Environmental Research 211 (2022) 113108

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Environmental Research
journal homepage: www.elsevier.com/locate/envres

Decolorization of safranin using Fissidens species and its ecotoxicological

assessments: An in vitro and in silico approach
T. Kiruthika a, M. Poonkothai a, *, K. Kalaiarasi b, Jamaan S. Ajarem c, Ahmed A. Allam d,
Jong Seong Khim e, C. Sudhakar f, T. Selvankumar f, M. Alaguprathana g
a
Department of Zoology, Avinashilingam Institute for Home Science and Higher Education for Women, Coimbatore, 641 043, Tamil Nadu, India
b
Department of Textiles and Clothing, Avinashilingam Institute for Home Science and Higher Education for Women, Coimbatore, 641 043, Tamil Nadu, India
c
Zoology Department, College of Science, King Saud University, Riyadh, Saudi Arabia
d
Zoology Department, Faculty of Science, Beni-Suef University, Beni-Suef, Egypt
e
School of Earth and Environmental Sciences, College of Natural Sciences, Seoul National University, Seoul, 08826, Republic of Korea
f
PG and Research Department of Biotechnology, Mahendra Arts and Science College (Autonomous), Kalippatti, Namakkal, 637501, Tamil Nadu, India
g
Department of Zoology, Adhiyaman Arts and Science College for Women, Uthangarai, Krishnagiri, 635 207, Tamil Nadu, India

A R T I C L E I N F O A B S T R A C T

Keywords: Decolorization of safranin was investigated using Fissidens species in a batch system under optimized conditions.
Fissidens species The decolorization efficiency was improved by optimizing the conditions such as initial pH (3–9), temperature
Adsorption (25–45 ◦ C), initial dye concentration (10–50 mg/L), biosorbent dosage (100–500 mg/L) and contact time (1–6
Optimization
days). Maximum decolorization (95%) was recorded at initial pH of 6 with dye concentration of 20 mg/L,
Characterization
Toxicological assessments
biosorbent dosage of 200 mg/L at 30 ◦ C and contact time of 2 days. Desorption studies revealed 0.1 N NaOH as
In silico study the best desorbing agent with 92% recovery on third day. Experimental data well fitted to Langmuir isotherm
and Pseudo-second order kinetic model. The negative values of ΔGo and positive value of ΔSo and ΔHo indicates
that the reaction is spontaneous, favorable and endothermic. The biosorbent - dye interactions were confirmed
using UV–Vis, FT-IR, XRD and FE-SEM with EDX studies. The detoxified nature of the dye degraded metabolites
was confirmed by the significant growth of green gram. The color fastness and color strength of the fabrics dyed
using Fissidens species treated dye solution were compared with the tap water dyed fabrics which indicated the
reuse potential of treated water in textile sector. The decolorization efficiency was further confirmed through in
silico approach, where safranin well docked with the active sites of Photosystem II protein D1 of the Fissidens
species. Thus, the present study proves that Fissidens species is a promising biosorbent for safranin decolorization
and will lay a platform for the control and management of environmental pollution.

1. Introduction The discharge of dye containing colored wastewater into the

ecosystem results in severe soil and water pollution, which consequently
Rapid technological development and urbanization leads to serious hinders the photosynthetic action of freshwater resources by reducing
impact on environment and causes ecological imbalance. Textile in­ light penetration. This in turn affects the trophic level of the ecosystem,
dustries involves the usage of enormous water for finishing process and causing depletion in the dissolved oxygen level, reduction in the growth
releases densely colored wastewater which contains salts, detergents, of the floral and faunal diversity. As a result, they pose a serious threat to
acids, synthetic dyes, and heavy metals into aquatic and terrestrial the biotic and abiotic components of the ecosystem and necessitate the
ecosystem (Khan et al., 2022; Hu et al., 2020; Imran et al., 2019). Textile removal before discharged into the environment (Elbanna et al., 2010;
dyes have high molecular weight, and the complex chemical structure Franciscon et al., 2012; Pathak et al., 2014).
confers the ability to remain in recalcitrant nature and enables them to In the present study, safranin is chosen as a candidate dye, since it is
endure in natural environment for a protracted period (Dasgupta et al., mainly used as a textile colorant, food dye in flavoring and coloring
2015; Rane et al., 2014). candies and cookies. It is also used for dyeing bastfibres, wool, silk,

* Corresponding author.
E-mail address: [email protected] (M. Poonkothai).

https://doi.org/10.1016/j.envres.2022.113108
Received 8 January 2022; Received in revised form 28 February 2022; Accepted 8 March 2022
Available online 18 March 2022
0013-9351/© 2022 Elsevier Inc. All rights reserved.
T. Kiruthika et al. Environmental Research 211 (2022) 113108

