Bioremediation potential of duckweed ( Lemna gibba ) in the decolorization of 1 C . I . Basic Green 4 and chlorophyll a fluorescence analysis : understanding plant 2 performance 3 4

27 In this research, the duckweed (Lemna gibba) potential has been investigated 28 spectrophotometrically as an obvious bioagent for the biological decolorization of organic dye 29 Basic Green 4 (BG4). Experiment result showed that L. gibba has a potent ability to extract BG4 30 from contaminated water. The study showed that for better results the temperature at 25-30 C and 31 pH 8.0 is considered to be optimum. A significant induction in SOD, GPOD and CAT activity was 32 observed in L. gibba treated with 15 and 30 mg / l BG4, respectively, after 24 hours of 33 biodecolorization process. It was observed during five repeated batch run that L. gibba showed 34 comparable efficiency in dye decolorization. The chlorophyll fluorescence analysis also shows 35 that photosynthetic apparatus of L. gibba is highly tolerant of BG4. The overall results of the 36 observations here demonstrate the duckweed L. gibba can be used as a potent biodegrading 37 organism for BG4. 38


Enzyme extraction and assay 108
About 300 mg Lemna fronds (fresh weight) were homogenized in 5 ml ice-cold potassium 109 phosphate buffer (0.1 M, pH 7.8) for prepare enzyme extract, the homogenate was centrifuged at 110 15,000 × g (4 O C) for 20 min (Remi ® , India). the supernatant was saved and used as the enzyme 111 extract. All the preparation for enzyme extract was carried out at 4 o C. 112 The SOD activity was determined by spectrophotometrically by measuring its ability to 113 inhibit the photochemical reduction of Nitro Blue Tetrazolium (NBT) at 560 nm 28 . The reaction 114 mixture containing 100 µl, L-methionine, 100 µl NBT, 10 µl riboflavin, and 100 µl enzyme 115 extract. Make up the volume 3 ml by adding 0.05M Na2CO3. 116 The CAT activity was measured by the consumption of H2O2 at 240 nm 29 . The reaction 117 mixture containing 120 µl enzyme extract, 80 µl H2O2 (500mM) and make final volume 3 ml by 118 adding 2.8 ml potassium phosphate buffer (50mM). 119 GPOD activity was determined by spectrophotometrically by measuring changes in 120 absorbance at 436 nm for 15 sec. up to 5 minutes 30 . Reaction mixture containing 1 ml guaiacol 121 (1%), 1.7 ml phosphate buffer (0.05M, pH 7.0). the reaction started by adding H2O2. Lynn, Norfolk, U.K.). Before the measurements, control and treated plants were adapted to 127 darkness in room for 1 h, and additionally, the measured spots were kept in darkness in the clip for 128 1 min just before measurement 31 . Fluorescence transients were induced over a leaf area of 4 mm 129 diameter by a red light (peak at 650 nm) of 3000 µmolm -2 s -1 (sufficient excitation intensity to 130 ensure closure of all PSII RCs to obtain a true fluorescence intensity of FM) provided by a high 131 intensity LED array of three light-emitting diodes. A total measuring time of one second was used 132 thought out the experiments 32 .

Statistical analysis 162
The data analysis was done using SPSS (v. 21.0) software and the graphs were prepared 163 using Microsoft office. All values presented in the paper are means of three independent replicates. 164 Statistical analyses of data were carried out by ANOVA tests and significant differences were 165 established by Tukey (HSD) tests at P ≤ 0.05. 166

Effects of temperature and pH 177
The temperature is an important environmental factor that alters various biological 178 processes. In the present study, temperature is one of the important and effective parameters. To 179 determine the effect of temperature on biological decolorization was studied at the range of 10-50 180 o C at an initial concentration of 30 ml/l. As displayed in (Figure 4a), with increasing the 181 temperature biological decolorization was also increased and the results also showed that the 182 thermal deactivation of dye decolorization was not observed. The reaction of biosorption between 183 duckweed and BG4 was an endothermic process prove through the above finding. The results are 184 similar to literature information that high temperature induces biological dye decolorization 185 capacity 13,33 . 186 pH is also one of the major environmental factors and biological decolorization of BG4 187 was highly regulated by pH. In the present study biodecolorization of BG4 was analyzed over a 188 range from 1.0 to 9.0 pH. It was observed that dye decolorization efficiency increases as the pH of 189 the solution increase up to 8 (Figure 4b).  (2500-500 cm -1 ) and after 6 and 12 h of treatments no significant alteration in fingerprint region 198 was observed which indicate that the mechanism involved in decolorization of MG by duckweed 199 is bio accumulation and not biodegradation.  (Figure 7a). 208 The activity of GPOD and CAT were also increased 120.45 % and 106.96 % respectively from 209 control with exposure by 15 mg/l of BG4. A significant increase was observed in GPOD and CAT 210 activity with exposure with 30 mg/l of BG4 after 24 hours (143.66 % and 113.91 % respectively 211 from control) (Figure 7b and c). As displayed in Figure 6  the entire duration (12h) of BG4 decolorization, which indicates that L. gibba has high potential 243 to maintain its photosynthetic efficiency even during/after the decolorization of BG4 by 244 modulating the specific, phenomenological fluxes, the density of active PSII RCs and Fv/Fm 245 (Figure 9b). Chlorophyll fluorescence analysis demonstrates that L. gibba has high physiological 246 adaption to sustain overall photosynthesis during the post-decolorization of BG4.

Conclusion 248
The results from present research work give a positive sign that L. gibba has remarkable 249 potential for decolorization of BG4. The L. gibba mediated BG4 decolorization depends on various 250 parameters that are assessed in this study. As increasing pH, Temperature, contact time, and plant 251 weight the BG4 decolorization capacity was also increased. The study revealed that the 252 temperature at 25-30 o C and pH 8.0 are considered as optimum for the best results. BG4 treatment 253 to L. gibba leads to activation of antioxidant activity which determined by the increased value of 254 SOD, CAT, and GPOD which usually activated when plants suffering unfavorable environmental 255 conditions. The repeated batch experiment confirms the reusability of L. gibba for BG4 256 decolorization. 257 The study of chlorophyll fluorescence also reveals that photosynthetic apparatus of L. 258 gibba is highly tolerant of BG4 and did not alter even during and after the dye decolorization. The 259 overall results of the present findings highlight that duckweed L. gibba can be used as a potent 260 organism for biodecolorization of BG4. 261

Funding: 263
This research did not receive any specific grant funding from agencies in the public, commercial, 264 or not-for-profit sectors. 265