Bioremediation of heavy metals using a novel species of ciliate Paramecium multimicronucleatum isolated from industrial wastewater

The introduction of heavy metals by industries in the aquatic ecosystem is a threatening alarm for living organisms. Bioremediation is an eco-friendly and inexpensive technique as an alternative to chemical methods for wastewater treatment. Wastewater samples were collected from ponds receiving effluents from the Kot Lakhpat Industrial zone, Lahore (Pakistan). Heavy metal-resistant ciliate, Paramecium multimicronucleatum was isolated and characterized with the help of 18SrRNA biomarker. The ciliate showed optimum growth at 25 °C ± 1 °C and pH 7. Growth patterns of P. multimicronucleatum were observed with and without metal stress in wheat grain medium. The minimum inhibitory concentration of cadmium, copper, zinc, and lead was 60, 70, 110, and 160 μg ml−1, respectively. The maximum uptake by Paramecia for Cd, Cu, and Zn was recorded as 90%, 82%, and 93% respectively after 96 h of exposure in each case. While 90% of lead ions were absorbed by Paramecium cells after 48 h of exposure. The order of uptake ability by Paramecium sp. was Zn2+ > Cd2+ > Cu2+ > Pb2+. This preliminary study of exploring bioremediation ability of this ciliate would be helpful for investigating it further using advanced molecular techniques.


Introduction
Environmental pollution is becoming a serious global concern due to rapid industrialization, modern agricultural development and urbanization. Pollution is the introduction of contaminants into the environment which causes instability, discomfort, and harm to the ecosystem [1]. Uncontrolled discharge of heavy metals in the environment can cause detrimental effects on humans, animals, and even plants [2]. Municipal sewage and industrial wastes add multiple heavy metals to the aquatic ecosystem. Metal accumulation in wastewater always depends on people's way of life, the disposal of waste materials in the environment, and the types of industries of the particular area [3]. Contamination by heavy metals and the problems generated in living organisms by them are well documented [4]. High concentrations of heavy metals like Cu, Hg, Cd, Cr, and Zn cause hazardous health effects, e.g., abnormalities in growth and development, mental retardation, kidney failure, and many other disorders [5]. In the aquatic ecosystem, the toxicological effect of heavy metals on organisms depends on exposure concentration and their nature as essential or non-essential metals [6]. For example, copper and zinc are the key elements for the growth of living organisms. While at large doses, they can inhibit cell growth and even lead to cell lysis [7].
Zinc is found in effluents from electroplating, galvanizing, battery manufacturing, and other metallurgical industries. This metal has limited bioavailability in its metallic form and poses no ecological risks. Moreover, zinc may react with other chemicals, e.g., acids and oxygen, to form toxic compounds that may cause hazardous damage to biological systems [8]. As an inorganic pollutant, zinc shows inhibitory effects on the growth of plants [9]. According to some reports, it is indicated that apoptosis may be inhibited by Zn [10]. Anthropogenic activities are the main source of lead accumulation in the environment. It has serious hazardous effects on the brain and spinal cord [11]. It falls into the category of non-essential metals and has no biological role in plants, animals, and even microbes. Cadmium is one of the most common heavy metals found in the environment. It's one of the most carcinogenic and toxic heavy metals to living organisms. It is widely used in industries that are the main cause of its exposure [12]. It has no role in the biological functioning of the cell and body and is known to be the most hazardous pollutant. It can cause mutations and cancer in plants and animals [13]. In natural water bodies, we rarely find copper, but it is found in man-polluted environments. Currently, the increasing concentration of this metal is mainly due to effluents from industries, water from refuse dumps, seepage, corrosive water from pipes containing copper, and pesticides [14]. The trace amount of this metal is essential for life. Moreover, it also catalyzes the synthesis of reactive oxygen species, leading to severe damage to DNA, RNA, and proteins by their oxidation [15]. Conventional methods for wastewater treatment like lime coagulation, chemical precipitation, solvent extraction, and ion exchange have many disadvantages like high energy, partially completed metal removal, and high cost [16]. Besides these techniques, adsorption processes are more common and reliable than the previous methods to remove metals from aqueous solutions, even at lower concentrations [17].
An efficient technique has been introduced as an alternative to these traditional methods known as bioremediation [18]. Bioremediation is using microorganisms to remove or degrade contaminants [19]. The bioremediation of metals is an effective and competent method due to its low cost, less energy requirement, and eco-friendly nature [13]. The water bodies receiving effluents from industries are rich in bacteria, yeast, algae, protozoa, and many fungi species. Some microbes are adapted to the toxic materials released in their environment. These microorganisms can tolerate, resist, metabolize and detoxify heavy metals [20]. The eukaryotic microorganisms, ciliates, have adapted their life in both soil and aquatic ecosystem. They feed on bacteria and other microorganisms and significantly improve the effluent quality in wastewater treatment systems [21]. Ciliates are widely known as early warning bioindicators for environmental risks due to their biological and ecological properties including high diversity, cosmopolitan distribution, short life cycles, ease of collection, and rapid responses to environmental changes. Furthermore, evidence are available for their role as model organisms for the assessment of the toxicological effects of the pollutants [22].
Paramecium and Tetrahymena are one of the most famous ciliates genera which include two top species with maximum gene annotation data i.e., Paramecium tetraurelia and Tetrahymena thermophila [23]. These model organisms have greatly helped us to understand the biological mechanisms occurring in the eukaryotic lineages. Paramecium, with about 87Mb genome size [24], is a universal laboratory organism due to its comparatively large body size [25]. It is widely used to study the morphogenetics of conserved structures, genome evolution, and regulated secretions [26]. Ciliates cope with heavy metal toxicity by developing several defensive mechanisms such as the increased production of antioxidant enzymes, genes like metallothionein induced by metal stress, and heat shock proteins' production for their survival in the polluted environment [27]. The present research work aimed at the molecular identification of metal-resistant Paramecium species and their uptake ability of different metals like Cd 2+ , Cu 2+ , Zn 2+ , and Pb 2+ with the help of an atomic absorption spectrometer.

