Applied Clay Science 87 (2014) 8186

Contents lists available at ScienceDirect

Applied Clay Science

journal homepage: www.elsevier.com/locate/clay

Research paper

Biodegradation and adsorption of crude oil hydrocarbons supported on

homoionic montmorillonite clay minerals
Uzochukwu C. Ugochukwu , Martin D. Jones, Ian M. Head, David A.C. Manning, Claire I. Fialips
School of Civil Engineering and Geosciences, University of Newcastle Upon Tyne, NE1 7RU, United Kingdom

a r t i c l e

i n f o

Article history:
Received 24 November 2012
Received in revised form 2 November 2013
Accepted 13 November 2013
Available online 13 December 2013
Keywords:
Interlayer cations
Hydrocarbons
Biodegradation
Microbial growth
Homoionic montmorillonite
Adsorption

a b s t r a c t
The role of homoionic montmorillonites in hydrocarbon removal during biodegradation was investigated in
aqueous clay/oil microcosm experiments with a hydrocarbon degrading microorganism community. The clay
mineral used for this study was montmorillonite which was treated with the corresponding metal chloride salt
to produce Na-, K-, Mg-, Ca-, Zn-, Al-, Cr-, and Fe-montmorillonites used in this study. The study indicated that
Zn- and K-montmorillonites were inhibitory to biodegradation of the crude oil hydrocarbons and appears to
do so as a result of extensive adsorption of the hydrocarbons. However, Na-, Ca- and Fe-montmorillonites with
relatively high surface area and cation exchange capacity (CEC) were stimulatory to biodegradation of the
crude oil hydrocarbons. This study reveals that whereas surface area and the local bridging effect were important factors from the clay minerals that conferred stimulatory effect on the biodegradation of the hydrocarbons,
the hydrolysis of the interlayer water by trivalent cations of the clay to generate protons and increase acidity of
the medium is suggested to be inhibitory to biodegradation of the crude oil hydrocarbons. The interlayer cations
(trivalent cations) that impart the highest local bridging effect which is stimulatory to biodegradation also impart
the highest hydrolysis of interlayer water which is inhibitory. This study showed that interlayer cations play a
crucial role on the ability of the clay mineral to inuence biodegradation of the hydrocarbons.
2013 Elsevier B.V. All rights reserved.

1. Introduction
Clay minerals have surface properties such as huge specic surface
area, cation exchange capacity (CEC) and surface acidity that confer adsorptive properties as well as catalytic and support capabilities to them.
These have resulted in several benecial uses of clay minerals such as
stimulation of biodegradation and sorption of toxic substances for the
purpose of environmental control (Bright and Fletcher, 1983; Fletcher
and Marshall, 1982; Kosita et al, 2002; Murray, 2000; Shelobolina
et al, 2004; Tuccillo et al, 1999; Van Loosdrecht et al., 1990; Warr
et al., 2009). The ability of some clay minerals to aid dispersion and enhance pseudosolubilization of oil leading to increase in interfacial area
that in turn enhances bioavailability of the hydrocarbons (that are the
main hydrophobic compounds of oil) has been reported (Owens and
Lee, 2003). Chaerun and Tazaki (2005) have reported the ability of
some kaolinites to aid the biodegradation of oil. The effect of different
clay minerals on the biodegradation of saturated fraction of crude
oil has been reported (Ugochukwu et al., 2013). Other clay minerals
have also been investigated with respect to their abilities to stimulate
biodegradation of crude oil. Warr et al. (2009) has reported that Naand Ca-montmorillonites, hectorite, and palygorskite stimulated

Corresponding author at: Drummond Building, School of Civil Engineering and

Geosciences, University of Newcastle Upon Tyne, NE1 7RU, United Kingdom.
E-mail address: ugouzochukwu@yahoo.com (U.C. Ugochukwu).
0169-1317/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.clay.2013.11.022

