The efficiency of Anabaena sp. was analyzed for the phytoremediation of wastewater loaded with organic matter and heavy metals like chromium. Simulated wastewater was contaminated with chromium. A side-stream membrane bioreactor was used for the treatment of wastewater. A feed tank of 20 L capacity was used with a stirring arrangement. A ceramic microfiltration membrane composed of clay and alumina was obtained from Johnson & Johnson. The removal efficiency of chemical oxygen demand, biochemical oxygen demand, and chromium was evaluated. The process was used for algae harvesting and wastewater treatment. About 92% of chemical oxygen demand (COD), 98% chromium, and oil and grease were completely removed. Membrane fouling was explained by the pore blocking and cake resistance model. Stress in algal cells was determined from the superoxide dismutase (SOD) and catalase (CAT) analysis. The lipid content of algal cells was measured.

  • A side-stream ceramic membrane bioreactor was used for the treatment of wastewater containing toxic heavy metals.

  • Application of algae-based bioremediation of chromium (Anabaena sp.) was done.

  • Reduction of COD (92%), Cr(VI) (98%), and complete removal of turbidity and oil was obtained in the process.

  • Algal harvesting and wastewater treatment were achieved in the process.

  • The biodiesel and lipid content of algae growing in wastewater are compared.

The generation and treatment of wastewater is a global problem. Pollution is created due to the discharge of huge amounts of untreated effluent from various sectors. Aquatic environments are directly exposed to these sorts of pollution as they are thought to be the most convenient sinks for disposal (Hiraoka 2021). Therefore, the need to treat and discharge toxic effluent has been in high demand. In this respect, the use of algae a potential microorganism for the remediation of wastewater is nowadays gaining importance. Conventional treatment of wastewater produces water that does not comply with stringent international laws set aside for the quality of discharge water. Microalgae are useful for the removal of phosphorus, nitrogen, toxic heavy metals, etc. (Priyadarshani et al. 2011). Substantial biomass production with simultaneous wastewater remediation has been obtained from microalgae (Cai et al. 2013). According to USEPA (2011) microalgae have been applied for wastewater treatment in ponds for the last 3,000 years (US EPA 2011). Algal treatment of wastewater compared to conventional treatment requires no additional chemicals, is cost-effective, metal removal, lesser generation of sludge, etc. Consortium of Chlorella sp. and Scenedesmus sp. was used for the treatment of domestic wastewater by several authors (Silambarasan et al. 2021; Vaz et al. 2023). The use of Chlorella vulgaris and Scenedesmus quadricauda for the removal of nutrients from wastewater has been studied. C. vulgaris removes more efficiently and S. quadricauda removes BOD and phosphate (Ayodhya & Kshirsagar 2013). Phytoremediation of sewage wastewater was carried out using different microalgae, i.e. Chlorella minutissima, Scendesmus sp. and Nostoc sp. The algae could reduce BOD, COD, NO3, NH4, PO4 , TDS, etc. Chlorella was the best among all the three algae and produced more manure (Sharma & Khan 2013). Five algal species, i.e. Anabaena, Diatoms, Spirogyra, Hyalophacus, and Monoraphidium were studied based on of their ability to reduce pollutants from wastewater. The algae use nitrogen and phosphate and reduce carbon dioxide (Velan & Saravanane 2013). Apart from nutrient pollution, wastewater also contains heavy metals such as chromium, lead, nickel, and zinc that enter the human body and accumulate in the food chain of the ecosystem causing severe health hazards. Treatment technologies for heavy metals, such as reverse osmosis, chemical precipitation ion exchange, and ultrafiltration were used. But these methods are ineffective at lower concentrations and are not cost-effective. The potential use of many living organisms such as algae, fungi, bacteria, yeast, and waste materials is done as biosorbents for the removal of heavy metals. Live organisms are also used for the uptake of metals. Chromium is highly toxic and carcinogenic and imparts hazardous effects on human health. Chromium in its hexavalent state is more toxic than its trivalent state. Chromium is widely used in electroplating industries, tanning industries, the manufacturing of chrome, etc. The ability of algae to take up chromium and accumulate in their cells is studied. Biomass thus produced can be used for the production of biofuel. Algae can bind metal on the surface of their cell wall. Metal-binding sites are present on their cell wall. Their cell is composed of lips, proteins, polysaccharides, etc. which are composed of functional groups such as amino, hydroxyl, and carboxyl groups that act as metal-binding sites. Moreover, algae can use the metal for their growth (Macfie & Welbourn 2000; Dayana et al. 2013; Jyoti & Awasthi 2014). The use of dried algae for chromium uptake was studied using Ulva lactuca with 92% removal efficiency (Gowda 2021). Removal of hexavalent chromium was studied using immobilized Chlorella pyrenoidosa with calcium beads. Removal efficiency was 75 mg/L at pH 3 (El-Sikaily et al. 2007). But very limited literature data are available for the application of live algal cells in metal sequestering (Mohsenpour et al. 2021). Therefore in the present study, Anabaena sp. was studied for the removal of organic matter and chromium from sewage water in a membrane bioreactor (MBR). The biomass produced was studied for the production of biofuel.