mordanted cotton, leather, and paper. Exposure of living organisms to

Percent decolourisation = (I − F) ​ /I × 100 (1)
safranin leads to health problems such as stomach pain, mouth, tongue
and throat irritations which may lead to vomiting, nausea and diarrhea
where, I and F signifies the initial (I) and final (F) absorbance of the dye
(Rejniak and Piotrowska, 1966). Since, safranin is pervasively used in
respectively (Phugare et al., 2011). The maximum rate of decolorization
textile and food industries as a colorant, it is considered as the repre­
was measured, and the bryophyte was inoculated into the dye solution
sentative in the effluent discharged into the ecosystem (Jain et al.,
under the optimized conditions to assess its decolorization efficiency.
2006). Various physicochemical methods have been reported for
decolorization of dyes, but those methods have severe restrictions in
terms of high cost, specificity for certain dyes, requirement of energy, 2.3. Adsorption isotherms, kinetics and thermodynamics
decreased environmental efficiency, generation of large amounts of
sludge, and above all due to its excessive chemical usage which results in Langmuir (1918) and Freundlich (1906) isotherm equilibrium
secondary pollution (Rawat et al., 2016; Akansha et al., 2019). models embody the relationship between the dye concentration in so­
Recently, biological methods using algae, bacteria, fungi, and plants lution and the amount of dye adsorbed onto the solid phase at equilib­
have become a global consideration as they are feasible economically rium. The linear form of Langmuir and Freundlich isotherms are
and ecofriendly for treating textile wastewaters (Gao et al., 2018). expressed as follows
Bryophyte, a perennial herb has played a major role in monitoring 1 1 1
changes in the Earth’s atmosphere and has the capacity to accumulate = + (2)
qe bqmCe qm
pollutants. They seclude the toxic substances by binding them to the cell
walls through the cation exchange process or accumulate within the cell 1
vesicles and protect cellular metabolism or may combine with other lnqe = lnKf + lnCe (3)
n
elements as insoluble compounds and renders them as harmless. Lack of
significant cuticle or epidermis on leaves, being only one cell thick Where, qe is the amount of safranin adsorption at equilibrium (mg g− 1),
makes the bryophyte well suited as a bioindicator and bio monitor. They qm is Langmuir monolayer adsorption capacity, Ce is the concentration
have also been used to monitor airborne pollution caused by emissions of safranin (mg L− 1), b and Kf is the Langmuir and Freundlich constant (L
from factories. The advantage of using bryophyte is that they can be g− 1) and 1/n is the heterogeneity factor describing adsorption intensity.
easily collected, transplanted and harvested at any time of the year and The Langmuir isotherm can be explained by separation factor (RL)
can be utilized for analysis for many years. Thus, bryophytes offer a which indicates
great promise in the cleanup of toxic contaminants from the environ­
1
ment (Satake et al., 1989; Taylor and Smith, 1980). Literature reveals RL = (4)
1 + bCe
that the decolorization of safranin using Fissidens species has not been
documented still now and the present study is the first report using the the shape of the isotherm to be either unfavorable (RL > 1), linear (RL =
above bryophyte. Photosystem II protein D1 of Fissidens species has 1), favorable (0 < RL < 1) or irreversible (RL = 0). The RL value between
broader substrate specificity and can degrade a wide range of dyes. 0 and 1 indicates the favorable adsorption of the dye onto the
Hence, the present study is formulated to assess safranin decolorization biosorbent.
using Fissidens species and to evaluate the reuse potential and toxicity of Pseudo first order and pseudo second order kinetic models were used
treated dye solution. Molecular docking technique aids in technology to study the rate and mechanism of safranin adsorption onto Fissidens
transfer by anticipating the nature and toxicity of the protein-dye species. The linear form of pseudo first and pseudo second order models
interaction. were expressed as prescribed by Mahmoodi et al. (2011) and Ho (2006).

2. Materials and methods ln(qe − qt ) = lnqe − k1 t (5)

/ /
2.1. Biosorbent and adsorbate preparation t
= 1 k2 q2e +t qe (6)
qt
The bryophyte (Moss) was collected from the campus of Avina­
where qe and qt (mg g− 1) are the amount of safranin adsorbed onto
shilingam Institute for Home Science and Higher Education for Women,
Fissidens species at equilibrium and at time t, k1 and k2 are the pseudo
Coimbatore, Tamil Nadu, India. The bryophyte was washed thoroughly
first and pseudo second order constants respectively.
to remove dirt and shade dried. It was identified macroscopically, and
The thermodynamic behavior between the adsorbate and biosorbent
further authentication was carried out in Botanical Survey of India,
during adsorption process was reflected based on the change in standard
TNAU, Coimbatore, Tamil Nadu, India. The dried bryophyte was ground
enthalpy (ΔH◦ ), entropy (ΔS◦ ) and free energy (ΔG◦ ) which was
well, sieved and the powdered bryophyte was used as a biosorbent for
calculated using the formula (Milonjić, 2007)
decolorization of safranin.
Safranin (Adsorbate) was procured from Himedia, Mumbai, India ΔG = − RTlnKD (7)
(Chemical formula = C20H19CIN4; Molecular weight = 350.85 g/mol;
Absorbance maximum (λmax) = 680 nm) and the stock solution of dye
◦ ◦
ΔS ΔH
ln KD = − (8)
was prepared to a concentration of 1000 mg/L. R RT

2.2. Biosorption studies ΔG∘ = ΔH ∘ − TΔS∘ (9)

where, KD is the thermodynamic equilibrium constant, R is the universal

Experiments were carried out in batch mode to study the effect of
gas constant (8.314 J mol− 1 K− 1), T is the absolute temperature (K).
initial dye concentration (10–50 mg/L), biosorbent concentration (100
mg–500 mg/L), pH (3–9), temperature (25–45 ◦ C) and contact time (1–6
days) on the removal of safranin from aqueous solution. After each in­ 2.4. Desorption study
cubation period, the percent decolorization of dye was determined in the
aqueous solution by measuring the absorbance at 680 nm and a control The dye-loaded biosorbent (200 mg/L) was amended into 1000 ml
was maintained. The decolorization efficiency was calculated using the Erlenmeyer flask containing the desorbing agents namely, 0.1 N HCl,
equation 0.1 N HNO3 and 0.1 N NaOH separately and agitated at 200 rpm at pH 6

