In-Situ and Ex-Situ Phycoremediation Competence of Innate Scenedesmus Sp. On Polluted River Water


 The present study was aimed to investigate the ex-situ and in-situ phycoremediation efficacy of native Scenedesmus sp. in polluted Thirumanimuthar River water sample. Based on the macroscopic and microscopic analysis the predominant microalgae culture from polluted water was identified as Scenedesmus sp. Most of the physicochemical parameters (EC: 3110.00 micro mho cm-1, turbidity: 17.10 mg L-1, total hardness: 812.00 mg L-1, BOD: 230 mg L-1, and COD: 352 mg L-1) and some minerals such as Ca: 232.00 mg L-1 and Sulfate: 532.00 mg L-1 were crossing the permissible limits. Among three sets of treatments (I, II, & III), 3% in ex-situ (treatment III), and 30% in in-situ (treatment III) were shown a better reduction in physicochemical properties of polluted river water in 14 days of treatment. The in-situ study has shown better pollutants reduction than ex-situ as it reduced BOD & COD 27.83% and 23.30%, respectively. Further, the chloride, sulphate, phosphate, sodium, calcium, and magnesium were reasonably reduced up to 40.00, 43.61, 31.03, 18.75, 70.26, and 33.93%, respectively. The FTIR analysis confirmed that the presence of pollutants absorbing functional groups in dried biomass of Scenedesmus sp. and the SEM analysis image confirmed the absorption of pollutants by resulting in morphology changes of Scenedesmus sp. The results concluded that a significant reduction was found in most tested physicochemical and minerals contents in the treated water through the in-situ study than the ex-situ approach. Through this sustainable phycoremediation strategy, the pollutant reducing Scenedesmus sp. could be used as feedstock for biofuel production.


Introduction
Globally, most of the rivers and freshwater streams are primarily polluted by industrial wastes which are derive from different industries such as petrochemicals, fertilizers, oil re neries, paper, textiles, sugar mills, steel, tanneries, distilleries, and pharmaceuticals, etc. (Liu et al. 2019; Leng et al. 2020). The untreated e uents released from textile industries contain different kinds of chemicals (acids, minerals, alkalis, and several dyes) that cause severe water pollution (Sarayu and Sandhya 2012). The more population-holding nations from Asian continents, the water pollution became a thoughtful issue since more population eruption leads to the discharge of more volume of untreated municipal wastewater to existing water reservoirs and create water pollution (Angelaalincy et al. 2017). Besides, in India, all types of pollutions were collectively accountable for 21% of demises from cerebrovascular disease, 26% of demises due to coronary artery disease, 23% by hemorrhagic stroke, 51% to respiratory infections, and 43% due to lung carcinoma. In developing countries, environmental pollution has been accountable for numerous demises as AIDS and tuberculosis collectively made (NGT 2019). Among the various pollution, water pollution is one of the signi cant causes of illness to humans and animals (Leng et al. 2020).
The rapid urbanization leads to releasing a huge volume of untreated sewage into the river water. According to the national green tribunal principal bench, New Delhi, India (NGT 2019), the untreated e uent releasing is the solitary cause of pollution of surface and groundwater. Further, about 60% of untreated urban sewage of India is directly disposed into the river and reduce the quality of water and made it un t for human utilization (consumption) and cause diseases, demises, and critical destruction to water, earth, and air (Murty 2011;Asselborn et al. 2015). The Central Pollution Control Board (CPCB) of India, performed a regular portfolio of water resource, polluted water creation, and it reported that the disposal of around 33,000 million liters of polluted water created every day from Class-I and Class -II municipalities (CPCB Report 2009). These unprocessed discharges from houses pollute the surface and groundwater resources.
The modern industrialization process and inappropriate discharge of residues contained e uents into water bodies caused heavy metal pollution in the surrounding environment and produced serious issues . Hence, nding the most suitable eco-friendly technologies with potential pollutants removing or minimizing agents is signi cant now. Traditional methods such as physical ( ltration, sedimentation, etc.), chemical ( occulation, precipitation, etc.), and biological (phytoremediation, bioremediation, etc.) treatment are reducing due to economic crisis, treatment time duration, and limitations in using a large scale (Li et al. 2013). The phycoremediation is an alternative, eco-friendly, e cient, and low cost (Gonçalves et al. 2017;Li et al. 2013) technique to treat polluted water. Since microalgae are one of the crucial parts of the polluted microbial niche, which act as a signi cant contributor in polluted water selfpuri cation (Gonçalveset al. 2017) and also used for the elimination of pollutants such as excess nutrients, xenobiotics, and CO 2 which is low cost-effective, non-intrusive and safer technology (Ajayan et al. 2015). Historically, microalgae have been employed to treat domestic polluted water through raceway ponds and photo-bioreactors. Besides that, they possess the e ciency to uptake heavy minerals from polluted water and serve as a signi cant role in heavy metal removal . For the past few decades, the two common algal species like Chlorella and Scenedesmus sp. are majorly used to treat polluted water (Krohn-Molt et al. 2017).
Recently, many countries, namely, Australia, the USA, Thailand, Taiwan, and Mexico, developed algal and fungal technology to remediate polluted waters in the environment ). The domestic sewage water in a warmer climate is the ideal habitats for the growth of Scenedesmus sp. and Chlorella sp. (Tripathi et al. 2019). The various species of microalgae viz, Chlorella sp., Scenedesmus sp., Phormidium sp., Botryococcus sp., Chlamydomonas sp., and Spirulina sp. have been utilized for treating domestic polluted water (Wang et al. 2015;Gonçalves et al. 2017;Liu et al. 2019). As per the recent National Green Tribunal report (2019), the physicochemical properties of Thirumanimuthar River were not meeting the India standards. With this brief background, the current study was focused on in-situ and ex-situ phycoremediation of contaminated water collected from highly polluted region of the "Thirumanimuthar" river (Salem, Tamil Nadu, India) using native microalgae species.