microbial degradation of crude oil hydrocarbons unlike high defect

kaolinites, which were inhibitory. The study reported that many of the
most successful clay minerals in stimulating the bacterial digestion of
the oil were dioctahedral smectites especially Ca-montmorillonite. The
study of Warr et al. (2009) indicated that exchangeable divalent cations
(with local bridging effect) were responsible for why some of the clay
mineral samples such as Ca-montmorillonite and palygorskite were
more successful in stimulating the biodegradation of crude oil hydrocarbons. As a result, it would be important to investigate the effect of other
monovalent, divalent cations (such as Mg, Zn) and trivalent cations such
as Al, Cr and Fe(III) as homoionic interlayer clays to be able to draw
adequate conclusions about the effect of interlayer exchangeable
cations. Also, the reports of Chaerun and Tazaki (2005) and Warr et al.
(2009) on the biodegradation of oil supported on clay minerals did
not quantify the effects of adsorption in their experiments (which
would need to be known), as well as those of volatilization in order to
accurately assess the losses due to biodegradation and abiotic processes.
The experimental design and procedure of the work in this study has
therefore been carried out to account for removal of hydrocarbons due
to abiotic processes such as volatilization and adsorption. Whereas a
previous study by Warr et al. (2009) used semi-quantitative thin layer
chromatographyame ionization detection (TLCFID) method to
quantitate the oil removal during the process of biodegradation, TLC
FID may suffer interference from the lipids present in the microbial
cells. Therefore this study used GCFID to quantitate TPH removed by
both biodegradation and adsorption.

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U.C. Ugochukwu et al. / Applied Clay Science 87 (2014) 8186

The objective of this study was: to understand the role of interlayer

cations of montmorillonites in the biodegradation and adsorption of
crude oil hydrocarbons.
2. Materials and methods
Bentonite (Berkbent 163) was supplied by Steetley Bentonite &
Absorbent Ltd (now Tolsa UK Ltd; www.tolsa.com). The bentonite
contains the following minerals: smectite (88%), quartz (1%), calcite
(5%), dolomite (3%), feldspar (2%) and opals (1%). The chemical composition is: SiO2 (53.8%), Al2O3 (17.28%), MgO (5.61%), CaO (4.98%), Fe2O3
(4.77%) and Na2O (3.71%).
Microbial communities responsible for biodegradation of the crude
oil hydrocarbons were isolated from beach sediment sample consisting
of ne sand particles collected in a sterilized glass bottle (Duran) from
a site at St Mary's Lighthouse near Whitley Bay, Newcastle upon Tyne
(N 55 04 18, W 01 26 59), United Kingdom and stored at 4 C
in cold room until commencement of the experiment. The microbial
community has been reported to contain mainly Alcanivorax spp. as
the dominant genera in another study carried out by Singh et al.
(2009) conducted with exactly the same beach sediments as source of
microbial cells.
The BushnelHaas (BH) broth as the nutrient source, nutrient agar
and all other chemicals were supplied by Sigma-Aldrich. The crude oil
was an undegraded North Sea crude oil originally supplied by British
Petroleum (BP). This oil was characterized by determining the saturate,
aromatic, resin and asphaltene components as 52%, 36%, 10% and 2%
respectively.
2.1. Preparation of the homoionic montmorillonites
The homoionic montmorillonite samples were generated from
exchanging the interlayer cations of the montmorillonite with Na+,
K+, Mg2+, Ca2+, Zn2+, Al3+, Cr3+ and Fe3+. Solution concentration of
0.5 M of the corresponding metal chloride salt was prepared and then
200 mL of each of the solution used to disperse 5 g of clay mineral to
have a clay suspension (Reddy et al., 2007). The suspensions were shaken for 24 h in a mechanical shaker and then subsequently centrifuged.
The supernatant was rejected and the clay mineral washed repeatedly
until the chloride level is not measurable.

2.2.3. Surface area

The surface area of the clay samples were measured by the ethylene
glycol monoethyl ether (EGME) method following the method of Carter
et al. (1965).
2.2.4. Cation exchanged capacity (CEC) of the clay samples
The CEC of the clay samples was determined following the standard
ammonium acetate method (Lewis, 1949).
2.2.5. pH
The pH of the clay suspension was measured with a pH meter.
2.3. Laboratory biodegradation
2.3.1. Microbial growth
The procedure adopted for the enrichment culture and isolation of
the microbial cells involved mixing 20 g of Whitley Bay sediments,
100 mL of BH medium (autoclaved) and 500 mg of crude oil in a
250 mL conical ask and incubating for 5 d while continuously being
stirred. Consequently, indigenous microbial cells of Whitley Bay sediments were isolated by collecting 5 mL of the incubated cell suspension
with which a fresh subculture was prepared. Hence the microbial cells
were proliferated via several subcultures prior to use for laboratory
biodegradation studies. Microbial growth during culture enrichment
was monitored by measuring absorbance at 600 nm using UVVisible
spectrophotometer. Given that the ninth subculture had high cell density, it was harvested on the fth day when the cell growth was still at the
exponential stage and used for carrying out biodegradation experiments in the presence of clay minerals. Microbial growth kinetics
in the presence of homoionic montmorillonites was measured via
standard plate count.