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T. Kiruthika et al. Environmental Research 211 (2022) 113108

for 5 days at 30 ◦ C. Desorption efficiency was determined by analysing respectively. Homology modeling of Photosystem II protein D1 was
the filtrate at every 24 h time interval (Vigneshpriya et al., 2017). performed using the SWISS MODEL server and the structure was further
validated for its stability using SAVES-PROCHECK Ramachandran Plot.
2.5. Analytical studies Docking study of the Photosystem II protein D1 with safranin (ligand)
was carried out using Autodock 4.2. The integrated depiction of docking
The change in the absorption spectrum of the decolourised dye so­ predictions provided by Bovia Discovery studio substantially facilitates
lution was recorded using UV–visible spectrometer (HITACHI, Japan) at the comprehension of docking data and their incorporation into existing
200–800 nm. To investigate the changes in the functional groups that research processes.
intervene the process of dye degradation, Fourier transform infrared
(FT-IR) spectroscopy (Shimadzu 8400 S, Japan) was performed at 800 - 3. Results and discussion
4000 cm− 1. The surface changes in the dye treated and untreated bio­
sorbent was analyzed using SEM (TESCAN, MIRA3 XMU, CZECH). The 3.1. Identification of the bryophyte
SEM equipped with an energy dispersion spectrometer (EDX) was
employed to analyze the chemical constituents of the biosorbent. The The selected bryophyte is identified as Fissidens species which be­
nature of the treated and untreated biosorbent was determined by per­ longs to the family Fissidentaceae. The voucher specimen number for the
forming X-ray diffraction (XRD) spectrometer (XPERT- PRO PAN­ identified bryophyte is BSI/SRC/5/23/2019/Tech./3359. It mainly
alytical, Netherlands). The particle size of the dye unloaded and loaded grows on low land with neutral and mild acidic soil, in woodland, on
biosorbent was determined from the 2θ values of the X-ray diffraction stream sides, aerial fields, in garden and also in clean to moderately
peaks using the following Debye-Scherrer’s equation. polluted river. The roots are the base which makes it get firmly attached
to the growth substrate. It grows well in winter with dark green color
D = Kλ/β cos θ (10)
whereas it turns brown in summer because of high temperature and
excessive light (Fig. 1a and b).
where K = constant, λ = wavelength of the X-rays, β = full width half
maximum of the XRD peak (radians), θ = Bragg’s angle of the XRD peak.
3.2. Biosorption studies at different process parameters
2.6. Toxicity studies
The percentage decolorization was noticed to be the maximum at a
Healthy seeds of surface sterilized green gram (Vigna radiata L.) seeds concentration of 20 mg/L (90%) by Fissidens species within two days. As
were watered regularly with tap water (control), untreated and treated the concentration of the dye increases (30–50 mg/L) the color removal
dye solution to assess the toxicity. On 7th day, the plants were uprooted efficiency of the dye by Fissidens species decreased gradually (79-62%)
and biometric parameters such as germination percentage, shoot and (Fig. 2a). Initial dye concentration offered a substantial driving energy
root length and vigour index were determined (Jadhav et al., 2010). The to overcome all mass transfer resistance of dye in aqueous and solid
toxicity of untreated and treated dye solution was assessed against phase. When the concentration of dye increases the sorption sites on the
selected bacterial (Pseudomonas aeruginosa, Streptococcus pyogenes, biosorbent gets unavailable, leading to the decline in color removal from
Staphylococcus aureus and Escherichia coli) and fungal (Aspergillus flavus, the aqueous solution. The total number of available sorption sites for a
Rhizopus sp, Alterneriasp, Tricoderma sp. And Aspergillus niger) isolates by biosorbent is constant and thereby they adsorb same amount of the
agar well diffusion technique and the zone of inhibition was recorded adsorbate (dye), resulting in a decreased dye removal with increase in
(Ventura et al., 2012). dye concentration. Lee and Low (1987) suggested that the moss
Calymperesde lessertii Besch as an efficient biosorbent to uptake methy­
lene blue and astrazone red violet 3 R, with the rate of decolorization
2.7. Reuse of treated dye solution for dyeing
being determined by a combination of surface adsorption and diffusion
within the moss.
A commercially bleached 100% cotton fabric was purchased from
The decolorization of safranin was high when the biosorbent con­
National Textile Corporation (NTC), Coimbatore District, Tamil Nadu,
centration was 200 mg/L (94%) and minimum at a concentration of 500
India. The cotton fabric was prepared for dyeing by treating it with 1%
mg/L (65%). As the concentration of the biomass was increased
non-ionic detergent, rinsed and dried at room temperature. The fabric
(300–500 mg/L) the color removal efficiency decreased (Fig. 2b).
was dyed with safranin (1%) using tap water (control) and treated dye
Higher concentration of biosorbent may lead to the aggregation and
solution (test sample) individually with a material-to-liquor ratio of
interference of dye molecules which in turn causes repulsion between
1:20 at 90 ◦ C for 60 min. The dyed fabrics were washed thoroughly,
the binding sites and the surface area of the biosorbent gets reduced, and
dried and analyzed for its color fastness to sun light, wet/dry crocking
results in decreased dye removal. Tabaraki and Sadeghinejad (2017)
and wet/dry pressing using standard grey scale rating (1–5).
reported that an increase in biosorbent dose leads to increase in binding
The fabrics dyed were assessed for its color co-ordinates value (L*, a*
sites and surface area thereby the dye removal efficiency increases. The
and b*) using Premier Color scan SS 5100H Spectrophotometer, Mum­
decrease in the dye removal capacity of the biosorbent with increase in
bai. Using Kubelka-munk equation the relative color strength (K/S) of
the biosorbent dose is mainly due to in saturation of binding sites
the dyed fabrics was calculated
through the sorption process (Tural et al., 2017).
/ /
K S = (1 − R)2 2R (11) The percentage decolorization of safranin by Fissidens species grad­
ually increased from the first day and reached maxima at second day of
where, R is the light reflectance, K and S are the absorption and scat­ incubation (90%) and thereafter the decolorization rate decreased (82-
tering coefficients respectively (Habibzadeh et al., 2010; Sisodia and 55%) (Fig. 2c). The biosorption of safranin to Fissidens species at early
Parmar, 2014). incubation period was quite rapid and gradually slows down reaching
equilibrium. The rapid adsorption observed initially is possibly due to
2.8. In silico study the availability of active sites on the surface of the biosorbent, and with
gradual occupancy of these sites, the biosorption becomes less efficient.
Molecular docking modeling provides a holistic strategy for suc­ The decrease in the color removal with increase in incubation period
cessful bioremediation through the interaction of substrate and binding may also be due to the aggregation of the dye molecules around the
sites. The protein sequences of Photosystem II protein D1 and the biosorbent which may hinder the migration of the dyes. The adsorption
structure of safranin was retrieved from UniProt and PubChem database sites become filled up, and resistance to diffusion of dye molecules

