Assessment of Iron Biosorption Potential by Live and Dead Biomass of Bacillus subtilis (MN093305) from Aqueous Solution

Abstract

Heavy metals polluted aquatic ecosystems and become a global environmental issue due to their toxic effect on all forms of ecosystems and further on all forms of life. Heavy metals are non- degradable and accumulated in different life forms by accumulating in the food chain; this increases the need for the development of a sustainable method for the removal of these metals. Biosorption is an eco-friendly and cost-effective convenient technique of heavy metal bioremediation from the contaminated aquatic ecosystem. The current investigation involves biosorption of iron using Bacillus subtilis strain (MN093305) isolated from Ganga river at different physical parameters with the highest rate of biosorption was 96.64%, 98.91%, 97.88%, and 99.44% at pH 5, 60 min incubation period, 35 °C temperature and 2.5 mg/ml of biomass respectively for dead biomass. Living biomass biosorption rate was 87.32%, 96.74%, 96.94% and 95.02% at pH 7, 72 h, 35 °C and 2.5 mg/ml respectively. Functional groups involved in the biosorption of iron by Bacillus subtilis were fitted to a second-order kinetic model. Langmuir and Freundlich’s isotherm are used to evaluate data; both isotherms indicate iron absorption as a favorable process.

Introduction

Heavy metals are naturally occurring elements with relatively high density i.e., 5 gcm⁻³ or above are highly carcinogenic in nature even at very low concentrations. They exist in an ecosystem by variation in the environment such as volcanic eruption, weathering of rocks, etc., and by various human activities such as mining, vehicle exhaust, smelting, coal combustion, etc. the major heavy metals of concern are arsenic, lead, cadmium, chromium, mercury because of their lethal effect on human health even in very minute quantity [38]. Some of them such as iron, copper, zinc, etc. were essential metals but any of these heavy metals above their permissible limit act as a toxin and result in functional group blocking, essential elements displacement, and conformation of biomolecule active form change.

According to EPA, in 1993 iron is the second most abundant metal in the world occupying 26th element position in the periodic table. Iron is an appealing metal for different organic redox forms because of its transformational property between ferrous (Fe²⁺) and ferric (Fe³⁺) ions [31] is a most urgent component the for development and survival of all living beings [44]. The wellspring of iron in the surface water is anthropogenic and mining exercises. The generation of sulphuric corrosive and the release of ferrous (Fe²⁺) happen due to the oxidation of iron pyrites (FeS2). Iron becomes unsafe when retained iron neglects to tie to the protein, and free iron prompts lipid peroxidation resulting in extreme harm to mitochondria, microsomes, and other cell organelles [3]. Iron delivered hydrogen free radicals that assault DNA, bringing about cell harm, mutation, a variety of infections, and a destructive impact on the gastrointestinal tract and organic liquids [21].

Currently, in order to minimize heavy metal’s impact on the environment, several physical and chemical processes of remediation are being adopted including precipitation, ion- exchange, leaching, electrolytic technologies, vapors extraction, stabilization, etc. but not all were deemed to be sufficiently efficacious (Sayqal and Ahmed, 2021). This raise the need for cheaper, feasible and eco- friendly methods, which shift the focus towards other absorbents such as microbes and plants [13] Biosorption has several advantages over other conventional methods as it is a biological method that involves inexpensive microbial material for heavy metal removal without producing any harmful by-products [29] many research has been conducted using the live and dead forms of microbes concluding that physical parameters such as pH, temperature, contact time, biomass concentration directly influence biosorption rate from aqueous solution [37], out of several microbes, bacteria such as Bacillus subtilis, Bacillus cereus, Bacillus safensis, Pseudomonas, etc. are considered to be more beneficial for heavy metal biosorption due to well-known resistance mechanisms and can survive in extreme environments. [33].

Bacillus subtilis is non-pathogenic gram-positive bacteria with GRAS status, presence of teichoic and teichuronic acids significantly increase electronegative charge density and functional group with oxygen, nitrogen, Sulphur or phosphorous in gram-positive bacteria [22] which play important role in heavy metal absorption (Fig. 1). Therefore, this study is aimed at determining the biosorption rate of iron from aqueous solution by Bacillus subtilis isolated from Ganga river an important aquatic ecosystem of India.

Fig. 1.