2.2.1. X-ray diffraction (XRD)

The basal spacings of the clay samples were measured by XRD. The
samples were prepared for XRD measurement by orienting the clays
in a glass slide following standard procedure. The slides were air dried
and placed in a desiccator containing silica gel to prevent rehydration.
Glycolated and heat treated (at 300 C) samples were also prepared
following standard procedure. The XRD was observed using Cu-K generated at 40 kV and 40 mA using PANalytical X'Pert Pro MPD tted with
an X'Celerator machine. The data was collected over a range of 2702
with a nominal step size of 0.01672 and nominal time per step of
1.00 s. Data were interpreted by reference to X'Pert accompanying
software program High Score Plus in conjunction with the ICDD
Powder Diffraction File 2 database (1999) and the Crystallography
Open Database (October 2010; www.crystallography.net).

2.3.2. Total petroleum hydrocarbon (TPH)

The effect of the homoionic montmorillonites on biodegradation of
the crude oil hydrocarbons was determined by measuring the residual
TPH after incubation of the aqueous clay/oil microcosm experiments
with hydrocarbon degrading microorganism community for 60 d.
Clay/oil ratio (w/w) of 5:1 was used in all the experiments. Hence, the
microcosm consists 250 mg of clay and 50 mg of oil in 10 mL of
BushnelHaas medium with the microbial cells. The BushnelHaas
(BH) medium supplied the nutrients required by the microbial cells
and was prepared by dispersing 3.2 g of BH broth in 1.0 L of deionized water and autoclaved at 15 lb and 121 C for 15 min. The BH
broth consisted of magnesium sulfate (0.2 g/L), calcium chloride
(0.02 g/L), monopotassium phosphate (1.0 g/L), dipotassium phosphate (1.0 g/L), ammonium nitrate (1.0 g/L), and ferric chloride
(0.05 g/L). Control experiments to account for abiotic processes due to
volatilization and adsorption in addition to the positive control were
in place. For the purpose of this study, the following descriptions hold
for the controls: Control-1 is the positive control containing BH medium, oil and microbial cells (no clay). Control-2 is the negative control
containing BH medium and oil (no clay and no microbial cells). This
control accounts for volatilization and is also used (in combination
with clay controls) to estimate adsorption of the hydrocarbons. Clay
controls were a set of controls containing BH medium, clay and oil
(no microbial cells) and were important in estimating biodegraded
and adsorbed hydrocarbons. All experiments were carried out in triplicates to ensure reproducibility and amenability to statistical analysis.

2.2.2. Fourier transform infra-red (FTIR)

The clay samples were prepared using KBr pellets at sample
concentration of about 1% and the Fourier transform infrared spectra
of the clay samples were recorded in Thermo Nicolet Nexus 870 tted
with a transmission accessory and equipped with a DTGS detector.
The spectrum of the clay sample was collected, by collecting 100 scans
over a wavenumber of 4004000 cm1 at 4 cm1 resolution.

2.3.3. Extraction of hydrocarbons and analysis

Three stages of extraction with 30 mL of dichloromethane (DCM)
for each stage were employed to extract the residual oil (EOM) subsequent to spiking with squalane as surrogate and TPH quantitation standard. The extract was further extracted on solid phase extraction (SPE)
columns following the procedure of Bennett et al. (2002) to separate the
hydrocarbons from the polars (resins and asphaltenes) of the residual

2.2. Characterization of the clay samples

Table 1
Basal spacing of 001 reections (XRD) and selected FTIR absorption bands of the clay samples.
Sample

