Tolerance and decolorization potential of duckweed (Lemna gibba) to C.I. Basic Green 4

With growing human culture and industrialization, many pollutants are being introduced into aquatic ecosystems. In recent years, dyes have become a major water pollutant used in the manufacture of paints and other production purposes. In this research, the potential of duckweed (Lemna gibba) plant was investigated spectrophotometrically as an obvious bioagent for the biological decolorization of the organic dye C.I. Basic Green 4 (Malachite Green, BG4). Photosynthetic efficiency analysis showed that the photosynthetic apparatus of L. gibba is very tolerant to BG4. Significant induction of reactive oxygen species (ROS) scavenging enzymes was observed after 24h of biodecolorization process in L. gibba treated with 15 and 30 mg/l BG4. The experimental results showed that L. gibba has a strong ability to extract BG4 from contaminated water and the best results were obtained at 25–30°C and pH 8.0. We conclude that duckweed L. gibba can be used as a potent decolorization organism for BG4.

www.nature.com/scientificreports/ swollen duckweed or fat duckweed. L. gibba belongs to the family Lemnaceae and its small size, high multiplication rate, and reduced anatomy makes it a good candidate for phytoremediation [21][22][23] . To assess the ability of L. gibba to decolorize BG4, various parameters such as initial biomass concentration, temperature and pH were implied. Here, we have studied the ROS scavenging enzymes status in L. gibba in order to get insights into the role of BG4 dye in bringing on oxidative stress. We examined the change in antioxidant activity by analyses three Reactive Oxygen Species (ROS) scavenging enzymes: superoxide dismutase (SOD; EC.1.15.1.1), catalase (CAT; EC 1.11.1.6), and guaiacol peroxidase (GPOD; EC 1.11.1.7). The repeated batch operation was also performed to determine the reusability of L. gibba for dye decolorization. Through the analysis of polyphasic chlorophyll fluorescence induction curves, efforts were made to study the impact of the dye decolorization process on photosystem II (PSII) photochemistry of L. gibba. The aim of this study was to check the potential of duckweed plants (Lemna gibba) as a powerful decolorization organism of Basic Green 4 (BG 4) dye, which is commonly used by various industrial sectors.

Materials and methods
Plant materials and growth condition. In the present investigation duckweed, L. gibba was used for phytoremediation of triphenylmethane dye BG4. Plant material (Fig. 1a) was collected from the region of Ayad river located at Udaipur, India (24°35′14.97"N, 73°42′38.75"E). Details about water properties and environmental condition of Ayad river are given in Table 1. BG4 was procured from HIMEDIA (India). The plants were rinsed with double distilled water to remove surface contamination and maintained in plastic pond under illumination provided by white fluorescent light with 6500-10,000 lx light irradiance, 14-h photoperiod, and 25/20 °C day/night temperature for three months as a pre-treatment before experiments (as per OECD guidline, 2002) 24 . A full-strength Jacob culture medium was prepared according to the Table 2 and pH maintained at 6.0. The medium was replaced every two months and the circulation was provided with an electric pump.
Experiment. Photosynthetic performance. Fast Chl a fluorescence kinetic transient. To determine changes in Chlorophyll (Chl) a fluorescence, O-J-I-P transient was recorded after 12 h of dye treatment by Plant Efficiency Analyzer, PEA (Hansatech Instruments, Kings Lynn, Norfolk, U.K.). Before the measurements, control and treated plants were adapted to darkness in room for 1 h, and additionally, the measured spots were kept in darkness in the clip for 1 min just before measurement 25 . Fluorescence transients were induced over a leaf area of 4 mm diameter by a red light (peak at 650 nm) of 3000 µmol m -2 s -1 (sufficient excitation intensity to ensure closure of all PSII RCs to obtain a maximal fluorescence intensity of F M ) provided by a high intensity LED array of three light-emitting diodes. A total measuring time of one second was used thought out the experiments 19 . Abbreviations, formulas, and definitions of the JIP-test parameters used in the current study are presented in Table 3 26,27 .
