Science of the Total Environment 550 (2016) 611618

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Science of the Total Environment

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Surface tailored organobentonite enhances bacterial proliferation and

phenanthrene biodegradation under cadmium co-contamination
Asit Mandal a,b, Bhabananda Biswas a,c, Binoy Sarkar a,c,, Ashok K. Patra b, Ravi Naidu c,d,
a

Future Industries Institute (formerly Centre for Environmental Risk Assessment and Remediation), University of South Australia, Mawson Lakes, SA 5095, Australia
Indian Council of Agricultural Research (ICAR), Indian Institute of Soil Science, Bhopal, India
Cooperative Research Centre for Contamination Assessment and Remediation of the Environment (CRC CARE), ACT Building, University of Newcastle, Callaghan, NSW 2308, Australia
d
Global Centre for Environmental Remediation (GCER), Faculty of Science and Information Technology, University of Newcastle, Callaghan, NSW 2308, Australia
b
c

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

Surface tailored organobentonite synthesised and characterised

Modied clay adsorbs Cd and reduces
toxicity to Mycobacterium gilvum.
It creates congenial microenvironment
for bacterial survival.
It enhances phenanthrene biodegradation in metal co-contaminated condition.

a r t i c l e

i n f o

Article history:
Received 28 August 2015
Received in revised form 19 January 2016
Accepted 24 January 2016
Available online 2 February 2016
Keywords:
Polycyclic aromatic hydrocarbon (PAH)
Heavy metal
Modied clay
Bioremediation
Clay-bacterial interaction

a b s t r a c t
Co-contamination of soil and water with polycyclic aromatic hydrocarbon (PAH) and heavy metals makes
biodegradation of the former extremely challenging. Modied clay-modulated microbial degradation provides a novel insight in addressing this issue. This study was conducted to evaluate the growth and phenanthrene degradation performance of Mycobacterium gilvum VF1 in the presence of a palmitic acid (PA)grafted Arquad 2HT-75-based organobentonite in cadmium (Cd)-phenanthrene co-contaminated
water. The PA-grafted organobentonite (ABP) adsorbed a slightly greater quantity of Cd than bentonite at
up to 30 mg L1 metal concentration, but its highly negative surface charge imparted by carboxylic groups
indicated the potential of being a signicantly superior adsorbent of Cd at higher metal concentrations. In
systems co-contained with Cd (5 and 10 mg L 1), the Arquad 2HT-75-modied bentonite (AB) and PAgrafted organobentonite (ABP) resulted in a signicantly higher (7278%) degradation of phenanthrene
than bentonite (62%) by the bacterium. The growth and proliferation of bacteria were supported by ABP
which not only eliminated Cd toxicity through adsorption but also created a congenial microenvironment
for bacterial survival. The macromolecules produced during ABPbacteria interaction could form a stable

Correspondence to: B. Sarkar, Future Industries Institute (formerly Centre for Environmental Risk Assessment and Remediation), University of South Australia, Mawson Lakes, SA
5095, Australia.
Correspondence to: R. Naidu, Cooperative Research Centre for Contamination Assessment and Remediation of the Environment (CRC CARE), ACT Building, University of Newcastle,
Callaghan, NSW 2308, Australia.
E-mail addresses: binoy.sarkar@unisa.edu.au (B. Sarkar), ravi.naidu@newcastle.edu.au (R. Naidu).

http://dx.doi.org/10.1016/j.scitotenv.2016.01.164
0048-9697/ 2016 Elsevier B.V. All rights reserved.

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A. Mandal et al. / Science of the Total Environment 550 (2016) 611618

clay-bacterial cluster by overcoming the electrostatic repulsion among individual components. Findings of
this study provide new insights for designing clay modulated PAH bioremediation technologies in mixedcontaminated water and soil.
2016 Elsevier B.V. All rights reserved.