Brief pro le about the study area
The study area Thirumanimuthar River located at the Poolavari village belongs to Salem District, Tamil Nadu, India (11° 36'40.8" N latitudes, and 78°06'16.9" E longitudes, Fig. 1). This area received an average annual rainfall was 800 mm. The geography of the study area revealed that the presence of archaean crystalline rocks and surrounded by several hillocks in all directions, namely, Shevaroys and Nagaramalai (North), Jarugumalai Hills (South), Kanjamalai (West), and Goudamalai (East). This river has been polluted by releasing untreated dyeing and bleaching (155 unauthorized dyeing and bleaching units and surface and groundwater and made them un t for drinking and irrigation (NGT 2019). The farmers depend entirely on this river for agriculture activities and drinking purposes due to insu cient annual rainfall.

Collection of polluted water
The polluted water sample was collected from Thirumanimutharu River, at Poolavari village, Salem district of Tamil Nadu. The samples were collected in sterile polyethylene bottles that added a few drops of HNO 3 to prevent loss of elements and transported to the laboratory for further investigation (Ajayan et al. 2015).
2.3. Isolation, culturing, and identi cation of microalgae Initially, 25 µm diameter sized phytoplankton net was used to lter the dust from the polluted river water. Later, 0. 45 size Whatman No. 1 lleter paper was used to lter the microalgae species from polluted water and immediately observed under a standard microscope and con rmed the presence of microalgae in lter paper. The lter paper was rinsed with sterile distilled water and 1 mL of microalgae suspended rinsed water was inoculated in petri plates containing 25 mL of semi-solid Bold's Basal Medium (BBM) with 200 µg mL -1 of ampicillin (to avoid bacterial growth) and incubated for 3 weeks at 28 ± 1 o C with day/night cycling illumination (40 µmol photons m -2 s -1 ; 15h light/ 9 h dark). The growth of green color colonies has appeared in the 2 nd and 3 rd weeks of the incubation period, and those algae were identi ed as Scenedesmus sp. (through the study of macroscopically and microscopically: using a compound binocular microscope) that transferred into a sterilized BBM containing new petri dish. A single colony of Scenedesmus sp. was isolated and inoculated in liquid BBM media. The growth of algae (after two weeks of incubation) was measured using a UV-visible spectrophotometer at 680 nm (Mehrabadi et al. 2015).

