganisms, oxidize and coagulate metal ions, degrade organic pollutants and remove suspended particulate matter. Its reduction product ferric ion is a green-friendly agent for its flocculation . Potassium ferrate can effectively remove some algae in water body. It is reported that the strong oxidizability of ferrate directly leads the break of the filaments of Oscillatoria and influences on its normal schizont breeding . It also can damage the cellular sheath of scenedesmus and chlorococoum and cause the loss of cellular internal components to kill the algae . Nevertheless, the investigation on the oxidation of potassium ferrate on other algae and the mechanism of action has been rarely performed. In this article, we investigate the physiological and biochemical changes occurring in FACHB-905 Microcystis aeruginosa, a kind of algae which has intimate relations with algae bloom, under the oxidation of potassium ferrate. This work also studied the possible mechanism of the action and aimed at providing experimental evidence to control the algal blooms in the future. II.
Material and methods
A. Microcystis aeruginosa culture Microcystis aeruginosa FACHB-905 was provided by the Culture Collections of the Freshwater Algae of the Institute of Hydrobiology, Chinese Academy of Sciences. They were grown in nutrient-rich BG-11 culture medium under 25 ȝmol photons m-2 s-1 photosynthetically available radiation in 12:12 h light/ dark cycle at 25±1 䉝㻚 B. Preparation of potassium ferrate The preparation of potassium ferrate was based on a procedure of hypochlorite oxidation according to Hrostowski and Scott  and the content of K2FeO4 was determined by the redox titration and the as prepared K2FeO4 was 89.6%. C. Experimental design and parametric measure All the experiments were carried out on a JTZ1-6 stirrer of coagulation-flocculation test. Generally, 250 mL Microcystis aeruginosa in Logarithmic phase was transfer to a conical flask and K2FeO4 at different dosage (theoretical value 5 mg, 10 mg, 15 mg, 20 mg and 25 mg) was added to oxidize the algal while vigorous stirring at a speed of 500 r/min for 10 minutes and then stirring slowly at a speed of 50 r/min for 50 minutes. After
that, the reaction mixture was sampled to analyze Chlorophyll-a, enzymatic activity, photosynthetic oxygen evolution and the parameters of fluorescence spectrometry.
increases. After 2 h, the decrease was not evident. There was no significant change even on the treatment of 12 h.
D. Determination of chlorophyll-a and protein content The samples were centrifuged and chlorophyll-a was extracted with 80% acetone, then placed in a refrigerator of 4䉝㻌 㼒㼛㼞㻌 㻞㻠㻌 㼔㼛㼡㼞㼟㻌 㼍㼚㼐㻌 㼒㼛㼘㼘㼛㼣㼑㼐㻌 㼎㼥㻌 㼏㼑㼚㼠㼞㼕㼒㼡㼓㼑㻚㻌 㼀㼔㼑㻌 㼟㼡㼜㼑㼞㼚㼍㼠㼑㻌 was used to determine the value of A663and A645 and the content of chlorophyll-a was calculated according to the equation of Arnon . The content of total protein was determined by the kits (provided by Jiancheng Bio. Co., Nanjing, China) using the principle of Bradford methods.
B. Effect of potassium ferrate on the photosynthesis of Microcystis aeruginosa The maximum electron transfer rates (ETRm) fluorescence ratio (Fv/Fm) was the reflection of the maximum photochemistry quantum yield and the maximum electron transfer rates (ETRm) was the reflection of the photosynthetic activity of photosynthetic organism. It turned out that the photosynthesis evolution rate decreased with the increase of
E. Measurement of photosynthetic characteristic The measurement of photosynthetic characteristic was performed on a Clark type oxygen electrode (Rank Brothers, Cambridge, UK) under the condition of 25䉝㻌㼍㼚㼐㻌㼕㼞㼞㼍㼐㼕㼍㼚㼏㼑㻌 level of 25ȝmol m-2s-1. The content of chlorophyll-a was recorded when the net photosynthetic rate and respiratory rate were measured. The maximum electron transport rate (ETRm) and maximum photochemical efficiency (Fv/Fm) was measured using PHYTO-PAM˄WalzˈEffeltrichˈGermany˅ and the details of measurement and calculation were describled in the literature [6, 7]. F. Measurement of malonaldehyde (MDA) and anti-oxygenic enzyme activity Typically, 10mL sample was centrifuged at 5000 rpm for 15 minutes to collect the algal cells, and 10% trichloroacetic acid was added and pestled in an ice-bath, then centrifuged at a speed of 8000 rpm, 4ć for another 15 minutes. The supernate was used to detect the content of MDA. For the enzymatic assays, the sample was prepared from 50 mL of cells harvested after each treatment by centrifugation at 7000 rpm for 10 min. The pellets were resuspended in 3 mL of 50 mM sodium phosphate buffer, pH 7.8, and extracted by ultrasonic and liquid nitrogen. Debris was removed from the extract by centrifugation at 8000 rpm for 15 min at 4ć. The supernatants were used as crude extract to detect the activity of enzymatic (SOD, CAT and GST) using kits. G. Detection of microcystin-LR (MC-LR) MC-LR was determined by solid phase extraction (SPE) combined with high performance liquid chromatography . III.
A. Effect of potassium ferrate on chlorophyll-a The effect of potassium ferrate on the content of chlorophyll-a in Microcystis aeruginosa was studied and the result was shown in Fig. 1. As indicated, chlorophyll-a decreased with the increase of potassium ferrate. After 12 h treatment, chlorophyll-a reduced 28.3% at a dose of 60 mgL-1, and 35.9% at 100 mgL-1. Moreover, there were also some relationships between chlorophyll-a and the treatment time. Within the initial 2 h, chlorophyll-a declined rapidly as time
Fig.1 Changes of Chl-a contents after K2FeO4 treated of Microcystis aeruginosa
K2FeO4. The photosynthesis evolution rate of algae treated in 20 mgL-1 K2FeO4 was only 74.1% (ANOVA, p0.05). With the increase of K2FeO4, the value of ETRm and Fv/Fm decreased correspondingly. Compared to the control, there was significant difference in ETRm and Fv/Fm (ANOVA, p