Specific and phenomenological fluxes. Specific activities of active PSII reaction centre i.e. antenna size of an active PSII (ABS/RC), electron transport flux from QA to PQ per active PSII (ET/RC) and Dissipated energy flux per reaction centre (DI/RC) and phenomenological fluxes i.e. Absorption flux per cross section (ABS/CS), Electron transport flux per cross section (ET/CS) and Dissipated energy flux per cross section (DI/CS) were calculated using following equations of JIP test 19 .  (2)  Enzyme extraction and assay. After the decolorization experiment (12 h) Lemna fronds treated with 15 and 30 mg/l BG4 were removed from the solution respectively. About 300 mg (fresh weight) fronds were homogenized in 5 ml ice-cold potassium phosphate buffer (0.1 M, pH 7.8) for preparing enzyme extract. The homogenate was centrifuged at 15,000 × g (4 °C) for 20 min (REMI, India). the supernatant was saved and used as the enzyme extract. All the preparation for enzyme extract was carried out at 4 °C and the enzyme activity was expressed in the term of U mg −1 fresh weight.
The SOD activity was determined spectrophotometrically by measuring its ability to inhibit the photochemical reduction of Nitro Blue Tetrazolium (NBT) at 560 nm 28 . The reaction mixture containing 100 µl L-methionine, 100 µl NBT, 10 µl riboflavin, and 100 µl enzyme extract and 2.7 ml Na 2 CO 3 (0.05 M). The tubes containing reaction mixture were placed below white fluorescent light for 10 min after that the reaction stopped by placing the Table 3. Abbreviations, formulas, and definitions of the JIP-test parameters used in the current study (Strasser et al. 2000(Strasser et al. , 2004. www.nature.com/scientificreports/ tubes in dark for 8 min and absorbance was measured at 560 nm. SOD enzyme to produce a 50% inhibition of the reduction of NBT was expressed as one unit of SOD enzyme activity. The CAT activity was measured by the consumption of H 2 O 2 at 240 nm 29 . The reaction mixture containing 120 µl enzyme extract, 80 µl H 2 O 2 (500 mM) and 2.8 ml potassium phosphate buffer (50 mM). One unit of catalase activity was defined as the amount of enzyme necessary for reduction 1 mM of H 2 O 2 per minute.
GPOD activity was determined spectrophotometrically by measuring changes in absorbance at 436 nm for 15 s. up to 5 min 30 . Reaction mixture contained 1 ml guaiacol (1%), 1.7 ml phosphate buffer (0.05 M, pH 7.0) and the reaction was initiated by adding H 2 O 2 . The enzyme required for the transformation of the substrate in 1 min is expressed as unit enzyme activity.
Dye removal experiment. The decolorization experiments were performed in the 250 ml beaker containing 200 ml solution of malachite green dye (Fig. 1b). Details about chemical structure and characterization of BG4 are given in Table 4. After every regular treatment interval, the sample was isolated and the remaining dye was determined with a UV spectrophotometer (ANALYTIKJENA SPECORD 200, Germany) at maximum absorbance wavelength (λ max ) = 619 nm. A linear correlation was established between the concentration of dye and the absorbance (A) at 619 nm (λ max ). in the range of C dye = 0 to 30 mg/l. Percent dye removal was calculated by using Eq. (10).
To evaluate the effects of environmental factors and operational parameters on the efficiency of dye removal, the decolorization experiments were carried out with pH values (2.0-9.0), temperature (10-50 C) and weights of plant (2.0-10 g) respectively. The pH of the dye solution was adjusted using 1 N NaOH and 1 N HCl and was measured by pH meter (Hanna HI98100, United States). To determine the reusability of L. gibba repeated-batch processes were performed to remove BG4 31 . When the decolorization process was completed, the same plants were used to decolorize another dye solution containing 30 mg/L BG4. This process was repeated four times. Statistical analysis. The data analysis was done using SPSS (v. 21.0) software and the graphs were prepared using Microsoft office. All values presented in the paper are means of three independent replicates. Statistical analyses of data were carried out by One-way ANOVA tests and significant differences were established by Tukey (HSD) tests at p ≤ 0.05.