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T. Kiruthika et al. Environmental Research 211 (2022) 113108

Fig. 1. a) Dark green colored Fissidens species (Winter) b) Brown colored Fissidens species (Summer). (For interpretation of the references to color in this figure
legend, the reader is referred to the Web version of this article.)

Fig. 2. Optimization parameters for safranin decolorization using Fissidens species a) Dye concentration b) Biosorbent concentration, c) Contact time, d) pH, e)
Temperature.

develops as the concentration of biosorbent increases (Gupta et al., of biosorption. Pipíška et al. (2018) also reported the biosorption of
2014). cationic dyes thioflavin T and methylene blue from single and binary
pH of dye solution influences the surface charge of the biosorbent solutions using the dried biomass of freshwater moss Vesicularia
and the degree of ionization of the dye molecule. Decolorization of dubyanaasa at different contact time, pH, and biomass/sorbate
safranin over a range of 3–9 was studied, and the percentage dye concentration.
removal was found to increase with increase in pH up to 6 (Fig. 2d). From Fig. 2e, it can be inferred that a temperature of 30 ◦ C (88%) is
Fissidens species removed maximum color at the optimum pH 6 (93%) most favorable for the decolorization of safranin and a significant
and minimum decolorization was observed at pH 9 (56%). The cell wall decrease was observed at higher temperatures. Temperature is one of
of mosses consists of polysaccharides such as lignin and cellulose which the essential factors that influence the rate of biological processes and
possess alcohol, aldehyde, ketone, carboxylic acids, and phenols as their consequently controls the rate of biosorption. At optimal temperature
main functional groups. The safranin dye gets electrostatically attracted there may be an increase in the enzyme activity which aids in maximum
to the surface of the biosorbent which in turn increases the dye removal dye removal as reported by Israni et al. (2002). The result of the present
capacity (Ringqvist et al., 2002). Hence ionic exchange, electrostatic study coincides with the findings of San Keskin and Uyar (2019) who
repulsion and structural properties of dye play a vital role in the process reported that 50 mg/L of bog moss, Sphagnum palustre L. removed 99.5%

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T. Kiruthika et al. Environmental Research 211 (2022) 113108

methylene blue within an hour at pH 4 at 25 ◦ C. An increase in tem­ Table 1

perature may alter the surface activity of biomass and results in a Adsorption isotherms, kinetics and thermodynamic parameters for biosorption
decreased removal of dye, thereby indicating the process to be of safranin onto Fissidens species.
exothermic in nature. The decrease in biosorption of dye onto the bio­ Adsorption isotherms
sorbent at higher temperature implies the weak adsorption interaction Langmuir isotherm Freundlich isotherm
between biosorbent surface and the dye (Hasan et al., 2009). The
KL (L mg− 1) RL R2 KF(L mg− 1) 1/n R2
decolorization rate of safranin using Fissidens species was observed to
1.60 0.74 0.99 2.47 0.025 0.82
increase to a maximum of 95% under optimum conditions. Adsorption kinetics

Pseudo-First order kinetics (min¡1) Pseudo-second order kinetics

3.3. Desorption study (g mg¡1 min¡1)

qecal K1 R2 qecal K2 R2
Desorption study helps to analyze the behavior of the adsorption and 1.16 0.013 0.86 11.29 0.40 0.90
the recovery of the dye from the aqueous solution. Desorption carried Thermodynamics
out with dye loaded biosorbent at optimized conditions showed that Temperature ΔG◦(kJ ΔH◦(kJmol− 1) ΔS◦(J R2
92% of the adsorbed safranin was desorbed on third day using 0.1 N (oC) mol− 1) mol− 1K− 1)
NaOH when compared with 0.1 N HNO3 (68%) and 0.1 N HCl (51%) 25 − 226.55 0.384 218.57 0.928
respectively. The desorption efficiency decreased with further increase 30 − 274.36
in incubation period when the dye loaded biomass was treated with the 35 − 317.17
40 359.16
eluents (Fig. 3). Hence, the test biosorbent can be a used as a sustainable −
45 − 396.57
resource in the recovery of pollutants from the textile sector.