Sample collection, purification, and enrichment
Microorganisms were collected in screw-capped sterile bottles from wastewater ponds located at Kot Lakhpat industrial zone, Lahore (Pakistan). Temperature (°C) and water pH were recorded immediately during sample collection. Paramecium species were isolated and raised in a wheat grain medium. The pH and temperature of the medium were adjusted at 7.2 and 25°C respectively. Algae was eliminated by placing the samples in semidarkness. Paramecium species were identified by their morphological features, specific body shape, behavior, and movements [28].
Different methods were used to purify the culture. The cell strainer and syringe filter techniques were used in which water sample was passed through different pore size syringe filters or cell strainers to get the smaller organisms in the filtrate. The larger microbes were on the upper surface of the membrane filter that was collected with the help of distilled water by reverse flow of a syringe [29]. The other method used was a depression slide for purification. A 10μl drop of water sample was placed on a glass slide and focused under the 40X power of the light microscope. After washing with distilled water several times, the drop having only one Paramecium was poured into the depression slide. Added 1 grain of wheat in each depression along with 200 μl distilled water. On a daily basis, a small drop of 5 μl water from the depression slide was taken, and observed the number of cells under the microscope [30].

Molecular marker-assisted identification of Paramecium
Genomic DNA was extracted from Paramecium isolated from the culture at its log phase. In 1.5 ml eppendorf, the purified culture was centrifuged at 4032 g for 10 min. The cells were lysed with lysis buffer (42% urea, 10 mM Tris HCl: pH 7.5, 0.3 M NaCl, 10 mM EDTA, and 1% SDS). The genomic DNA was extracted with phenol: chloroform in 1:1 concentration [31]. Genomic DNA was amplified by using a universal eukaryotic 18S rRNA primer pair (F: AATATGGTTGATCCTGCCAGT and R: TGATCCTCCTGCAGGTTCACCTAC). PCR was performed in a 25 μl mixture containing 40 ng of genomic DNA, 1 μl of 10 mM of each primer, 3 μl of 10 × PCR buffer, 5 μl MgCl 2 , 3 μl of 10 mM dNTP mix, and 1μl of 2.5 U Taq DNA Polymerase (Thermo Scientific Cat. # 00855243). Initial denaturation was performed for 5 min at 94°C followed by 5 cycles, each of 1 min denaturation at 94°C, 2 min annealing at 42°C and 2 min extension at 72°C and then 30 cycles, each of 1 min at 94°C, 2 min at 54°C and 2 min at 72°C. The amplified product was visualized and confirmed using 1% agarose gel under UV light. The bi-directional Sanger sequencing of the amplicon was performed commercially at Macrogen (South Korea). The sequence obtained was submitted to the NCBI database with accession number MW024128.