Collection and culture of algae

Algal strains of Anabaena sp. were purchased from National Collection of Industrial Microorganism (NCIM), Pune, India. The culture was then aseptically transferred to a culture tank containing Fogg's media (Fogg & Thake 1987) and maintained in a clean atmosphere inside laminar airflow (temperature maintained at ±25 °C), with 12-h photoperiod. The algal species was identified as Anabaena cylindrica with an optical microscope (50× magnification). All the chemicals used for media preparation were purchased from Merck, India. The glass wares used for algal culture and experiments as well as media were properly sterilized before and after each use to minimize contamination.

Since algae require carbon dioxide for growth, wastewater, and control (media) were saturated with carbon dioxide at 50 cc/min from a CO2 cylinder. The saturation level was detected by a CO2 hand analyzer.

The initial content of the culture was 25 mg dry weight/L and the initial optical densities at OD650 were 0.042. The cell density of alga can be quantified in two basic ways, i.e. as grams of dry or wet weight per litre of the sample or as the number of viable/dead cells per mL. For measuring dry weight, the alga is separated from the solution and dried before weighing which account for dry weight per litre of the sample. To measure the wet weight of algal cells, biomass was detected spectrophotometrically using a UV–Vis spectrophotometer and measured at a wavelength of OD 650. Light absorbance is proportional to the change of cell number for microalgae like Anabaena sp. Fourier transform infrared spectroscopy (FTIR) analysis of dried algal biomass was carried out to understand the functional groups present in the cellular structure.

Collection and characterization of wastewater

Wastewater was collected from a nearby sewage pumping station in Kolkata. Immediately after collection, the water was characterized into organic and inorganic matter. Simulated wastewater was prepared by adding hexavalent chromium (5–20 ppm concentration). Algal cells were allowed to grow in wastewater keeping all other culture factors the same.

Side-stream MBR study

A large-scale study was carried out in 1 L sterilized glass cylinders covered with cotton plugs. Wastewater was saturated with carbon dioxide and algae (0.5 g/L) were added. Wastewater was drawn at regular intervals for analyzing various parameters and measuring dissolved carbon dioxide. The growth rate was measured at regular intervals by weighing the complete set. Evaporation loss was calculated by setting up an experimental set-up without algae that served as blank. Algae collected after 7 days of culture were subjected to characterization for different biochemical parameters and stress enzymes. These algae-containing effluents were subjected to a microfiltration study.

Ceramic microfiltration study

A ceramic microfiltration study was conducted using a single-channel ceramic microfiltration membrane obtained from Johnson & Johnson, Kolkata. The membrane was 300 mm in length and 10 mm/mm OD/ID. It was of single-channel configuration. The average pore size of the membrane was about 0.6 μm. The feed tank was of 20 L capacity made of stainless steel 304 L with stirring arrangement. The set-up has two-way valves for reverse connection and the total set-up was mounted on a moveable trolley. Flux was studied at a constant transmembrane pressure of 2 bar for 180 min and 3 LPM (L min−1) cross-flow velocity (CFV). Permeate was collected at regular intervals and was characterized for organic and inorganic components. The membrane fouling constant was obtained by fitting the flux data in the cake resistance and pore blocking models – both complete and intermediate blocking models. The equations are depicted in Equations (1)–(3).
formula
(1)
formula
(2)
formula
(3)
where Jt is the flux at time t and J0 is the initial flux, kc, kb, and ki are the fouling constant for cake resistance and complete pore blocking and intermediate pore blocking model, respectively, and t is the time (Bella & Trapani 2019).