3.4. Adsorption isotherm during safranin adsorption. Contreras et al. (2007) reported that the
equilibrium experiment results for the adsorption of Basic Blue 3 and
The results of adsorption isotherms, kinetics, thermodynamics and Basic Orange 2 onto Sphagnum magellanicum peat fitted to the Langmuir
the respective correlation coefficients for the adsorption of safranin onto isotherm and pseudo first and second order models at optimized con­
Fissidens species was depicted in Table 1. The attained correlation co­ ditions. The thermodynamic parameters obtained by Aksu and Tezer
efficient of Langmuir isotherm (R2 = 0.99) was higher than the (2005) in the biosorption of reactive dyes onto Chlorella vulgaris and by
Freundlich isotherm (R2 = 0.82), representing the experimental data Dotto et al. (2011) in the adsorption of acid blue 9 and food yellow 3
well fitted the Langmuir isotherm and supported the postulation that onto chitosan coincide with the results of the present study. Thus, the
safranin was homogeneously adsorbed onto the surface of Fissidens present study indicated that the Fissidens species has promising safranin
species (Fig. 4a and b). The RL (0.74) and 1/n (0.025) values proves to be decolorization capacity and highlighted its possible exploration in dye
favorable and unfavorable for safranin adsorption onto Fissidens species polluted habitat.
under the optimized conditions, thereby does not obey the Freundlich
isotherm model. Fig. 5a and b depicts the plot of ln (qe -qt) and t/qt 3.5. Analytical studies
against time for pseudo first and pseudo second order models. The R2
value (0.903) of the experimental data well fitted with pseudo second Complete disappearance of peak was observed at 524 nm and 279
order kinetics at varied time than those for pseudo first order model nm in the UV–Vis spectrum which strongly depicts the degradation or
(0.86), thereby depicting the former more suitable for the adsorption decolorization of safranin due to Fissidens species (Fig. 6). The dye gets
behavior of safranin onto Fissidens species. The negative values of ΔGo adsorbed on the surface of the bryophyte where enzymatic degradation
revealed the spontaneous and favorable nature of safranin adsorption involves in the formation of new compounds.
onto Fissidens species and also suggested the absence of energy barrier The nature of the chemical bonding and functional groups of the
during the decolorization. The positive value of ΔHo and ΔSo indicated unloaded and dye loaded bio sorbents were observed from FT-IR spec­
the endothermic nature and the incidence of increased randomness in troscopy. The FT-IR spectrum of dye unloaded biomass revealed char­
the solid-liquid interface during decolorization process. Moreover, the acteristic peaks at 3857.56, 3863.50, 2308.79, 1461.42, 1367.53,
positive values of entropy depicted structural changes in the biosorbent 1217.08 and 1062.78 cm− 1 (Fig. 7a). After the sorption of the safranin
dye by Fissidens species the peak was shifted to 3390.86, 2125.56,
1640.40, 1543.05, 1228.66 and 1050.76 cm− 1 (Fig. 7b). The shift in the
peaks revealed the possible assignments of the absorption bands such as
N–H stretching vibrations of amino groups coupled with O–H stretching,
C–H stretching, C– – C stretching and C–H bending. The results of FT-IR
analysis proved that the dye adsorption might have occurred on the
biosorbent by the presence of hydroxyl, amine and carboxylic acid
groups which resulted in the decline of adsorption frequencies.
The XRD spectrum of dye unloaded biosorbent displayed charac­
teristic diffraction peaks of 2θ corresponding to 20.96, 22.04, 26.65,
27.70, 29.35, 35.32, 39.46, 47.35 and 52.79 and displayed amorphous
nature (Fig. 8a). The XRD spectrum of the dye unloaded biosorbent at 2θ
revealed a shift in the diffraction peaks to 23.00, 27.57, 29.42, 35.14,
36.61, 47.57, 50.18, 55.58, 58.74, 60.05, 64.67, 67.81 and 71.51 and
revealed crystalline nature (Fig. 8b). The changes in the peak intensity
might be due to the diffusion of dye molecules into the pores of the
biosorbent through physisorption and chemisorption with alteration in
carbon skeleton (Namasivayam and Kavitha, 2004). The particle size of
the biosorbent was recorded as 51.99 nm. The surface area existing for
Fig. 3. Desorption efficiency of dye loaded biosorbent using different eluents. adsorption is significantly determined by particle size of the biosorbent

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T. Kiruthika et al. Environmental Research 211 (2022) 113108

Fig. 4. Langmuir (a) and Freundlich (b) isotherm plot for the biosorption of safranin onto Fissidens species under optimized conditions.

Fig. 5. Pseudo-first order (a) and Pseudo-second order (b) kinetic model for safranin biosorption onto Fissidens species.

Fig. 7. FT –IR spectra of dye unloaded (a) and dye loaded (b) Fissidens species.
Fig. 6. UV–Vis spectra of safranin before (a) and after (b) decolorization by
Fissidens species.
the dye unloaded biosorbent showed cavities, pores, rough and irregular
surface providing large surface area for ion interaction (Fig. 9a),
and is an essential controlling factor in the process of adsorption. The
whereas when the dye molecules get adsorbed on the pores of the bio­
smaller particle size of biosorbent provides larger total surface area
sorbent, the surface becomes smoother (Fig. 9b). EDX is executed to
which might have resulted in better adsorption of dye, since the sorption
analyze the elements or chemical characterization of a biosorbent in
capacity of the biosorbent is directly proportional to the exposed surface
conjunction with SEM. The EDX spectrum of biosorbent unexposed to
area and inversely correlated to the particle diameter (Wanyonyi et al.,
dye consists of various elements namely K, C, Ca, N, O, Na, Mg, Si, P, S
2014).
and Cl (Fig. 9c). The EDX analysis confirmed the penetration of dye into
The morphological changes in the surface of the bryophyte before
the pores and surface of the biosorbent and thereby results in the
and after biosorption was portrayed through SEM. SEM micrographs of

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T. Kiruthika et al. Environmental Research 211 (2022) 113108

Fig. 8. X – Ray Diffractogram of dye unloaded (a) and dye loaded (b) Fissidens species.