Maximum Ion biosorption process occurs at optimum conditions such as pH, temperature, time of contact, biomass concentration, etc. by the functional groups present on the bacterial cell wall

Material and Methods

Description of Sampling Sites

The study was carried out in the Haridwar district of Uttarakhand which is located in the lower foothills of the Himalayas having geo coordinate of 28°44' to 31°28' N and 77°35' to 81°01' E [30]. The holy river Ganga appears firstly in the land of Haridwar after traveling a long kilometer in the mountain. While traveling such a long distance, intakes lots of ingredients from the adjoining areas thus making a lot of changes in the water quality. Keeping this in view different sites were undertaken in the study. The description of the sites is well illustrated in Table 1 and Fig. 2.

Table 1.

Description of the sampling sites

S.No	Sampling Sites	Sampling Site Code	Geo coordinates	Source of Water
1,	Bhimgodha	SS1	29°60′25.9’’N;7814′28.1’’E	Canal water
2	Kankhal	SS2	29°63’’92.6’’N;78°14′6.3E’’	Canal water
3	Premnagar	SS3	29°55′48.8’’N;78°08′10.3’’E	Canal water
4	Jwalapur	SS4	29°71′92.7 N; 78°10′8.0’’ E	Canal water

Fig. 2.

Satellite map of the sampling sites

Methodologies for Sampling

The samples were collected using a grab sampling method from four different locations in Haridwar. The sampling was done in the morning hours and the samples were collected in the previously well sterilized Tarson bottles. In order to retain the heavy metal characteristics, a few drops of Ultrapuric acid were added to the bottles and stored in the ice boxes at a temperature of 4ºC. The samples were brought to the laboratory for further analysis following the standard methodologies as given by APHA (2012) [11, 41]

Isolation

For the isolation of bacteria serial dilution technique was used (Waksman and Fred). One ml of water sample was appended in nine ml of sterile distilled water blanks and sample was diluted up to 10⁻³dilutions; 0.1 ml of suspension was spread over nutrient agar plates and plated were incubated at 30±2°C for 24 to 48 hours.

Screening of Iron-Absorbing Bacterial Isolates

Well diffusion method was subjected to screen iron tolerant bacterial isolates, 0.1 ml of 20, 40, 60, 80, and 100 mg/L of iron standard solutions were added in well prepared in nutrient agar plates that were spread with 24 hrs. old isolated bacterial culture After incubation, zone of inhibition was measured for different heavy metals at different concentration. A zone less than 1mm scored as resistant strains [40].

Molecular Identification of Iron-Resistant Bacteria

The iron tolerant bacteria were identified by 16SrDNA Sequencing which was done by Eurofins Genomics India Private Limited. Further strain confirmation was done by software program Clustal W. Distance matrix and phylogenetic tree was constructed by MEGA.

Biosorption Assay

Preparation of Metal Solution

Stock solution was prepared by dissolving iron sulphate and distilled water. It was of 100mg/l [42].

Preparation of heavy metals stock solution (1000 ppm)

$$StocksolutionofmetalX=\frac{\mathit{MolecularweightofmetalX}}{\mathit{MolecularweightofanysaltofmetalX}}$$ 1

Preparation of different concentrations of a metal solution from stock solution

$${\text{C}}_1{\text{V}}_1={\text{C}}_2{\text{V}}_2$$ 2

where C1 = concentration of a stock solution (1000 ppm); C2 = concentration of the solution that had to be prepared; V1 = volume of stock solution and V2= volume of solution to be prepared.

Biosorbent Preparation

Dead Biomass preparation

Pure culture of Bacillus subtilis was grown in nutrient broth at 30 ± 2 °C for 24 h and cells were harvested by centrifuging at 10,000 rpm for 10 min at 4 °C. Recovered cell pellets were washed with distilled water so that no media remain attached to the cell pellets, after this cell pellets were dried in hot air oven at 80 °C for 6 Hours.

Live Biomass preparation

Identified culture of Bacillus subtilis was preserved on nutrient agar slants and pure culture was revived in sterile nutrient broth at 30 ± 2 °C in a shaker incubator which was further used as live Biomass.

