2.1 Sample collection and growth condition

S. polyrhiza was collected from a local pond of Charada (23°29′03.89″N and 72°38′49.18″E) Gujarat, India. Dry weight and wet weight of S. polyrhiza is mentioned in Supplementary Table 1. After sterilization with 1% NaOCl for 5s, stock culture was maintained in Schenk-Hildebrandt growth medium at pH 5.6 with 12/12 h light/dark cycle in the 50 mmol m⁻²s⁻¹ irradiance. Plants were subculture at 2 week intervals. Pre-cultivation was developed ten days before experiments in SH growth medium. Experimental procedure was started by inoculating 3–4 fronds/m² in small artificial pond with dimensions 100 × 100 × 10 cm³ containing sewage wastewater in the environmental condition. The plants were exposed to 25–200 mM NaCl, 25–200 ppm RV5 and 25–200 ppm NaCl + RV5 for determination of enzyme activities, proline, MDA, pigments and starch contents after 96 h exposure. All experiments were repeated at least 3 times. Plant growth inhibition was estimated according to following formula,

2.2 Enzyme extraction and assay

0.2 g biomass of S. polyrhiza was homogenized in mortar and pestle with liquid N₂. Then the biomass was suspended in 0.8 ml 50 mM potassium phosphate buffer (pH-7.0) having 1 mM EDTA and 1 mM phenyl methyl sulfonyl fluoride. The homogenate was centrifuged at 15,000g for 30 min. at 4 °C and the supernatant was stored at 4 °C temperature for further enzyme assay. The APX activity was determined by monitoring the decomposition of ascorbic acid absorbance at 290 nm (Nakano and Asada,1981). The SOD activity was assayed by monitoring inhibition of photochemical reduction of NBT (Beauchamp and Fridovich,1971). Hydrogen peroxide content was measured (Sumithra et al. (2006). The O^2•− was assayed by measuring the reduction of exogenously NBT (Doke 1983).

2.3 Purification of APX

Crude extract of S. Polyrhiza was precipitated with (NH₄)₂SO₄ at 40–70% of saturation. The precipitates were then collected by centrifugation at 17,000g for 20 min and suspended in 100 ml 25 mM Tris at pH 7.8. The dialysis of the precipitate was carried out overnight against 1L of same buffer. Further the ion exchange chromatography was performed in 2.5 × 23 cm column of DEAE cellulose. Purified enzyme was subjected to non-denaturing, discontinuous mini-gel electrophoresis (Davis 1964). The gel was rinsed and washed with phosphate buffer (pH-7.0), followed by stained in 1 mM 3,3-diaminobenzidine and 0.03% H₂O₂ for detecting the APX activity (Abdelbasset et al. 1995).

2.4 Preparation and characterization of CuNPs

Copper nanoparticles were synthesized by mixing 0.07 mg of purified AOX having 0.1U activity with 0.1 M CuSO₄·5H₂O solution in 1:3 ratios. The samples were taken at regular interval for measuring the spectrum over the wavelength range of 200–800 nm till 24 h. The synthesized CuNPs were characterized using DLS, SEM-EDX, XRD and FTIR techniques. All the samples were washed with the ethanol for removing the NaCl contents before XRD analysis. XRD was analyzed in Philips X’Pert Pro instrument with source of radiation was Cu-K (ʎ = 1.54A). Samples were scanned within the 2Θ range from 20 to 70°. CuNPs were sprinkled on adhesive carbon tapes supported on metallic plate, the images and elemental contents were recorded at different magnification by Philips XL-30S FEG type instrument. Functional groups present on the surface of the CuNPs were determine by the FTIR. Spectra was scanned in the range of 4000–400 cm⁻¹ using Varian Carry 50 spectrophotometer with 32 times scan and given one reflection that is equal to 21 resolutions.

2.5 Decolorization experiments

Photocatalytic degradation of different reactive dyes was carried out in aqueous medium to evaluate the efficiency of CuNPs. Rectangular borosilicate glass photoreactor having a dimension of 23 × 23 × 18 cm³ was used for the experiments. A total 400 mg of CuNPs were added per 100 ml of the dye solution. The experiment was started after the addition of the 0.2 ml 30% H₂O₂ per 100 ml in the reaction mixture. The samples were withdrawn and analyzed after every 15 min. for the determination of change in absorbance at ʎmax of dyes. Hach DR 2010 spectrophotometer and Hatch COD reactor was used for the COD analysis. Percentage of decolorization and rate of decolorization was calculated according to Patel et al. (2013).