Results and discussion
Photosynthetic performance. L. gibba exhibited no profound effect on photosynthetic efficiency during the decolorization process. However, the activities of specific fluxes (ABS/RC, TR/RC, and ET/RC) were found more sensitive to BG4 ( Table 5). The BG4-induced decline in ABS/RC and TR/RC 19.19% and 17.96% respectively. Similarly, the ET/RC reduced 9.62% during the complete decolorization of BG4. To compensate for the BG4-induced reduction in specific fluxes, the plants increased the RC/CS. The density of active RCs increased by 6.88% in BG4-treated plants as compared to controls (Fig. 2a). Transformation of inactive PSII RCs into active form displays physiological adaptation in L. gibba against BG4-induced chemical stress 25 .  Overall electron transport rate per cross-section (ET/CS) remained unchanged in duckweed during decolorization of BG4, which indicated that L. gibba enhanced the concentrations of active PSII RCs to maintain the rate of ET/CS. Similarly, no significant variations in Fv/Fm were observed during the entire duration (12 h) of BG4 decolorization, which indicates that L. gibba has high potential to maintain its photosynthetic efficiency even during/after the decolorization of BG4 by modulating the specific, phenomenological fluxes, the density of active PSII RCs and Fv/Fm (Fig. 2b). Chlorophyll fluorescence analysis demonstrates that L. gibba has high physiological adaption to sustain overall photosynthesis during the post-decolorization of BG4.
Tolerance to the dye (antioxidative enzymes). Plant enzymes play a crucial role in the biodecolorization of pollutants 13 and directly participate in the decolorization of synthetic dyes 33 .
SOD is an important enzyme of plant antioxidant defense system and it converts two superoxide radicals (O 2 .-) to water and O 2 . Subsequently, the products of SOD were furthers detoxified by other enzymes CAT and GPOD and convert into less toxic compounds 34 . After 24 h of biodecolorization process, a significant induction (116.67% and 164.76%) in SOD activity was observed in L. gibba treated with 15 and 30 mg/l of BG4, respectively (Fig. 3a). The activity of GPOD and CAT were also increased 120.45% and 106.96% respectively from control with exposure by 15 mg/l of BG4. A significant increase was observed in GPOD and CAT activity with exposure with 30 mg/l of BG4 after 24 h (143.66% and 113.91% respectively from control) (Fig. 3b,c). As displayed in Fig. 3 antioxidant enzyme activity increases significantly (p ≤ 0.05). The presence of pollutants in the environment around the plants leads to oxidative stress and produces a high amount of reactive oxygen species (ROS) and perhaps these antioxidant enzymes directly involved in the conversion of these harmful ROS into a less toxic product 35 . The amount of ROS increased due to high concentration of dye 31 which activates the antioxidant system to protect the plant from these deleterious components 34 . Enzymes SOD, GPOD, and CAT are major components of the defense system of the plant against stress 33 .

The effects of different parameters on removal rate. Effect of duckweed biomass on dye removal
rate. The initial concentration of dye, temperature, and pH were kept constant in order to make a comparative study for biogenic decolorization of BG4 dye in the presence of different amounts of duckweed (2-10 g). The dye removal efficiency was significantly increased with increasing the biomass (Figs. 4, 5a) until it reached a value  www.nature.com/scientificreports/ of 74.72% with the biomass of 6 g. The decolorization of BG4 was spectrophotometrically analyzed and the UV spectra were shown in (Fig. 5b). These results indicate that the duckweed biomass of 6 g would be the minimum desired biomass for removing dye from contaminated water (Fig. 4c). Increases in duckweed biomass provided more surface area for absorption of the dye molecule 10,31 . Another possibility of our finding is that high initial concentration of dye (30 mg/L) will provide high probability to contact between dye molecules and plant surface areas. Daneshvar et al. (2007) and  reported that increased initial concentration of pollutants gives results in high rate of decolorization 36,37 .