Biosorption Experiment:

Biosorption of iron ions on Bacillus subtilis live and dead biomass was executed at a variant value of different physical parameters, one target parameter was varied by keeping the other constant such as pH (3, 5, 7, 9, and 11), temperature (25 °C, 30 °C, 35 °C, 40 °C, and 45 °C), biomass concentration (0.5, 1, 1.5, 2, and 2.5 mg/ml) and contact time (20, 40, 60, 80, and 100 min) for dead biomass (24, 48,72,96, and 120 h) for live biomass after incubation, the biomass and supernatant were obtained by centrifugation at 9000 rpm for 10 min.

Determination of Absorbed Iron Ions

The percentage of iron residual in aqueous solution after the process of biosorption was analyzed by the AAS technique [26] at Deepa Enterprises, Faridabad. At equilibrium conditions percentage of biosorption, is obtained by the following equation

$$Q=V(C_i-C_f)/m$$ 3

where Q = Metal uptake, V = liquid sample volume, Ci = Initial concentration of metal, Cf = Final concentration of metal in supernatant and m= biosorbent added (mg/l).

Absorption Isotherm

Langmuir and Freundlich isotherm model was used to characterize the biosorption process of iron with the Bacillus subtilis biomass.

Langmuir model

This working principle of this model is that maximum absorption occurs when a solute’s saturated monolayer which is present on the absorbent surface, energy is constant with no migration of absorbate molecules in the surface plane. Langmuir model is represented by following equation

$$\frac{C_e}{q_e}=\frac{C_e}{q_{\mathit{max}}}+\frac{1}{b_{\mathit{qmax}}}$$ 4

where C_e and q_e show the metal concentration in solution and the q _max and b are .langmuir and adsorption constant.

Freundlich model—This was given by following equation

$$log{q}_e=log{K}_f+\frac{1}{n}log{C}_e$$ 5

where Kf = constant for relative biosorption capacity of the biosorbent, 1/n = biosorption intensity parameters, which was calculated by the linear plot of log qe versus log Ce.

Kinetic studies

Pseudo first order and Pseudo second order kinetic study were applied to determine the nature of biosorption process. Varying biosorbent doses i.e., 0.5, 1.0, 1.5, 2.0, and 2.5 gm was used for the biosorption of 100 mg/l of iron in aqueous solution. First order kinetics expression was given by [39].

$$\ln \left(q_e,-,q_t\right)=ln{q}_e-k_{1}t$$ 6

where q = amount of iron absorbed.

q_e = amount of absorbed iron at equilibrium.

t = iron absorbing time in minutes.

K₁ t and q_e = first order rate constant determined by slopes and intercepts of plots of log (q_e—q) versus t at.

different biomass dosages.

In 1999, Ho and Mckay give second order kinetics expression as

$$\frac{t}{q_t}=\frac{1}{K_2(q_e^2)}+\frac{t}{q_e}$$ 7

where, q_e and q_t represent adsorption of metals ions adsorbed at equilibrium level, and t, K₂ denote time and constants of second order kinetics, respectively

Fourier Transforms Infrared (FT-IR) Spectroscopy

This technique is used for the study of biochemical nature of biosorbent before and after iron absorption. For the analysis of FT-IR, iron loaded and unloaded Bacillus subtilis pellet were obtained and dried at 80⁰ C for 2 h in hot air oven. In this process 0.1 g fine grounded biomass pellets were mixed with KBr [40].

Scanning Electron Microscopy

Elemental and morphological characterization of biosorbent before and after iron absorption was examined by scanning electron microscope at an accelerated voltage of 20.0kV by LEO 435VF. Unloaded and metal loaded Bacillus subtilis biomass mounted on aluminium tab sequenced by covering with a dainty layer of gold under vacuum to expand the electron conduction and to improve the quality of the micrographs. Procedure was carried out at IIT Roorkee, Uttarakhand, India.

Result and Discussion

In total 15 isolates were isolated from divergent Ganga samples, screened for iron tolerance through well diffusion method (Table 2). 11 iron tolerant isolates with either no or very small zone of inhibition up to 100 mg/l were screened, further study was conducted only by focusing on HGB1 which was molecularly identified by 16 S rDNA sequencing as Bacillus subtilis (Fig. 3).

Table 2.