1. Introduction
Polycyclic aromatic hydrocarbon (PAH) is one of the commonly encountered contaminants in the environment posing carcinogenic, mutagenic, and teratogenic effects on human health (Lemieux et al., 2015).
This group of contaminants frequently coexists with toxic heavy metals
at sites like former gasworks (Duan and Naidu, 2013; Olaniran et al.,
2013; Thavamani et al., 2012). Finding novel clean-up technology for
these contaminants is urgently needed because of the increased awareness among community about their harmful effects and stringent regulations evolving around the globe.
PAHs, specially the 45 ring compounds, are challenging to remediate because of the compounds' toxicity to microorganisms and their
limited bioavailability. Where PAHs are present together with metals
as mixtures, metals may exert additional toxic stress to microorganisms
and make microbial degradation extremely difcult (Lu et al., 2013; Vig
et al., 2003). One possible strategy in such scenario would be reduction
of metal toxicity and improvement of PAH bioavailability.
A range of natural or modied adsorbents including clay minerals
could be used for reducing metal bioavailability and hence toxicity to
bacteria (Gupta and Bhattacharyya, 2006; Gupta et al., 2014; Mohan
et al., 2014). However, preconditions of successful remediation in a
mixed-contaminated scenario include the following: (a) the adsorbent
would reduce metal bioavailability but improve PAH bioavailability
and degradation, and (b) the adsorbent is fully compatible to bacterial
growth and proliferation (Biswas et al., 2015a; Sarkar et al., 2012).
Clay minerals can be modied in several ways for adsorbing environmental contaminants including PAHs and heavy metals (Sarkar
et al., 2012). Adsorbent prepared by grafting long chain fatty acid
chelates, e.g., palmitic acid (PA) and stearic acid (SA), on clay minerals
and the product's application in reducing heavy metal (cadmium)
toxicity to Pseudomonas putida was reported previously (Malakul
et al., 1998b). The degree of biocompatibility of such organoclay adsorbents is likely to vary according to the organoclay constituents (Sarkar
et al., 2013; Sarkar et al., 2010a) and individual bacterial species. However, report on such interactions and their plausible effect on bacterial
proliferation and PAH biodegradation under mixed-contaminated situation is scant in the literature.
The current study therefore was conducted to evaluate the
growth performance of Mycobacterium gilvum VF1, a well-known
PAH degrading bacterium (Kstner et al., 1994; Mutnuri et al., 2005;
Pagnout et al., 2007), in the presence of a PA grafted di(hydrogenated
tallow) dimethylammonium based organobentonite (Sarkar et al.,
2011) under an aqueous condition co-contaminated with cadmium
(Cd) and phenanthrene. The adsorption of Cd and biodegradation
of phenanthrene were thoroughly investigated under the above
conditions.
2. Materials and methods
2.1. Preparation of adsorbents and their characterisation
An Australian bentonite (B) (CEC = 85.8 cmol [p+] kg1; conductivity = 3.74 dS m1; clay content = 83.4%; silt content = 2.5%) was used
in this study. This clay is a montmorillonite-type bentonite mixed with
traces of dolomite and halite (see the X-ray diffraction (XRD) pattern in
Fig. 1); the material is mainly composed of 47.06% SiO2, 25.28% Al2O3,
6.51% TiO2, 2.34% Na2O, 2.81% Fe2O3, 0.67% CaO, 0.23% K2O (analysed

by Energy Dispersive X-ray Absorption Spectroscopy (EDAX), image

not shown).
Bentonite (B) was modied with the surfactant (Arquad 2HT-75
which is a di(hydrogenated tallow) dimethylammonium type cationic
surfactant; purchased from Sigma-Aldrich, Australia) at a loading rate
100% of CEC of the clay (Sarkar et al., 2011). The surfactant modied
bentonite was denoted as AB.
In the next step, the surfactant modied bentonite (AB) was added
to palmitic acid (PA) (purchased from Sigma-Aldrich, Australia; purity
N98%; dissolved 10.99 g (100% CEC of the clay) in 1000 mL ethanol
water mixture (1:1 v/v) at pH 8.08.5) (Biswas et al., 2015a). The
above mixture was agitated gently over 4 h on a magnetic stirrer. The
nal product was collected by centrifugation at 3400 g for 20 min,
washed thoroughly with Milli-Q water (resistivity = 18.2 cm 1),
dried at 60 C and stored in an air-tight container. The PA-grafted
organobentonite was termed as ABP.
XRD patterns of the original bentonite (B) and its modied products
(AB and ABP) were obtained using CuK radiation ( = 1.540598 ) on
a PANalytical Empyrean X-ray diffractometer equipped with PIXcel3D detector (PANalytical Inc., The Netherlands). The instrument was operated
at 40 kV and 40 mA between 2 and 50 2 at a step size of 0.0263. The
basal spacing (d) was calculated from the 2 value using Bragg's equation
(n = 2dsin, where, n = an integer, = wavelength and = the scattering angle).
Fourier Transform Infrared (FTIR) spectra of the adsorbents were
collected by using an Agilent Cary 600 Series FTIR Spectrometer.
Finely powdered samples were mixed with dehydrated KBr and
discs were prepared with the aid of a hydraulic press. Spectra were
recorded in the range of 4000 cm 1 to 400 cm 1 with a 4 cm 1
resolution.
2.2. Adsorption of Cd
In a batch experiment, 0.1 g of each of the adsorbents (B, AB, ABP) in
duplicate was added into 20 mL of Cd solutions (prepared with
Cd(NO3)2 (Scharlab) of various concentrations (075 mg L 1) in
Milli-Q water in a 50 mL polypropylene tube. The screw-tight tubes
were then incubated in an end-over-end shaker for 24 h at 25 C temperature. The Cd concentration was measured by ICP-MS (Model
7500c, Agilent Technologies, Japan) in the clear supernatant obtained
by centrifugation at 3400 g for 20 min. The quantity of Cd adsorbed
was calculated using the following Eq. (1):
qe V

C i C e
M  1000

Where, qe is the amount of solute adsorbed on the adsorbent

(mg g 1), Ci the initial liquid phase concentration of the solute
(mg L1), Ce the equilibrium liquid phase concentration of the solute
(mg L1), V the volume of liquid phase (mL) and M the mass of the
adsorbent (g).
2.3. Bacterial culture and inoculum preparation
M. gilvum VF1, which is able to degrade PAHs, was purchased from
the German collection of microorganisms and cell cultures (DSMZ,
Germany) (Kstner et al., 1994). Freeze-dried cells were revived,
grown and stored at 80 C until further use. Prior to conducting