Fig. 9. SEM Micrograph (9a, 9 b), EDX spectrum (9c, 9 d) and overlay of elements (9e, 9f) of dye unloaded and dye loaded Fissidens species.

increase and decrease of carbon and oxygen content by weight after its non-toxic nature. The results of the study were in agreement with the
adsorption (Fig. 9d). The weight percentage of elements on dye unloa­ findings of Shanmugam et al. (2020) who reported that the stimulation
ded and loaded biosorbent is portrayed in Fig. 9e and f. in plant growth might be due to the discharge of utilizable metabolites
or minerals on the degradation of dyes.
3.6. Toxicity studies The zone of inhibition exhibited by Pseudomonas aeruginosa (30 ±
0.4), Streptococcus pyogenes (29 ± 0.2), Staphylococcus aureus (32 ± 0.5),
Generally, untreated effluents are discharged into aquatic bodies, Escherichia coli (39 ± 0.3), Aspergillus flavus (13 ± 0.2), Aspergillus niger
and they are utilized for agricultural purposes. This in turn affects the (13 ± 0.4), Rhizopus (12 ± 0.6), Alternaria (14 ± 0.4) and Trichoderma
plant growth and the fertility of the soil and also causes health problems. (15 ± 0.3) species against untreated dye solution reveals the toxic effect
Hence, it is of primary concern to check the toxicity of dyes before and of the dye against microbes, whereas no zone of inhibition was recorded
after treatment. The germination percentage of green gram was 86% in treated dye solution indicating its non-toxic nature.
when watered with treated and untreated dye solutions, whereas only
33% of the seedlings germinated in untreated dye solution. There was 3.7. Fabric evaluation
93% germination in green gram seeds grown using tap water (control).
The shoot length, root length and the vigor index of the green gram The dyed fabrics exhibited good color fastness to sunlight, rubbing
seedlings grown using treated dye solution (8.49 cm, 5.05 cm and 1164) and excellent fastness to pressing in both dry and wet conditions. The
showed significant results when compared with untreated dye solution results revealed that the color fastness properties of the fabrics dyed
(1.78 cm, 0.9 cm and 87). Plants grown with tap water exhibited a using treated dye solution was on par with that of the fabrics dyed using
remarkable growth (8.77 cm, 5.56 cm and 1333). The results thereby tap water which establishes that the treated dye solution can be
reveals that the green gram grown using untreated dye solution affects employed for dyeing.
germination, shoot and root length, whereas those grown with treated The color strength and color coordinate’s values of dyed fabrics are
dye solution significantly alleviated the plant growth thereby indicating presented in Table 2. L* represents lightness value of the fabrics, where

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T. Kiruthika et al. Environmental Research 211 (2022) 113108

Table 2 whereas 90.9% with 269 residues was observed in the accompanying
L*, a*, b* and K/S values for fabric dyed using untreated and treated dye region which depicts the stability of the protein model (Fig. 10). Thus,
solution. the study would afford an insight to various types of azo dyes which can
S. Samples L* a* b* K/S Color be degraded by the photosystem II protein D1 and the conformational
No. Obtained changes that take place in the protein structure can be further analyzed.
1. Fabric dyed 30.659 31.533 6.267 117.421 The docking affinity of safranin (ligand) with the photosystem II
using tap water protein D1 (substrate) was assessed based on its binding energy, ligand
2. Fabric dyed 31.092 33.115 6.737 95.921 efficiency, intermolecular energy, hydrogen bond and van Der Waals
using interactions. The results of the docking study revealed that there was a
decolourised good binding (− 5.23 kcal/mol) and intramolecular (− 6.13 kcal/mol)
dye solution
energy between the ligand and the receptor site of the target protein.
The efficiency of the ligand to bind with the target protein was also
more negative and positive value of L* denotes darker and lighter shade significant (− 0.22 kcal/mol). Moreover, the negative values revealed
respectively. The tone of color is represented by a* and b* where, the high feasibility of interaction between the ligand and target. The
negative and positive value of a* indicates green and red color respec­ hydrogen and alkyl bond interaction with the amino acid His 272 and
tively. Similarly, the negative and positive value of b* indicates blue and Trp 142, Ala 146, Ala 276, Val 280 play a vital role as the catalytic site
yellow color respectively (Zulrushdi et al., 2016). The L* value of fabric residue in the photosystem II protein D1 of Fissidens species (Fig. 11a and
dyed using tap water was found to be 30.66 and 31.09 for fabric dyed b). Alaguprathana et al. (2020) reported a good interaction between the
using treated dye solution. The a* value of fabrics dyed using tap water ligand, methyl orange and azoreductase, the receptor protein which
(31.53) and treated dye solution (33.11) shows that both the fabrics lie plays a pivotal role in dye degradation. Thus, the rate of decolorization
in the redder region. The b*value of fabrics dyed using tap water (6.27) for safranin could likely afford enhanced information on the molecular
and treated dye solution (6.74) reveals that both the fabrics falls under mechanisms involved in catalysis, as well as a heuristic origin for
yellowish region. The values of a* and b* indicate that both the dyed envisaging catalytic residues in unknown functional enzymes.
fabric falls under reddish and yellowish category. The color strength of
tap water dyed fabric (117.42) and fabric dyed using treated dye solu­ 4. Conclusion
tion (95.92) indicate that the treated water could be effectively used for
dyeing, which minimize water consumption in textile industries. From Fissidens species proved to be a potent biosorbent in the decoloriza­
the above results, it could be concluded that the color strength and color tion of safranin from aqueous solution under different experimental
coordinates of the fabrics dyed using Fissidens species treated water is conditions. The equilibrium data for safranin on Fissidens species well
comparable with that of tap water dyed fabric proving the utilization of fitted with the Langmuir isotherm. The kinetic data confirms the inter­
treated water in textile industries. action between adsorbate and adsorbent by chemisorption process. The
interaction between the dye and biosorbent was revealed morphological
3.8. In silico interaction between safranin and photosystem II protein D1 changes in the biosorbent. Phytotoxicity and microbial toxicity studies
revealed that the untreated dye solution was non-toxic as compared with
The stereo chemical validation of the protein structure was assessed the candidate dye, safranin against green gram and selected microbes
for its stability through PROCHECK that generated the Ramachandran respectively. Reuse potential of the treated dye solution for dyeing the
plots where φ-ψ torsion angles of the amino acid residues were plotted fabrics further minimize the rate of pollution. In silico studies provides
against each other. The Ramachandran Plot reveals 8.4% and 0.7% with the theoretical overview on the behavior and nature of the protein and
25 and 2 residues in the additional and generously allowed region ligand interaction which supported the in vitro studies. Thus, the present

Fig. 10. Homology-modeled structure and Ramachandran plot of Photosystem II protein D1of Fissidens species.

