3 Biotech (2021) 11:491

[Link]

ORIGINAL ARTICLE

Characterization of an exopolysaccharide produced by Enterobacter

sp. YU16‑RN5 and its potential to alleviate cadmium induced
cytotoxicity in vitro
Bythadka Erappa Dhanya1 · Athmika1 · Punchappady Devasya Rekha1

Received: 18 May 2021 / Accepted: 18 October 2021 / Published online: 9 November 2021
© King Abdulaziz City for Science and Technology 2021

Abstract
Natural biopolymers have gained remarkable attention for bioremediation particularly in heavy metal removal and oil
degradation due to their non-toxic nature and lack of secondary pollution. The exopolysaccharides (EPS) produced by the
bacteria have become an important class of biopolymers that are employed in bioremediation. The bacteria isolated from
the rhizospheric soil have higher metal tolerance and their EPS are effective in biosorption of heavy metals. Here, we report
the characterization of an EPS (EPS-RN5) isolated from the root nodule-associated bacteria, Enterobacter cancerogenus
strain YU16-RN5 and its heavy metal biosorption abilities. The bacteria isolated from the West coast of India was cultured
in yeast extract mannitol (YEM) medium for EPS extraction and to study the production kinetics on a temporal scale. The
biochemical composition, rheological properties and thermostability of EPS-RN5 was characterized by standard methods.
The biosorption potential of EPS-RN5 against the selected heavy metals was analyzed by employing the inductively cou-
pled plasma atomic emission spectroscopy (ICP-AES) technique. Further, cell culture experiments were used to test the
role of EPS-RN5 in reducing the cytotoxicity exerted by the heavy metals in vitro using a human embryonic kidney cell
line (HEK 293T). The bacteria showed good growth in YEM media and the maximum EPS yield was 1800 mg/L at 96 h.
The molecular weight of EPS-RN5 was 0.7 × ­106 Da and it contained 61.5% total sugars and 14.5% proteins. The monosac-
charide composition of the EPS included glucose, sorbose and galactose in the ratio 0.25:0.07:1.0. The EPS-RN5 showed
high thermal stability with a degradation temperature of 273 °C. Rheological analysis revealed the non-Newtonian behavior,
with pseudoplastic characteristics. The EPS-RN5 efficiently absorbed cadmium and other heavy metals such as mercury,
strontium, copper, arsenic, and uranium. In vitro studies revealed significant protective effect against the cadmium-induced
cytotoxicity in HEK 293T cells. These results indicate the potential applications of EPS-RN5.

Keywords Exopolysaccharide · Enterobacter cancerogenus · Biosorption · Heavy metals · Cytotoxicity

Introduction copper, nickel, cobalt, chromium, zinc and arsenic are toxic
to living organisms. For instance, cadmium is known to have
Heavy metals are stable in nature and persist in the envi- numerous undesirable effects on both humans and animals.
ronment for longer periods due to their nondegradability. It exerts toxic effects on multiple organs including the liver,
The recalcitrant property of the heavy metals leads to the kidney, pancreas and can adversely affect their functions
accumulation or biomagnification in the food chain caus- (Zhang et al. 2017). Several strategies such as reverse osmo-
ing serious health and ecological risks (Gutnick and Bach sis, ion exchange, chemical coagulation and ultra filtration
2000). Heavy metals and metalloids such as cadmium, among others are adopted for the removal of heavy metal
ions from the contaminated environment (Renu and Singh
2017; Sun et al. 2020a, b).
* Punchappady Devasya Rekha Biological methods such as phytoremediation, bioac-
rekhapd@[Link] cumulation, biocoagulation, bioleaching, biosorption and
1 bioimmobilization are introduced to remove heavy metals
Yenepoya Research Centre, Yenepoya (Deemed
to be University), University Road, Deralakatte, from the contaminated sites as they are environmentally
Mangalore 575018, India safer than the chemical methods (Rahman and Singh 2019;