BU
B-Na
B-K
B-Mg
B-Ca
B-Zn
B-Al
B-Cr
B-Fe

XRD

FTIR

d-Spacing ()

Absorption band (cm1)

Ethylene
glycolated

Air dried

Heat treatment
(300 C)

OH-stretch

Carbonate

17.1
17.2
17
17
17
17
17
17
17

12.5
12.7
12.6
14.5
14.8
14.6
14.4
14.4
14.4

10.6
10
10
10.1
10.2
10.2
10.3
10.3
10.3

3623
3623
3623
3623
3623
3623
3623
3623
3623

1430
1430
1430
1430
1430

BU = unmodied montmorillonite, B-Na = sodium montmorillonite, B-K = potassium

montmorillonite, B-Mg = magnesium montmorillonite, B-Ca = calcium montmorillonite,
B-Zn = zinc montmorillonite, B-Al = aluminum montmorillonite, B-Cr = chromium
montmorillonite, B-Fe = ferric montmorillonite.

oil (EOM). Prior to GCFID analysis, the samples were spiked with
internal standards namely, heptadecylcyclohexane, 5-androstane
and 1,1-binaphthyl. The relative response factor (RRF) of the surrogate
standard varied between 0.78 and 0.8 which is acceptable however,
for computing the percentage recovery of the surrogate standard, RRF
of unity was assumed. The percentage recovery of the surrogate
standard lied between 70% and 120% which is within acceptable range
(USEPA method 8270).

viable cells (CFU/ml)/10^9

U.C. Ugochukwu et al. / Applied Clay Science 87 (2014) 8186

83

1.6

Control-1
B-Na
B-K
B-Mg
B-Ca
B-Zn
B-Al
BU-250
B-Cr
B-Fe
Control-2

1.4
1.2
1
0.8
0.6
0.4
0.2
0
0

10

12

14

Time (days)
Fig. 1. Effect of homoionic montmorillonites on the growth of hydrocarbon degrading
bacteria. Control-1 is the positive control (BH + oil + cells) without clay; Control-2 is the
negative control (BH + oil) with neither clay nor cells. BU-250 = unmodied montmorillonite, B-Na, B-K, B-Mg, B-Ca, B-Zn, B-Al, B-Cr and B-Fe represent sodium-, potassium-,
magnesium-, calcium-, zinc-, aluminum-, chromium-, and ferric-montmorillonites respectively. There was no growth on clay controls, CBU-250, CB-Na, CB-K, CB-Mg, CB-Ca, CB-Zn,
CB-Al, CB-Cr and CB-Fe (not shown on chart). Values are reported as mean one standard
error of the mean.

2.5.1. Differentiating between TPH biodegraded from those adsorbed

The following equations were used to determine the weight and
percentage of hydrocarbons removed by biodegradation and adsorption.

TPHas biodegraded mg TPHcy TPHr

h


i
TPHas biodegraded % TPHcy TPHr =TPHcy  100

where:

2.4. Analytical instrumentation for the hydrocarbons

2.4.1. GCFID
The GC instrument used was an HP 5890 series II gas chromatograph
equipped with a split/splitless injector and ame ionization detector
(FID). The sample was injected using a HP 7673 autosampler. The
separation of the crude oil hydrocarbon compounds was carried out
on an Agilent HP-5 capillary column (30 m 0.25 mm) coated with
5% phenylmethyl-polysiloxane (0.25 m thick) stationary phase. The
GC oven temperature was programmed from 50 C for 2 min and then
ramped at 4 C/min, up to 300 C where it was held for 20 min. The
carrier gas used was hydrogen at a ow rate of about 2 mL/min at an
initial pressure of 100 kPa. The GC data was acquired using Atlas
software on HP computer desktop.

TPHcy = TPH (mg) of the clay control (BH + oil + clay; no cells),
TPHr = TPH (mg) of clay test sample (BH + oil + clay + cells).