A. Mandal et al. / Science of the Total Environment 550 (2016) 611618

biodegradation experiments, the cells were cultured and subcultured

three times in minimal salt media which was enriched with phenanthrene (98%; Sigma-Aldrich, Australia) (20 mg L1) as the sole carbon
source (Supplementary Information). Phenanthrene was added into the
culture media from dimethylformamide (DMF)-dissolved 5000 mg L1
stock solution. A preliminary experiment conrmed that the amount of
DMF required to obtain a 20 mg L1 phenanthrene concentration in the
culture media did not have any growth-limiting effect on M. gilvum VF1.
At the late log phase of the culture (Supplementary Information, SI
Fig. 1), the cells were harvested by centrifugation at 10,000 g for
10 min at 4 C and washed twice with 0.02% PBS. The washed cells
were passed through sterile packed glass wool lter and washed again
with 0.02% PBS to remove any phenanthrene crystal likely to originate
from the culture inoculum. This was required to avoid any overestimation of phenanthrene in the suspension during the biodegradation
experiment (Jimenez and Bartha, 1996). The cells were then resuspended in minimal salt media and OD600 was measured. Following achieving
a desired cell concentration (OD600 = 0.9) (ca. 1.7 109 cells), it was
inoculated in the biodegradation vials.
2.4. Biodegradation experiment
Predetermined volume of acetone soluble phenanthrene
(10,000 mg L 1 ) was taken into the bottom of dark glass vials
(40 mL capacity) to achieve a nal 20 mg L 1 phenanthrene concentration in 10 mL aqueous suspension. All vials had a Teonlined screw cap. Acetone was rst allowed to evaporate inside a
laminar ow and then minimal salt media were added into the
vials. The mixture was shaken vigorously and sonicated for
15 min in a water jacket at room temperature to facilitate
solubilisation of phenanthrene in the aqueous media. The clay adsorbents (B, AB and ABP) were added into the media at a 0.5% loading rate and mixed thoroughly. In order to test Cd toxicity to
M. gilvum VF1, two levels of Cd (5 and 10 mg L 1 ) as Cd(NO 3 ) 2
were added to the above suspension. Control without clay addition
(NC) and Cd spiking (0 mg L 1 ) were also maintained. Inoculum
(ca. 1.7 109 cells) of bacteria was then added into the suspension.
Abiotic system was also maintained (without adding inoculum of
bacteria). The newly inoculated vials in duplicate were incubated
at room temperature on a rotary shaker at 150 rpm in dark to prevent any photo-degradation of phenanthrene and the time was
counted as hour 0. Two individual sets of experiments were
conducted; one to record the bacterial colony forming unit (CFU) periodically (0, 168, 216, 264 and 312 h), and the other for phenanthrene
extraction and measurement (Section 2.4.1). To avoid any error from
sub-sampling, a destructive sampling of the suspension was required
for obtaining reproducible phenanthrene concentrations.
Phenanthrene biodegradation was calculated by deducting the control (abiotic) value from the phenanthrene concentration obtained at
each sampling event, and expressed as percentage.
2.4.1. Phenanthrene extraction and measurement
Phenanthrene was extracted from the suspension periodically at 0,
72, 168 and 312 h by sacricing individual vial at each sampling event.
A modied protocol of exhaustive solvent extraction (Ni et al., 2011)
was employed. In brief, hexane (5 mL) was rst added into the sample
vial, and it was vortexed for 10 s. At this stage, methanol-dissolved
benzo[b]uorene (1 g mL1) was added as a surrogate for calculating
the extraction efciency. The mixture was again vortexed for 10 s
followed by sonication (80 MHz) for 15 min in a water bath. The mixture was then transferred into a 50 mL Teon centrifuge tube. To ensure
a complete transfer of the analyte (portion likely to be retained by the
glass vial), the above process was repeated by adding a fresh 5 mL
hexane, and the wash-out was added into the same Teon tube. The
tube was shaken for 30 s followed by 15 min sonication. Organic
analytes, which are likely to partition in the hexane phase, was

613

separated by centrifugation at 10,000 g for 10 min. A clear hexane

phase (4 mL) was pipetted out into a dark glass vial and the solvent
was evaporated by using N2 ow cold-trap solvent evaporator (Dionex
SE 500). The fraction remaining after evaporation was concentrated in
methanol. Following ltration through a 0.2 m PTFE membrane, the
methanol phase was analysed by High Performance Liquid Chromatography (HPLC) equipped with a Diode Array Detector (DAD) (at 254 nm
wavelength) (Model: 1200, Agilent technologies, Japan). Phenanthrene
concentration was determined and adjusted against the peak of the internal standard (benzo[b]uorene). A Zorbax Eclipse XDB-C18 HPLC
column (4.6 150 mm, 5 m particle size) was used, while the mobile
phase was methanol:water (85:15 v/v).

2.4.2. Quality control for phenanthrene analysis

All phenanthrene concentrations in water samples following extraction into solvent were calculated based on the calibration curve obtained
from the ratio of the chromatographic peaks of highly pure phenanthrene
and the surrogate (benzo[b]uorene) standards (Supelco, Sigma-Aldrich)
(Supplementary Information, SI Fig. 2). About 65 to 85% recovery of phenanthrene was achieved under different clay treatments (Supplementary
Information, SI Figs. 3, 4).