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Rebello et al. 2021). Natural biopolymers have a greater β-glucan production (Nakanishi et al. 1976). Taxonomic
ability to immobilize heavy metals due to the presence of identification was done by 16S rRNA gene sequencing. The
chelating groups. Biopolymers from the bacterial origin such sequence data were aligned and compared with available
as exopolysaccharides (EPS) have metal-binding proper- standard sequences of bacterial lineage in the web-based
ties (Kaplan et al. 1987; Aryal et al. 2010; Raklami et al. EzTaxon-e server (Kämpfer et al. 2018). The 16S rRNA
2020). Some of the important bacterial EPS that facilitate sequence was submitted to GenBank under the accession
heavy metal biosorption are alginate (Pseudomonas aer- number MH191375.
uginosa, Azotobacter vinelandii), gellan (Sphingomonas
paucimobilis), hyaluronan (Pseudomonas aeruginosa, Pas- Extraction and purification of EPS
teurella multocida, Streptococci attenuated strains), xan-
than (Xanthomonas campestris), galactopol (Pseudomonas For the EPS production, the isolate was grown in YEM broth
oleovorans) and fucopol (Enterobacter A47) (Öner 2013). at 37 °C for 96 h using a shaker set at 100 rpm. After incu-
Carboxyl, amino and sulfonate groups in alginate are respon- bation, the culture broth was centrifuged at 8000 rpm for
sible for the metal ion binding by ion exchange mechanisms 10 min to separate the cells and the cell-free supernatant
(Bertagnolli et al. 2014). The positively charged metal ions was mixed with thrice its volume with chilled ethanol. The
are chelated more efficiently by the anionic biopolymers. contents were kept overnight at 4 °C and the precipitated
This is one of the primary mechanisms involved in metal EPS was separated by centrifugation at 8000 rpm for 10 min
biosorption. (Quesada et al. 1994). The collected EPS was dissolved in
This study reports the potential heavy metal biosorp- MilliQ water and re-precipitated to remove the remnants of
tion property of an EPS (EPS-RN5) produced by a coastal media contaminants. The precipitated EPS was separated by
rhizosphere-associated bacterium Enterobacter sp. strain centrifugation and dialyzed using a 12 kDa cut-off dialysis
YU16-RN5. The Inductively coupled plasma atomic emis- membrane (HiMedia, India) against MilliQ water for 48 h
sion spectroscopy (ICP-AES) and FT-IR were used to char- with intermittent changes of water. The dialyzed EPS was
acterize the heavy metal absorption mechanisms of EPS. lyophilized and the yield was determined (dry weight basis).
In addition, using the in vitro cell culture experiments the The EPS obtained was designated as EPS-RN5 and used for
possible protective role of EPS-RN5 in alleviating the cyto- further studies.
toxicity of cadmium on HEK 293T cells was established.
Biochemical and structural analysis of EPS

Materials and methods Total sugar content in EPS-RN5 was measured by phenol
sulfuric acid method (Dubois et al. 1956) and protein content
Isolation and identification of an exopolysaccharide by Lowry’s method (Lowry et al. 1951). Uronic acid was
producing marine bacterial strain estimated by treating the EPS with tetraborate (12 mM) in
concentrated sulfuric acid and m-hydroxy diphenyl reagents
The bacterial strain YU16-RN5 was isolated from the root (Blumenkrantz and Asboe-Hansen 1973). For structural
nodules of a coastal dune plant, Derris elliptica from the characterization, FT-IR spectral analysis was used by scan-
coastal region of Mangalore (12°47′ 10.3524″ N 74˚51′ ning in the wavenumber range of 4000–400 ­cm−1 using an
12.4344″ E). The root nodules were separated from the Alpha e-Bruker spectrometer (Bruker Optik GmbH, Ettlin-
roots of the plant, washed with sterile water thoroughly to gen, Germany).
clean the dirt, immersed in 70% ethanol for 30 s and washed
twice with sterile distilled water (Mendes et al. 2007). Later, Estimation of molecular weight by gel permeation
it was macerated and the suspension obtained was serially chromatography
diluted (ten-fold) in sterile normal saline (0.9% NaCl).
From this 100 μL sample suspensions were inoculated into For the determination of molecular weight, gel permea-
yeast extract mannitol (YEM) agar plates (Himedia, India) tion chromatography (GPC) technique was used. Briefly,
(Reis et al. 2004). The plates were incubated at 37 °C up to the Sephacryl S-500 HR column (XK 26 mm/100 cm) was
5 days, and colonies showing mucoidal morphology were eluted with 0.1 M Tris–HCl buffer (pH 7.2). The column was
pure cultured and preserved in 30% (v/v) glycerol at −80 °C calibrated using a mixture of blue dextran standards (Sigma,
(Rosalam and England 2006). As an initial screening test USA) with a range of molecular weights (50, 150, 670 and
for β-glucan production, the bacterial isolate was streaked 20,000 kDa). The lyophilized EPS-RN5 solution (1.0 mg/
in YEM agar plates supplemented with 0.05% aniline blue mL) (0.50 mL) was added to the column and eluted with
and incubated at 37 °C for 48 h. The plates were observed 0.1 M Tris HCl buffer at a flow rate of 1.0 mL/min. Eluted
for blue-colored colonies which indicate the presence of fractions (0.5 mL each) were collected and analyzed for total