TPHas biodegraded mg for Control1 TPHc2 TPHcx

TPHas biodegraded % for Control1

TPHc2 TPHcx =TPHc2   100

where TPHc2 is the residual TPH (mg) of Control-2 (BH + oil: with
neither clay nor cells) and TPHcx is the residual TPH (mg) of Control-1
(BH + oil + cells: without clay).
TPHas adsorbed mg TPHc2 TPHcy

2.5. Total petroleum hydrocarbon (TPH)

The basis for the assessment of biodegradation in this study is the
determination of the total petroleum hydrocarbons (TPHs) using GC.
This TPH as determined by GC is the residue of the original TPH after
incubation. The determination of the TPH concentration was done by
measuring the total GC area between 10 and 70 min and subsequent
quantitation with squalane.

Table 2
EGME-surface area, pH and cation exchange capacity (CEC) of the clay samples.
Sample

pH

Surface area (m2/g)

CEC (meq/100 g)

BU
B-Na
B-K
B-Mg
B-Ca
B-Zn
B-Al
B-Cr
B-Fe

9.0
7.8
7.8
7.8
7.6
5.9
4.7
4.8
4.9

645
570
455
522
598
525
513
489
646

83.3
83
76.4
81.7
79.1
73.8
65.9
83
88

TPHas adsorbed %

h


i
TPHc2 TPHcy =TPHc2  100:

Table 3
Maximum cell yield, specic growth rate, % TPH biodegraded and % TPH adsorbed due to
the effect of homoionic montmorillonites. Values are reported as mean one standard
error of the mean.
Sample

Maximum cell yield

(CFU/mL) / 10^8

Specic growth
rate (h1) 10^3

% TPH
biodegraded

% TPH
adsorbed

BU-250
B-Na
B-K
B-Mg
B-Ca
B-Zn
B-Al
B-Cr
B-Fe
BU-250
Control-1

11.2
12.0
8.2
10.0
14.1
6.5
10.1
8.1
13.2
11.2
8.1

10.2
10.1
8.8
9.5
11.3
7.5
10.1
8.8
11.0
10.2
9.1

72
71
41
61
79
44
64
47
78
72
50

27
17
49
20
18
46
17
29
16
27

0.4
0.8
0.6
1
0.5
0.7
0.5
0.3
0.7
0.4
0.3

0.4
0.4
0.4
0.6
0.4
0.5
0.4
0.3
0.4
0.4
0.2

2
1.8
2.3
3.2
0.5
4.5
1.0
2.0
2.0
2.0
1.0

3.7
2.7
4.2
2.8
2.4
2.9
1.3
3.4
1.7
3.7

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96 (96,1)
Acquired 30 July 2010 08:52:22

Atlas,nrg_ch03.ugo23-7-10,96,1,1

C28

C29

C30

C27

C26
squ

hdch
C25

C23

11bi nap

C24

C21

C22

C19

andro

23.80

100

pyt

pris

C20

C15

150

C14

Response

18

17

C16

200

50
0

10

15

20

25

30

35

40

45

50

55

60

65

70

Retention time
Fig. 2. Chromatogram (of TPH fraction) showing no biodegradation-sample Control-2. Control-2 = negative control (BH + oil) no clay and no cells. Squ = squalane;
hdch = heptadecylcyclohexane; 1,1-binaph = 1,1-binaphthyl; andro = 5-androstane. The hydrocarbon peaks are labeled: C14C30.

3. Results and discussion

3.1. Characterization of the clay samples
Na-montmorillonite and K-montmorillonite have almost identical
d-spacing (001) on air drying (12.7 and 12.6 ) whereas divalent
and trivalent cations in the interlayer of montmorillonite caused the
d-spacing on air drying to shift to between 14.4 and 14.8 as a result
of increased hydration energy which favors attraction of two water
layers (Table 1). The FTIR absorption band at 3623 cm 1 is due to
OH-stretch of AlAlOH typical of montmorillonites. Samples B-Zn, B-Al,
B-Cr and B-Fe did not show any absorption band at 1430 cm 1
(which is normally assigned to carbonates such as calcite) as the carbonates must have been digested during the cation exchange reactions.
These samples have relatively lower pH (Tables 1 and 2).
The EGME-surface area, CEC and pH of the clay samples are as shown
in Table 2 below.