Monitoring of bacterial isolates for arsenic tolerance ability (Average of triplicates)

ZOI	Isolates
	HGB1	HGB2	HGB3	HGB4	HGB5	HGB6	HGB7	HGB8	HGB9	HGB10	HGB11	HGB12	HGB13	HGB14
10 ppm	–	–	–	–	–	–	–	–	–	–	–	–	–	–
20 ppm	–	–	–	–	–	–	–	–	–	–	–	–	–	–
40 ppm	–	–	–	–	–	4.1 ± 0.01	–	–	–	–	–	–	–	–
60 ppm	–	4.4 ± 0.8	–	–	–	6.9 ± 0.3	–	–	5.3 ± 0.3	–	–	–	–	–
80 ppm	–	6.6 ± 0.3	–	–	3.2 ± 0.01	10.8 ± 0.33	–	–	8.6 ± 0.33	–	–	–	–	–
100 ppm	–	7.2 ± 0.1	–	–	4 ± 0.01	13 ± 0.01	–	–	10.09 ± 0.01	–	–	–	–	–

Fig. 3.

Phylogenetic tree showing the relationship on the basis of 16S rDNA sequencing

Biosorption Studies

The effect of different experimental parameters at various conditions was studied as discussed below.

Effect of pH of a Solution

pH is a unit of moles per liter of hydrogen ions that contributes to biosorption availability of biosorbent by controlling overall charge of the biomass surface [16]. High and low pH both effect the biosorption in different ways,high pH deprotonates functional group such as carboxyl, sulfhydryl, hydroxyl, amino group, etc. resulting in the increase of over all negative charge consequently increase the biosorption of metal cations [15], low pH protonates biomass and increase positive charge and biosorption of metal anions [27] Darnall et al., 1986, categorized metals into 3 classes in regarding their optimum pH range class I metal ions include Al (III), Cu (II), Cr (III), Fe (III), Ni (II) strongly bound at or near neutral pH i.e. 7, class II metal ions PtCl4²⁻, CrO4²⁻, SeO4²⁻ binds strongly at low pH and class III metal ions binding is strongest and pH independent (Ag²⁺, Hg²⁺ and AuCl⁻). Experiments on different pH i.e., 3, 5, 7, 9, and 11 showed maximum iron i.e., class I metal biosorption was at pH 7 for both dead (96.64%) and live (87.32%) respectively (Fig. 4). Congeevam et al., 2007, and [25] studied the effect of pH on biosorption and show positive agreement with the present study.

Fig. 4.

Effect of pH on iron biosorption by dead and live biomass of Bacillus subtilis

Effect of Time of Contact

Time of contact is an important parameter for isotherm studies, biosorption rate increases with an increase in time of contact while the rapid kinetics was obtained during contact as the availability of active site was more. Cell physiology (live or dead) decides the dependence of biosorption on time of contact as on living cells, the initial metal concentration may affect the time of the stationary phase [34]. In the present study, the time of contact at which rate of biosorption reaches to stationary phase were 60 min (98.66%) and 72 h (96.74%) for dead and live biomass respectively (Fig. 5. Garcia et al., 2016; [28] examined the optimum time of contact to be 60 min for dead biomass and 72 h for live biomass during heavy metal biosorption.

Fig. 5.

Effect of time of contact on iron biosorption by dead and live biomass of Bacillus subtilis

Effect of Temperature

Temperature effect on biosorption efficiency varies widely, [28],Ahmed and Kibert, 2013; [46] observed that an increase in temperature above 35 °C decreases the biosorption rate concluding that biosorption is an exothermic reaction. The present study also shows the same effect of temperature on iron biosorption, as maximum biosorption of iron was at 35 °C for both dead (97.88%) and lives (96.94%) respectively (Fig. 6). Al-Gheethi et al., 2017 observed an increase in biosorption efficiency with an increase in temperature within a range from 25 to 35 °C and with a further increase in temperature to 45 °C reducing the efficacy of heavy metal biosorption suggesting that the process become exothermic in nature above 35 °C.

Fig. 6.

Effect of temperature on iron biosorption by dead and live biomass of Bacillus subtilis

An increase in temperature to a certain point increases the metal biosorption by decreasing solution viscosity consequently increasing the diffusion rate of metal ions across the membrane of biosorbent (Barka et al., 2013). After reaching equilibrium metal uptake reduces due to the damage of active binding sites [34] or the increasing tendency of biosorbent to desorbed metal at very high temperatures (Saltah et al., 2007).