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sugars using phenol–sulfuric acid method (Dubois et al. the shear stress (Pa), K is the consistency index (Pa sn), γ
1956). The molecular weight was estimated from the graph is the shear rate (­ s−1), and n is the flow index (dimension-
plotted using the blue dextran molecular weight standards less) (Steffe 1996). The flow curve was constructed using
against the elution volume as previously described elsewhere GraphPad Prism 8.0.2.
(Insulkar et al. 2018; Sajna et al. 2013).
Quantification of heavy metal biosorption
Thermogravimetric analysis
Heavy metal biosorption ability of the EPS-RN5 was evalu-
Thermogravimetric analysis (TGA) of EPS-RN5 was car-
ated against mercury, cadmium, strontium, copper, arsenic
ried out in a thermal analysis system (TG–DTA/DSC Model:
and uranium ions. For this, mercuric sulfate, cadmium chlo-
Q600 SDT). For this, about 7 mg of lyophilized EPS sample
ride, strontium chloride, copper chloride, sodium arsenate
was used and subjected to a temperature range of 30–700 °C
and uranyl nitrate solutions of 100 mg/L concentration was
with a temperature increment of 10 °C/min under the flow of
prepared in MilliQ water. All the chemicals employed in this
nitrogen. The degradation pattern was studied by plotting the
study were of analytical grade. EPS-RN5 at a concentration
weight (percentage) loss and heat flow against temperature
of 1% (w/v) was added to metal ion solutions and allowed to
(Wang et al. 2010). The graph was constructed using Origin
react for 30 min. The solution was centrifuged at 8000 rpm
2017 SR2 software.
for 10 min and the precipitate obtained due to metal interac-
tion with the EPS was digested with nitric acid and diluted
Monosaccharide analysis of the EPS‑RN5 by Gas with MilliQ water. The concentrations of absorbed metal in
chromatography – Mass spectrometry the precipitate and metal ions in the supernatant were deter-
mined using Inductively Coupled Plasma Atomic Emission
The monosaccharide composition of the EPS-RN5 was
Spectroscopy (ICP-AES) (ARCOS, M/s. Spectro, Germany).
analyzed by Gas chromatography—Mass spectrometry
The metal uptake (qe) was determined as follows according
(GC–MS). For this, 1.0 mg purified sample was subjected
to previously described methods (Zewail and Yousef 2015;
to methanolysis using methanolic TFA (3 M) for 4 h at
Omorogie et al. 2012; Zhao et al. 2015):
100 °C. The mixture was dried under nitrogen and dis-
solved in dichloromethane (DCM) and dimethylacetamide. V(Ci − Cf )
qe =
The content was evaporated under nitrogen and re-dissolved W
in N, O-bis (trimethylsilyl)—trifluoroacetamide-trimethyl
where V = the volume of mixture solution (L), Ci = the initial
chlorosilane (BSTFA-TMCS) in DCM and kept at 60 °C for
concentration of metal in mixture solution (mg/L), Cf = the
1 h. The derivatized sample was analyzed using a GC–MS
equilibrium concentration of metal in mixture solution
system (Agilent 7890 GC with 5975 C MS, USA) with DB
(mg/L), W = the dry weight of EPS (g), The graph was con-
5 ms 30 × 0.25 mm column (Agilent Technologies). For MS
structed using GraphPad Prism 8.0.2.
analysis, 1.0 µL sample was injected using a splitless injector
with injector temperature maintained at 260 °C. Oven pro-
gramming included an initial column temperature of 50 °C In vitro studies to evaluate the protective effect
for 2 min followed by a ramp at 10 °C/min to 310 °C and of EPS‑RN5 against the cytotoxicity of cadmium
held at 310 °C for 7 min. Monosaccharides were identified in HEK 293T cells
using a MS detector in full scan mode in the molecular mass
range of 350–700 m/z. Monosaccharides were identified by For in vitro studies, a human embryonic kidney cell line
comparing them with the standard mass spectra available in (HEK 293T) obtained from National Centre for Cell Sci-
the NIST 11 mass spectral library (Blau and Halket 1993). ence, Pune, India was used. The cells were maintained in
Inositol was used as an internal standard. Dulbecco’s minimum essential medium (DMEM) contain-
ing 1.0 mM sodium pyruvate, l-glutamine, 4.5 g/L glucose
Rheological characterization and 1.5 g/L sodium bicarbonate supplemented with 10%
fetal bovine serum (FBS) in 5% ­CO2 atmosphere at 37 °C
To evaluate the rheological properties, EPS-RN5 was dis- (Forma STERICYCLE 371, C ­ O2 incubator, Thermo Sci-
solved in MilliQ water and diluted to 0.5% (w/v) concen- entific, USA). Cells were used for the experiments within
tration. The viscosity of the solution was measured over a three passages. The exponentially growing HEK 293T cells
range of shear rates (96–768 ­s−1) at 25 °C using a Brookfield were trypsinized and seeded to 96-well plates at a density
DV3T Rheometer (Brookfield, USA) equipped with spin- ­ O2 incu-
of 5000 cells/well and incubated for 24 h in a C
dle CP-51. The flow (n) and consistency (K) index were bator (5% ­CO2, 37 °C). To study the effect of EPS-RN5
determined using the Power-law model τ = Kγn where τ is in reducing the cytotoxic effect, EPS solutions at 25 and