Estimation of optimum growth conditions
The optimum growth of Paramecium was checked at different temperatures and pH. The purified culture in its log phase was used as inoculum for both parameters. Bold-basal medium [NaNO [32][33][34] and wheat grain medium (∼20 boiled wheat grains in 100 ml d.H 2 O, used day after tomorrow) [30,35,36] were used to check the growth patterns of Paramecium. 25 ml of each media with pH 7.1 ± 0.1 was kept at temperature 20°C, 25°C, 30°C, and 35°C for 8 days. Likewise, flasks were prepared the same as for temperature but with 5, 6, 7, 8, and 9 pH for determination of optimum pH and kept at 25 ± 1°C. The Paramecia cells per ml were counted after every 24 h under the lens of a light microscope at 40X for both parameters. All experiments described were performed in triplicates.

Growth Assay under different metal stress
Purified culture of Paramecium (∼100 cells) was inoculated in a 250 ml conical flask having 30 ml wheat grain medium. In the log phase of the organism, different increasing concentrations of salts of heavy metals were added to the flasks to determine the minimum inhibitory concentration of these metals. Cadmium stress was given in the form of CdCl 2 with varying concentrations (10,20,30,40, and 50 μg ml −1 ), copper stress was given in the form of CuSO 4 .5H 2 O with (10,20,30,40,50, and 60 μg ml −1 ) concentrations, zinc stress was given in the form of ZnCl 2 having concentrations of (20, 40, 60, 80, and 100 μg ml −1 ) while lead stress was given in the form of Pb 2 (CH 3 O 2 ) 2 with varying concentrations of (30, 60, 90, 120, and 150 μg ml −1 ). Flasks were kept at optimum temperature and pH. No more metal ions were added when Paramecium species became dead, which determined the minimum inhibitory concentration (MIC) for each metal. The morphological changes occurring due to metal stress were observed and the number of diving cells was counted by taking a drop of 5 μl observed under the light microscope. The growth patterns with metal stress and without metal stress (control) samples were plotted with the mean values estimated for 1 ml of the medium. The experiment was performed in triplicates.

Estimation of metal uptake ability of Paramecium species
In the log phase of the Paramecia, metals were added in flasks (prepared with 30 ml of wheat grain medium) with varying concentrations, as described in (section 2.4). The positive control with medium and metal stress only, was also run along with the experiment for comparison for each metal. The experiment was done in triplicates. Samples were taken after 0, 24, 48,72, and 96 h and centrifuged at 7168 g for 10 min. Pellets were washed with 0.9% NaCl (normal saline), air-dried, and digested in 40 μl concentrated nitric acid (HNO 3 ). The final sample was diluted with molecular-grade water according to the requirement. The supernatant was used to calculate the remaining metal ions in the culture medium. Metal concentration in pellets and supernatants for Cd 2+ , Cu 2+ , Zn 2+ , and Pb 2+ was measured at 228.8 nm, 324.8 nm, 213.9 nm, and 217 nm wavelengths, respectively, using ThermoUnicam-Solar atomic absorption spectrophotometer.

Statistical analysis
All values were recorded for three independent replicates. Means and standard deviation (SD) were calculated and expressed as error bars.

Isolation and identification
The strain was identified as Paramecium due to its slipper-shaped body rounder at the front and pointed at the back. The purification was successfully done with the depression slide, cell strainer, and syringe filter technique (figure 1). PCR amplification reaction yielded a fragment of approximately 1.8 kb, as shown in figure 2. The Blast analysis showed a 99% resemblance with Paramecium multimicronucleatum. The phylogenetic tree was built using MEGA-11 software.

Growth characteristics of Paramecium multimicronucleatum
A growth pattern of P. multimicronucleatum in BBM (Bold-basal medium) and wheat grain medium was observed. The best temperature for the growth of Paramecium species was noted as 25 ± 1°C for both media used; any temperature change showed a sharp fall in the growth of organisms, with 35°C appearing as the most adverse temperature in the case of both media ( figure 3).
The strain showed growth in a pH range of 5-9, but the log phase of the organism was observed under pH 7.0 in both types of media used. No growth was observed below pH 5 and above pH 9. The organism showed better growth and behavior in the wheat grain medium as compared to the Bold-basal medium, so we preferred to use the wheat grain medium for further experiments ( figure 4).

