Phytoremediation of Cd 2 + by Marine Phytoplanktons , Tetracelmis chuii and Chaetoceros calcitrans

The use of marine phytoplankton, Tetracelmis chuii and Chaetoceros calcitrans as phytoremediator has already been reported. However their use as phytoremediator on the cadmium polluted marine has not yet well understood. Therefore, this study was conducted to evaluate the influence of the Cd concentration, interacting time, and medium pH on accumulation of Cd in the phytoplankton. Methods of analysis and data collection were carried out on 1) the growth rate, the number of phytoplankton cells, and the content of chlorophyl-A; 2) the Cd concentration in phytoplankton at various interacting times and medium pHs; and 3) infrared spectra of phytoplankton biomasses before and after interaction with Cd. The growth of phytoplankton and the content of chlorophyl-A after the addition of Cd into the T. Chuii medium decreased, while that after the addition of Cd into the C. Calcitrans medium increased. The optimum accumulation occurred after 15 min at the pH of 8, i.e. 13.46 mg Cd per g T. Chuii and 1055.27 mg per g C. Calcitrans. The functional groups of T. chuii involved in the bioaccumulation of Cd are OH, -CN, S=O, N-O, S-S and M-S, while that of C. Calcitrans are OH, C=O, S-S, M-S and C=C.


Introduction
Marine phytoplankton, T. Chuii and C. Calcitrans, have sizes of 7-12 and 6-8 m, respectively (Falkowski & Raven, 2007).In order to use phytoplankton as phytoremediator of heavy metals in the marine waters, the study of toxicity, the ability to accumulate heavy metals and the potential functional groups that play an important role in the interaction in the phytoplankton from classes of dinoflagellata and diatom need to be investigated (Abe, 2001).In this research, cadmium was used as the object of research because the metal is not required in the growth process of living organism and is classified as a toxic metal.T. chuii and C. Calcitrans representing classes of dinoflagellata and diatom, respectively, were used as phytoremediators for metals.
Many researchers stated that organisms synthesize metal chelating proteins in order to give a respond to the effect of toxic heavy metals.The main small molecules in plants, algae, and fungi are considered as rich peptides called phytochelatin (Grill et al., 1985;Kawakami et al., 2006).The molecules have a structure of (Y-Glu-Cyc) n -Gly, where n can be 2-11 depending on species from which the peptides were isolated and also on the induction condition with the primary structure, as shown in Figure 1 (Hirata et al., 2001).
Phytochelatin was synthesized from a tripeptide derivative (glutathione) composed by glutamate, cysteine, and glysine.Glutathione can be found in all cells, often in a high level (Schat et al., 2002).If the environment is mediated by metal ions, glutathione will form metal chelating peptides, a phytochelatin.The phytochelatin binds metal ions to form phytochelatin-M and then enters into the vacuole (Mercado et al., 2009;Burcu, 2004).The pure culture of T. chuii and C. calcitrans was cultivated in a 500 mL erlenmeyer using the Arschat medium.Lighting with a Neon lamp of 80 watt, was given continuously under flowing CO 2 gas supplied by an air aerator at the temperatures between 20 and 22 and the medium pH.To determine the growth pattern of ℃ Phytoplankton, calculation of the number of cell per mm medium was performed.A sample about 0.1-0.5 mL was taken by a sterile drop pipette, dropped into Haemocytometer, and then observed under a microscope (Seafdec, 1985).

Growth rate and inhibition percentage of the growth of T. chuii and C. calcitrans before and after addition of Cadmium ion (Cd 2+ ) in arschat medium
Results of determination of the specific growth rate () for each concentration of the Cd 2+ ion calculated using equation ( 1) are presented in Tables 1 and 2. ln ln N t = Cell density at t (cells/mL)c N o = Cell density at the beginning (cell/mL)  = specific growth rate t = time (days) (Doshi et al., 2007) The growth of T. Chuii at the culture medium added by 0.15 mg•L -1 of Cd 2+ ion tends to be similar to that of the control.The higher the concentration of Cd 2+ ion added causes the lower the growth pattern.This phenomenon proves that the existence of Cd 2+ ion in the culture medium of T. Chuii can decrease the growth of phytoplankton as can be seen in Table 1.
Table 2 shows that generally the growth rate of C. calcitrans increases with the increase of the exposure time until the third day except for the addition of Cd 2+ ion at the concentration above 1 ppm showing the highest growth rate at the forth day.It is interested to be noted that the addition of the Cd 2+ concentration in the culture medium increases the specific growth rate until the concentration of Cd 2+ ion equals to 1 ppm.However, further addition of the Cd 2+ concentration causes the decrease in the specific growth rate of C. calcitrans.