2.4.3. Microbial count (CFU)

Serially diluted clay-bacterial suspension (dilution factor 106 and
107) was spread on nutrient agar plates in duplicate, and incubated
for 37 days at room temperature for CFU counting. No CFU was observed in the control without bacterial inoculation (NB).

2.4.4. Scanning electron microscopy (SEM)

At the end of incubation, the claybacteria aggregation from the
PA-modied organobentonite with a higher level of Cd treatment
(ABP-10 mg L1 Cd sample) was observed under SEM. A glutaraldehyde
(2.5%)-xed suspension (10 L) was diluted with 20 mL of doubleltered (using 0.45 m syringe lter) Milli-Q water and passed through
0.4 m polycarbonate membrane. The claybacteria complex retained
on the membrane was observed under SEM following a 40 nm pulsed
carbon coating on a Quorum QT150ES coating system. The samples
were examined on a FEI Quanta 450 FEG Environmental Scanning
Electron Microscope operating at 5 kV accelerating voltage, 280 Pa pressure and 10 C temperature by using a gaseous secondary electron detector (GSED).

2.4.5. Surface charge and pH measurement

Zeta potential () values of the bacteria, clay and claybacteria suspensions were measured by using a Nicomp 380 ZLS (USA) zeta potential analyser. Bacteria was grown separately in minimal media with
phenanthrene as the carbon source, harvested at late log phase and
washed twice with 0.02% PBS. The washed cells were then resuspended
in Milli-Q water for measurement. Similarly, a 0.05% clay suspension in
water was used to measure the values of the adsorbents (B, AB and
ABP). The bacterial and clay suspensions were then mixed at 50:50
ratio and values of the mixed suspensions were also measured.
The pH values of different clay-bacterial treatments were also recorded before and at the end of incubation using a standard pH probe.

2.5. Graphical presentation and statistical analyses

Microsoft spreadsheet (version 10) was used for graphical presentation of the data. One-way ANOVA (at 95% condence level; Duncan's
test for post-hoc analysis) was performed to determine the effects of
adsorbents on biodegradation of phenanthrene as well as microbial proliferation using IBM SPSS 20.0 software package.

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A. Mandal et al. / Science of the Total Environment 550 (2016) 611618

3. Results and discussion

3.1. Characteristics of the modied clays
3.1.1. X-ray diffraction (XRD)
The XRD patterns indicated modication of the montmorillonite
crystal structure (in B) due to intercalation of Arquad 2HT-75 (in
AB) and further incorporation of PA (in ABP) into the clay interlayer
(Fig. 1). Due to insertion of the surfactant molecules (Arquad 2HT75) in the interlayer, the d spacing increased from 12.7 to 30.3 in
AB in addition to appearance of a secondary diffraction reection at
13.4 (Fig. 1). Thereafter, due to further inclusion of PA in the interlayer
structure, the d spacing increased to 40.3 in ABP with appearance of
the secondary and tertiary diffraction reections at 19.9 and 12.9 , respectively. All clay products showed the presence of quartz as an impurity, while NaCl present in B washed out during its modication with
organic molecules in aqueous media (Fig. 1). The cationic surfactant
molecules entered into the clay interlayer through a cation exchange
reaction by replacing the hydrated cations (Sarkar et al., 2010b),
which was followed by inclusion of the chelating agent (PA) through a
hydrophobic interaction between the long alkyl chains of surfactant
and PA molecules (Biswas et al., 2015a). Thus, grafting of PA in the
clay structure took place in a two-step synthesis process with the aid
of the surfactant molecules.
3.1.2. Fourier transform infrared spectroscopy (FTIR)
The IR bands at 663 cm1, 1002 cm1 and 34003600 cm1 regions
(Fig. 2), which are attributed to Si\\O stretching, Al\\O + Si\\O out of
plane vibration and broad OH-stretching, respectively (Madejov,
2003; Sarkar et al., 2011), remained unaltered in all the adsorbents.
These are the key characteristic IR bands of bentonite. However,
two additional bands at 29152931 and 2854 cm 1 region in the
organoclays indicated the anti-symmetric and symmetric stretching
vibrations of CH2, respectively (Fig. 2). These bands conrmed the
incorporation of CH2 group into the bentonite structure from the
alkyl chain of the surfactant molecules in AB, and both of the surfactant molecule and PA in ABP (Sarkar et al., 2011). The CH2 antisymmetric stretching band in ABP shifted notably to a lower wave

number (2915 cm 1) in comparison to AB (2931 cm 1), which indicated a highly ordered all-trans conformation of PA molecules in the
bentonite structure and hence a successful grafting reaction (Sarkar
et al., 2011). Additionally, the IR spectra of PA-grafted organoclay

Fig. 1. X-ray diffraction patterns of (a) bentonite (B), (b) surfactant-modied bentonite
(AB), and (c) palmitic acid-grafted bentonite (ABP).

Fig. 3. Adsorption isotherms of Cd on bentonite (B), surfactant-modied bentonite (AB),

and palmitic acid-grafted bentonite (ABP). The scatter points show the experimental
data, solid lines represent the Langmuir model ttings. The inset gure shows the
adsorption scenario at low input Cd concentration range.

Fig. 2. FTIR spectra of (a) bentonite (B), (b) surfactant-modied bentonite (AB), and
(c) palmitic acid-grafted bentonite (ABP). The number with arrow shows the
wavenumber of the selective band.