Growth observations under the stress of different metals
The growth of Paramecium multimicronucleatum was observed under the stress of different concentrations of metal ions at 0, 24, 48, and 96 h. In the case of cadmium, the maximum number of cells was 3.5 × 10 3 cells ml −1 at 20 μg ml −1 . A decrease in the rate of diving cells was observed at 50mM with 5 × 10 2 cells ml −1 ( figure 6(a)). In the case of copper, the maximum number of cells was 2.5 × 10 3 cells ml −1 reported at 20 μg ml −1 . While a minimum decrease in the number of diving cells was observed at 60 μg ml −1 with 6 × 10 2 cells ml −1 ( figure 6(b)). It is illustrated in (figure 6(c)) that with the stress of zinc metal on Paramecium multimicronucleatum, the maximum number of cells observed at 60 μg ml −1 was 3.5 × 10 3 cells ml −1 . The increase in metal concentration showed a gradual decrease in the cell count. The maximum numbers of cells reported in the case of lead were 3.5 × 10 3 at 30 μg ml −1 after exposure of 96 h ( figure 6(d)). With the increase in metal stress, the slow movement of cells, more vacuole formation (figure 5), loss of cell integrity and cell inflammation were observed.

Cadmium uptake ability of Paramecia
An increase in the uptake of cadmium was noticed by P.multimicronucleatum with the increasing concentration of metal ions from 10 μg ml −1 to 40 μg ml −1 ( figure 7). The maximum metal uptake by cells was 90% at the concentration of 30 μg ml −1 after 96 h of exposure ( figure 7(c)). The concentration of removal of cadmium ions by Paramecia from the culture medium increased gradually from 10 μg ml −1 up to 40 μg ml −1 . The lowest uptake values were recorded at 50 μg ml −1 ( figure 7(d)). At the highest concentration of 50 μg ml −1 of cadmium ions, the absorption (figure 7(e)) and the number of cells decreased ( figure 6(a)). Descending values of the supernatant represent the remaining amount of cadmium ions in the culture medium at each concentration.

Copper uptake ability of Paramecium multimicronucleatum
The copper-treated Paramecium removed 82% of cadmium ions at 10 μg ml −1 after 96 h of exposure ( figure 8(a)). At higher concentrations of metal, a gradual decrease in uptake concentration was observed like 72.5% at 20 μg ml −1 after 96 h exposure ( figure 8(b)) and 63% at 30 μg ml −1 ( figure 8(c)) again after 96 h of exposure. The number of cells in the medium was also counted ( figure 6(b)) and the least count was noted at higher concentrations of metal at 50 μg ml −1 and 60 μg ml −1 (figures 8(e), (f)).
3.6. Zinc uptake ability of Paramecium cells Paramecium multimicronucleatum removed 93% of maximum zinc ions from the culture medium at 60 μg ml −1 metal concentrations after 96 h of exposure ( figure 9(c)). As in the case of cadmium, it was observed that zinc  metal uptake ability by Paramecia increased gradually with increasing metal concentration. At 20 μg ml −1 the cells uptake 80% metal ( figure 9(a)), at 40 μg ml −1 , the uptake was 87% (figure 9(b)) and 93% as described earlier at 60 μg ml −1 , each after every 96 h of exposure to the metal. At 80 μg ml −1 and 100 μg ml −1 concentration of metal ions, a gradual decrease in metal absorption (figures (d), (e)) as well as in the number of cells was observed ( figure 6(c)).

Lead uptake ability of Paramecia
The maximum uptake of lead ions by Paramecium species was 90% at 90 μg ml −1 after 48 h of exposure ( figure 10(c)). Besides this highest removal rate, the organism also showed the best results at 30 μg ml −1 with 83% uptake after 72 h exposure (figure 10(a)) and at 120 μg ml −1 after 72 h of exposure ( figure 10(d)). The lowest absorption by the cells was observed at 60 μg ml −1 and150 μg ml −1 (figures 10(b), (e)). The decline in the number of cells can also be seen in figure 6(d).