2.6 Enzymatic hydrolysis of starch for bioethanol production

Biomass slurry remain after CuNPs synthesis, mix with 80% ethanol at 70 °C for 15 min for starch extraction. Further the biomass was heated at 100 °C for 10 min and hydrolyzed by optimized process with α-amyloglucosidase Sigma A7095 and α-amylase Sigma A4582. A 0.5 g of extracted starch was mixed with 1%, 2% and 5% v/v amylases in fix volume (30 ml) of sodium acetate buffer (pH 4.8), incubated at 37 °C for 4 h on an orbital shaker and centrifuged for 5 min. at 12,000g. The concentration of glucose was analyzed by water HPLC system equipped with C18 column and Uv–Vis detector. The starch content was determined by following equation;

Saccharomyces cerevisiae MTCC180 was enriched with peptone (5 g/L) and yeast extract (3 g/L) and used for the fermentation of glucose to ethanol. The fresh yeast culture having 200 CFU/mL was inoculated in the fermentation broth at 28 ± 2 °C on orbital shaker at 180 rpm for 48 h. Samples were withdrawn after 12 h interval for analyzed ethanol yield and re were withdrawn at 12 h interval for analysis of ethanol generated and remaining residual reducing sugar by GC–MS and HPLC method, respectively.

3.1 S. polyrhiza growth and nutrient removal from sewage

S. polyrhiza was grown in the sewage keeping the facts in the mind that India is generating above 62,000 MLD sewage and discharging in the environment as it is containing high concentration of nitrogen and phosphorus resulting eutrophic condition in receiving water bodies. The sewage containing TN-46.7 ± 4.2 mg/L, TP-18.3 ± 2.3 mg/L and other contaminants as shown in Table 1. The concentration Ammonia, Nitrate, Nitrite was significantly higher in sewage as 20.4 ± 1.9 mg/L, 19.6 ± 1.6 mg/L and 4.9 ± 0.7 mg/L respectively. After 15 days' experiment, the reduction in concentration of TN, and TP were 89.51% and 95.16% respectively (Table 1). NH₄⁺–N concentration gradually decreased during the initial days of the incubation from 20.4 ± 1.9 to 2.4 ± 0.19 mg/l, on the other hand Nitrate and Nitrite concentration was increase by 52.04% and 91.84% respectively. it might be due to dominance of nitrifying bacteria (Ammonia to Nitrate to Nitrite) over denitrifying bacteria (Nitrate to Nitrite to Nitrogen gas) in the rhizosphere of S. polyrhiza. The rate of the biomass generation of S. polyrhiza was 21.6 ± 1.63 g/m²d. The computational yield of 3.25 ton of fresh biomass after 15 days’ incubation by treating 1 MLD of sewage was achieved.

3.2 Effect of exogenous exposure of NaCl and RV5 on macromolecules of S. polyrhiza

It was observed that NaCl and RV5 stress significantly reduce the chlorophyll and carotenoids concentration and growth inhibition of S. polyrhiza (Supplementary Figs. 1, 2). Stress induced S. polyrhiza fronds were analyzed for H₂O₂, ROS, MDA and proline assay. H₂O₂ and ROS content increased significantly as compared to the control plants. The accumulation of H₂O₂ and ROS were 2.34 ± 0.41 and 2.58 ± 0.57 µmol/g FW respectively, which is 5750 and 3585% higher compare to control, 387 and 529% higher compare to only NaCl treatment and 317 and 396% higher compare to only RV5 treatment respectively (Fig. 1a). An increase in H₂O₂ and ROS content trigger higher lipid peroxidation of polyunsaturated fatty resulted in increase the MDA concentration. Maximum concentration of MDA was 2.56 ± 0.1 nmol/mg protein found at 150 ppm combine exposure of RV5 + NaCl, which was 526% higher compare to control, 164% higher compare to only NaCl exposure and 62% higher compare to only RV5 exposure (Fig. 1b). Further increasing in the concentration up to 200 ppm, decrease the MDA concentration by 84%. In the present study, a dose-dependent increase in MDA concentration compare to control was also found, which indicated that NaCl and RV5 stress damages membrane systems, resulted in cellular integrity loss in S. polyrhiza. Previous research also supports the peroxidative damages caused by exogenous hormones in plant and microbes (Belhaj et al., 2017). Accumulation of proline in stress condition is common characteristic of monocot plants and metal stressed common duckweed but not found in giant duckweed. As shown in Fig. 1b the proline was not significantly accumulated compare to control plants. Such phenomena were observed by several researchers, might be due to the accumulation of more proline was found in water deficit plant compare to salt-stressed cells (Ashraf and Harris, 2004). Yamaya and Matsumoto (1989) had observed same results for barley plants. In other words, duckweed have enzymatic complexes or non-enzymatic antioxidants for osmotic adjustment through the under NaCl or RV5 stress (Ashraf and Harris, 2004).