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50 μg/mL were sterilized using 0.22 µ syringe-driven fil- the requirement of complex media composition are one of
ters. These were added to the cells exposed to cadmium (10, the interesting features of Enterobacter strains.
20, and 40 μg/mL). The contents were incubated for 48 h.
The wells containing HEK 293T cells with only EPS were
maintained as positive control and those with only cadmium Biochemical and structural characteristics
was maintained as a negative control. Following incubation, of EPS‑RN5
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazo-
lium bromide) at a concentration of 1.0 mg/mL was added The total sugar content of the EPS-RN5 on a dry weight
to the assay wells and incubated at 37 °C for 4 h in dark. basis was 61.4 ± 2.3% that is comparable with the EPS pro-
The formazan crystals were solubilized in DMSO and the duced by Enterobacter sp. PRIM-26 (62%) (Priyanka et al.
absorbance was recorded using a multimode plate reader 2015). The EPS-RN5 contained 14.5 ± 2.9% proteins and
(FLUOstar Omega) at 570 nm (Mosmann 1983). Cytotoxic- small amounts of uronic acid (8.3 ± 0.3%). The presence of
ity was measured using the following equation: uronic acid groups in the polysaccharides imparts bioac-

Absorbance of the control − Absorbance of the test

Cytotoxicity(%) = × 100
Absorbance of the control

For visualization of the data, a graph was constructed tivity having implications in regenerative medicine, tissue
using GraphPad Prism 8.0.2 and the micrographs obtained engineering and also act as anti-thrombotic and anti-arthritic
were arranged using CorelDraw Technical Suite. agents (Silvi et al. 2013). The isolate Enterobacter sp.YU16-
RN5 produced EPS with a molecular mass of 0.7 × ­106 Da.
However, the EPS produced by Enterobacter sp.YG4 had a
molecular mass of 11.99 × ­106 Da (Nagaraj et al. 2016), the
Results and discussion EPS of Enterobacter cloacae and Enterobacter strain A47
had a molecular mass of 1.7 × ­106 and 5.8 × ­106 Da, respec-
EPS production by the bacterial isolate tively (Meade et al. 1994; Freitas et al. 2009). It has to be
noted that the genus Enterbacter produces high molecular
The isolate YU16-RN5, was a gram-negative bacterium
identified by 16S rRNA gene sequencing as Enterobacter
sp. (MH191375) with 100% similarity to Enterobacter can-
cerogenus. The species used for the study was isolated from
the urban coastal region of Mangalore (South-West coast
of India) that is exposed to heavy metal contamination by
various anthropogenic activities. The bacteria isolated from
these regions generally have adaptations to such hostile envi-
ronment and the EPS produced by them act as metal chelat-
ing agent rendering a natural survival mechanism against
heavy metal exposure. There has been a similar study that
reports the heavy metal tolerance by a few bacterial com-
munities associated with the monazite sand of Someshwara
beach (Shreedhar et al. 2014). The isolate Enterobacter
sp. YU16-RN5 grown on YEM agar plates showed copi-
ous mucoidal pale white colonies at 37 °C at 96 h of incu-
bation (Fig. 1a). On the YEM agar media supplemented
with aniline blue, the bacteria formed blue-colored mucoid
colonies indicating the EPS as β-glucan-like biopolymer
(Fig. 1b). The β-glucans are industrially important biopoly-
mers with a range of therapeutic applications (Moscovici
2015). The maximum yield of the EPS in the YEM media Fig. 1  EPS production by the bacterial isolateYU16-RN5. a Mucoid
was 1800 mg/L at 96 h (Fig. 1c). This yield is compara- colonies of YU16-RN5 grown on yeast extract mannitol (YEM) agar.
