Accepted Manuscript

Biosorption for removal of hexavalent chromium using microalgae Scenedesmus

sp.

Debabrata Pradhan, Lala Behari Sukla, Bibhuti Bhusan Mishra, Niharbala Devi

PII: S0959-6526(18)33321-3

DOI: 10.1016/j.jclepro.2018.10.288

Reference: JCLP 14690

To appear in: Journal of Cleaner Production

Received Date: 30 January 2018

Accepted Date: 27 October 2018

Please cite this article as: Debabrata Pradhan, Lala Behari Sukla, Bibhuti Bhusan Mishra,
Niharbala Devi, Biosorption for removal of hexavalent chromium using microalgae Scenedesmus
sp., Journal of Cleaner Production (2018), doi: 10.1016/j.jclepro.2018.10.288

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Total wordcount of the whole document: 7988

Biosorption for removal of hexavalent chromium using microalgae

Scenedesmus sp.
Debabrata Pradhan, Lala Behari Sukla, Bibhuti Bhusan Mishra, Niharbala Devi*

Biofuels and Bioprocessing Research Center, Institute of Technical Education and Research,
Siksha ‘O’ Anusandhan (Deemed To Be University), Bhubaneswar-751030, India

Abstract

Biosorption was carried out in this study using the biomass of microalgae Scenedesmus sp. as
adsorbent for the removal of hexavalent chromium or Cr(VI) from solution. Different relevant
parameters like initial pH, contact time, initial Cr(VI) concentration, biosorbent dosage, particle
size, and temperature were examined to evaluate their effect. They were found to be effective
with the maximum of 92.89% Cr loading onto the biomass. The FTIR analysis revealed the
presence of easily hydrolyzed functional groups like aldehydes, amides, carboxylic acids,
phosphates, and halides, which charged positively below the point of zero charge at pH 2.65. The
biosorption proceeded through the anionic adsorption mechanism and followed the pseudo-first
order kinetic model. Its feasibility was confirmed as the experimental data closely fitted to both
Langmuir and Freundlich isotherms. The biosorption was spontaneous. Its spontaneity increased
with the increase of temperature. The positive entropy change suggested the randomness at the
solid-liquid interface. This study recommended that the toxic Cr(VI) could be removed from
different contaminated water samples in the nearby chromite mines using the microalgae
biomass. However, it requires attention in some other sides like activation of biomass,
interference of other ions, elevation of the experimental pH, the large biomass generation and
harvesting, desorption of the Cr-loaded biomass, and recovery of the energy value of the waste
biomass.

Keywords: Biosorption; Microalgae; Adsorption parameters; Adsorption isotherms; Kinetics;

Thermodynamics

*Corresponding author: Niharbala Devi, Email: niharbaladevi@soa.ac.in, Phone: +91-

9437968279
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1. Introduction

The chemical properties of Cr(VI) favor the increasing concentration level in water and make it a
recognized waterborne pollutant(Pradhan et al., 2017). Various anthropogenic activities make it
waterborne(Bielicka et al., 2005). During propagation of the environmental nutrient cycle, it
becomes the intake of various plants and animals(Gheju, 2011; Kimbrough et al. 1999; Tseng et
al., 2018). It shows toxicity in the solution even at the level measuring in parts per billion
(ppb)(Barrera-Díaz et al., 2012; Gibb et al., 2000; Langard, 1990). Owing to its toxicity, the
growing concern and the relentless pursuit in combating it have led to development of different
physical and chemical treatment options(Zhu et al., 2018). However, they have been conducted
through different intensive and complex sub-processes which utilize sophisticated plant set-up
and substantial chemical reagents. Furthermore, they generate different toxic sludges which fear
the possibility of recontamination to the environmental resources (Malaviya and Singh, 2016;
Yang et al., 2014). In-situ operations of implementing them look far reaching due to the
complicated sub-processes. If the cost of these processes would be included in the production
cost of different chromium appliances, overall cost of the products would reach approximately
twice of the only production cost(Pradhan et al., 2017). Therefore, an alternative treatment way
is utmost necessary for its removal.

It is not surprising that the biosorption technique occupy a prominent rank for the abatement of
heavy metals from different wastewaters(Volesky and Holan, 1995). The biosorption is a passive
process and has shown the credibility of simple way for the removal of different heavy metals at
a low-cost operation (Kim et al., 2009; Liu et al., 2016). The abundant raw and waste biomasses
are generally used as adsorbent of the process (Dittert et al., 2012). Specific to the Cr(VI)
removal, the biomass of bacteria, fungi, plants and their nuts, and algae have shown high
biosorption capacity (Jena et al., 2012; Kim et al., 2010; Mandal et al., 2017; Pradhan et al.,
2018). Microalgae are photosynthetic microphytes generally found in the aquatic environments.
They have the high growth rate compared to the terrestrial plants and can complete an entire
growing cycle in every few days(Sukla et al., 2013). Combined with their high productivity, they
are possibly the rich candidate to mitigate Cr(VI). The microalgae tissues made of different
polysaccharides attach to the inner fibrous skeleton as well as the outer amorphous embedding
matrix within the cells (Dittert et al., 2012). They contain the large amount of deprotonated

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sulfate and carboxyl groups as well as the monomeric alcohol and laminaran, which are most
likely responsible for the biosorption (Bertagnolli et al., 2014). Multiple functional groups, such
as hydroxyl, carboxylic acid, amide, carbonyl, ether, present in the microalgae biomass attract
both cationic and anionic species of different heavy metals from the solution(Nouha et al., 2016).
Other properties like humidity, zeta potential, and density of the biomass add its favour(Gupta
and Rastogi, 2009). Numerous studies have demonstrated the excellent removal of Cr(VI) from
the solution through different adsorption mechanisms using biomass of various micro- and
macro- algal species such as Chlorella vulgaris (Chen et al., 2016), Sargassum cymosum
(deSouza et al., 2016), Sargassum filipendula (Bertagnolli et al., 2014), Pelvetia canaliculata
(Hackbarth et al., 2016), Halimeda gracilis (Jayakumar et al., 2014), Cystoseira indica (Basha et
al., 2008), Laminaria digitata (Dittert et al., 2014), and Oedogonium hatei (Gupta and Rastogi,
2009).