Ex-situ experimental setup for phycoremediation
The Scenedesmus sp. culture was spun at 7000 rpm for 12 min and obtained pellet (biomass) was rinsed with sterile distilled water, and seed inoculum was prepared with three different density (5 × 10 4 cells mL -1 , 7.5 × 10 4 cells mL -1 , and 10 × 10 4 cells mL -1 ) using spectrophotometer (680 nm) and a hemocytometer. These three different cell densities The treatment setups were incubated at 28±1°C for 14 days under sterile laboratory conditions. Illumination was provided to cultures using casual white luminous lamps at 4000 Lux with a dark/light period of 16:8 h with periodical manual shaking (Verma et al. 2020).

In-situ phycoremediation of polluted water
In-situ phycoremediation study was performed near the bank of the Thirumanimutharu River at Poolavari village, Salem District, Tamil Nadu by setting up a waste stabilization pond system (5.75 x 2 x 2 m in size), and the treatment of polluted water was conducted for 14 days with (periodical manual blending) following experimental setups Treatment I: 5 × 10 4 cells mL -1 of Scenedesmus sp. in 100 L of polluted river water sample supplemented with 200 µg mL -1 of ampicillin Treatment II: 7.5 × 10 4 cells mL -1 of Scenedesmus sp. in 100 L of polluted river water sample supplemented with 200 µg mL -1 of ampicillin Treatment III: 10 × 10 4 cells mL -1 of Scenedesmus sp. in 100 L of polluted river water sample supplemented with 200 µg mL -1 of ampicillin Control : 100 L of polluted river water sample supplemented with 200 µg mL -1 of ampicillin without culture Triplicate setup was performed, and at the end of the 14 th day, both the treated and control polluted river water sample was collected from the respective ponds and analyses the physicochemical properties of them and calculated the percentage of phycoremediation occurred by Scenedesmus sp. (Sutherland and Ralph 2019). Before the sample was taken, the suspected amount of water evaporated during the treatment process was lled with elements free sterile distilled water.

Growth kinetics of Scenedesmus sp. during in-situ and ex-situ phycoremediation process
During the in-situ and ex-situ treatment, the pH of water and growth rate (density: number of cells mL -1 ) of Scenedesmus sp. were measured at 48 h intervals up to 14 days of treatment (Derakhshandeh and Tezcan Un 2019), using a pH meter, hemocytometer, and spectrophotometer (at 680 nm). Later, the biomass of Scenedesmus sp. from the treated (in-situ and ex-situ) water samples were harvested and dried in an oven for further analyses like FTIR and SEM. 2.7. Analysis of physicochemical properties of pre and post-treated river water sample The various physicochemical characteristics of pre and post-treatment of in-situ and ex-situ polluted water were carried out in terms of odor, turbidity, electrical conductivity, pH, total dissolved solids (mg L -1 ), total alkalinity, total hardness, Biological Oxygen Demand (BOD), Chemical Oxygen Demand (COD), Dissolved Oxygen (DO), free ammonia, and nitrite along with the quanti cation of minerals (calcium, magnesium, sodium, iron, chloride, sulfate, and phosphate according to the standard protocols developed by American Public Health Association (APHA 2005) using Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES Agilent 5900 SVDV). All the analyses were carried out at District Water Testing Laboratory (TWAD), Govt. of Tamil Nadu, Salem District, Tamil Nadu, India.

Fourier Transforms Infrared Spectroscopy (FTIR) and Scanning Electron Microscope (SEM) analysis
The dry Scenedesmus sp. biomass was subjected to the FTIR spectroscopy and SEM analysis. The Infrared spectra were documented in the range of 4000 cm -1 to 400 cm -1 using an IR spectroscopy (IR-200 The algal growth in the ex-situ and in-situ based phycoremediation process with various concentration (Fig. 3a, b, c, & d) of Scenedesmus sp. (Treatment I, II, & III) culture on the treatment of polluted river water was measured and counted by a spectrophotometer and hemocytometer. Out of three, the treatment III results showed that algal growth was increased gradually in both ex-situ and in-situ conditions on the 6 th day onwards. Conversely, the algal growth in the labscale was lower than the in-situ trial. Further, signi cant pH changes (from 6.99 to 8.44) were observed in both ex-situ and in-situ study on the 12 th day of treatment ( Fig. 4a & Fig. 5a) and it was statistically signi cant at P < 0.005. The results showed that Scenedesmus sp. growth in polluted water continued up to the 14 th day of treatment from 6 th day onwards (Fig. 4b & 5b). Entirely, the Scenedesmus sp. growth was considerably higher in in-situ treatment than ex-situ study, and it was statistically signi cant at P < 0.005. Since, in the in-situ study, high light intensity 25,000 lux in the morning, 75,000 lux in the noon, and 15,000 lux in the evening were recorded. The better aeration was observed in an entire day and, thus, might be a possible reason for the high growth of algae in in-situ than ex-situ study (Fig. 4b & 5b) (