3.2. Microbial growth and biodegradation of crude oil hydrocarbons in the

presence of homoionic montmorillonite clay samples
The effect of homoionic montmorillonite clay samples on the growth
of hydrocarbon degrading bacteria is presented in Fig. 1.
The growth of the hydrocarbon degrading bacteria as evidenced by
maximum cell yield and specic growth rate varied with the type of
the interlayer cation of the montmorillonite (Fig. 1 and Table 3).
The gas chromatograms of selected samples are as shown in Figs. 24.
The nC17/pristane and nC18/phytane ratios for the sample, Control2 are 2.0 and 2.1 respectively.

The nC17/pristane and nC18/phytane ratios for samples B-K and B-Ca
(Figs. 3 and 4) are not measurable as a result of biodegradation.
The effect of the clay mineral samples on microbial growth, biodegradation and adsorption of crude oil hydrocarbons assessed by the
maximum cell yield, specic growth rate, % TPH biodegraded and %
TPH adsorbed is presented in Table 3.
Biodegradation of the hydrocarbons occurred most in samples B-Ca
and B-Fe among all samples (Table 3) whereas they occurred least in
samples B-K, B-Zn and B-Cr. This suggests that the modication of the
interlayer cation to calcium- and ferric-montmorillonites leads to improvement on the biodegradation of the hydrocarbons. The 2-sample
t-test at 95% condence interval (CI) with respect to maximum cell
yield, specic growth rate constant and percentage biodegradation of
TPH indicates that B-Ca and B-Fe are signicantly different from B-K,
B-Zn, B-Cr and Control-1 (P-values of b 0.05). However, samples B-K
and B-Zn adsorb hydrocarbons signicantly more than all the other
samples (Table 3; P-values b 0.5) and is believed to be due to their
surface chemistry. It appears that the implication of having for example,
K+ in the interlayer of the clay is that the attraction of water in the interlayer will be reduced due to the lower hydration energy of K+ as its
charge/radius ratio is relatively low (Jaynes and Boyd, 1991). This
makes the interlayer of K-montmorillonite to exhibit a relatively reduced hydrophilicity. This, coupled with the fact that K+ has a relatively
large size (that will expose the hydrophobic siloxane surface), causes
this clay sample to possess the ability to host hydrocarbons which are
hydrophobic. The same reason of exposure of the hydrophobic siloxane
surface accounts for why the EGME-surface area of K-montmorillonite
is the lowest among the homoionic montmorillonites as the EGME
(which is a polar compound) would have difculty accessing the exposed hydrophobic siloxane surface in the interlayer of the clay sample

59 (59,1)
Acquired 28 July 2010 08:11:48

Atlas,nrg_ch03.ugo23-7-10,59,1,1

heptadecylcyclohexane

squ

150

11bi nap

100
andro

Response

200

50
0

10

15

20

25

30

35

40

45

50

55

60

65

Retention time
Fig. 3. Chromatogram (of TPH fraction) showing moderate to heavy biodegradation-sample B-K. B-K = K-montmorillonite.

70

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144 (1,1)
Acquired 04 August 2010 17:36:58

85
Atlas,nrg_ch03.ugo1-8-10,1,1,1

hdch

200

squ

1,1-binaph

100

41.49

andro

Response

150

50
10

15

20

25

30

35

40

45

50

55

60

65

70

Retention time
Fig. 4. Chromatogram (of TPH fraction) showing very heavy biodegradation-sample B-Ca. B-Ca = calcium-montmorillonite.

of the hydrocarbons. Whereas local bridging effect will lead to

enhanced biodegradation of the crude oil hydrocarbons, hydrolysis of
the interlayer water by the interlayer cations is suggested to lead to
inhibition of biodegradation of the crude oil hydrocarbons. However,
the role of polarization of water by trivalent cations in inhibiting biodegradation would be investigated in further studies by monitoring the pH
of the system as the volume of water decreases during the biodegradation process.

4. Conclusion
Homoionic montmorillonites affect the biodegradation of crude oil
hydrocarbons, though whether the biodegradation will be inhibited or
stimulated depends on the type of interlayer cation. Potassium-, zinc-,
and chromium-montmorillonites seem to be inhibitory to biodegradation of crude oil hydrocarbons and the inhibition appears to be as a
result of their ability to cause extensive adsorption of the hydrocarbons.
Calcium- and iron(III)-montmorillonites appear to be more stimulatory
to biodegradation of the hydrocarbons than unmodied montmorillonite. Surface area and the local bridging effect have been identied as important factors that determine the stimulatory role of the clay minerals.
Adsorption of hydrocarbons by the clay samples during biodegradation
of crude oil hydrocarbons is believed to take place via cation interactions and its extent is a function of the interlayer cation. This study indicates that Fe-montmorillonite, Ca-montmorillonite and unmodied
montmorillonite would serve a very useful purpose in the bioremediation of crude oil spill sites.

