Report for using aquatic plant as phytoremediation for removing heavy metals Vu Thi Dieu Huong (M2) 1. INTRODUCTION Charophytes are submerged macrophytes grown in wide range of water bodies and its existence is considered as indicator of clean water ecosystems. Many forms of Charophytes are subjects to calcification a process which refers to the precipitation of CaCO 3 within or on the cell wall at the alkaline band. Calcification in charophytes is not dispersed and associated with the plants themselves. Under hard water conditions (high Ca concentration), heavily calcified plants showed high rate of photosynthesis due to a lower amount of leakage of CO 2 from cells to alkaline zones (1). Many heavy metals may inhibit the growth of plants and the encrustation formation at high content. Such inhibition is sometimes known as crystal poisoning. For example, in the presence of Mg, Ca absorption from the water was greatly reduced (2) Thanks to calcification, the plants can absorb heavy metals to create less bioavailable form or precipitation compounds (immobile), even when these plants become senescence and die, these components are still kept in Charophyte meadows for long time and found at sediment layer. It helps to avoid accumulated heavy metals or contaminants release into the water column. Therefore, charophyte can be known as cost effective, non-invasive alternative to the presently available physicochemical pollutant remediation techniques. 2. OBJECT OF THIS RESEARCH - To evaluate the effect of Calcium on the growth of Chara braunii at various concentration (role of calcium for the growth) - To evaluate the effect of heavy metals (in the case of Zinc) on the growth because Zn can act either in a stimulant element for plant metabolism or in an inhibitory depending on its level of availability. - To evaluate the effect of calcification process on the Zn absorption as well as other heavy metals, position of encrustation formation and heavy metals absorption in the cell. 3. MATERIAL AND METHOD 3.1. Experiment setup Chara braunii grown in different Ca concentration (4mg/l; 40mg/l and 80mg/l) for about 5 weeks and there was Zn exposure
Temporal variation of elongation (% relative to initial length) at 0.15mg/l; 0.5mg/l; 1mg/l in 4 weeks and Zn exposure at 2mg/l; 5mg/l and 10mg/l in 2 weeks. The experiment were at 25 0 C, using fluorescent lamps with the photoperiod of 12hour light and 12h dark, ph 7 (±0.2) and cultured solution of Forberg (3) b) Absorption of heavy metal in water and plant after harvest using Atomic Absorption Spectroscopy c) Scanning electron microscope (SEM) and dispersive X-ray (EDX) analysis 4. RESULTS 4.1. Effect of Ca and Zn concentration in water on the growth of Chara braunii a) Effect of Ca on the growth 3.2. Methods Designed model for the experiment Effect of Ca on the growth of Chara braunii 1,2 1 0,8 0,6 0,4 0,2 0 Wo W1 W2 W3 W4 W5 W6 Time (week) Ca 4mg/l Ca 40mg/l Ca 80mg/l a) Temporal variation of elongation This parameter was used to evaluate the effect of Ca and Zn to the growth and it was calculated by this equation: Et = (Lt -Lo) x 100/Lo in which: Et: % relative to the initial length at week t Lt: average length for primary and branches in t th week andcalculated by Lt = a1+a2+a3+b1 Lo: average initial shoot length Figure 1: Effect of Ca on the growth of Chara braunii after 5 weeks of exposure Because under hard water conditions, heavily calcified plants showed high rate of photosynthesis, therefore at concentration of 80mg/l Ca, the temporal variation of elongation showed the highest value compared to lower concentration (4 and 40mg/l Ca). b) Effect of Zn on the growth At the low concentration of Zn (0.15 and 0.5mg/l Zn), the level effect was insignificant, even though it is growth stimulating factor.