Characterization of the membrane

The membrane support before and after the application was subjected to SEM analysis (Inspect F50, FEI, USA) to understand the surface morphology of the membrane.

Assay of superoxide dismutase and catalase

Oxidative stress analysis was conducted on algal cells exposed to wastewater and media to compare the induced stress on the cells that might be used for biodiesel production. Algal tissues (0.5 g) were homogenized in a 50 mM cold phosphate buffer (pH 7.0) and centrifuged (Kubota, 6,500, Japan) at 14,000 rpm for 15 min at 4 °C. The supernatant was used for further analysis. For superoxide dismutase (SOD) measurement, the method described by Giannopolitis & Ries (1977) was used and expressed as U/mg protein/min (Giannopolitis & Ries 1977). 1.3 μM riboflavin, 13 mM methionine, 63 μM NBT, and 0.05 M sodium carbonate (pH 10.2) were added to the reaction mixture. Distilled water was added to make the volume up to 3 mL. Samples were properly illuminated and the blank was not exposed to light. After 30 min, samples were measured spectrophotometrically at 560 nm. Beauchamp and Fridovich explained 1 unit of SOD as the amount that inhibits the NBT reduction by 50% (Beauchamp & Fridovich 1971). Catalase (CAT) activity was measured by the disappearance of H2O2 when added to enzyme extract and expressed as U/mg protein/min (Aebi 1983). Measurement was carried out spectrophotometrically at 240 nm. The reaction mixture contained phosphate buffer and hydrogen peroxide. Chlorophyll content was measured as per the method described in APHA and measured spectrophotometrically at 664 nm (Greenberg et al. 2005).

Statistical analysis

Each set of experiments was in three replicates and the standard deviation (S.D.) was determined and expressed as X ± S.D. in GraphPad InStat 3 software (GraphPad, San Diego, CA, USA) by one-way analysis of variance (ANOVA). The means of different treatments were determined using p ≤ 0.05 and p ≤ 0.01 as levels of significance.

Algal lipid and biodiesel content

Biodiesel and lipid content of algae were measured. Lipid content was measured by extraction in methanol and the biodiesel content was measured by transesterification with alkali (Velan & Saravanane 2013). The algal culture was taken in chloroform solvent (1:2) and set in an orbital shaker for 15 min. It was then heated in a water bath for 1 h at 75–80 °C and then filtered. The solvent extract was esterified with 0.9% NaCl and collected in a separating funnel. Two separate layers were formed where lipid was separated in the lower part and collected in a container (Hamouda et al. 2023).

Chromium removal study

Algal cells were cultured in a chromium solution in batch mode to observe the removal efficiency of algae. 100 mg/L potassium dichromate solution was prepared as stock solution and further dilutions (0–20 mg/L) were carried out for batch mode study. pH of the solution was adjusted using 0.1 N HCl and 0.1 N NaOH. Chromium removal was measured spectrophotometrically at ƛmax 540 nm using a 1,5 diphenylcarbazide solution. Removal capacity was calculated from the following formula:
formula
(4)

A batch adsorption study was carried out at varying metal concentrations (0–20 mg/L), pH (3–5), and algal cell density (0–1.23 × 108) at 25 °C ± 2 °C.

Algal growth

Algal growth was monitored for 7 days and live cells were observed spectrophotometrically at ƛmax 650 nm. Dry weight was measured by collecting algal cells and drying them at 80 °C for 24 h. Growth of both methods was compared and a good fit (R2 – 0.925) was observed. The growth curve was determined (Figure 1) and calculated from the following equation:
formula
(5)
where L = growth rate (μ day−1), N1, N2 are dry biomass (g L−1) at time t1 and t2, respectively (Krzeminska et al. 2014). The growth rate was maximum after 7 days of culture.
Figure 1

Growth curve of Anabaena sp. in media over 7 days.

Figure 1

Growth curve of Anabaena sp. in media over 7 days.