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T. Kiruthika et al. Environmental Research 211 (2022) 113108

Fig. 11. a. Docking interaction of safranin and Photosystem II protein D1. b. Amino acid interactions with safranin dye.

study on decolorization of safranin using Fissidens species will lay a Gupta, V.K., Bhushan, R., Nayak, A., Singh, P., Bhushan, B., 2014. Biosorption and reuse
potential of a blue green alga for the removal of hazardous reactive dyes from
platform and would provide a benchmark for scientific society in the
aqueous solutions. Bioremediat. J. 18, 3179–3191.
control and management of environmental pollution in an ecofriendly Habibzadeh, S., Tayebi, H., Eerami, E., Shams, A., Nateri, M., Bahmai, M., 2010. Silk
manner. dyeing using saw wood of the zelkoa forest tree. World Appl. Sci. J. 9 (3), 295–299.
Hasan, S.H., Srivastava, P., Talat, M., 2009. Biosorption of Pb (II) from water using
biomass of Aeromonas hydrophila: central composite design for optimization of
Declaration of competing interest process variables. J. Hazard Mater. 168, 1155–1162.
Ho, Y.S., 2006. Review of second-order models for adsorption systems. J. Hazard Mater.
136 (3), 681–689.
The authors declare that they have no known competing financial
Hu, H.J., Wageh, S., Al-Ghamdi, A.A., Yang, S.B., Tian, Z.F., Cheng, B., Ho, W.K., 2020.
interests or personal relationships that could have appeared to influence NiFe-LDH nanosheet/carbon fiber nanocomposite with enhanced anionic dye
the work reported in this paper. adsorption performance. Appl. Surf. Sci. 511, 145570.
Imran, M., Ashraf, M., Hussain, S., Mustafa, A., 2019. Microbial biotechnology for
detoxification of azo-dye loaded textile effluents: a critical review. Int. J. Agric. Biol.
Acknowledgement 22, 1138–1154.
Israni, S., Koli, S.S., Patwardhan, A., Melo, J., Melo, S.F., 2002. Phenol degradation in
The authors wish to place their record of thanks to the authorities of rotating biological contactors. J. Chem. Technol. Biotechnol. 77 (9), 1050–1057.
Jadhav, J.P., Kalyani, D.C., Telke, A.A., Phugare, A.A., Govindwar, S.P., 2010.
Avinashilingam Institute for Home Science and Higher Education for Evaluation of the efficacy of a bacterial consortium for the removal of color,
Women, Coimbatore, and DST CURIE (No. SR/CURIE/PHASE II/01/ reduction of heavy metals, and toxicity from textile dye effluent. Bioresour. Technol.
2014) for the support given to conduct the study successfully. The au­ 101 (1), 165–173.
Jain, R., Sharma, N., Jadon, N., Radhapyari, K., 2006. Electrochemical studies on a
thors acknowledge Researchers Supporting Project number (RSP-2021/ textile azine dye: safranin T. Int. J. Environ. Pollut. 27 (1/2/3), 121–134.
149), King Saud University, Riyadh, Saudi Arabia. Khan, S., Naushad, M., Govarthanan, M., Iqbal, J., Alfadul, S.M., 2022. Emerging
contaminants of high concern for the environment: current trends and future
research. Environ. Res. 207, 112609.
References Langmuir, I., 1918. The adsorption of gases on plane surfaces of glass, mica and
platinum. J. Am. Chem. Soc. 40, 1361–1403.
Akansha, K., Chakraborty, D., Sachan, S.G., 2019. Decolorization and degradation of Lee, C.K., Low, K.S., 1987. The removal of cationic dyes by a natural moss: I. Adsorption
methyl orange by Bacillus stratosphericus SCA1007. Biocatal. Agric. Biotechnol. 18, sites. Pertanika 10 (3), 327–334.
101044. Mahmoodi, N.M., Hayati, B., Arami, M., Lan, C., 2011. Adsorption of textile dyes on pine
Aksu, T., Tezer, S., 2005. Biosorption of reactive dyes on the green alga Chlorella cone from colored wastewater: kinetic, equilibrium and thermodynamic studies.
vulgaris. Process Biochem. 40 (3 - 4), 1347–1361. Desalination 268 (1–3), 117–125.
Alaguprathana, M., Poonkothai, M., 2018. Impact of untreated and bioremediated Milonjić, S.K., 2007. A consideration of the correct calculation of thermodynamic
methyl orange on seed germination, growth and yield of flower plants - marigold parameters of adsorption. J. Serb. Chem. Soc. 72 (12), 1363–1367.
(Yellow and orange), Celosia argentea. Int. J. Innov. Res. Sci. Technol. 5 (7), 22–28. Namasivayam, C., Kavitha, D., 2004. Adsorptive removal of 2, 4-dichlorophenol from
Contreras, E.G., Martinez, B.E., Sepulveda, L.A., Palma, C.L., 2007. Kinetics of basic dye wastewater by low- cost carbon from an agricultural solid waste: coconut coir pith.
adsorption onto Sphagnum magellanicum peat. Adsorpt. Sci. Technol. 25 (9), Separ. Sci. Technol. 39, 1407–1425.
637–646. Pathak, H., Soni, D., Chauhan, K., 2014. Evaluation of in vitro efficacy for decolorization
Dasgupta, J., Sikder, J., Chakraborty, S., Curcio, S., 2015. Remediation of textile and degradation of commercial azo dye RB-B by Morganella sp. HK-1 isolated from
effluents by membrane based treatment techniques: a state of the art review. dye contaminated industrial landfill. Chemosphere 105, 126–132.
J. Environ. Manag. 147, 55–72. Phugare, S.S., Kalyani, D.C., Surwase, S.N., Jadhav, J.P., 2011. Eco-friendly degradation,
Dotto, G.L., Pinto, L.A.A., 2011. Adsorption of food dyes acid blue 9 and food yellow 3 decolourisation and detoxification of textile effluent by a developed bacterial
onto chitosan: stirring rate effect in kinetics and mechanism. J. Hazard Mater. 187 consortium. Ecotoxicol. Environ. Saf. 74, 1288–1296.
(1–3), 164–170. Pipíška, M., Valica, M., Partelová, D., Horník, M., Lesný, J., Hostin, S., 2018. Removal of
Elbanna, K., Hassan, G., Khider, M., Mandour, R., 2010. Safe biodegradation of textile synthetic dyes by dried biomass of freshwater moss Vesicularia dubyana: a batch
azo dyes by newly isolated lactic acid bacteria and detection of plasmids associated biosorption study. Environments 5 (1), 10.
with degradation. J. Bioremed. Biodegrad. 1 (3), 1000112. Rane, N.R., Chandanshive, V.V., Khandare, R.V., Gholave, A.R., Yadav, S.R.,
Franciscon, E., Grossman, M.J., Paschoal, J.A.R., Reyes, F.G.R., Durrant, L.R., 2012. Govindwar, S.P., 2014. Green remediation of textile dyes containing wastewater by
Decolorization and biodegradation of reactive sulfonated azo dyes by a newly Ipomoea hederifolia L. RSC Adv. 4, 36623e32.
isolated Brevibacterium spp. strain VN-15. SpringerPlus 1, 1–10. Rawat, D., Mishra, V., Sharma, R.S., 2016. Detoxification of azo dyes in the context of
Freundlich, H.M., 1906. Over the adsorption in solution. J. Phys. Chem. 57, 385–470. environmental processes. Chemosphere 155, 591–605.
Gao, Y., Yang, B., Wang, Q., 2018. Biodegradation and decolorization of dye wastewater:
a review. IOP Conf. Ser. Earth Environ. Sci. 178, 012013.