The biosorption of Cr(VI) depends on different physical, chemical and environmental parameters
like biosorbent feed, temperature, Cr(VI) concentration, pH of the solution, contact time,
agitation, redox stimulating reagents, immobilizers, and free cell reductase (Hackbarth et al.,
2016; Pradhan et al., 2008). Optimization of the parameters helps the prediction of mechanism
and designing of reactors(Dittert et al., 2012). The feasibility of biosorption has been examined
by different standard adsorption isotherms like Langmuir, Freundlich, BET, Redlich-Peterson,
Tempkin, Koble-Corrigan, and Toth models (Saha and Orvig, 2010). Of them, Langmuir and
Freundlich are found to be more suitable(Maleki et al., 2015; Sibi, 2016). They characterize the
interactions Cr(VI) ions on the biomass surface. They establish the relationship between Cr(VI)
concentration and the biomass dose at the equilibrium point. The Langmuir isotherm model
describes the monolayer adsorption of adsorbate such as Cr(VI) ions on the biomass surface
without any chemical interaction occurs among them (Miretzky and Cirelli, 2010). The
Freundlich isotherm model explains the multilayer adsorption of Cr(VI) ions on the biomass
surface with appropriate interactions among adsorbate ions (Sun et al., 2009). The kinetic model
describes the reaction paths and the equilibrium of the process, which further defines its
feasibility for the pilot scale operations. The faster rate process reduces the reactor size, set-up
area and human resources, and increases production and economy (Basha et al., 2008).
Biosorption of Cr(VI) follows different standard kinetic models like pseudo-first order, pseudo-
second order, intra-particle diffusion, and Elovich kinetic models; however, they are specific to

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different biomass(Elangovan et al., 2008). Further the thermodynamic interpretation describes

the randomness at the interface of heterogeneous phases and spontaneity of the process towards
equilibrium(Yan et al., 2017).

In this study biomass of a microalgae species, namely Scenedesmus sp., was used for the purpose
of Cr(VI) removal from a synthetic K2Cr2O7 solution with an aim to remove Cr(VI) from the
contaminated mine water sample collected from the seepage of chromite overburden (COB)
located in the Sukinda area of the Odisha province, India.

2. Materials and methods

2.1. Chemicals

Chemicals, such as K2Cr2O7 (Merck), H2SO4 (CDH) and 1,5-diphenylcarbizide (Sigma-Aldrich),

were used as required by the biosorption experiments. K2Cr2O7 was dissolved to prepare a stock
solution of concentration 1000mg.L-1 Cr(VI). This stock solution was diluted and used as
required by the experimental conditions.

2.2. Collection of microalgae biomass

About one kilogram pre-grown raw microalgae biomass of the Scenedesmus sp. was collected
from the CSIR-IMMT, Bhubaneswar, India. The Scenedesmus sp. (IMMTCC-13) was isolated
from the brackish water of Chilika Lake of Odisha, India, and further characterized and stored in
the culture collection center of the CSIR-IMMT (Sukla et al., 2013). The microalgal species
were grown in a race way pond set up at the CSIR-IMMT campus. The illuminated surface area,
depth, and capacity of the raceway pond were 100m2, 0.3m and 40m3, respectively. Other
conditions for growth of the species have been described by Sukla et al. (2013).

2.3. Preparation of biosorbent

Unwanted sediments deposited on the as collected biomass were removed by thorough washing
in the tap water. The cleaned biomass was then dried in the open air for one week followed by in
a hot oven at 50oC until a constant mass obtained. The moisture free biomass was ground to
powder using a laboratory blender and then sieved to different size fractions. The sieve size
distributions were -45, -75+45, -100+75 and +100µm, and the respective average particle sizes
were assigned as 45, 60, 87.5 and 100µm. The average particle sizes were assigned only for the

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convenience in discussing the results. These biomass powders of different particle size
distributions were stored in a desiccator and further used as biosorbent for the purpose of
biosorption study.

2.4. Biosorption experiment

The biosorption experiments were conducted in 200mL glass beakers containing 100mL of the
specified Cr(VI) solution as stated by the experimental condition. The stirring speed was fixed at
300rpm using a laboratory hotplate-cum-magnetic stirrer. The temperature of the solution was
maintained by using a thermocouple connected to the hotplate. Just prior to start the experiment,
required amount of the specified biomass was added into the content. Before adding the biomass,
the initial pH of the content was maintained by adding dilute H2SO4 (10%) solution drop wise
with the help of a glass tube. Samples were collected according to the predetermined time
intervals in order to analyze the Cr(VI) concentration and parallely pH of the content was
measured. Each time about 2mL of liquid sample was drawn from the content with the help of a
pipette and centrifuged at 3000rpm for 5 min using a small Remi centrifuge. Then 1.0mL sample
was taken from the supernatant with the help of a micro pipette and analyzed for the Cr(VI)
concentration by the 1,5-diphenylcarbizide method (Tandukar et al., 2009). The following
biosorption conditions were maintained unless otherwise stated: contact time,120 min;
Cr(VI),10mg.L-1; solid-liquid ratio,10%(w/v); initial pH,1.0; temperature,30oC; particle size,-
75+45μm (average 60μm). All the experimental runs were triplicated and the standard deviations
of the results were within ±5%.

2.5. Analysis

A bench top pH meter (PC510, EUTECH) was used to measure the pH of the solution during the
experiment. Cr(VI) concentration of the samples was analyzed spectrophotometrically by the
standard 1,5-diphenylcarbizide method (Tandukar et al., 2009). The absorbance of pink coloured
Cr(VI)-complex was taken at the wavelength 543 nm by using an UV-Visible spectrophotometer
(Evolution-201,Thermo Scientific).