Close modal

Reduction of organic matter and chromium removal

Algal cells grown in wastewater, when observed under a microscope (100 × , Olympus, Japan), had cylindrical cells with no other cells being observed suggesting the growth of a single strain of algae (Figure 2). From Table 1 it was observed that about 93% reduction in COD was observed after algal treatment with considerable reduction of BOD, dissolved solids and turbidity. 97% chromium removal was also observed. Microalgae can grow in wastewater generating biomass that can be harvested for various valuable by-products. Wastewater contains many organic and inorganic nutrients such as phosphate, ammonia, carbon, and nitrate that supports algal growth. Anabaena sp. as cyanobacteria can thrive in highly polluted water and use contaminants for its growth simultaneously remediating the waste.
Table 1

Characteristic of domestic and industrial effluent before and after algal treatment

ParametersSimulated sewage water
Raw effluentAfter algal treatmentAfter side-stream MBR
COD (mg/L) 330 21.5 11.2 
BOD (mg/L) 54 BDL BDL 
Turbidity (NTU) 39 1.4 <1 
pH 7.1 8.0 7.2 
TDS (mg/L) 272 160 24 
Cr+6 (mg/L) 9.8 0.25 0.2 
ParametersSimulated sewage water
Raw effluentAfter algal treatmentAfter side-stream MBR
COD (mg/L) 330 21.5 11.2 
BOD (mg/L) 54 BDL BDL 
Turbidity (NTU) 39 1.4 <1 
pH 7.1 8.0 7.2 
TDS (mg/L) 272 160 24 
Cr+6 (mg/L) 9.8 0.25 0.2 
Figure 2

Microscopic image of Anabaena sp. as observed under a microscope (100×).

Figure 2

Microscopic image of Anabaena sp. as observed under a microscope (100×).

Close modal
FTIR of dried algal biomass was conducted to study the functional groups present that might take part in chromium removal (Figure 3). The broad peak observed at 3,846 cm−1 is due to the –OH bond (Sheng et al. 2004). Sheng et al. (2004) confirmed that the removal of heavy metals such as lead, cadmium, copper, zinc, and nickel by marine algal biomass of Sargassum sp. and Padina sp. was due to carboxyl, ether, alcoholic, and amino groups, as observed from FTIR. Stretching vibrations observed at 2,924; 2,849; 2,147; and 2,023 cm−1 are due to –CH2 groups. Similar results were obtained in dried biomass of Anabaena sphaerica used as the biosorbent for lead removal (Abdel-Aty et al. 2013). Peaks detected at 1,643 and 1,827 cm−1 correspond to the C–O stretching (Fourest & Volesky 1996). The bands observed at 1,522; 1,599; and 1,445 cm−1 are due to stretching vibrations of amide groups of protein chains (CO–NH) (Sheng et al. 2004). Polysaccharides present in algae can be observed from peaks at 1,323 and 1,043 cm−1. Peaks at 993, 901, and 777 cm−1 are C–O–C glycosidic linkage of polysaccharides (Nikonenko et al. 2000). Bands at 592 and 546 cm−1 are due to –OH groups whereas the peak observed at 501 cm−1 was due to C–N–S stretching of polypeptide (Lodeiro et al. 2006; Murphy et al. 2007; Sari & Tuzen 2008). Murphy et al. (2007) observed that copper ion removal was possible due to the interaction of Cu2+ ions with carboxyl, amino, sulphonate and hydroxyl groups present in seaweed.
Figure 3

FTIR spectra of dried algal biomass.

Figure 3

FTIR spectra of dried algal biomass.

Close modal
The growth rate of algae increases with time, i.e. within the first 3–5 days, after which the growth attained a steady state (Figure 1). A batch study, using algae for chromium removal, is shown in Figures 4 and 5. Microalgae operate in a wide pH range of 1–10 and in this study chromium removal was facilitated at pH 5. Algae can remove heavy metals both in life form and as adsorbent. In this study, live algal cells were used for the removal of hexavalent chromium from wastewater. About 97% removal of metal was observed within 7 days of culture. Algal growth inevitably increased in the presence of metal and it may be deduced that metal ions were biosorbed on the active sites present in the cell structure. The cell wall contains proteins, lipids, polysaccharides, etc. Other functional groups include carboxyl, hydroxyl, phosphate, amino, etc. that interact with Cr+6 ions and binds with them, using for cell growth, thereby removing toxic metals in the process (Fernando et al. 2017; El-Said et al. 2019; Grace et al. 2020; Shobier et al. 2020).
Figure 4

(a) Effect of Cr+6 removal with increasing algal growth and (b) the effect of pH on Cr+6 removal.

Figure 4

(a) Effect of Cr+6 removal with increasing algal growth and (b) the effect of pH on Cr+6 removal.