Fitted Line Plot

TPH(%) = - 55.70 + 0.2155 Surface area (square meter/ g)
S
R-Sq
R-Sq(adj)

80

% TPH biodegraded

(Table 2). It is not clear yet how Zn2 + in the interlayer of the clay
mineral accomplishes relatively high adsorption of the hydrocarbons.
The results of the stimulatory role of the Ca-montmorillonite and
Na-montmorillonite appear to be consistent with the studies of Warr
et al. (2009) although the study (Warr et al., 2009) did not cover the
effect of other homoionic montmorillonites such as K-montmorillonite,
Zn-montmorillonite Al-montmorillonite, Cr-montmorillonite and Femontmorillonite on microbial growth and biodegradation of crude oil
hydrocarbons. With respect to adsorption, this study has taken the
study of Warr et al. (2009) a step further by quantifying adsorption of
hydrocarbons during biodegradation of the oil. The absence of a clay
control (i.e. an experiment with only clay and oil) in the study of Warr
et al. (2009) made it impossible for their study to estimate losses due
to adsorption. This study indicates that adsorption could be signicant
depending on the interlayer cation (Table 3). The microbial growth
and percentage biodegradation of crude oil hydrocarbons in the
presence of the clay samples appear to be high with the samples of
high surface area (Fig. 5, Tables 2 and 3). Surface area of the clay samples
therefore appears to play a vital role during biodegradation of the
hydrocarbons (Warr et al., 2009).
Interlayer cations are believed to cause local bridging effect
(effective delivery of nutrients to cells due to reduction of zeta potential
or electrical double layer repulsion) and this together with high surface
area has been suggested to account for why some clays stimulate
biodegradation (Bright and Fletcher, 1983; Fletcher and Marshall,
1982; Warr et al., 2009). However, clays with interlayer cations that
favor adsorption would render the hydrocarbons unavailable and
hence inhibit biodegradation of the hydrocarbons. It appears that local
bridging effect is not the only major factor that determines the extent
of biodegradation of the hydrocarbons in the presence of clay. If it
were, the trivalent cations such as Al3+, Fe3 + and Cr3 + would cause
the most microbial growth and consequently, the highest extent of biodegradation. Also, Na-montmorillonite would have effect the least extent of hydrocarbon biodegradation given that sodium is monovalent.
In addition to the adsorption factor during biodegradation, another factor such as polarizability of the interlayer cations that leads to hydrolysis
of the interlayer water could occur. Interlayer cations have the ability to
hydrolyze shells of water in the interlayer of clay minerals producing
protons that can increase the Bronsted acidity of the clay suspension
(Laszlo, 1987; Pinnavaia, 1983; Soma and Soma, 1989). The ability of trivalent cations to hydrolyze the water in the interlayer of the clay mineral is a function of the charge/size ratio of the cation. Trivalent cations
have the highest ability to polarize (hydrolyze) the interlayer water
(Table 2). This is expected to have the effect of reducing the pH of the
system especially as the volume of water in the medium decreases
which will ultimately reduce microbial activity (Alexander, 1999).
This ability of trivalent cations to generate protons by polarizing interlayer water is therefore suggested to be inhibitory to biodegradation

7.85618
76.9%
73.1%

70

60

50

40
450

500

550

600

650

Surface area (square meter/g)

Fig. 5. Effect of surface area of the homoionic montmorillonites on the extent of biodegradation of the crude oil hydrocarbons.

86

U.C. Ugochukwu et al. / Applied Clay Science 87 (2014) 8186

Acknowledgments
We thank Berny Bowler, Paul Donohue, Phil Green and Ian Harrison
for the laboratory support received from them. Generally, we are
grateful to Petroleum Technology Development Fund (PTDF) of the
Federal Republic of Nigeria for funding this project and the School of
Civil Engineering and Geosciences for providing the facilities used in
this study.
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