2.6. Characterization

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Since biosorption favorably a physisorption process, the functional groups present in the biomass
are responsible for the biosorption kinetics. Therefore, the functional groups present in the
microalgae biomass used as biosorbent was analyzed using a Fourier Transmission Infrared
(FTIR) spectrophotometer (JASCO-4600). Also the FTIR analysis was performed for the Cr(VI)
loaded biomass obtained from the experimental runs. Pellets for the FTIR analysis was prepared
by mixing 100 mg KBr with 1 mg microalgae biomass. The FTIR spectrum was taken from 500-
4000 cm-1. The SEM-EDX analysis of the microalgae biomass was performed by using a Zeiss
Olympus FESEM (Carl Zeiss, Supra gemini55) attached with an EDS microprobe.

2.7. Kinetic Study

Kinetic interpretation provides the mechanism of biosorption. The kinetic parameters help in
deciding the design of a reactor in terms of the residence time and dimension of the reactor.
Therefore, predicting the differential order of the process is very important.

The biosorption capacity(qt in mg.g-1) of Cr(VI) at different time interval was evaluated using

the formulae given in Eq.1 (Maleki et al., 2015).

(C0 ‒ Ct) × V
qt = m
(1)

where, Co=initial Cr(VI) concentration(mg.L-1); Ct=final Cr(VI) concentration(mg.L-1); m=mass

of biosorbent(g); V=volume of solution(L).

The formulae given in following Eq.2 was used to calculate the equilibrium biosorption capacity
(qe in mg.g-1):

(C0 ‒ Ce) × V
qe = m
(2)

where, Ce=Cr(VI) concentration(mg.L-1) at equilibrium.

The efficiency of biosorption(%) was calculated from the formulae in Eq.3 (Pradhan et al.,
2009).

(
(C0 ‒ Ct)
%= C0 ) × 100 (3)

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The biosorption data were calculated using the above formulae. They were interpreted with
different adsorption kinetic models. Standard adsorption isotherm models like Langmuir and
Freundlich models were used to interpret the data. Further, the thermodynamic feasibility of the
biosorption experiment was evaluated.

3. Results and Discussion

3.1. FTIR analysis

Different chemical groups like hydroxyl, carbonyl, sulfhydryl, sulfonate, etc. have been
identified for the biosorption of heavy metals (Dittert et al., 2012). In order to find out the
functional groups present in the microalgae biomass and their behavioral changes after Cr(VI)
adsorption, the FTIR analysis was performed for both original and Cr(VI) loaded biomass. Fig.1
shows the FTIR spectral data of both biomasses. The Fig. 1 displays many peaks and valleys,
indicating the complex nature of the biomass (Elangovan et al., 2008). The wave number (cm-1)
of the major absorption peaks obtained from the FTIR analysis and the predicted functional
groups of both original and Cr(VI) loaded biomass are given in Table 1. The peak at 3543.56cm-1
may be related to N-H stretching of the amide (Basha et al., 2008). The peak at 3400.85cm-1
shows the presence of O-H stretching with hydrogen bonding in either alcohols or phenols
(Sheng et al., 2004). The natural proteins and cellulose present in the microalgae cell wall
generally contain the functional groups like N-H and O-H (Dittert et al., 2012). The peak at
2958.27cm-1 may be related to the aliphatic C-H stretching which represents the aliphatic organic
chains of the cellulose (Sathvika et al., 2016). The peak at 2924.52cm-1 may represent the O-H
stretching of the carboxylic acid (Dittert et al., 2012). This defines the presence of an acidic
group such as –COOH in the cell wall of the biomass as the algae generally contains alginic acid
which is a hyper chemical compound for the biosorption of different multivalent metals
(Bertagnolli et al., 2014). The peak at 2852.21cm-1 represents the C-H stretching in different
aldehydes (Silverstein et al., 1981). The peak at 1658.42cm-1 may be related to the >C=O
stretching in the conjugated aldehyde/amide bend proteins which are key factors for the
biosorption (Silverstein et al., 1981). The peak at 1536.98cm-1 represents the >C=O stretching in
different carboxylic acids (Dittert et al., 2012). The peaks at 1454.064 and 1402.96cm-1 may be
related to the >C=O stretching in the amide groups (amide I and II bands) (Elangovan et al.,
2008). The peak at 1148.41cm-1 may represent either the characteristic absorption peak of

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phosphate group or the C-O stretching in the alcohols/aldehydes (Silverstein et al., 1981). The
peaks at 1117.54, 668.21 and 599.75cm-1 may be related to the organic halide compounds
(Silverstein et al., 1981). From the Fig.1 and Table 1, it can be observed that the microalgae
biomass contained different functional compounds like aldehydes, amides, acids, phosphates,
and halides. They might compensate the Cr(VI) uptake onto their host biomass from the solution.
By comparing the FTIR data of the original biomass with the Cr-loaded biomass residue (Fig.1
and Table 1), it can be observed that the peaks of some functional groups shifted towards the
lower energy. When a metallic ion attached to a functional group of the organic compounds, it
attracts the electron cloud from the functional group resulting in the decrease of electron density
and the increase of the bond length (Elangovan et al., 2008; Sathvika et al., 2016). Therefore, the
absorption peaks of the Cr-loaded biomass residue sifted towards the lower energy (Bertagnolli
et al., 2014).

3.2. SEM-EDX analysis

Figs. 2(a&b) show the surface texture and morphological characteristics of the microalgae
biomass before and after biosorption, respectively. They show the biomass surface are irregular
and porous empowering the large interface for the heterogeneous biosorption. The biomass
surface became hazy after the Cr(VI) adsorption (Fig.2(b)). The qualitative SEM-EDX analysis
of the biomasses before and after biosorption are shown in Fig.3(a&b). Elemental composition of
the biomass was found to be C, O, Mg, Al, Si, P, S, Ca and Cr with C and O together 83.57%.
From Fig.3(a&b) it can be observed that the peaks for Cr ranged between 5 and 6 keV. The Cr
peaks are broadened in case of the biomass after biosorption compared to that of the before
biosorption. Also the amount of Cr in the biomass increased from 0.38 to 1.14% during the
biosorption. This indicated that substantial amount of Cr(VI) was loaded onto the microalgae
biomass (Sathvika et al., 2016). The surface areas of the original and Cr-loaded biomass were
analyzed using a BET method and they were respectively found to be 27.18 and 24.92 m2.g-1.
The decrease in surface area of the Cr-loaded biomass described the occupancy of Cr(VI).