Close modal
Figure 5

(a) Removal of Cr+6 in response to increasing metal concentration and (b) the effect of time on the removal of Cr+6 by algae.

Figure 5

(a) Removal of Cr+6 in response to increasing metal concentration and (b) the effect of time on the removal of Cr+6 by algae.

Close modal

Table 2 depicts the values of stress enzymes like superoxide dismutase and catalyze. Since algae were cultured in wastewater and media served as control, an assay of stress enzymes was carried out. Algae generate antioxidant enzymes to cope with reactive oxygen species (ROS) generated in a stressful environment to reduce cellular damage. SOD is the first line of defence that living cell produces to combat cellular damage. It is a metalloprotein that catalyses superoxide dismutation into hydrogen peroxide and oxygen (Rezayian et al. 2019). CAT converts peroxide radicals generated under stress into water and oxygen. The values generated from a 3-day (72 h) experimental study suggest that stress generated within 24 h gradually decreased with time and data were mostly comparable to the control (Mate et al. 1999). Thus algae were able to survive and grow under stress conditions. Chlorophyll content was measured as it is an important parameter that indicates wastewater toxicity. Chlorophyll content was less than control, suggesting stress shock to cells (Table 2).

Table 2

Oxidative stress biomarkers in the tissue of Anabaena cylindrica exposed to different solutions

Oxidative stress biomarkersExperimental groupsAnabaena sp. exposure time
24 h48 h72 h
SOD (U/mg protein) Control 3.4± 0.04 3.3± 0.05 3.0± 0.07 
Simulated sewage water 4.7± 0.11 4.5± 0.07 4.1± 0.08 
CAT (U/mg protein/min) Control 4.9 ± 0.08 4.7 ± 0.06 4.3± 0.05 
Chlorophyll (mg/g) Simulated sewage water 5.8± 0.06 5.4± 0.07 5.0± 0.12 
Control 4.5± 0.14 4.3± 0.10 4.1± 0.04 
Simulated sewage water 3.9± 0.08 3.8± 0.11 3.4± 0.10 
Oxidative stress biomarkersExperimental groupsAnabaena sp. exposure time
24 h48 h72 h
SOD (U/mg protein) Control 3.4± 0.04 3.3± 0.05 3.0± 0.07 
Simulated sewage water 4.7± 0.11 4.5± 0.07 4.1± 0.08 
CAT (U/mg protein/min) Control 4.9 ± 0.08 4.7 ± 0.06 4.3± 0.05 
Chlorophyll (mg/g) Simulated sewage water 5.8± 0.06 5.4± 0.07 5.0± 0.12 
Control 4.5± 0.14 4.3± 0.10 4.1± 0.04 
Simulated sewage water 3.9± 0.08 3.8± 0.11 3.4± 0.10 

Values are mean ± S.D.; p < 0.05.

Lipids are mainly fatty acids and their derivatives. Fatty acids produced by microalgae generally contain combinations of zero to five cis double bonds. Increased lipid content in untreated wastewater might be because under stress conditions algae reduce their cell growth and accumulate available carbon for the synthesis of lipid and other high energy density compounds to combat carbon limiting conditions. This result can be established from the growth rate obtained in this study where, after the 5-day growth, almost attained a steady state. Since wastewater is a nutrient-rich medium, it becomes light and carbon limiting for algae growing in it. But some algae thrive well in the light-limiting condition as well as utilize organic carbon for their growth (Ota et al. 2009; Onyeaka et al. 2021). The lipid content of algae growing in media was 3.7% (±0.03) whereas that of algae growing in wastewater was 5.3% (±0.03). Biodiesel content was on the lesser side than the control (Table 3).

Table 3

Lipid and biodiesel content of algae cultured in media as control and simulated sewage water

ParametersControl (media)Simulated sewage water
Lipid content (%) 3.7 ± 0.03 5.3 ± 0.03 
Biodiesel content (%) 4.7 ± 0.5 4.42 ± 0.3 
ParametersControl (media)Simulated sewage water
Lipid content (%) 3.7 ± 0.03 5.3 ± 0.03 
Biodiesel content (%) 4.7 ± 0.5 4.42 ± 0.3 

Note: Data represent the average of three experiments.