A. Mandal et al. / Science of the Total Environment 550 (2016) 611618

showed bands at 1727 cm 1 (Fig. 2) which could be attributed to the

presence of COOH group in ABP contributed by the long fatty acid
(PA) (Malakul et al., 1998a). Thus, the FTIR ndings further conrmed the XRD results that PA was successfully grafted into the
organobentonite to develop a product which could potentially
immobilise heavy metals through the grafted organic functional
groups.
3.2. Adsorption of Cd on clay products
The adsorption behaviour of the adsorbents changed over the
concentration range of the adsorbate (Cd) (Fig. 3).The Arquad 2HT75-grafted organoclay (AB) adsorbed signicantly smaller amount of
Cd than B and ABP over the input concentration (075 mg L1)
(Fig. 3). This was due to the formation of a positive charge on its surface
as a result of cationic surfactant intercalation, which was not favourable
to attract Cd2 + (Biswas et al., 2015a; Sarkar et al., 2011). However,
grafting of PA into the organoclay changed the adsorption capacity of
the material (ABP) signicantly, which was either similar to the raw
bentonite (B) or even higher at a smaller input concentration of Cd
(030 mg L1) (Fig. 3). The adsorption isotherms of B, AB and ABP
were best tted to the Langmuir type model as expressed by the following Eq. (2) (Fig. 3):
qe

Q mKLCe
1 K LCe

Where, qe is the adsorbed Cd per unit mass of adsorbent at equilibrium (mg g1), Ce the initial concentration of Cd (mg L1), Q m the maximum adsorption capacity by the adsorbent (mg g1), KL the Langmuir
adsorption constant related to the adsorption energy.
Within the input concentration range (075 mg L1), the maximum
adsorption of Cd was predicted in the presence of B (Q m = 14.2 mg g1)
followed by ABP (Qm = 11.4 mg g 1) and AB (Q m = 8.2 mg g1)
(Table 1). However, in the biodegradation experiment, the Cd concentration was maintained below 10 mg L1 in aqueous suspension; within
this range, B and ABP showed a similar adsorption performance. While B
adsorbed Cd mostly through coulombic interactions and cation exchange (Kaoser et al., 2004), ABP tended to adsorb Cd through complex
formation by the surface carboxylic groups (as appeared in the FTIR
study). The Cd adsorption capacity of ABP could be attributed to the
presence of COOH groups (Section 3.1.2) on the adsorbent's surface.
As discussed in the subsequent sections, ABP would facilitate phenanthrene biodegradation by inducing microorganism proliferation and
reducing metal (Cd) toxicity in the aqueous environment. Also, it
was earlier reported that metal immobilising PA-grafted organoclay
could reduce the bioavailability of Cd in a Cd-phenanthrene cocontaminated soil (Biswas et al., 2015a).
3.3. Phenanthrene biodegradation under Cd co-contamination
The percentage degradation of phenanthrene increased as the incubation period progressed (Fig. 4). The highest degradation (79.5%) was
observed at 312 h in the system in the absence of Cd and no clay
Table 1
Langmuir parameters of Cd adsorption on bentonite (B), surfactant-modied bentonite
(AB), and palmitic acid-grafted bentonite (ABP).

B
AB
ABP

Qm

KL

RMSE

14.1894
8.2344
11.3685

2.6818
0.0961
5.6578

0.9577
0.7904
0.8653

1.0834
1.2651
1.7502

qe: exp:n qe:model:n

R2 = 1  n1
n
2 .

q
n
1
RMSE (Root Mean Square Error) = n1
n1 qe: exp:n  qe:model:n 2 ; where, qe.exp. is
the qe of the experimental data; qe.model the qe of the isotherm model data; n the number of
observation.
n1 qe: exp:n qe: exp:n