3.3. Effect of contact time

The biosorption of Cr(VI) using the microalgae biomass was conducted by varying different
contact times, such as 30, 60, 90, 120, 150, 180, 210, 240, 270 and 300 min, while other

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parameters kept constant. Fig.4 shows the plot of Cr(VI) concentration and biosorption
efficiency with respect to the contact time. It can be observed that the biosorption rate was
initially very fast up to 90 min with the efficiency of biosorption just above 90%. Further
increasing the contact time up to 300min it added only 3% more Cr(VI). The fast rate accounted
for 90.18% of the Cr(VI) biosorption within 90min only. The biosorption rate significantly
decreased after 90 min and accounted 93.1% removal up to 120min, thereafter the biosorption
rate was too slow to quantify. Hence, the biosorption time 120min was considered as the
equilibrium point of the biosorption experiment. The quick equilibrium time may be due to the
finer particle size of the biomass (Basha et al., 2008). Therefore rest of the biosorption
experiments varying other parameters were conducted for 120min. Since the biosorption process
is a biphasic system, the initial fast rate was observed due to the adsorption of Cr(VI) onto the
functional groups present at the biomass surface (Sibi, 2016). Initially the surface binding sites of
the biomass were vacant and the Cr(VI) concentration was high, which accelerated the Cr(VI)
uptake up to 90min and vice versa. However, slow rate of biosorption attributed to the
cytoplasmic membrane transportation of Cr(VI) followed by its diffusion onto the internal
binding sites of the biomass (Basha et al., 2008).

3.4. Effect of initial pH

The initial pH of the solution is important for the biosorption of Cr(VI) as protonation of the
biosorbent configures the active ion-exchange sites and surface activity (Sibi, 2016). Also pH
regulates the charge of the adsorbate. As discussed in the FTIR analysis, the biomass contained
easily hydrolyzed groups like amine and aldehydes. These two groups are easily protonated and
they develop more binding sites on acidification of the biomass(Elangovan et al., 2008).
Therefore, the effect of initial pH on the Cr(VI) biosorption was examined by varying pH of the
solution from 0.5 to 5.0, while other parameters kept constant. The biosorption capacity and
efficiency during the pH variation experiment were evaluated for different initial pHs. Fig.5
shows the graph of biosorption capacity and efficiency plotted versus initial pH of the solution.
The biosorption capacity reached a maximum of 0.093mg.g-1 at pH 1.0. With the increase of pH
from 1.0 to 5.0 it decreased steadily and reached 0.028mg.g-1 at the pH-5.0. Saha and Orvig,
(2010) described that the anionic adsorption favored the maximum Cr(VI) upload onto the
protonated biomass surface at the lower pH. The maximum biosorption efficiency was 92.9% at

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the pH-1.0. At the lower pH the biomass surface containing amide, carboxyl, halide and
hydroxyl groups protonated and became positively charged(Sathvika et al., 2016). At the same
time anionic species of Cr(VI), such as tetraoxohydrochromate (HCrO4-), chromate (CrO42-) and
dichromate (Cr2O72-) ions, were exist in the acidic solution at lower pH (Pradhan et al., 2017).
The positively charged biomass surface attracted the anionic species of Cr(VI) electrostatically,
resulted a strong physisorption of Cr(VI) onto the biomass at the lower pH range (Sathvika et al.,
2016). When pH of the solution was gradually increased, surface of the biomass became
negatively charged due to the decreasing in the proton concentration. The negatively charged
biomass competed with the anionic chromate ions due to the electrostatic repulsion, resulting in
the decrease of biosorption efficiency at the higher pH range (Saha and Orvig, 2010). This was
further confirmed by the evaluation of point of zero charge from the pHZPC curve which is shown
in Fig.6. The point of zero charge pH was found to be 2.65. It explained that the positive charge
of the biosorbent sustained until pH 2.65 and above which the biosorbent charge became
negative (Tyagi and Khandegar, 2018). The amount of acid consumed(g) at different initial pH
of the solution was evaluated. Fig.7 shows the graph of the amount of acid consumed plotted
versus the equilibrium biosorption capacity at different initial pHs. The biosorption capacity was
directly proportional to the amount of acid consumed for the experiments conducted at the lower
pH range (e.g. 0.5<pH<3.0). However, the biosorption conducted at the higher pH range (e.g.
3.0<pH<5.0) did not consume acid significantly.

3.5. Effect of biosorbent dosage

The biosorbent dosage in terms of solid-liquid ratio or S/L (the amount of biomass in gram added
to 100mL solution,%(w/v)) was varied as 2, 5, 10, 15, 20, and 30%(w/v). The graph of
biosorption capacity and efficiency plotted versus S/L is shown in Fig.8. The Cr(VI) removal
efficiency increased rapidly from 45.9 to 92.9% with the increase of S/L from 2 to 10%(w/v),
and on further increase up to 30%(w/v) it slightly increased to 96%. Bermúdez et al., (2012)
described that the higher Cr-loading observed at lower biosorbent dosage is beneficial for the
industrial application. The increase of biosorption rate with the increase of S/L credited the
increasing surface area while more amount of biosorbent was added; however, the observed flat
curve for the biosorption rate, when biosorbent dosage above 15%(w/v), was due to the partial
agglomeration of the biomass particle resulting in the reduction of the effective surface area

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(Gupta and Rastogi, 2009). Maleki et al., (2015) described that the overlapping or aggregation of
biosorbent resulted the decrease of overall biphasic interface. Hence, the S/L of 10 %(w/v) was
chosen as an optimum biosorbent dosage for the Cr(VI) removal. The biosorption capacity
decreased steadily from 0.229 to 0.032 mg.g-1 with the increase of S/L from 2 to 30%(w/v),
respectively. There was an obvious result of decreasing in the biosorption capacity with the
increase of the biosorbent dosage (Fig.8) as it was calculated on the basis of amount of Cr(VI)
adsorbed per gram of biosorbent (Eq.2).