Ceramic microfiltration study

FESEM images of unused and used membranes are shown in Figure 6. Unused membrane displays visible pores of about ∼0.6 μm but after a microfiltration study, pores were blocked partially by algal cells.
Figure 6

FESEM image of ceramic membrane before and after use.

Figure 6

FESEM image of ceramic membrane before and after use.

Close modal
For algal cultures in untreated sewage water, cake resistance and pore blocking models fitted well with the experimental values. For cake resistance, complete and intermediate pore blocking model initial flux values were 66.70 LMH, 65.76 LMH, and 66.18 LMH, respectively, and the experimental initial flux value was 66 LMH (Figure 7). The cake resistance (kc) and complete pore blocking (kb) and intermediate pore blocking (ki) were , 0.00223 min−1, and , respectively (Table 4). From Figure 8(a)–8(c), it was also observed that the decrease in flux was not significant after 180 min of operation at 2 bar pressure. A ceramic microfiltration membrane can effectively be used for microalgae harvesting without any subsequent deterioration in membrane properties like chemical stability, etc. The pore blocking and cake filtration models fit, it might be observed that fouling behaviour could be due to the combined effect of two fouling mechanisms, viz. pore blocking and cake formation (Matsumoto et al. 1992; Bolton et al. 2006; Li et al. 2011; Nakamura et al. 2012; Iritani et al. 2015). For ceramic membrane, fouling was reversible and the membrane can be regenerated by back washing after each run (Bhattacharya et al. 2011, 2013, 2014).
Table 4

Complete pore blocking, intermediate pore blocking, and cake filtration model fitting

Model nameFunctionInitial flux (J0 inLMH)R2Blocking constant
Complete blocking  65.76 0.9286 0.00223 
Intermediate blocking  66.18 0.9306  
Cake filtration  66.70 0.9426  
Model nameFunctionInitial flux (J0 inLMH)R2Blocking constant
Complete blocking  65.76 0.9286 0.00223 
Intermediate blocking  66.18 0.9306  
Cake filtration  66.70 0.9426  
Figure 7

Effect of time on permeate flux.

Figure 7

Effect of time on permeate flux.

Close modal
Figure 8

Model fitting data for (a) complete pore blocking, (b) intermediate pore blocking, and (c) cake filtration for a side-stream algal MBR study.

Figure 8

Model fitting data for (a) complete pore blocking, (b) intermediate pore blocking, and (c) cake filtration for a side-stream algal MBR study.

Close modal

The fate of algal biomass

The produced algal biomass can be utilized in many ways. The synthesis of nanoparticles from algal biomass has been widely used because of its smaller size, greater surface-to-volume ratio, and higher reactivity (Cheng et al. 2019). The removal of heavy metals using algae-based adsorption process is studied (Li et al. 2019; Goswami et al. 2022). The produced algal biomass can also be used for the production of biofuel. Studies were conducted using algal biomass where after the adsorption of Pb(II) Chlorella sorokiniana was subjected to biodiesel production. The GC–MS analysis of algae showed that fatty acids and SFA are required for biodiesel production (Nanda et al. 2021). Algal-based treatment of wastewater and heavy metal remediation were done using produced biomass for the generation of biocrude oil or bioenergy-like value-added products (Goswami et al. 2020).

The study shows that algae can efficiently phyto-remediate wastewater-containing hexavalent chromium and organic matter. The growth rate data of algae suggested that the nutrients in wastewater served the growth requirements. 97% removal of chromium was obtained with 93% COD removal. The FTIR analysis proved the presence of polysaccharides that might take part in chromium uptake by algae. An assay of stress enzymes, such as SOD, CAT, and cholorophyll content, showed the generation of ROS in response to toxic wastewater and heavy metal compared to the control in algae within 24–48 h in which the algae were able to combat within 72 h as evident from reduced values of SOD and CAT. Fouling behaviour was observed with 66 LMH flux values. When pore blocking and cake filtration models agreed with fouling data they showed that fouling behaviour was reversible and a membrane can be regenerated by back washing after each run. Thus, a side-stream MBR study using algae showed promise in remediating toxic metal-laden wastewater and treated water could be reused and biomass can be harvested for the production of biofuel.

The financial support from the Department Of Science And Technology, Government of India vide Grant no. DST/WOS-B/WWM-2/2021 (G) dated 17.09.2021 is gratefully acknowledged.

Data cannot be made publicly available; readers should contact the corresponding author for details.

The authors declare there is no conflict.

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