Physicochemical and metal analysis
The pretreated physicochemical properties of polluted Thirumanimuthar River water sample results showed that speci c parameters (EC: 3110.00 micro mho cm -1 , turbidity: 17.10, total hardness: 812.00, BOD: 230, and COD: 352 mg L -1 ) and some minerals (Ca: 232.00, Sulfate: 532.00 mg L -1 ) were crossing the permissible limits of Indian standards like Central Pollution Control Board (CPCB), Indian Council of Medical Research (ICMR), and Indian Standards Institution (ISI) of India. Further, a considerable amount of phosphate, nitrite, ammonia, sodium, magnesium, and chloride was also present in the river water (Table 1) situ and in-situ methods were presented in Table 1. The treatment III of ex-situ and in-situ results show better phycoremediation than I and II. Initially, both the ex-situ and in-situ (stabilization pond system) remediation of the polluted water appeared as small black (Fig. 6a) in color with an odor which turned into green color and odorless at the end of treatment (Fig. 6b). The BOD and COD values in the in-situ study were found as 166 (mg L -1 ) and 270 (mg L -1 ) while labscale 205 (mg L -1 ) and 310 (mg L -1 ), respectively. The BOD and COD level in the in-situ study was reduced to 27.83% and 23.30%, whereas in the lab-scale, 10.87% and 11.93% compared with the control respectively. Tang et al. (2018) noted a similar amount of BOD and COD reduction in the treated municipal sewage polluted water used C. vulgaris and S. obliquus. This might be high growth of microalgae and photosynthesis mechanism. Gupta and Rastogi (2008) explained microalgae role in polluted water treatment, which reduced the signi cant number of contaminants in various physicochemical parameters like COD, BOD, and turbidity. Biological oxygen demand is especially useful in evaluating the auto-puri cation capacity of streams, which serves as a measure to assess the quantity of polluted water, safely assimilated by a stream (Rahman and Hasegawa 2011). Similarly, Gonçalves et al. (2017) reported that the Dundiella sp. has the highest removal of BOD, COD, and ammonium from polluted water.
Signi cantly increased pH values (up to 8.44, 14% -20%) were found in both ex-situ and in-situ phytoremediation processes (Fig. 4a, 5a, and 7). The present results were agreed with Scenedesmus sp., shown a remarkable effect on the electrical conductivity value of polluted water which has been reduced to 32% (2110 mho cm -1 ) in both ex-situ and in-situ. The EC value of water is quite linear with thevolume of dissolved ions were reported by Mata et al. (2012). Mencio et al. (2016) stated that the increased total hardness level in water was unsuitable for agricultural purposes. The present study observed the total hardness of in-situ and ex-situ analysis were reduced to 316 mg L -1 (61.08%) and 328 mg L -1 (59.61%) from the control value 812 mg L -1 by Scenedesmus sp., due to the e cient uptake of the nutrients, respectively.
The ex-situ phycoremediation study by Scenedesmus sp. on minerals removal in polluted river water was analyzed. The result states that the treatment III has effectively performed the phycoremediation I and II. In the III treated water sample, the presence (mg L -1 ) of 252, 300, 2, 260, 69, and 37 of chloride, sulphate, phosphate, sodium, calcium, and magnesium respectively were occurred and that was equivalent to 40.00%, 43.61%, 31.03%, 18.75%, 70.26%, and 33.93% reduction (Fig. 7) while compared to those found in control as 420, 532, 2.90, 320, 232, and 56 mg L -1 , respectively. Whereas, 248, 295, 1.5, 260, 67, and 36 mg L -1 , of chloride, sulfate, phosphate, sodium, calcium, and magnesium, were found in the in-situ study (in-situ treatment III) that expressed more mineral reduction potential of Scenedesmus sp. than the ex-situ. An increased level of potassium (33.33% -55.56%) was noted in the treated river water, and it might be derived from as an output of metabolic activity of Scenedesmus sp.
The amount of DO concentration was increased signi cantly from the control value 2.86, and 3.80 mg L -1 in the ex-situ and in-situ study after the treatment (III) that corresponds to 73.33% and 130.30% surge, respectively. Since the microalgae in polluted water treatment provide oxygen during the phycoremediation process, it could enhance the native bacterial breakdown of organic pollutants and minimize their toxicity (Cheah et al. 2018). After treatment, 100% nitrate (NO 3 ) removal was obtained in both ex-situ and in-situ trials. Ammonia (NH 3 ) concentration was drastically reduced from 3.5 to 0.00 mg L -1 (100%) in ex-situ and in-situ treatment. In general, the total amount of minerals was remarkably reduced in the treated polluted water. Cheah et al. (2018) reported that the total hardness (due to Na, K, Ca, etc.) was reduced drastically (69%) when the sewage water was treated with Scenedesmus sp. Pham and Bui (2020) reported that the algal-based remediation of river water found a reduction in calcium (67.1 %) and potassium (67.4%) contents from polluted river water in a short duration of the treatment process. The sulfate content of treated e uent with Scenedesmus sp. was reduced to 43.9%. Similarly, Liang et al. (2013) reported the reduction of Sulfate in the sewage e uent by microalga Chlorella sp. Lee et al. (2013), reported that the alkalinity of e uent was reduced to 44% by Scenedesmus dimorphus and noted high alkalinity, which harms aquatic organisms. In-situ phycoremediation mainly focused on treating the polluted water in eld-based waste stabilization ponds system has proven to be for treating polluted water in terms of the reduced physicochemical level parameters than ex-situ phycoremediation. Similarly, Whitton et al. (2016) studied aerobic, facultative, and anaerobic pond methods to remediate the polluted water by nutrient removal. The results indicate an e cient reduction of BOD, COD, and TSS. Live algae possess intracellular and extracellular polyphosphates that are involved in metal sequestration and metals binding process (Zhao et al. 2015). The cell wall of microalgae consists of several functional groups such as carboxyl, hydroxyl, phosphate, amino, and sulphydryl moieties from polysaccharides, proteins, and lipids molecules. Functional groups bestow a net negative charge to the cell surface and are reported to have an outstanding metal binding nature (Gupta and Rastogi, 2008).