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Fig. 1

Effects of different concentrations of NaCl, RV5 and NaCl combine with RV5 on Reactive Oxygen species and H2O2 (a), Proline and MDA (b), APX and SOD enzyme activities (c) and Accumulation of the starch (d) in Spirodela polyrhiza.

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Proline accumulation was not significantly increase but APX and SOD enzymes activity was significantly increase under NaCl and RV5 stress condition and maintain the osmotic balance in the duckweed. The maximum concentration of APX activity was 741.8 ± 65.36 Unit mg⁻¹ protein observed at 150 ppm concentration of NaCl + RV5, which is 333% higher compare to control, 67% higher compare to only NaCl treatment and 31% higher compare to only RV5 treatment. Further increasing in the concentration up to 200 ppm decrease the proline concentration was decrease by 71% (Fig. 1c). The maximum concentration of SOD activity was 345.8 ± 61.64 Unit mg⁻¹ protein observed at 150 ppm concentration of NaCl + RV5, which is 275% higher compare to control, 175% higher compare to only NaCl treatment and 55% higher compare to only RV5 treatment. Further increasing in the concentration up to 200 ppm decrease the SOD activity by 70% indicating an antioxidative response in plants (Fig. 1c). Higher activity of SOD enzyme indicates the presence of reactive oxygen species in plants exposed to salt or other organic toxicants. APX & SOD have play vital roles during abiotic and biotic stress for growth, respiration, transpiration and gas exchange in plants (Gill and Tuteja, 2010; Doğanlar and Atmaca, 2011). SOD play important roles in neutralizing ROS toxicity by converting O²⁻ to H₂O₂, sequentially POD reduce H₂O₂ into H₂O and O₂ (Ruiz-Lozano and Aroca, 2010). Result shown in Fig. 1a–c, indicate that the S. polyrhiza tolerated larger oxidative stress by exposed to higher NaCl and RV5 concentrations, which could explain by dose-dependent increase in the concentration of H₂O₂, MDA, ROS, SOD and APX activity. Similar results were also obtained for SOD and APX activity in duckweed exposed to both organic and inorganic pollutants, C. demersum (under Pb and Zn stress), L. minor (exposed to Cd, Pb and electroporating wastewater) (Torbati et al., 2015, Mishra et al., 2006, Singh and Singh, 2006).

Starch was accumulated 11.25 ± 0.97% and 12.36 ± 0.74% in biomass exposed with 100 mM NaCl and 100 ppm RV5 respectively.

But as shown in Fig. 1d the maximum concentration of starch 16.57 ± 2.35% was achieved from biomass exposed with 150 ppm concentration of NaCl + RV5. It is 354% higher compare to control, 47% higher compare to only NaCl treatment and 34% higher compare to only RV5 treatment. It might be due to some stress factors in stress condition S. polyrhiza suppress the vegetative growth and utilize non-utilized photosynthetic molecular carbohydrate in form of the accumulated starch (Sree and Appenroth 2014, Appenroth et al., 2010). Further increasing in the concentration up to 200 ppm decrease the starch accumulation by 62%, might be due to the antioxidative response in plants.