b Bacteria colonies grown in YEM media with 0.05% aniline blue are
ble with the EPS produced by Enterobacter cloacae WD7 stained blue due to the presence of β-glucan. c Graph showing the
(1680 mg/L) (Prasertsan et al. 2006) and Enterobacter A47 growth pattern of YU16-RN5 in YEM broth and EPS yield at differ-
(1810 mg/L) (Marques 2017). Higher EPS yields without ent time points

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weight EPS which often leads to increased viscosity and is weight loss of 12% within 153 °C and in the second step
considered as one of the most desirable rheological features depolymerization at 273 °C, with 49% loss was observed.
(Herbst et al. 1992). This depolymerization temperature of EPS-RN5 is compa-
The FT-IR spectrum of the EPS-RN5 showed a charac- rable to that of xanthan gum (278 °C with 30% of weight
teristic peak of polysaccharide at 3289–3742 ­cm−1, repre- loss), KF5 EPS (279.6 °C) and locust gum (278.5 °C)
senting O–H stretching of hydroxyls (Fig. 2a). The band (Wang et al. 2010). The stability of EPS-RN5 at higher
at 3420 ­cm−1 represents the presence of N–H group. The temperatures makes it a promising additive as a stabilizer,
well-defined peaks found between 1200 and 900 ­cm−1 rep- thickener and gelling agent in value-added products.
resents skeletal C–O and C–C vibration of glycosidic bonds
and pyranoid rings. The peak at 1250 ­cm−1 may also be
attributed to the C–O–C vibration of acyls. The two strong Monosaccharide composition
bands around 1607 and 1405 ­cm−1 in the spectrum can be
attributed to the asymmetric and symmetric stretching of From the GC–MS analysis the monosaccharide composition
carboxylates, respectively (Synytsya et al. 2003). The band of EPS-RN5 was identified as glucose, sorbose and galactose
at 1720 ­cm−1observed is attributed to the C = O stretching of in the ratio 0.25: 0.07: 1.0 (Fig. 3). The EPS also contained
carbonyls in acyl groups (Alvarez-Manceñido et al. 2008). low amounts of mannoonic acid, pentanoic acid, ribitol, tri,
However, the ionizable functional groups such as carboxy, octa and hexadecanoic acids. Whereas, EPS of Enterobacter
carbonyl and amide groups can contribute to the binding sp. A3CK showed mannose, glucose and galactose and that
mechanism of cadmium by EPS (Camacho-Chab et al. of E. cloacae strain P2B was reported to contain mannose,
2018). The peak at 894 ­cm−1 is assigned to β-glycosidic glucose, galactose and xylose (Naik et al. 2012). However,
linkage and a peak at 1721 ­cm−1 indicates the presence of the sugar monomers of EPS of Enterobacter sp. MS16 was
uronic groups (Wang et al. 2015). composed of glucose, galactose, and arabinose (Jhadhav
et al. 2011) and that of Enterobacter A47 contained glucose,
Thermal stability of the EPS‑RN5 galactose and fucose, (Torres et al. 2014). Thus, it shows
that EPS produced by most of the Enterobacter sp. possess
The thermostable property of the biopolymers plays an glucose and galactose monomers in common. Whereas, the
essential role in its diverse industrial utilization (Marinho- other monomers might be strain specific or expressed in dif-
Soriano and Bourret 2005). The TGA analysis (weight loss ferent environmental conditions (Lloret et al. 1998). Fur-
versus temperature) showed that EPS-RN5 degrades in two thermore, the EPS produced by Enterobacter sp. A47 DSM
steps wherein the first phase represents the decrease in 23139 composed of fucose, glucose, galactose, glucuronic
weight due to loss of water molecules and in the second acid, pyruvate, succinate, acetate (Freitas et al. 2011; Huang
phase depolymerization occurs (Fig. 2b). In the first step,