FTIR and SEM analysis of Scenedesmus sp.
The FTIR analysis of microalgae is a real preference for tedious documentation of a functional group of dried microalgae (Xiong et al. 2016). In the present study, the FTIR spectra of control biomass (without treated) of Scenedesmus sp., and the polluted river water ex-situ and in-situ treated biomass are shown in Figure 8a, b, & c respectively, and the characteristic functional groups were presented in Table 2. The prominent region between the 3200 cm -1 -3600 cm -1 represented the stretching vibration of -OH groups. The peak value at 3311.42 cm -1 was shown the -OH groups stretching vibration. The intensive peaks between 2800 cm -1 -3000 cm -1 showed the presence of alkanes (CH) groups. The untreated biomass indicated the curve at 2922 cm -1 of C-H unequal vibration of aliphatic (CH 2 ) e cient clusters. The curve at 1655 cm -1 re ects a sharper due to the C=C stretching of carbonyl clusters. The IR region between 1600-1585 cm -1 indicating the prominent peaks of C-C vibrational stretching of 1551 cm -1 aromatic clusters. 1100-1058 cm -1 were P=O or C-O stretching is maybe polysaccharides.
FTIR spectra of untreated biomass curves were expressed at 720 cm -1 show the occurrence of C-Cl stretching. The IR spectra of polluted water treated biomass represented an unstable of the peaks at 698 cm -1 and 690 cm -1 . Similarly, Yu et al. (2019) reported the FTIR transmittance of dried biomass of Scenedesmus sp. as an aliphatic character at 400-800, phenols, and alcoholic groups at 1,000-1,400, carboxyl group at 1,500-1,700 cm -1 , hydroxyl group: 3,200-3,400 cm -1 . These chemical groups could act as an agent to absorb the pollutants present in the polluted environment (Xiong et al. 2016), and the spectrum of polluted e uent (Pb, Cr, Co, Ni, and Cu) treated biomass (in-situ and ex-situ) revealed broad peaks in frequency observed at 3,386 cm -1 and 3,380 cm -1 , but these peaks were absent in control biomass. Few functional group changes were perceived in the spectra of treated biomass, which indicates that the -OH groups might be involved in the metal absorption mechanism of Scenedesmus sp. Similarly, Castrillo et al. (2013) stated that the Chlorella sp. treated with minerals has interacted with COOH, -OH, and C=O clusters that have absorbed the pollutants effectively from the polluted water.
The results of the SEM analysis of the present study showed that the untreated algal cells shown standard shape with a smooth, transparent external layer found in the outer cell surface (Fig. 9a). After treating the water, the cells become slightly rough and crenelated textures, and some elements were found on the surface of the cell wall ( Fig. 9b and 9c). Chiu et al. (2015) explained that surface protuberance on the algal cell surface due to the deposition of crystallized salts absorbed from polluted water. The approximate size of the Scenedesmus sp. was recorded as 6.04 to 8.01 μm (Magni cation of 4000×). Similarly, Gour et al. (2016) reported the size of Scenedesmus quadricauda and Scenedesmus dimorphus ranged from 6.21 to 8.3 μm and 8.28 to 8.74 μm, respectively