3.6. Effect of initial Cr(VI) concentration

Since biosorption is a physical process of mass diffusion at the interface of two phases, the initial
concentration of Cr(VI) as adsorbate has a major role in defining the biosorption capacity (Basha
et al., 2008). Fig.9 shows the graph of biosorption capacity and efficiency plotted versus the
initial concentration of Cr(VI). The biosorption rate was not affected significantly by the initial
Cr(VI) concentration increment (5 to 10mg.L-1) as it decreased slightly from 95 to 92.9%;
however, it decreased steadily from 92.9 to 50.2% with the increase of initial Cr(VI)
concentration from 10 to 50mg.L-1. Hence, the optimum initial Cr(VI) concentration was
10mg.L-1. The biosorption capacity increased from 0.04 to 0.12 mg.g-1 with the increase of initial
Cr(VI) concentration from 5 to 15mg.L-1, and to further increase up to 50mg.L-1 there was hardly
any change. This may be due to inadequate active sites in the biosorbent for the diffusion of the
increasing amount of Cr(VI) or their mutual collisions hindered the diffusion at the interface of
two phases or competing repulsion among them (Rezaei, 2016). Bermúdez et al., (2012)
described that the biomass surface has limited binding sites; upon completing the adsorption at
those sites further Cr-loading is not possible.

3.7. Effect of particle size

Since biosorption is a surface phenomenon, variation of the particle size of the biosorbent is
necessary for designing the bed dimension during scaling up of the process. The coarse sized
biosorbent particles favour the strength of adsorption bed; however, they have less active surface
area. Therefore, optimization of the particle size distribution of the biosorbent is an important
parameter. Four particle size distributions, such as -45, -75+45, -100+75 and +100µm,
respectively averaged to 45, 60, 87.5 and 100µm were used to evaluate the particle size effect on

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the biosorption process. The graph of the biosorption capacity and efficiency plotted versus the
average particle size of the biosorbent is shown in Fig.10. Both the biosorption capacity as well
as efficiency decreased with the increase of the particle size. Since the biosorption process was a
mass diffusion phenomenon at the interface of two phases, the finer particle size favored the
biosorption rate due to the larger exposure of potential active sites in the finer biosorbent
particles.

3.8. Effect of temperature

According to the adsorption mechanism, the physical adsorption is triggered thermodynamically

at the interface of two phases (Bermúdez et al., 2012). It depends on the randomness of adsorbate
species at the surface of the biosorbent. Therefore, variation of temperature has a major role in
defining the randomness at the interface of a biphasic system. The temperature was varied from
25 to 50oC in order to evaluate its effect on the Cr(VI) loading onto the microalgae biomass. Fig.
11 shows the graph of biosorption capacity and efficiency plotted versus the temperature. Both
the biosorption capacity as well as efficiency increased with the increase of temperature. The
biosorption rate steadily increased from 84.7 to 97.8% with the increase of temperature from 25
to 50oC. This may be due to the endothermic nature of the biosorption process (Gupta and
Rastogi, 2009). The rising temperature benefitted the biosorption rate due to either the
enhancement of binding sites on the biomass surface or the suppression of boundary layer
thickness on the cell surface making Cr(VI) easily approachable towards the active binding sites
or combination of both (Meena et al., 2005).

The biosorption kinetics depended on various parameters, i.e. initial pH, biosorbent dosage (S/L
ratio), initial Cr(VI) concentration [Cr(VI)], particle size (PS) and temperature (T). Therefore,
the rate equation can be written as follow (Pradhan et al., 2009):

n1 n2 n3 n4 n5
Rate = k × (pH) × (S/L) × [Cr(VI)] × (PS) × (T) (4)

where, n1, n2, n3, n4 and n5 are the dependence factors for initial pH, S/L, [Cr(VI)], PS and T,
respectively. The logarithm form of Eq.4 gives following Eq.5:

log(Rate) = log(k) + n1log(pH) + n2log(S/L) + n3log[Cr(VI)] + n4log(PS) + n5log(T) (5)

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The slopes of the logarithmic plots of five parameters plotted individually should give the
dependence factors. They were evaluated accordingly and are given in Table 2.

3.9. Kinetic studies

3.9.1. Pseudo-first order

The equation (Eq.6) gives the differential form of the pseudo-first order kinetic equation (Maleki
et al., 2015).

‒ ln (qe ‒ qt) = k1t ‒ ln(qe) (6)

where k1(in min-1) is the pseudo-first order rate constant. The values of k1 and –ln(qe) can be
evaluated from the slope and intercept a graph plotted between -ln(qe-qt) and t. The experimental
data were fitted in the plot. The equilibrium biosorption capacity was calculated from the
intercept and compared with the experimental value (Table 3).

3.9.2. Pseudo-second order

The differential form of the pseudo-second order kinetic equation is shown in Eq.7 (Gupta and
Rastogi, 2009).

1 1
qe ‒ qt
= k 2t + qe
(7)

1
where, k2(in g.mg-1.min-1) is the pseudo-second order rate constant. A graph of qe ‒ qt
plotted
1
versus t was plotted using the experimental data, which gave the slope and intercept as k2 and qe

, respectively. From the intercept the equilibrium biosorption capacity is calculated. All the data
are given in Table 3.

3.9.3. Elovich model

The Elovich equation is important for the qualitative characterization of the chemisorption
process (Saha and Orvig, 2010). The linear equation of the Elovich model is given in Eq.8.

1 1
qt = βln(αβ) + βln(t) (8)

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 ACCEPTED MANUSCRIPT

where, α= biosorption rate(mg.g-1.min-1); β= activation energy for the chemisorption(g.mg-1).