Wet pulp remaining after nanoparticles synthesis containing 16.57 ± 2.35% Starch and 0.73 ± 0.05% cellulose. The fractionation or traceable process minimizes 59 to 66% of the chemical use and reduce environmental damage by eliminating defatting treatments compare to conventional starch extraction. Starch was hydrolyzed by α-amyloglucosidase Sigma A7095 and α-amylase Sigma A4582 with 2% optimized dosage, 48 h of hydrolysis period and 45 °C temperature. The hydrolysate was analyzed by HPLC, confirmed formation of glucose 941.21 ± 72.36 mg/g starch, which corresponds to 94.28 ± 1.25 conversion efficiency (Fig. 2a). Fermentation of hydrolysates with S. cerevisiae MTCC 180 for 12 h produce ethanol 425.23 ± 45.84 mg/g glucose, which correspond to 87.24 ± 1.24% conversion efficiency (Fig. 2b and Supplementary Fig. 3). The conversion efficiency of duckweed sugars to ethanol remained between 87 and 89% which is 4.82 to 11.25% higher than those reported for algae using an engineered microbial platform.

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Fig. 2

HPLC analysis of hydrolysates (a), GC–MS analysis at different time interval of fermented broth (a).

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Effect of cell free extract of S. polyrhiza after treating with different concentration of NaCl + RV5 on size and zeta potential of nanoparticle was one of the major finding of the study. Result shown in the Fig. 3 significant decreasing in size of nanoparticle synthesized and smallest size 21.35 ± 11.23 nm of CuNPs were achieved at 150 ppm NaCl + RV5, 16-fold reduction in the size compare to control. While further increasing in the concentration NaCl + RV5 up to 200 ppm, 22-fold increase the size of CuNPs. It might be due to duckweed exposed to oxidative stress greater increase of SOD/POD activity play key role for the synthesis for CuNPs. Synthesized silver nanoparticle using lignin peroxidase of the Acinetobacter sp. (Singh et al., 2017). Zeta potential is defining stability and balance between the repulsive and attractive forces of nano-particles. The magnitude of zeta potential directs the electrostatic repulsion between two similar charged particles. So the colloids of nano-particles with high positive or negative zeta potential are electrically more stable. Results of zeta potential was −79.54 ± −5.69 mV at room temperature, confirm the CuNPs is highly stable (Fig. 3).

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Fig. 3

Effects of cell extract of S. polyrhiza treated with different concentrations of NaCl combine with RV5 on size of nano particles and the Zeta potentials.

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3.3 Synthesis of nanoparticle and characterization

The cell free extract with 0.226 Umg⁻¹ specific activity of APX was used as crude enzyme for the partial purification by (NH₄)₂.SO₄ precipitate. The crude enzyme was maximum precipitate at 55% saturation with 2.4-fold purification. The fraction of the best yield was loaded on DEAE-Sephadex, G-200-Sephadex, CM-Sephadex column, G-200-Sephadex, Ammonium sulphate 35% and crystallization sequentially yielding 6.35-fold purification with 1.44 Umg-1 specific enzyme activity (Table 2). Single band correspond to 37 kDa on SDS-PAGE, confirm the enzyme was homogenous, monomeric protein (Fig. 4). Reaction of purified APX with copper sulphate resulted in color change of reaction mixture from greenish blue to brownish green clearly indicate the formation of CuNPs. The spectrophotometric peaks at 547 nm and 638 nm in the visible region also support formation of CuNPs from cupper ions (Fig. 5a). The peak at 528 cm⁻¹ in FTIR spectra is corresponds to the Cu-O bond also confirm the formation of CuNPs (Rahman, et al., 2013) (Fig. 5b). XRD measurements were carried out for the determination of crystal phase composition of titanium dioxide nanoparticles synthesized using plant extracts. The measurements were carried out over the diffraction angle (θ) 0–80° (Fig. 5c). Line broadening of the diffraction peaks indicates that the synthesized materials are in nanometer range. The diffraction pattern showed that nanoparticles synthesized using different plant extract were from anatase phase. As, the main peaks in the range of θ = 43.31°, matches the (1 0 1) crystallographic plane of anatase of CuNPs, indicating that nanoparticles structure dominantly correspond to anatase crystalline. The crystallite sizes of different synthesized nanoparticles were calculated using Scherrer equation (4) for the main peak,

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Fig. 4

SDS–PAGE of homogenous monomeric APX corresponding to 37 kDa purified from S. polyrhiza treated with 150 ppm NaCl + RV5 concentration.

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Fig. 5

Characterization of CuNPs, Uv–vis. Spectroscopic analysis (a), FTIR analysis (b), XRD graph (c) and SEM-EDXA analysis (d) synthesized by purified APX.