Fig. 2  FT-IR and thermogravimetric analysis of EPS-RN5. a FT-IR radation pattern of EPS-RN5 showing three phases of degradation at
spectra of EPS-RN5 (pink lines) and EPS-RN5 chelated with cad- 153, 273 and 576 °C
mium (green lines) showing major peak. b Thermogravimetric deg-

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Fig. 3  Gas chromatogram of EPS-RN5 (a), the mass spectra of the peaks with retention time 30.297 (b), 18.975 (c) and 14.568 (d)

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Fig. 4  Rheological behaviour of

EPS-RN5 at a 0.5% concentra-
tion and b 0.5% xanthan taken
as a positive control

et al.2015) is reported with hypoglycemic and hypolipidemic (K) was 187 cP and flow behavior index (n) was 0.6 for the
activities in type 2 diabetic mice (Huang et al. 2015). EPS-RN5 solution. The EPS-RN5 solution also showed thix-
otropic behavior wherein, the viscosity lost during increased
Rheological properties of EPS‑RN5 shear stress was regained upon the removal of stress-induc-
ing forces. Shear-thinning phenomenon occurs when the
To evaluate the rheological properties, the steady flow polymer chains align themselves in the direction of flow,
behavior of an aqueous solution of EPS-RN5 (0.5% w/v) reducing the chemical interaction in the chain entanglement
over a range of shear rates were measured. The EPS-RN5 (Vardhanabhuti and Ikeda 2006). EPS-RN5 is water soluble
showed a slight decrease in the viscosity at increasing shear and forms non-Newtonian solutions that forms a gel-like
rates of 96–768 ­s−1, indicating its shear-thinning behavior substance. Such polysaccharides with unique rheological
(Fig. 4a). Xanthan gum (0.5%) that shows shear-thinning properties are used as a thickening, gelling, or stabilizing
behavior was used as the control (Fig. 4b). The shear stress agents in the food industry (Jindal and Khattar 2018). The
versus shear rate data for EPS-RN5 at different concentra- pseudoplastic property contributes to better sensory qualities
tions at 25 °C, fitted well to the power-law model. According of food and also helps in different levels of food process-
to the power-law model, the value of the consistency index ing (Moreno et al. 2000). Many applications in cosmetics,