1 1
The slope and intercept of a graph of qt plotted versus ln(t) gives the values of β
and βln(αβ),

respectively. The experimental data were fitted with the graph and the values of β and α were
evaluated. The value of qe was evaluated using the formula in Eq.8. The data are given in Table
3.

3.9.4. Intra-particle diffusion model

The biosorption experiment of Cr(VI) using the porous biomass may follow the systematic steps,
i.e. (i) diffusion taking place in the solution; (ii) diffusion at the interface of biosorbent and
adsorbate; (iii) intra-particle diffusion within biosorbent particles; (iv) biosorption within the
particles (Pradhan et al, 2009). Since the biosorption was carried under a dynamic condition, the
step-(i) cannot be the rate determining step. Similarly the step-(iv) does not determine the rate as
the resultant accumulation of Cr(VI) was very fast. The steps- (ii)&(iii), which are related to the
intra-particle diffusion of the biosorbent particles, must be the rate determining step (Elangovan
et al., 2008). Therefore, the amount of Cr(VI) adsorbed should directly proportional to t0.5 as
related mathematically in Eq.9 (Gupta and Rastogi, 2009).

0.5
qt = kid × t (9)

where, kid is the intra-particle diffusion coefficient. The Eq.9 can be linearised to an equation
(Eq.10) as follows:

log(qt) = log(kid) + 0.5log(t) (10)

A graph of log(qt) plotted versus 0.5log(t) should give a straight line. The experimental data

were fitted with the plot and the value of kid was evaluated from the intercept. Also the value of
qe was calculated using the formula in Eq.10. The data are given in Table 3. The correlation
coefficient(R2) value suggested that the intra-particle diffusion mechanism at the boundary layer
controlled the biosorption process (Sathvika et al., 2016). The kid value demonstrated that owing
to the intra-particle diffusion mechanism, the enhancement in the biosorption rate sustained
(Elangovan et al., 2008).

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Comparing the correlation coefficient(R2) values in Table 3, the possibility of pseudo second
order kinetics was eliminated. The calculated values of the equilibrium biosorption capacity are
0.101, 0.134 and 0.177mg.g-1, respectively, for the pseudo-first order, Elovich model and intra-
particle diffusion model. However, the calculated value of the equilibrium biosorption capacity
for the pseudo-first order closely fitted to the experimental value i.e. 0.093mg.g-1.

3.10. Adsorption isotherm

Adsorption isotherm correlates the metal uptake per unit mass of biosorbent at the constant
temperature and metal concentration within the biphasic system at the equilibrium. It defines the
distribution and interaction of metals in the biphasic system at the equilibrium (Gupta and
Rastogi, 2009). It is defined by different parameters, such as initial adsorbate concentration in
the solution, biosorbent dosage, the relative adsorb abilities, and competition among the solutes
(Elangovan et al., 2008).

The Langmuir adsorption isotherm describes a monolayer biosorption of different metals onto
biosorbents. It is the common model used to characterize the heterogeneous metal biosorption. A
derived form of the Langmuir adsorption isotherm is shown in Eq.11 (Maleki et al., 2015).

Ce 1 Ce
qe
=K + (11)
LQ0 Q0

where, Q0= the maximum biosorption capacity(mg.g-1); KL= Langmuir constant(L.mg-1). In

order to find out the feasibility of the Langmuir isotherm for the Cr(VI) biosorption onto
Ce
microalgae biomass, a linear graph of qe
versus Ce was plotted using the experimental data

(Fig.12(a)). The values of Q0, and KL (Table 4) were calculated from the slope and intercept,
respectively. The Langmuir isotherm was further characterized by interpreting the Langmuir
equilibrium parameter (RL) as shown in Eq.12.

1
RL = 1 + K (12)
LC0

The value of RL was calculated using the values of KL and C0 according to the equation (Eq.17)
and found to be 0.146. The nature of biosorption process is characterized by the RL value, e.g.
unfavorable(RL>1); linear(RL=1); favorable(0<RL<1); irreversible(RL=0) (Maleki et al., 2015).

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The calculated RL value (0.146) indicated that the Cr(VI) biosorption of onto the microalgae
biomass was a favorable process. Further the RL value below unity suggested that the biosorption
process was reversible process (Sathvika et al., 2016).

The Freundlich adsorption isotherm describes the distribution of metals in the biphasic system of
solid and liquid at the point of saturation. The model is expressed in the form of a linear equation
(Eq.13) as follows (Maleki et al., 2015):

1
log(qe) = log(KF) + nlog(Ce) (13)

1
where, KF= biosorption capacity at unit concentration(mg.g-1) and n
= biosorption intensity. In

order to investigate its applicability for the Cr(VI) biosorption, a linear graph of log(qe) versus
1
log(Ce) was plotted using the experimental data (Fig.12(b)). The values of n
and KF (Table 4)
1
were respectively calculated from slope and intercept of the plot. The value of n
characterizes
1 1 1
the feasibility of the isotherm, e.g. irreversible( n=0); favorable(0< n <1); unfavorable (n >1). The
1
calculated n
value (0.447) favoured the Freundlich isotherm model.

The correlation coefficients(R2 values) were used as the fitting criteria for both adsorption
isotherm models. They were found to be above 0.99 for both models suggesting that the data of
isotherm interpretations well fitted to both models (Gupta and Rastogi, 2009). Further the RL
1
value for the Langmuir isotherm and the n
value for the Freundlich adsorption isotherm, as

shown in Table 4, confirmed the feasibility of the biosorption of Cr(VI) onto the microalgae
biomass.

3.11 . Thermodynamic interpretation

Thermodynamic terms, such as Gibbs free energy(ΔG), enthalpy(ΔH), and entropy(ΔS),

altogether define the spontaneity of a biphasic biosorption process (Gupta and Rastogi, 2009).
The relation among ΔG, ΔH, ΔS, and absolute temperature(T) is shown in Eq.14 (Bermúdez et
al., 2012).

∆G = ∆H ‒ T∆S (14)

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The ΔG values at different temperature can be computed by the law of thermodynamics as given
in Eq.15 (Maleki et al., 2015).