615

treatment was added. At the end of incubation (312 h), phenanthrene

degradation was found to be signicantly higher (7278%) in case of
modied clays (AB and ABP) than the unmodied bentonite (B) (62%)
in systems which contained Cd (5 and 10 mg L1) (p b 0.05). At higher
Cd level (10 mg L1), the trend of phenanthrene degradation changed
remarkably only due to the ABP treatment (Fig. 4). Instead of a rapid depletion at rst 72 h, comparatively lower amount and slow degradation
of phenanthrene were observed in the aqueous suspension of ABP.
However, it was notable that the degradation graph for ABP was still
progressing steeply at 312 h, whereas the graphs for B and AB already
attained a plateau (Fig. 4). These results indicated that only ABP
would possibly have the chance to achieve a complete removal of phenanthrene in this system. This prediction was well supported by the
maintenance of a greater bacterial proliferation (without any cell depletion phase) by ABP at the end of 312 h (see Fig. 5, Section 3.4.1). Previous study also showed that a greater bioavailability of phenanthrene
was maintained in the soil which was amended with ABP (Biswas
et al., 2015a). Many studies reported that soil microorganisms were capable of degrading adsorbed organic compounds through multiple
mechanisms including catabolic enzymatic activity (Ortega-Calvo and
Saiz-Jimenez, 1998). Bacteria could mineralise organic substrate at a
higher rate in an adsorbent microenvironment different from that of
the bulk aqueous phase. This rate could be faster than that of a conventionally predicted rate governed by spontaneous partitioning behaviour
of the compound. Similar observations were reported for a P. putida
strain able to degrade naphthalene sorbed to soil particles (Guerin and
Boyd, 1992) and a surfactant-modied clay (Malakul et al., 1998b).
3.4. Viability of bacteria
3.4.1. Microbial count
At each Cd level, the number of M. gilvum VF1 (CFU) was signicantly higher in ABP-treated medium over the entire incubation period
(Fig. 5). The proliferation of bacteria dropped dramatically in the control
(NC) treatment and also in B- and AB-treated suspensions following
168 h of incubation (Fig. 5). The only exception was noted in the ABP
treatment where the cell number was maintained and improved
(Fig. 5). Along with producing a congenial growth microenvironment,
ABP would also have held phenanthrene in available form and supplied
food to the bacterium over a longer period of incubation. Since ABP was
able to adsorb a higher quantity of Cd, the enhanced survival of
M. gilvum VF1 in the ABP-treated suspension could also be correlated
with the adsorbent's ability to reduce Cd bioavailability to the bacterium
(Biswas et al., 2015a). Earlier reports also indicated that smectite clay,
especially bentonite, was a growth stimulator of bacterium and could
reduce cadmium toxicity (Babich and Stotzky, 1977). For example,
(Ugochukwu et al. (2014)) reported that bentonite acted as an absorbent of hydrocarbon and had positive inuence on the microbial counts.
The expandable clay also would play a role in protecting the microbial
cells by shielding them from the spilled toxic materials or by buffering
the toxic materials into less toxic compounds (Biswas et al., 2015b;
Ueshima et al., 2000). The current study furthermore suggested that
PA-grafted organoclay had a remarkably enhanced effect on the survival
and growth of PAH-degrading bacteria in comparison to an unmodied
or surfactant modied bentonite, which could be correlated to phenanthrene biodegradation in a Cd-co-contaminated aqueous media (Fig. 4).
3.4.2. Interaction between modied clay and bacteria
Aggregation of bacteria and ABP adsorbent was clearly visible under
SEM and a prominent microbial cluster was found (Fig. 6). The clear visibility of claybacteria cluster indicated that the bacterium M. gilvum
VF1 was protected from the external conditions, which provided it a
congenial microenvironment to grow and proliferate. Such a supportive
growth condition might have enabled an enhanced phenanthrene biodegradation rate in the system. The claybacteria cluster was not prominent in the unmodied bentonite and organobentonite treatments at

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A. Mandal et al. / Science of the Total Environment 550 (2016) 611618

Fig. 4. Degradation of phenanthrene (%) under Cd-co-contamination in aqueous suspension of bentonite (B), surfactant-modied bentonite (AB), and palmitic acid-grafted bentonite
(ABP). Different alphabets above the graphs indicate signicantly different values among adsorbents under similar Cd level at each incubation time (95% condence level). NC =
control treatment without clay addition; ns = not signicant. p-value indicates the overall difference based on ANOVA.

the end of the incubation period (image not shown). This could be the
unfavourable microenvironment for the bacteria as indicated by the
lower microbial count in B and AB treatments (Fig. 5). Moreover, the
ABP-aggregates were comparatively less stable under the electron
beam and yielded fewer electrons during the SEM analysis, which indicated a greater loading of organic molecules in this aggregate. Thus, SEM
results also indicated a better initiation of biolm formation around ABP
particles, which warrants a detail investigation.
3.4.3. Surface charge and pH of clay-bacterial interaction
At pH value around 7, M. gilvum VF1 cells, B, AB and ABP exhibited a
zeta potential value of 33.6, 27.9, +37.9 and 53.8 mV, respectively. The negative surface charge of bentonite (B) got converted to a positive value due to the intercalation of cationic surfactant molecules in
the clay structure (Biswas et al., 2015a; Sarkar et al., 2011), following
which grafting of PA into the organoclay brought back the particle's surface charge to a value even more negative than the unmodied bentonite. These charge properties of ABP were earlier reported over a wider
range of pH values (Biswas et al., 2015a). A greater negative zeta

potential of ABP makes it a potentially superior adsorbent of heavy

metals than unmodied bentonite. In the current study, pH changes of
the clay-bacterial suspensions over the incubation period were insignificant (Supplementary Information, SI Table 1).
Bacterial cluster formation and biolm formation may largely depend on the surface charge behaviour of the microorganisms and colloidal particles present in the system (Biswas et al., 2015b; Cao et al., 2011;
Rong et al., 2008). The bacterial cells, B and ABP contained negative surface charges (Fig. 7). Therefore, occulation of colloidal clay suspension
by bacterial exo-polysaccharides would require cancellation of the electrostatic repulsion force, which allows the surfaces to come together by
the adsorbing macromolecules via a bridging effect of other cations (Cao
et al., 2011). The current study revealed that the maximum positive
interaction took place between the bacterial cells and PA-grafted
organobentonite (ABP). This interaction showed an overall zeta potential value around 42.5 mV (Fig. 7). In comparison, the value was
18.5 mV for the unmodied bentonitebacteria interaction. Therefore, the macromolecules produced during ABP and M. gilvum VF1 interaction was able to overcome the electrostatic repulsive forces and thus

Fig. 5. Bacterial count in phenanthrene-Cd mixed-contaminated suspensions treated with bentonite (B), surfactant-modied bentonite (AB), and palmitic acid-grafted bentonite (ABP).
Different alphabets above the graphs indicate signicantly different values among adsorbents under similar Cd level at each incubation time (95% condence level). NC = control
treatment without clay addition; ns = not signicant.