9
T. Kiruthika et al. Environmental Research 211 (2022) 113108

Rejniak, L., Piotrowska, H., 1966. Effect of malachite green, Congo red and safranin on Taylor, J.A., Smith, R.T., 1980. Peat - a resource reassessed. Nature 288 (5789),
cell division in gemmae of Allium cepa. Nature 209 (5022), 517–518. 319–320.
Ringqvist, L., Holmgren, A., Oborn, I., 2002. Poorly humified peat as an adsorbent for Tural, B., Ertaş, E., Enez, B., Fincan, S.A., Tural, S., 2017. Preparation and
metals in wastewater. Water Res. 36 (9), 2394–2404. characterization of a novel magnetic biosorbent functionalized with biomass of
San Keskin, N.O., Uyar, G., 2019. Methylene blue dye removal using Sphagnum palustre L. Bacillus subtilis: kinetic and isotherm studies of biosorption processes in the removal
Bog-moss as a reusable biosorbent. Anatol. Bryol. 5 (1), 1–7. of Methylene Blue. J. Environ. Chem. Eng. 5, 4795–4802.
Satake, K., Takamatsu, T., Soma, M., Shibata, K., Nishikawa, M., Say, P.J., Whitton, B.A., Ventura, S.P., de Barros, R.L., Sintra, T., Soares, C.M., Lima, A.S., Coutinho, J.A., 2012.
1989. Lead accumulation and location in the shoots of the aquatic liverwort Scapania Simple screening method to identify toxic/non-toxic ionic liquids: agar diffusion test
undulata (L.) Dum. in stream water at Greenside Mine, England. Aquat. Bot. 33 (1–2), adaptation. Ecotoxicol. Environ. Saf. 83, 55–62.
111–122. Vigneshpriya, D., Krishnaveni, N., Renganathan, S., 2017. Marine brown macroalga
Shanmugam, L., Ahire, M., Nikam, T., 2020. Bacopamonnieri(L.) Pennell, a potential Sargassum wightii as a novel biosorbent for removal of brilliant green dye from
plant species for degradation of textile azo dyes. Environ. Sci. Pollut. Res. 27, aqueous solution: kinetics, equilibrium isotherm modeling and phytotoxicity of
9349–9363. treated and untreated dye. Desalination Water Treat. 78, 300–312.
Sisodia, N., Parmar, M.S., 2014. Dyeing behavior and fastness properties of corn (PLA) Wanyonyi, W.C., Onyari, J.M., Shiundu, P.M., 2014. Adsorption of Congo red dye from
fiber, 01–07 J. Polymer. Text. Eng. 1 (2). aqueous solutions using roots of Eichhornia crassipes: kinetic and equilibrium studies.
Tabaraki, R., Sadeghinejad, N., 2017. Biosorption of six basic and acidic dyes on brown Energy Proc. 50, 862–869.
alga Sargassumilicifolium: optimization, kinetics and isotherm studies. Water Sci. Zulrushdi, N.A.F., Hassan, R.M., Yusoff, A.M., 2016. Microwave-assisted extraction of
Technol. 75, 2631–2638. natural colorant extracted from mesocarp and exocarp of Cocus nucifera (coconut
palm), 01–05 Eur. J. Biotechnol. Biosci. 4 (4).

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