Effect of Biomass Concentration

It is one of those factors that strongly influence the rate of biosorption of metal ions, an increase in biomass concentration also increases the biosorption of metal ions increase in biomass concentration also increases the biosorption of metal ions [47]. [32] observed that with the increase in biomass dose, biosorption per unit of biosorbate decreases as the number of binding sites increases. In the present study, the highest biosorption was shown at 2.5 mg/ml biomass concentration i.e., 98.44% and 95.02% for dead and live biomass respectively whereas maximum biosorption per unit of biosorbate was attained at 1.5 mg/ml concentration for both physical forms (Fig 7). [7] reported that biosorption efficiency of R. opaques for Pb (II), Cr (III), and Cu (II) increases with an increase in biomass concentration from 0.5 to 1 g/L, but with a further increase in concentration to 2.5 g/l, rate of biosorption reduces.

Fig. 7.

Effect of temperature on iron biosorption by dead and live biomass of Bacillus subtilis

Overall high biosorbent concentration increases the specific surface area and consequently, the number of active sites increased removal efficiency [6, 9, 40]. However excessive increase in biosorbent concentration above optimum value reduces biosorption capacity (Gupta et al., 2008).

Application of Live and Dead Biosorbent in Metal Removal from Aqueous Solution

Both live and dead biomass of microorganisms have been used for heavy metal biosorption [49] various comparative studies on the biosorption ability of live and dead biomass were conducted by many researchers showed that dead biomass of biosorbents exhibits high biosorption activity in compare to live biomass, the present study also observed high biosorption efficiency of dead biomass over live biomass for all physical parameters (Table 3). Dead biomass involves only rapid extracellular metal binding (Geisweid and Urback, 1983) while living biomass involves both extracellular and intracellular metal absorption by adapting to environments below the toxicity level (Silver and Phung, 2005).

Table 3.

Biosorption efficiency of dead and live biosorbent

Heavy metals	Micro-organism	Initial concentration (mg/L)	Parameters	% of metal removal
			pH	Dead		Live
Iron (Fe)	Bacillus subtilis	100	3	59.92		1.88
			5	90.82		21.90
			7	96.64		87.32
			9	36.97		21.02
			11	17.24		4.10
			Temperature ( °C)
Iron (Fe)	Bacillus subtilis	100	25	86.99		81.14
			30	89.07		87.78
			35	97.88		96.94
			40	67.92		35.44
			45	58.11		7.99
			Biomass Concentration
Iron (Fe)	Bacillus subtilis	100	0.5	79.84		51.91
			1.0	82.22		64.82
			1.5	90.01		86.49
			2.0	94.17		91.44
			2.5	98.44		95.02
			Time of incubation (min)		Time of incubation (hours)
			Dead		Live
Iron (Fe)	Bacillus subtilis	100	20	89.92	24	84.17
			40	93.87	48	90.23
			60	98.91	72	96.74
			80	98.94	96	96.89
			100	99.04	120	97.04

Isotherm Analysis

It is a quantitative analysis of adsorbed per unit adsorbent. In present study 2 isotherm model Langmuir isotherm and Freundlich isotherm were studied for 100 mg/L at different biomass concentrations. The analytical factor of Langmuir isotherm is RL and processes are favorable when RL is 0 < RL < 1, linear when RL = 1, unfavorable when RL > 1, and irreversible when RL = 0 [1]. Freundlich Isotherm: Represent that the energy of sorption decreases exponentially as the adsorbent active sites are completed [14]. K_f and n are the constant of Freundlich isotherm and indicate adsorption capacity and adsorption intensity respectively if n < 1 then adsorption is a chemical process, n = 1 then adsorption is a linear process, n > 1 then adsorption is a physical process. In the present study 100 mg/L iron aqueous solution manifested the value of R2 (Langmuir) as 0.592 and r² (Freundlich) as 0.013 (Fig. 8) (Table 4) i.e., less than 1 for Bacillus subtilis, supporting both isotherm models concluding that absorption is favorable.

Fig. 8.

Langmuir (a) and Freundlich (b) isotherm for iron by Bacillus subtilis at pH 5, 2.5 mg/ml biomass and 30 °C temperature

Table 4.