Temporal variation of elongation(% relative initial length) Temporal variation of elongation Temporal variation of elongation (% relative initial length) 140% 120% 100% 80% 60% 40% 20% 0% Figure 2: Effect of Zn on the growth of Chara braunii at treatment of Ca 4mg/l Figure 3: Effect of Zn on the growth of Chara braunii at treatment of Ca 80mg/l However, with long period of Zn exposure, value Fv/Fm - quantum efficiency of the PSII photo-chemistry (used as a screening parameter for stress response with value 0.8 being in fully healthy plants) reduced. The result in Table 1 showed that at high Ca content (80mg/l), the plant illustrated more tolerance than Ca 4mg/l and Ca 40mg/l. Treatment: Ca 4mg/l Add Zn 1 2 3 4 5 6 7 8 9 Time (Week) Treatment: Ca 80mg/l Add Zn 140% 120% 100% 80% 60% 40% 20% 0% 1 2 3 4 5 6 7 8 9 Time (week) Zn 0.15mg/l Zn 0.5mg/l Zn 1mg/l Control Zn 0.15mg/l Zn 0.5mg/l Zn 1mg/l Control Ca concentration (mg/l) 4 Treatment Zn concentration (mg/l) Fv/Fm 0.50-0.53 40 0.15 0.47-0.55 80 0.62-0.70 4 0.22-0.26 40 0.5 0.35-0.39 80 0.65 4 0.14-0.2 40 1 0.29-0.34 80 0.46-0.5 Table 1: Fv/Fm under Zn stress condition at different Ca content. Otherwise, at cultured media with higher Zn content (5mg and 10mg/l), it caused toxicity for the plant as its temporal variation of elongation declined rapidly and the plant die after about 10days of Zn exposure 120% 100% 80% 60% 40% 20% 0% Treatment: Ca 80mg/l Add Zn W0 W1 W2 W3 W4 W5 W6 W7 Time (week) Zn 2mg/l Zn 5mg/l Zn 10mg/l Figure 4: Effect of high Zn concentration on the growth at treatment of Ca 80mg/l
Temporal variation of elongation (% relative initial length) Treatment: Ca 4mg/l Add Zn 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% W0 W1 W2 W3 W4 W5 W6 W7 Time (Week) Zn 2mg/l Zn 5mg/l Zn 10mg/l Figure 5: Effect of high Zn concentration on the growth at treatment of Ca 4mg/l 4.2. Zn accumulation in plant - At low Zn content, there was Zn accumulation at all concentration of this heavy metal (0.15mg/l; 0.5mg/l and 1mg/l) as using treatment of Calcium 40 and 80mg/l. However at Ca 4mg/l, the accumulation of Zn was only found in treatment of Zn 1mg/l Figure 7: Zn accumulation in plant after 10days of exposure 4.3. Ultra-structural view observed by Scanning electron Microscope (SEM) and dispersive X- ray (EDX) analysis a) Images of calcification observed by Light Microscope After 4 weeks of Zn exposure Figure 6: Zn accumulation in plant after 4weeks of Zn exposure The calcification was seen by naked eye with appearance of white band. The calcification position was at alkaline region where there was formation of calcite precipitation. - At high Zn content, the capacity of Zn accumulation was more significant in the higher Ca concentration (80mg/l) when compared to Ca 4mg/l.
Calcification in internodal part observed by light microscope at magnification 4x b. Ultra-structural view of calcification position c. Heavy metal absorption peak and relation with calcification. + Zn absorption: The ability of Zn accumulation was carried out and evaluated at content Zn 2mg/l. Zn appeared at extracellular site along with its high absorption peak. Calcification formation of Chara braunii at treatment of 40mg/l Ca The excessive influx of Ca from water containing high concentration of Ca avoids calcification inside cells (4) SEM image and X-ray spectrum analysis showed the position of calcification (in white region) distributed outside of cell wall and Ca absorption peak also was detected. Zn ababsorption peak Ca absorption peak The result pointed out that both Ca and Zn were at extracellular site and hypothesis of
heavy metal absorption by encrustation region could be related + Diatom and Silica (Si) absorption: During the experiment, there was presence of diatom attached on the surface of the plants. Diatom enclosed within a cell wall made of silica (hydrated silicon dioxide) called a frustule. Main element mapping (Ca, C, O, Si) showed the absorption of Si with clear background (green) was at extracellular site and Si absorption peak in EDX analysis. Si absorption peak at area 4 This image illustrated the diatom attached in the plant with brown spots as observed by light microscope + Manganese (Mn) appearance and morphological change in the plant. In SEM image, diatom was seen in filaments and marked site of area 4 was used for X-ray spectrum analysis.