∆G =‒ RTln(Kc) (15)

where Kc is the dimensionless equilibrium constant. Since the Langmuir costant (KL=0.584
L.mg-1) was derived in the previous section, the dimensionless value of Kc can be computed by
the formulae given in Eq. 16 (Tran et al., 2017).

KC = 51996 × 55.5 × KL (16)

The value 51,996 is the atomic weight of Cr in mg.mol-1. The value 55.5 is the concentration of
water mol.L-1. The value of Kc was computed using the formulae in Eq.16 and found to be
1,685,294. The ΔG values at different temperature were calculated using the formulae in Eq.15
and are given in Table 5. Generally the ΔH and ΔS are calculated using the van’t Hoff equation
(Liu, 2009). However, they were calculated using Eq.14 in this article. A graph of ΔG plotted
versus T gives a straight line, and the slope and intercept are respectively -ΔS and ΔH. The
experimental data were fitted in the plot and the values of ΔH and ΔS were evaluated
accordingly and are given in Table 5.

The ΔG values were found to be negative at the experimental range of temperatures, which
suggested the spontaneity of the biosorption (Bermúdez et al., 2012). Also the negative ΔG value
suggested that the Cr(VI) concentration on the biomass was much more than in the solution
(Sathvika et al., 2016). Further, its values proved that the spontaneity elevated with the increase
of temperature. Maleki et al. (2015) described that the range of ΔG for the physisorption should
be between -20 and 0kJ.mol-1; however, that in the chemisorption are ranging from -80 to -
400kJ.mol-1. Therefore, the ΔG values at different temperature, as given in Table 5, suggested
that the biosorption process followed a mixed physico-chemical adsorption mechanism (Liu,
2009). The much less value of ΔH did not have any significance on the biosorption; however,
The positive value of ΔS suggested that the Cr(VI) biosorption ensued owing to the randomness
at the interface of adsorbate and biosorbent.

4. Conclusion

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The biomass of microalgae Scenedesmus sp. was found to be worthy in the biosorption of Cr(VI)
from solution. At the optimum condition (e.g. contact time,120min; S/L ratio,10%(w/v);
Cr(VI),10mg.L-1; pH,1.0; temperature,30oC; particle size,-75+45μm; stirring,300 rpm), 92.89%
of Cr(VI) was removed. The FTIR analysis revealed the presence of different functional groups
like aldehydes, amides, carboxylic acids, phosphates, and halides in the biomass. The point of
zero charge was found to be 2.65. It means the functional groups of biomass became positively
charged below the pH 2.65, which favoured the anionic adsorption. The SEM-EDX analysis
demonstrated the attachment of Cr(VI) onto the biomass. The variation of contact time showed
an initial fast rate followed by a slow rate. The high biosorption rate at lower biosorbent dosage
described the industrial applicability of the process at low cost. The mutual collisions among the
adsorbate ions at higher Cr(VI) concentration hindered the diffusion at the biphasic interface. It
means the process preferred the lower chromium concentration with the higher efficiency, which
is an advantage for in-situ application. The biosorption was endothermic nature. The pseudo-first
order kinetic model closely fitted to the experimental data describes no complication when it is
scaled up. The process was thermodynamically spontaneous and proceeded due to the
randomness of Cr(VI) ions at the biphasic interface. The feasibility of adsorption was confirmed
by both Langmuir and Freundlich isotherm models. However, more attention require in the
future for the implementation of the Cr(VI) biosorption in the pilot scale and in-situ operation
process.

Acknowledgements

The authors are grateful to Prof(Dr.)M.R.Nayak,President, SOA (Deemed to be University), for

providing the infrastructures and encouragement throughout. The authors have thanked
Dr.N.Pradhan of CSIR-IMMT, Bhubaneswar for providing the microalgae biomass.

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Table 1. Assignment of FTIR absorption peaks for the original and Cr(VI) loaded microalgae
biomass.

Characteristics absorption (cm-1)

Original Residue Types of vibration
Peak Intensity Peak Intensity
3543.56 Broad, - - N-H stretching amide
medium
3400.85 Broad, 3385.42 Broad, strong O-H stretching with hydrogen
strong bonding alcoholic/phenolic
2958.27 weak 2956.33 weak C-H stretching aliphatic
2924.52 Broad, 2913.91 Broad, strong O-H stretching carboxylic acid
strong
2852.21 weak 2856.06 weak C-H stretching aldehyde
1658.42 Broader, 1631.48 Broader, C=O stretching conjugated
strong strong aldehyde/amide
1536.98 Broader, 1532.16 Broader, C=O stretching in carboxyl
strong 1402.96 strong
1454.064 weak 1455.02 weak C=O stretching in carboxyl or amide
1402.96 -groups (amide I and II bands)
1148.41 Broad, 1138.75 Broad, strong Phosphate group characteristic
strong absorption peak or C-O stretching
alcohol/aldehyde
1117.54 Broad, 1117.54 Broad, strong C-F stretching
strong
668.21 medium 666.28 medium C-Cl stretching
599.75 medium 596.86 medium C-Br stretching

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Table 2. Value of dependence factors ‘n’ for the biosorption of Cr(VI) at different parameters.

Dependence factors ‘n’ Value

n1 -0.47
n2 0.25
n3 -0.61
n4 -0.76
n5 0.19

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Table 3. Data of the kinetic coefficients for Cr(VI) biosorption onto the microalgae biomass.

Coefficients Value
Kinetics model
qe experimental in mg.g-1 0.093
k1 in min-1 0.038
Pseudo first order qe calculated in mg.g-1 0.101
R2 0.992
k2 in g.mg-1.min-1 3.725
Pseudo second order qe calculated in mg.g-1 0.222
R2 0.772
α in mg.g-1.min-1 0.028
β in g.mg-1 35.71
Elovich model
qe calculated in mg.g-1 0.134
R2 0.991
kid 0.016
Intra-particle diffusion model qe calculated in mg.g-1 0.177
R2 0.982

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Table 4. Adsorption isotherm coefficients for the Cr(VI) biosorption onto the microalgae
biomass.