A. Mandal et al. / Science of the Total Environment 550 (2016) 611618

617

Fig. 6. SEM images of (a) bacteria without any clay material, and (b) clay-bacterial complex from ABP-treated aqueous suspension at 10 mg L1 Cd concentration, at the end of incubation.

form stable clay-bacterial aggregates, which was also supported by the

SEM observation. Through a positive surface charge AB might have
adsorbed more number of cells at the initial stage of incubation, but
ABP provided a more congenial environment for the bacterium to
grow, proliferate and degrade phenanthrene in the long run (Fig. 5).

overcome the electrostatic repulsion among the components and thus

formed a stable clay-bacterial aggregate, which provided a microenvironment for congenial bacterial growth, proliferation and phenanthrene biodegradation. Findings of this study provide new insights for
designing clay modulated PAH bioremediation technologies in mixedcontaminated water and soil.

4. Conclusions
This study developed a surface tailored organobentonite by grafting
PA in bentonite structure with the aid of an organic surfactant Arquad
2HT-75. XRD, FTIR and surface charge properties of the material conrmed a successful modication of the bentonite clay (B). The PAgrafted organobentonite (ABP) adsorbed a slightly greater quantity of
Cd than bentonite at up to 30 mg L1 metal concentration, but its highly
negative zeta potential value imparted by the grafted carboxylic groups
indicated that it would remain a signicantly superior adsorbent of Cd
at a metal concentration above 30 mg L1. In systems co-contained
with Cd at 5 and 10 mg L1 levels, the surfactant-modied bentonite
(AB) and PA-grafted organobentonite (ABP) resulted in a signicantly
higher (7278%) degradation of aqueous phenanthrene than bentonite
(B) (62%) by the bacterium M. gilvum VF1. This was achieved through a
better growth and proliferation of bacteria supported by ABP which not
only eliminated Cd toxicity to the microorganism but also enabled creation of a congenial microenvironment for better bacterial growth. As
supported by the SEM and surface charge studies, the macromolecules
produced during ABP and M. gilvum VF1 interaction was able to

Contributions
This study was conducted as a part of AM's post-doctoral research
training and BB's PhD research work under the supervision of BS and
RN. AM and BB contributed equally to this work.
Acknowledgements
AM is grateful to the Indian Council of Agricultural Research for
funding his research training programme under the National Agricultural Innovation Project. BB thanks the University of South Australia
for providing him an International President's Scholarship. The authors
also acknowledge the nancial and infrastructural support from the
University of South Australia and CRC CARE for doing this research.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.scitotenv.2016.01.164.
References

Fig. 7. Zeta potential values of bacteria (M. gilvum VF1), bentonite (B), surfactant-modied
bentonite (AB), palmitic acid-grafted bentonite (ABP), and the clay-bacterial interactions.

Babich, H., Stotzky, G., 1977. Reductions in the toxicity of cadmium to microorganisms by
clay minerals. Appl. Environ. Microbiol. 33, 696705.
Biswas, B., Sarkar, B., Mandal, A., Naidu, R., 2015a. Heavy metal-immobilizing organoclay
facilitates polycyclic aromatic hydrocarbon biodegradation in mixed-contaminated
soil. J. Hazard. Mater. 298, 129137.
Biswas, B., Sarkar, B., Rusmin, R., Naidu, R., 2015b. Bioremediation of PAHs and VOCs: advances in clay mineralmicrobial interaction. Environ. Int. 85, 168181.
Cao, Y., Wei, X., Cai, P., Huang, Q., Rong, X., Liang, W., 2011. Preferential adsorption of
extracellular polymeric substances from bacteria on clay minerals and iron oxide.
Colloids Surf. B: Biointerfaces 83, 122127.
Duan, L., Naidu, R., 2013. Effect of ionic strength and index cation on the sorption of phenanthrene. Water Air Soil Pollut. 224, 117.
Guerin, W.F., Boyd, S.A., 1992. Differential bioavailability of soil-sorbed naphthalene to
two bacterial species. Appl. Environ. Microbiol. 58, 11421152.
Gupta, S.S., Bhattacharyya, K.G., 2006. Removal of Cd(II) from aqueous solution by kaolinite, montmorillonite and their poly(oxo zirconium) and tetrabutylammonium derivatives. J. Hazard. Mater. 128, 247257.