The plants absorbed Mn in cultured media and resulted in morphological change with appearance of dark band at area of main shoot. This site was also observed by light microscope and used for SEM and EDX analysis. with Mn absence, morphology of plant was not changed as compared to dark band. Point 1 Point 2 Point 3 Point 4 Green band observed by light microscope At extracellular site (point 1 and point 4), there were element composition of encrustation (Carbon - C, Oxygen - O, Calcium - Ca and Manganese - Mn) found in the cross-section of calcium-exposed Chara braunii with normalized concentration (wt.%). However, at intracellular site (point 2and 3), Mn and Ca composition of encrustation was not detected. In four points marked at this image including outside part and inside part of cell wall, there was only Ca absorption at outside part (point 1, point 3 and point 4) while Mn absorption was not detected with any point. The result proved that Mn caused dark band formation in the plant and in green area, Mn absorption was not seen. SEM analysis was also carried out at green band to give the evidence that in such regions
6. RECOMMENDATION FOR FURTHER STUDY 5. CONCLUSION 5.1. Chara braunii has potential capacity of calcification formation with the position of calcification was observed at outside of cell wall. 5.2. Calcium is generally considered to be a fast growing factor. The more Ca concentration is, the higher temporal variation of elongation is. The highest value is at 80mg/l Ca. 5.3. High Ca concentration helps cell wall structure to become stronger, so these plants cultured in such conditions become more tolerance in the long period of exposure with toxic Zn content. 5.4. Level of Zn at 0.15mg/l and 0.5mg/l is growth stimulator. At Zn 5mg/l and Zn 10mg/l, there is rapid decrease of elongation, inhibition of growth and death of plants. 5.5. At concentration of 80mg/l Ca showed high Zn absorption capacity in comparison with lower one. 5.6. The absorption of Zn as well as other heavy metals such as Si and Mn is at extracellular site along with Ca + Although Mg is a constituent of chlorophyll and aiding growth (5), Mg suppress the growth of calcite crystals because Mg 2+ incorporate into the calcite structure, which is supposed to be more soluble than the pure calcite phase and the lateral crystal growth (c-axis) is prevented in the presence of Mg 2+ ion. Therefore, it is necessary to study relation between Mg/Ca rate to formation and inhibition of calcification. + Calcite encrustation plays a vital role in the phytoremediation process. Therefore, further studies are required with analysis of Ca and/or carbonate bound Zn speciation +Evaluate and compare phytoremediation capacity of Chara braunii with other heavy metals REFERENCE (1), (4). T. McConnaughey, Calcification in Chara corallina: CO 2 hydroxylation generates protons for bicarbonate assimilation, Limnol. Oceanogr, 36 (1991), pp. 619 628 (2). T. Asaeda, Effect of Calcium and Magnesium on the growth and calcite encrustation of Chara fibrosa, Aquatic Botany, 113 (2014), pp. 100-106 (3). C. Forberg, Nutritional studies of Chara in axenic cultures, Physiol. Plant., 18 (1965), pp. 275 290. (5) I.A. Pattiyage, Impact of Calcium and magnesium on growth and morphological acclimations of Nitella: implications for calcification and nutrient dynamics, Chemistry and Ecology, 26(2010), pp. 479-491
Research result: Vu Thi Dieu Huong, Effect of nutrients and heavy metal on growth of Charophytes and phytoremediation capacity, Thesis.