Discussion
In the present research work, the heavy metals have significantly decreased the growth of the Paramecium species. Cadmium ions were more toxic than copper and zinc while lead ions were reported as least toxic. Metals affect the growth of ciliates in several ways, i.e., accumulating into their cell membranes by destroying their integrity, and ultimately, lysis of cells occurs [37]. In wastewaters, many species of protozoa have been reported [38]. The microbes have a high affinity for heavy metals and may absorb them inside their bodies through several mechanisms [39]. The small microbes give a large surface area to volume ratio, providing a big contact area showing interaction with heavy metals in their environment [40].
Vorticella microstoma was reported with its ability to pick up zinc metal ions from the culture media with a maximum of 99% removal after 92 h of exposure to metal [41]. A recent investigation showed Paramecium multimicronucleatum removed 93% zinc ions at 60 μg ml −1 concentration after 96 h of exposure. The microorganisms actively participate in improving effluents' quality since most feed upon the bacterial species present in wastewater [42]. Bioremediation has become extremely important due to consistently worsened environmental situations in some developing countries, including Pakistan. Conventional techniques, including   chemical reduction and adsorption, use many chemical reagents and high energy for heavy metals treatment. Treating toxic heavy metals with the help of microbes has more advantages over conventional methods as it is cheap and requires less energy [43]. One previous research mentioned that Euplotes mutabilis grown in the media containing copper has reduced 60% of copper from the medium after 48 h, 82% after 72 h, and 95% after 96 h of exposure [44]. Recent research reported that Paramecium multimicronucleatum removed about 88% of copper ions from the medium after 96 h of exposure [45]. In the present study, Paramecium species reduced 82% copper ions from the culture medium at 10 μg ml −1 after 96 h of exposure.
Cell growth decreased to 6.0 × 10 2 in the copper-treated sample compared to the control (4.5 × 10 3 cells ml −1 ) after 96 h of exposure. In the case of zinc and lead, the cell growth was also reduced at higher concentrations, with the least growth of 1.0 × 10 3 in both cases. Paramecia removed cadmium ions from the culture medium in a very interesting pattern as the uptake percentage of metal increased with increasing metal concentration, while the maximum 90% uptake was observed at 30 μg ml −1 after 96 h of exposure. In some cases, the growth of Paramecium at higher concentrations increases compared to lower concentrations. The organism is predicted to show resistance and develop the capability to grow and reproduce in a stressed environment. Metal's effect on organisms' growth and their metal resistance has also been reported in Colpidium colpoda, Euplotes aediculatus, and Halteria grandinella [38]. Textile industries are also introducing azo dyes in the aquatic environment in their effluents which are also life-threatening. Paramecium caudatum was used in the degradation of azo dyes with a maximum 90.86% ability of decolorizing [46].
Previous studies reported the growth of heavy metal-resistant ciliates (Euplotes) in molasses, LB, wheat grain, and Bold-basal medium, and in the present research work, it was found that ciliate P.multimicronucleatum cultured in wheat grain medium showed a maximum life span when compared to another medium i.e., Boldbasal medium. The availability of trace metals can impact the culture productivity, performance, and product quality. Some trace metals work as cofactors of enzymes and hence their presence regulates cellular metabolism. Moreover, they can also affect the availability of some other trace metals and also on the stability of other medium components. These factors are necessary to be considered in the formulation of trace metal concentrations in culture medium [33].
Activities of cellular enzymes are affected by temperature, while pH changes the ionization of biomolecules, specifically proteins that perform the activities in the cell. P.multimicronucleatum grows in a broader range of temperatures. They could grow between 20°C and 35°C with an optimum temperature of 25 ± 1°C [44]. Paramecium species grew in a large range of pH with optimum 7 pH noticed in our experiment, while growth was in a decline phase below pH 6.0 and above pH 8.0. Weisse and Stadler (2006) studied the effect of temperature and pH on the growth of freshwater ciliates of the genus Urotricha showing that pH tolerance is specie's specific. Heavy metal removal from domestic and industrial waters has become important in wastewater treatment systems. Due to their tolerance to heavy metals and ability to remove from the medium, P. multimicronucleatum can be a good option for microorganisms' consortium for the bioremediation of metalcontaminated water bodies.

Conclusion
Treating wastewater bodies for toxic pollutants, specifically heavy metals, is important in ensuring human and environmental health. In the present study, we highlighted the advantages of using Paramecium species in ecotoxicological studies. The multiple uptake potential of Paramecium multimicronucleatum, which is resistant to toxic metal ions like cadmium, copper, zinc, and lead was reported. As discussed in our study, these unique organisms can remove or eliminate heavy metals from wastewater bodies using different mechanisms, including absorption. The metal uptake ability of Paramecium sp. may be utilized for metal detoxification and up-scale environmental clean-up operations.