Langmuir isotherm model Freundlich isotherm model

Q0 KL KF 1
RL R2 R2
in mg.g-1 in L.mg-1 in mg.g-1 n
0.301 0.584 0.146 0.995 0.11 0.447 0.996

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Table 5. Thermodynamic parameters for the Cr(VI) biosorption onto microalgae biomass.

ΔG in kJ.mol-1 ΔH ΔS
Temperature in K in kJ.mol-1 in J.mol-1.K-1 R2
298 303 308 313 318 323

-35.5 -36.1 -36.7 -37.3 -37.9 -38.5 -5×10-12 119.2 0.99

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Transmittance (a.u.)

Original biomass
Cr(VI) loaded biomass

4000 3500 3000 2500 2000 1500 1000 500

Wavenumber in cm-1

Fig.1. FTIR analysis of the original and Cr(VI) loaded microalgae biomass.

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(a) (b)

Fig.2. FESEM analysis of the microalgae biomass: (a) before biosorption; (b) after
biosorption.

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(a)

(b)

Fig.3. EDX analysis of the microalgae biomass: (a) before biosorption; (b) after
biosorption.

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10 100
Cr(VI) concentration, mg.L-1

Removal of Cr(VI), %
8 80

Equilibrium concentration
6 60
% of removal

4 40

2 20

0 0
0 50 100 150 200 250 300

Time, min

Fig.4. Effect of contact time on the Cr(VI) removal. (Conditions: initial Cr(VI) conc., 10
mg.L-1; solid-liquid ratio, 10 %(w/v); initial pH, 1.0; Temp., 30 oC; particle size, -75+45
µm; stirring speed, 300 rpm)

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Adsorption capacity, mg.g-1

Removal of Cr(VI), %

100 0.250000004
% of removal
Adsorption capacity, mg/g 0.200000003
80

0.150000002
60
0.100000001

40
0.050000001

20 0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5
pH

Fig.5. Effect of initial pH on the Cr(VI) removal. (Conditions: initial Cr(VI) conc., 10 mg.L-
1; solid-liquid ratio, 10 %(w/v); contact time, 120 min; Temp., 30 oC; particle size, -75+45
µm; stirring speed, 300 rpm)

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6
Equilibrium pH-Initial pH

5 Initial pH
Equilibrium pH-Initial pH
4

1
2.
65
0
0.5 1 1.5 2 2.5 3 3.5 4 4.5 5
Initial pH

Fig.6 . pHZPC curve of biosorbent. (Conditions: initial Cr(VI) conc., 10 mg.L-1; solid-liquid
ratio, 10 %(w/v); contact time, 120 min; Temp., 30 oC; particle size, -75+45 µm; stirring
speed, 300 rpm)

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0.900000015
Acid cosumed, g

0.700000012

0.500000009

0.300000006

0.100000003

-0.1
0.02 0.04 0.059999999 0.079999999 0.099999998

Adsorption capacity, mg.g-1

Fig.7. Consumption of acid during Cr(VI) removal. (Conditions: initial Cr(VI) conc., 10
mg.L-1; solid-liquid ratio, 10 %(w/v); contact time, 120 min; Temp., 30 oC; particle size, -
75+45 µm; stirring speed, 300 rpm)

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Adsorption capacity, mg.g-1

100 0.250000004

0.200000003
Removal of Cr(VI), %

80
% of removal
Adsorption capacity, mg/g
0.150000002
60
0.100000001

40
0.050000001

20 0
0 5 10 15 20 25 30

Solid-liquid ratio %(w/v)

Fig.8. Effect of biosorbent dosage on the Cr(VI) removal. (Conditions: initial Cr(VI) conc.,
10 mg.L-1; initial pH, 1.0; contact time, 120 min; Temp., 30 oC; particle size, -75+45 µm;
stirring speed, 300 rpm)

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 ACCEPTED MANUSCRIPT

Adsorption capacity, mg.g-1

100 0.250000004
Removal of Cr(VI), %

% of removal
Adsorption capacity, mg/g 0.200000003
80

0.150000002
60
0.100000001

40
0.050000001

20 0
0 10 20 30 40 50 60

Initial Cr(VI) conc., mg.L-1

Fig.9. Effect of initial Cr(VI) concentration on the Cr(VI) removal. (Conditions: solid-
liquid ratio, 10 %(w/v); initial pH, 1.0; contact time, 120 min; Temp., 30 oC; particle size, -
75+45 µm; stirring speed, 300 rpm)

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100 0.250000004

Adsorption capacity, mg.g-1

Removal of Cr(VI), %

0.200000003
80
% of removal
Adsorption capacity, mg/g
0.150000002

60

0.100000001

40
0.050000001

20 0
40 60 80 100

Particle size, µm

Fig.10. Effect of particle size on the Cr(VI) removal. (Conditions: initial Cr(VI) conc., 10
mg.L-1; initial pH, 1.0; contact time, 120 min; Temp., 30 oC; solid-liquid ratio, 10 %(w/v);
stirring speed, 300 rpm)

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 ACCEPTED MANUSCRIPT

Adsorption capacity, mg.g-1

Removal of Cr(VI), %

100 0.250000004

0.200000003
80 % of removal
Adsorption capacity, mg/g
0.150000002
60
0.100000001

40
0.050000001

20 0
20 25 30 35 40 45 50 55
Temperature, oC

Fig.11. Effect of temperature on the Cr(VI) removal. (Conditions: initial Cr(VI) conc., 10
mg.L-1; initial pH, 1.0; contact time, 120 min; particle size, -75+45 µm; solid-liquid ratio, 10
%(w/v); stirring speed, 300 rpm)

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(a) 25

Ce/qe
20

15

10

5
0 1 2 3 4 5 6
Ce

-0.399999988
(b)
Log(qe)

-0.799999994

-1.2
-0.2 3E-9 0.200000006
0.400000009
0.600000012
0.800000015
Log(Ce)

Fig.12. Adosorption isotherm curves: (a) Langmuir and (b) Freundlich.

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