Fig. 5  Quantification of heavy metal biosorption and cell protective toxicity on the HEK cells evaluated using MTT assay (c). Data are
effect of EPS-RN5 against cytotoxicity of [Link] of represented as mean ± SD (n = 3). Significance of difference between
EPS-RN5 at 1.0% concentration (w/v) against selected heavy metals the groups are indicated by *p ≤ 0.05; **p ≤ 0.01; ****p ≤ 0.0001; ns-
(a). Cell proliferative activity of EPS-RN5 tested using MTT assay non significant (p > 0.05)
(b). Protective effect of EPS-RN5 against the cadmium-induced cyto-

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foodstuffs, or the petroleum industry require the EPS with excellent cadmium binding activity and removed 88.9% of
pseudoplastic qualities. cadmium from the solution and this could be related to its
distinct degree of specificity and affinity. The electro-neg-
Metal biosorption ativity and radius of metal ion also influence its affinity for
the EPS binding sites (Can and Jianlong 2007).
The heavy metal ions of mercuric sulfate, cadmium chlo-
ride, strontium chloride, copper chloride, sodium arsenate Role of EPS‑RN5 against cytotoxicity induced
and uranyl nitrate in an aqueous solution (100 ppm) mixed by cadmium
with EPS-RN5 (1% w/v) resulted in the precipitation. The
metal content in the precipitate was measured by ICP-MS Toxicity of cadmium can cause damage to different organs
and showed that, cadmium absorption was the highest including kidney, lung, liver and bone. However, the primary
(1555 mg/g) followed by mercury (218 mg/g). EPS-RN5 target organ of cadmium toxicity is the kidney. Moreover,
also showed absorption of strontium, copper, arsenic and cadmium is considered as a model cumulative nephrotoxi-
uranium ions but at comparatively lower levels (Fig. 5a). cant (Klaassen et al. 2009). Hence, HEK 293T (human
The absorption behavior could be attributed to the metal embryonic kidney cells) cell lines were chosen for the study.
ion acting as a complexation agent of protein or such other Cadmium tested at different concentrations on HEK 293T
compounds (Yang et al. 2015) present in the EPS-RN5. The cells showed a dose-dependent toxicity with more than 80%
presence of uronic acids, pyruvate, and inorganic residues cell killing at 40 µg/mL. On the other hand, the β-glucan-like
such as phosphate or sulfate, acidic amino acids, and phos- EPS-RN5 showed the highest biocompatibility and exhibited
phate-containing nucleotides along with ionisable functional the cell proliferative activity up to 40 µg/mL (Fig. 5b). Based
groups and non-carbohydrate substituents like acetamido on these results, the EPS-RN5 was tested against cadmium-
groups in the EPS can also render negative charge to the induced cytotoxicity. The EPS-RN5 treatment reduced the
EPS that in turn interact with positively charged metal ions cell killing effect of the cadmium significantly and the cell
playing an important role in metal binding property (Mohite viability of the cadmium exposed (10 µg/mL) HEK 293T
et al. 2017). The amino acid and protein-like substances in cells showed 50.7% cell viability at 25 µg/mL from 27.7%
the EPS is also believed to assist in heavy metal absorption (Fig. 5c). However, the EPS at higher concentration (50 µg/
mechanism (Cui et al. 2020). The chelation was confirmed mL) showed a lower cytoprotective effect. This may be due
by comparing the metal concentration in the supernatant that to the higher chelation of metal ion by the EPS changing
showed a relative decrease due to its precipitation from the the cell microenvironment due to the higher viscosity of
solution. The comparison of IR spectrum of the EPS-RN5 the culture media. At higher cadmium concentration (40 µg/
and metal-bearing EPS-RN5 from the precipitate showed mL), no significant difference in the cell viabilities between
marked changes in the peak positions and peak intensity. The the EPS-RN5 concentrations was observed. The capacity of
EPS-RN5 containing cadmium showed a sharp peak shift at EPS containing β-glucans to reduce heavy metal-induced
2934 ­cm−1, representing the –CH stretching vibration and cytotoxicity under in vitro conditions has been reported
3400 ­cm−1 of –OH vibration. Involvement of such groups in Enterobacter sp. YG4 (Nagaraj et al. 2018). From our
in the complexation of cadmium has earlier been reported study, it is evident that the treatment of HEK 293T cells
(Zeng et al. 2020). There are also reports indicating the con- with EPS-RN5 could reduce the cytotoxicity of cadmium
tribution of hydroxyl (O–H) and carbonyl (C = O) groups chloride. A few studies have also attributed these effects to
of the polymer in metal chelation (Shuhong et al. 2014). the antioxidant activity of the EPS (Nagaraj et al. 2016) as
Moreover, the absorption rate may also be dependent on well as the ability to neutralize the reactive oxygen species
the metal ion concentration and/or absorbent concentration thereby protecting the cells against oxidative stress and pre-
(Feng et al. 2012; Chakraborty et al. 2018)and the shift in venting apoptosis (Nagaraj et al. 2018). Further studies on
FT-IR peaks correspond to the metal-binding process occur- understanding the exact mechanisms of metal chelation will
ring on the surface of the biosorbent (Pavasant et al.2006). provide opportunities for utilizing the EPS-RN5 for specific
The formation of metal cation complex by the EPS results applications in cadmium toxicity and remediation.
in metal immobilization within the EPS (Gupta and Diwan
2017). In view of these properties there is a great interest
in the application of EPS as bioflocculants, bioabsorbents,
drug delivery and heavy metal removal agents (Salehizadeh Conclusion
and Yan 2014). The EPS from cadmium-tolerant bacteria E.
ludwigii LY6 and Enterobacter sp. DNB-S2 have shown a The EPS-RN5 produced by E. cancerogenus strain YU-
similar biosorption mechanism of the heavy metals (Biswas 16RN5 showed prominent shear thinning, thermostable,
et al. 2020; Sun et al. 2020a, b). The EPS-RN5 exhibited cell proliferative and metal chelation properties. These