Chlorophyl-A content in T. chuii and C. calcitrans grown in the medium added with Cd 2+ ion at various concentration
The content of Chlorophyl-A in T. chuii and C. calcitrans grown in Arschat medium containing Cd 2+ ion at various concentration can be seen in Table 3, Figure 2 and Figure 3.
It is clear from Figure 2 that the increase of the Cd 2+ concentration in medium causes the decrease of the chlorophyl-A content.This result is in parallel with the results reported by Ho (2003) and Reinfelder (2000) who studied the effect of Cd 2+ ion on the chlorophyl content of Chlorella ellipsoidee.Wang and Dei (2001), Baryla et al. (2001) and Inthorn (2001) reported that the addition of Cd 2+ ion caused the decrease of photosynthesis process and the nutrient uptake.
Figure 3 shows that the addition of Cd 2+ ion into the medium of C. calcitrans results in the increase of the chlorophyl-A content.This occurs because the Cd 2+ ion can stimulate the growth of phytoplankton (C.calcitrans).

Effect of the medium pH on the accumulation capacity of Cd 2+ ion on T. chuii and C. calcitrans
The ability of phytoplankton in accumulating Cd 2+ ion as a function of the medium pH has been studied.Results of the pH effect are presented in Table 4 and Figure 4. 4, it is obvious that the accumulation of Cd 2+ by both T. chuii and C. calcitrans increases gradually with the increase of the medium pH and at the pH of 8 the accumulation of Cd 2+ achieves optimum.Unlike in T. chuii where the increase of the amount of Cd 2+ accumulated as a function of the medium pH is relatively small, the increase of the amount of Cd 2+ accumulated in C. calcitrans is relatively high until the optimum pH as can be seen in Figure 4.

Identification of functional groups of T. chuii and C. calcitrans Involved in the accumulation process of Cd 2+ ion
Identification of functions groups of T. chuii and C. calcitrans was conducted by using FTIR before and after addition of the Cd 2+ ion into both living and non-living phytoplanktons.Results showed that there were 20 and 17 absorption bands for living and non-living T. chuii, respectively.Functional groups which are likely to be involved in the bioaccumulation of Cd 2+ in T. chuii are O-H, C=-N, S=O, N-O, S-SA, M-S and M-N.
After addition of Cd 2+ ion into the culture medium of living and non-living C. calcitrans, there were 16 absorption bands observed for both samples.For interaction of Cd 2+ ion with the non-living biomass of C. calcitrans, there were some new bands observed and some absortion bands had different wavelengths.This indicates that the interaction of Cd 2+ ion with living and non-living C. calcitrans is not the same.The functional groups of C. calcitrans involved in the bioaccumulation of Cd 2+ ion are O-H, C=C, C=O, S-S and M-S.

Conclusion
From this study, it can be concluded that: 1) Addition of cadmium ion with the concentration of 0.5 ppm into the culture medium of T. chuii decreases the growth rate, the number of cells, the dried weight and the chlorophyl-A content of the phytoplankton.Addition of the ion with the concentration of 1 ppm into the culture medium of C. calcitrans increases the growth rate, the number of cells, the dried weight and the chlorophyl-A content of the phytoplankton.
2) Bioaccumululation of cadmium by T. chuii and C. calcitrans increases gradually with the increase of the medium pH used and achieved the optimum at the pH of 8.
3) Identification of functional groups on the biomass of T. chuii before and after addition of Cd 2+ ion shows that functional groups of N-O, O-H, S=O, C-N, M-S and S-S plays an important role in the bioaccumulation process of cadmium.The functional groups involved in the bioaccumulation of cadmium in C. calcitrans are C=C, C=O, M-S, O-H and S-S.

Table 1 .
Constant specific growth rate (μ) Tetracelmis chuii without addition of metal ions Cd 2+ at various concentrations

Table 2 .
Constant specific growth rate (μ) Tetracelmis chuii with addition of metal ions Cd 2+ at various concentrations

Table 3 .
The content of Chlorophyl-A in T. chuii and C. calcitrans as a function of the concentration of Cd 2+ ion added in the medium

Table 4 .
Accumulation of Cd 2+ ion on T. chuii and C. calcitrans as a function of the medium pH (initial concentration of Cd 2+ ion = 0.25 ppm)