Conclusion
Based on the macroscopic and microscopic study, the predominant native microalgae culture was identi ed as Scenedesmus sp. The physicochemical properties such as EC (3110.00 micro mho cm -1 ), turbidity (17.10 mg L -1 ), total hardness (812.00 mg L -1 ), BOD (230 mg L -1 ), and COD (352 mg L -1 ) and some minerals such as Ca (232.00 mg L -1 ), Sulfate (532.00 mg L -1 ) were not under the acceptable limits of Indian standards. Three sets of treatments (I, II, & III) were performed with various percentages of the biomass of Scenedesmus sp. in ex-situ and in-situ conditions on polluted river water samples. The treatment III showed a better reduction in river water pollution load in 14 days of treatment in ex-situ and in-situ study. The FTIR and SEM analyses con rmed that the presence of active functional groups in biomass of Scenedesmus sp. and SEM image described the absorption of pollutants resulting from the changes in the morphology of Scenedesmus sp. under 4000× Magni cation. The present study ndings concluded that a signi cant reduction was found in most of the tested physicochemical and basic minerals in the treated polluted water using microalgae Scenedesmus sp. in the in-situ study than ex-situ approach. The cultivation of algae in polluted water could be offering duel bene ts: 1. Environmental welfares with the fabrication of algal biomass as phycoremediation. 2.
Which serves as a raw material for the production of biofuel and provides a valuable solution for contaminated water with possible obtaining of valued added raw material for biofuel production.

Declarations
The authors declare the following consent Funding Not applicable

Competing interests
The authors declare that they have no competing interests.

Not applicable
Availability of data and materials The detailed methodology and analytical data of the present ndings are available from the corresponding author on reasonable request.  In-situ   TI  TII  TII  TI  TII  TII  CPCB  ICMR  ISI   Odor  --    The mentioned values are mean and standard error (±) of triplicates. **: Statistically signi cant at P < 0.005 (a) The changes in pH value of polluted water during the ex-situ treatment. (b) Growth kinetic of Scenedesmus sp. during the phycoremediation process on river water: ex-situ analysis Figure 5 The mentioned values are mean and standard error (±) of triplicates. **: Statistically signi cant at P < 0.005 (a) The changes in pH value of polluted water during the in-situ treatment (stabilization pond system). (b) Growth kinetic of Scenedesmus sp. during the phycoremediation process on river water: in-situ analysis