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characteristics of EPS-RN5 could prove it to be an ideal can- biofilms. Int J Env Res Public Health 15(2):314. [Link]
didate for food, pharmaceutical and biomedical industries. 10.​3390/​ijerp​h1502​0314
Can C, Jianlong W (2007) Correlating metal ionic characteristics with
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Author contributions DBE and PDR contributed to the study concep- Cui D, Tan C, Deng H, Gu X, Pi S, Chen T, Zhou L, Li A (2020)
tion and design. Material preparation, data collection and analysis were Biosorption mechanism of aqueous P ­ b2+, ­Cd2+, and N
­ i2+ ions on
performed by DBE, Athmika. The first draft of the manuscript was extracellular polymeric substances (EPS). Archaea. [Link]
written by DBE. The manuscript was reviewed and edited by DBE, org/​10.​1155/​2020/​88915​43
Athmika and PDR. The work was supervised by PDR. All authors read Dubois M, Gilles KA, Hamilton JK, Rebers PT, Smith F (1956) Colori-
and approved the final manuscript. metric method for determination of sugars and related substances.
Anal Chem 28:350–356. [Link]
Funding Athmika acknowledges the Department of Biotechnology, Feng M, Chen X, Li C, Nurgul R, Dong M (2012) Isolation and iden-
India (BT/PR15730/AAQ/3/791/2016) for the funding. tification of an exopolysaccharide-producing lactic acid bacte-
rium strain from Chinese Paocai and biosorption of Pb (II) by its
Data availability The datasets generated during and/or analyzed dur- exopolysaccharide. J Food Sci 77:T111–T117. [Link]
ing the current study are available from the corresponding author on 1111/j.​1750-​3841.​2012.​02734.x
reasonable request. The 16S rRNA gene sequence has been deposited Freitas F, Alves VD, Carvalheira M, Costa N, Oliveira R, Reis MA
in the GenBank with the accession number MH191375. (2009) Emulsifying behaviour and rheological properties of the
extracellular polysaccharide produced by Pseudomonas oleo-
vorans grown on glycerol byproduct. Carbohydr Polym 78:549–
Declarations 556. [Link]
Freitas F, Alves VD, Torres CA, Cruz M, Sousa I, Melo MJ, Ramos
Conflict of interest The authors declare no competing interests. AM, Reis MA (2011) Fucose-containing exopolysaccharide pro-
duced by the newly isolated Enterobacter strain A47 DSM 23139.
Carbohydr Polym 83:159–165. [Link]
2010.​07.​034
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