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Crystallite size of synthesized nanoparticles was found to be in the range of 11.08 to 98.69 nm. A very sharp intense peak at 43.31°, 50.44° and 74.12° 2θ in XRD graph were identified as 111, 200 and 220 (Fig. 4c) planes confirm the formation of zero-valent copper (Kamali et al 2020). Further, the size range of the CuNPs were 5–35 nm with an average size of ∼23–26 nm were observed under SEM (Fig. 5d). The elemental Cu, C and Oxygen were observed in the SEM-EDX spectra of CuNPs.

3.4 Efficiency of CuNPs

As shown in Fig. 6a different reactive dyes were decolorized with varying the concentration from 100 to 1000 mg/l. The maximum degradation rate was 17.24 mg/L min. found for RV5 at 1000 mg/l after 1 h of incubation. However, the maximum rate of decolorization was 12.30, 11.56, 8.93 and 9.86 mg/L min achieved for RB222, RR31, RB-5 and RB220 respectively after 1.5 h incubation at 750 mg/L dye concentration, further increase in the concentration decrease % decolorization and the rate of decolorization (Fig. 6a). The present results revealed that the biogenic CuNPs possess excellent photocatalytic potential for the degradation of each dye up to 750 mg/L dye concentration. Significant increase in the degradation rate from 4.8 to 17.24 mg/L min was observed between 100 and 1000 mg/L concentration of RV5 dye, indicates potential of CuNPs for remediation of wide range of different reactive dyes. Various studies reported the role of CuNPs in photocatalytic degradation of azo dyes (Sasikala et al., 2018). It also possesses excellent photocatalytic potential and decolorized 91.53%, 73.89% and 84.89% of the initial concentration of methylene blue, methyl red and Congo red respectively under solar irradiation (Fathima et al., 2018).

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Fig. 6

Efficiency of CuNPs for; mineralization of wide spectrum of dyes (a), repetitive application of CuNPs for RV5 mineralization (b) effect of organic loading rate of RV5 on rate of decolorization, rate of COD and BOD reduction (c).

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The stability of the CuNPs for complete decolorization was observed up to the 10th cycle with 250 mg/L RV5 with in 26 min. Afterward up to the 15th cycle the decolorization percentage was drastically fall up 40% with increase the incubation time up to 45 min. (Fig. 6b).

CuNPs were also tasted for the different RV5 concentrations in the experiment ranged from 50 to 25,000 mg/l. The results showed that the increasing the concentration of RV5 increased the rate of decolorization, COD and BOD reduction rate from 3.0 ± 0.19 to 80 ± 4.23 mg/L min., 2012.7 ± 407.61 to 55263.3 ± 3298.5 mg/m³min. and 899.25 ± 210.98 to 30560.3 ± 1987.5 mg/m³min. respectively (Fig. 6c). The maximum the rate of decolorization, COD and BOD reduction rate was achieved at 15,000 mg/l RV5 concentration, further increase in the concentration, decrease 18.06% rate of decolorization and 22.15% COD reduction rate and 17.66% BOD reduction rate. It might be due to the quantity of intermediates increased as well, competing through side reactions with the parent dye decomposition. It is suggested that the increased RV5 concentration resulted in increased capacity of the solution, thereby blocking more sun light from reaching the CuNPs, reduce the catalytic efficiency of CuNPs.

RV5 degradation products and intermediates were also analyzed, which resulted in a number of compounds that could be fitted to proposed degradation pathways. The predominant intermediate products formed in the photocatalytic degradation of RV5 in the solution during the initial phase of degradation were 6-amino naphthalene sulfonic acid, 4-amino benzenes sulfonic acid and unknown product. Subsequent increase in incubation time concentration of 2,4-Hepta-2,4-dienedioic acid, 2-Carboxy Benzoic acid, Trans o-hydroxy benzylidene pyruvate, 3-Pent-3-enoic acid and 3-[(3-Hydrazinophenyl)sulfonyl] propionic acid was clearly indicate (Fig. 7), broken the ring structure in to the lower molecular weight aliphatic hydrocarbons led to complete mineralization (Patel and Bhatt, 2015, Patel et al., 2020). Further it is clear that the degradation of some auxochrome/chromophore group which was only modifications phenyl ring configurations with the help of OH^• random collision with RV5 and its intermediates products. Aliphatic acid intermediates readily might be converted into CO₂/H₂O or even smaller aliphatic hydrocarbons which cannot detected by the GC–MS analysis.

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Fig. 7

Proposed mechanism for RV5 mineralization pathway.

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