Effects of temperature and pH on decolorization potentials:. The temperature is an important environmental factor that alters various biological processes. The effect of temperature on biological decolorization process is one of the important and effective parameters. To determine the effect of temperature on biological decolorization was studied at the range of 10-50 °C at an initial concentration of 30 ml/l. As displayed in (Fig. 5c), high temperature triggers the removal rate of dye and results showed that the thermal deactivation of dye decolorization was not observed. The reaction of biosorption between duckweed and BG4 was an endothermic process prove through the above finding. The results are similar to literature information that high temperature induces biological dye decolorization capacity 36,38 . Biological decolorization of dye using plants is highly pH dependent 39 . In the present study, biodecolorization of BG4 was analyzed over a range from 1.0 to 9.0 pH. It was observed that dye decolorization efficiency increases as the pH of the solution increase up to 8 (Fig. 5d). It can be understood by the concept of zero-point discharge for biomass. An isoelectric point of around 3-4 pH is determined for plant biomass 38,40 . The plant surface charged positively in acidic solution and negatively in alkaline solution. However, BG4 is a cationic dye and the high pH solution enhances the bio-adsorption of dye, hence the dye removal potential increases as reported by Vasanth et al. 16 . www.nature.com/scientificreports/ FT-IR analysis. Figure 6a illustrates FT-IR spectra of BG4 treated by L. gibba at different reaction times. FT-IR spectrum of Malachite Green before removal showed the specific peaks in fingerprint region (2500-500 cm −1 ) benzene rings which is supporting to the peak at 1635 cm -1 for the C--C stretching of the benzene ring 41 . FT-IR spectrum of extracted product at 6 and 12 h reaction time displayed no significant alteration in fingerprint region which indicated that the mechanism involved in decolorization of BG4 by L. gibba is bio accumulation and not biodegradation. FT-IR studies are helpful in understanding the mechanism of remediation of BG4 by L. gibba. With the help of FT-IR studies, it can be concluded that L. gibba initially adsorbed the dye on its surface through electrostatic and hydrogen bond interactions among functional groups of L. gibba and BG4 dye. Similar results were found by Mahajan and Kaushal (2020) during the removal azo dye methyl red (MR) by macroalgae Chara vulgaris L 42 . However, further studies are required, which throws light on the plant's enzymatic mechanism for decolorization of BG4.
Recyclability of live L. gibba. To examine the reusability of L. gibba in Basic Green 4 decolorization a repeated batch operation was performed. During five repeated batch run it was recorded that L. gibba showed equal dye decolorization efficiency (Fig. 6b). No adverse effect of BG4 on biomass was observed upto 5 times reusability of plants. From these results, we can conclude that L. gibba possesses a great ability to recycle or reusability in repetitive decolorization processes. The results also indicate that the removal of the BG4 by the duckweed is a biological decolorization process. Earlier studies demonstrate that L. minor 43 and Chara 15 degrade dyes AB92 www.nature.com/scientificreports/ and BG4 respectively. In contrast, present findings reveal that L. gibba has high potential of taking up BG4 and can be used as a potent biological tool to remove BG4 from polluted water.

Conclusion
The results from present research work give a positive sign that L. gibba has remarkable potential for decolorization of BG4. Chlorophyll fluorescence analysis reveals that photosynthetic apparatus of L. gibba is highly tolerant to BG4. Plants photosynthetic efficiency did not alter even during and after the dye decolorization. BG4 treatment to L. gibba leads to activation of antioxidant activity which determined by the increased value of SOD, CAT, and GPOD which usually activated when plants suffering unfavorable environmental conditions. The L. gibba mediated BG4 decolorization depends on various parameters that were assessed in this study. As increasing pH, temperature, contact time, and plant weight the BG4 decolorization capacity was also increased. The study revealed that the temperature at 25-30 °C and pH 8.0 are considered as optimum for the best results. The repeated batch experiment confirms the reusability of L. gibba for BG4 decolorization. The FT-IR spectra of BG4 solution during biological decolorization under the optimized conditions revealed that this process is bioaccumulation. The overall results of the present findings highlight that duckweed L. gibba can be used as an effective organism for biodecolorization of BG4 thus, protecting the Earth's hydrosphere. Figure 6c shows the over view of L. gibba mediated BG4 decolorization.