618

A. Mandal et al. / Science of the Total Environment 550 (2016) 611618

Gupta, V.K., Nayak, A., Bhushan, B., Agarwal, S., 2014. A critical analysis on the efciency of
activated carbons from low cost precursors for heavy metals remediation. Crit. Rev.
Environ. Sci. Technol. 45, 613668.
Jimenez, I.Y., Bartha, R., 1996. Solvent-augmented mineralization of pyrene by a Mycobacterium sp. Appl. Environ. Microbiol. 62, 23112316.
Kaoser, S., Barrington, S., Elektorowicz, M., Wang, L., 2004. Copper adsorption with Pb and
Cd in sand-bentonite liners under various pHs. Part II. Effect on adsorption sites.
J. Environ. Sci. Health. Part A 39, 22412255.
Kstner, M., Breuer-Jammali, M., Mahro, B., 1994. Enumeration and characterization of the
soil microora from hydrocarbon-contaminated soil sites able to mineralize polycyclic aromatic hydrocarbons (PAH). Appl. Microbiol. Biotechnol. 41, 267273.
Lemieux, C.L., Long, A.S., Lambert, I.B., Lundstedt, S., Tysklind, M., White, P.A., 2015. Cancer
risk assessment of polycyclic aromatic hydrocarbon contaminated soils determined
using bioassay-derived levels of benzo[a]pyrene equivalents. Environ. Sci. Technol.
49, 17971805.
Lu, M., Xu, K., Chen, J., 2013. Effect of pyrene and cadmium on microbial activity and community structure in soil. Chemosphere 91, 491497.
Madejov, J., 2003. FTIR techniques in clay mineral studies. Vib. Spectrosc. 31, 110.
Malakul, P., Srinivasan, K.R., Wang, H.Y., 1998a. Metal adsorption and desorption characteristics of surfactant-modied clay complexes. Ind. Eng. Chem. Res. 37, 42964301.
Malakul, P., Srinivasan, K.R., Wang, H.Y., 1998b. Metal toxicity reduction in naphthalene
biodegradation by use of metal-chelating adsorbents. Appl. Environ. Microbiol. 64,
46104613.
Mohan, D., Kumar, H., Sarswat, A., Alexandre-Franco, M., Pittman Jr., C.U., 2014. Cadmium
and lead remediation using magnetic oak wood and oak bark fast pyrolysis bio-chars.
Chem. Eng. J. 236, 513528.
Mutnuri, S., Vasudevan, N., Kaestner, M., 2005. Degradation of anthracene and pyrene
supplied by microcrystals and non-aqueous-phase liquids. Appl. Microbiol.
Biotechnol. 67, 569576.
Ni, H.-G., Zeng, H., Zeng, E.Y., 2011. Sampling and analytical framework for routine environmental monitoring of organic pollutants. Trends Anal. Chem. 30, 15491559.
Olaniran, A., Balgobind, A., Pillay, B., 2013. Bioavailability of heavy metals in soil: impact
on microbial biodegradation of organic compounds and possible improvement
strategies. Int. J. Mol. Sci. 14, 1019710228.

Ortega-Calvo, J.-J., Saiz-Jimenez, C., 1998. Effect of humic fractions and clay on biodegradation of phenanthrene by a Pseudomonas uorescens strain isolated from soil.
Appl. Environ. Microbiol. 64, 31233126.
Pagnout, C., Frache, G., Poupin, P., Maunit, B., Muller, J.-F., Frard, J.-F., 2007. Isolation and
characterization of a gene cluster involved in PAH degradation in Mycobacterium sp.
strain SNP11: expression in Mycobacterium smegmatis mc2155. Res. Microbiol. 158,
175186.
Rong, X., Huang, Q., He, X., Chen, H., Cai, P., Liang, W., 2008. Interaction of Pseudomonas
putida with kaolinite and montmorillonite: a combination study by equilibrium adsorption, ITC, SEM and FTIR. Colloids Surf. B: Biointerfaces 64, 4955.
Sarkar, B., Megharaj, M., Xi, Y., Krishnamurti, G.S.R., Naidu, R., 2010a. Sorption of quaternary ammonium compounds in soils: implications to the soil microbial activities.
J. Hazard. Mater. 184, 448456.
Sarkar, B., Xi, Y., Megharaj, M., Krishnamurti, G.S.R., Rajarathnam, D., Naidu, R., 2010b.
Remediation of hexavalent chromium through adsorption by bentonite based
Arquad 2HT-75 organoclays. J. Hazard. Mater. 183, 8797.
Sarkar, B., Megharaj, M., Xi, Y., Naidu, R., 2011. Structural characterisation of Arquad
2HT-75 organobentonites: surface charge characteristics and environmental application. J. Hazard. Mater. 195, 155161.
Sarkar, B., Xi, Y.F., Megharaj, M., Krishnamurti, G.S.R., Bowman, M., Rose, H., Naidu, R.,
2012. Bioreactive organoclay: a new technology for environmental remediation.
Crit. Rev. Environ. Sci. Technol. 42, 435488.
Sarkar, B., Megharaj, M., Shanmuganathan, D., Naidu, R., 2013. Toxicity of organoclays to
microbial processes and earthworm survival in soils. J. Hazard. Mater. 261, 793800.
Thavamani, P., Megharaj, M., Naidu, R., 2012. Multivariate analysis of mixed contaminants
(PAHs and heavy metals) at manufactured gas plant site soils. Environ. Monit. Assess.
184, 38753885.
Ueshima, M., Mogi, K., Tazaki, K., 2000. Microbes associated with bentonite. J. Clay Sci. Soc.
Japan (Nendokagaku) 39, 171183.
Ugochukwu, U.C., Jones, M.D., Head, I.M., Manning, D.A.C., Fialips, C.I., 2014. Biodegradation of crude oil saturated fraction supported on clays. Biodegradation 25, 153165.
Vig, K., Megharaj, M., Sethunathan, N., Naidu, R., 2003. Bioavailability and toxicity of
cadmium to microorganisms and their activities in soil: a review. Adv. Environ. Res.
8, 121135.