Variable of isotherm models for Iron

Biosorbent	Langmuir isotherm			Freundlich isotherm
	Qo	B	R2	r2	Kf	N
Bacillus subtilis	3.56	1.005	0.592	0.013	0.24	0.16

Kinetic Analysis

Suitable kinetic model for iron uptake was calculated by plotting metal uptake against time, the linear line shows a favorable biosorption process, and iron biosorption in the present study fitted in pseudo-second order kinetics (Table 5) (Fig. 9).

Table 5.

Kinetic for iron biosorption

Biosorbent	Pseudo first order			Pseudo second order
	K1	qe	R2	K1	qe	R2
Bacillus subtilis	0.00017	1.978	0.082	0.00019	1.09	0.999

Fig. 9.

First order (a) and second order (b) kinetics for iron by Bacillus subtilis at pH 5, 2.5 mg/ml biomass and 35 °C temperature

FT-IR Analysis

FT-IR analysis discloses those functional groups that take parts in the biosorption process Fig. 10a shows a control biomass spectrum; A strong and broad peak was observed in control biomass spectrum which indicates the presence of hydroxyl group, another peak lies in a range of 2700–2950 which indicates the presence of C-H variable group, an amino group indicating peak was observed at 1.636 cm⁻¹.

Fig. 10.

a FT-IR for surface analysis of Bacillus subtilis control. b FT-IR for surface analysis of Bacillus subtilis in presence of iron

The spectrum of metal-loaded biomass (Fig. 10b) indicates a peak at 3405.84 cm⁻¹ showing sharp and strong broad stretching of OH and N–H group 1654.68 cm⁻¹, 1541.03 cm⁻¹, and 1441.07 cm⁻¹ shows variable N–H stretching, 1242.17 cm⁻¹, and 1051.48 cm⁻¹ shows variable bending of OH, C–N, and C–O stretching. Comparing the FT-IR spectrum of B. subtilis biomass before and after heavy metal biosorption it was found that bands shift toward lower frequency, which occurs when metal ions co-ordinate with carbonyl groups (Arvindhan et al., 2011). The change in the frequency of these functional groups indicate that they participate in biosorption process, similar trends in FT-IR spectrum i.e. at 3411 cmcm⁻¹, 2929 cmcm⁻¹, 1239 cmcm⁻¹ were also observed in the studies of [12, 23, 36, 43, 45] while studying biosorption of different heavy metals.

SEM Analysis

To understand the surface morphology of microorganisms during the biosorption of heavy metals, SEM analysis of B. subtilis was carried out, SEM provides both elemental and topographical information about biosorbent, SEM produces a clear picture of biosorbent surface as it magnification and resolution power is very high (Ramya and Joseph Thatheyus 2018). Bacillus subtilis in absence of metal ions; proper rod shape of bacteria was observed (Fig 11a) but in the presence of iron, SEM image reveals a denser and spongier cell surface (Fig 11b). Similar patterns were obtained by Sethuraman and Balasubramanian in 2010, during biosorption of Cr (VI) by B. subtilis and Enterobacter cloacae. Rezaei in 2013 while studying biosorption by Spirulina sp. [11] during study of arsenic resistant bacteria and Ramya and Thatheyus in 2018 when they review bacterial biosorption.

Fig. 11.

SEM analysis of Bacillus subtilis Control (a), in presence of iron (b)

Conclusion

The present research was conducted to investigate a Fe absorbing bacterium from Ganga river. The selected bacterial isolate HGB 1 identified as Bacillus subtilis. The biosorption behavior was investigated at a different range of variant physical parameters by live and dead biomass, dead biomass of Bacillus subtilis has a better iron uptake capacity at pH 5, biomass concentration 2.5 mg/ml, 35 °C at 60 min of incubation. FT-IR demonstrated interaction of bacterial –OH and –C=O group with iron, SEM analysis indicate the Fe ions interaction with bacterial surface resulting in a significant structural difference when metals were bios orbed onto bio sorbent. Both Langmuir and Freundlich adsorption isotherm and pseudo second order kinetic model fit for the experimental data.

This work thus illustrated use of Bacillus subtilis biomass alternative techniques for the water management that is more convenient to utilize at low costing.

Authors Contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Vani Sharma. All authors read and approved the final manuscript.

Funding

This work was supported by DST INSPIRE Fellowship (Grant Numbers IF140001).

Availability of Data and Material

Not applicable.

Declarations

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors have no relevant financial or non- financial interest to disclose.

Ethical approval

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Consent to Participate

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Constant to Publish

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