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Compositional changes of crude oil SARA fractions due to biodegradation and

adsorption on colloidal support such as clays using Iatroscan

Article  in  Environmental Science and Pollution Research · April 2013

DOI: 10.1007/s11356-013-1635-8 · Source: PubMed

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Compositional changes of crude oil SARA
fractions due to biodegradation and
adsorption on colloidal support such as
clays using Iatroscan

Uzochukwu C. Ugochukwu, Martin

D. Jones, Ian M. Head, David
A. C. Manning & Claire I. Fialips

Environmental Science and Pollution

Research

ISSN 0944-1344

Environ Sci Pollut Res

DOI 10.1007/s11356-013-1635-8

1 23
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1 23
 Author's personal copy
Environ Sci Pollut Res
DOI 10.1007/s11356-013-1635-8
RESEARCH ARTICLE
Compositional changes of crude oil SARA fractions
due to biodegradation and adsorption on colloidal
support such as clays using Iatroscan
Uzochukwu C. Ugochukwu & Martin D. Jones &
Ian M. Head & David A. C. Manning & Claire I. Fialips
Received: 25 November 2012 / Accepted: 12 March 2013
# Springer-Verlag Berlin Heidelberg 2013
Abstract The compositional changes of saturates, aromatics,
resins and asphaltenes (SARA) fractions in aqueous clay/oil
microcosm experiments with a hydrocarbon-degrading micro-
organism community were analysed using Iatroscan. The clay
mineral samples used in this study were organomontmorillonite,
acid-activated montmorillonite and K, Ca, Zn and Cr montmo-
rillonites produced by modifying the original montmorillonite
sample. The evaluation and quantification of biodegradation
and adsorption were carried out using a combination of the
Iatroscan and gravimetric analysis. The SARA compositions in
the presence of organomontmorillonite and acid-activated
montmorillonite after incubation follow the same pattern in
which the aromatic fraction is higher than the other fractions
unlike in the presence of unmodified, K, Ca and Zn montmo-
rillonites, where the saturates fraction is higher than the other
fractions. Changes in SARA fractions due to biodegradation
seemed to occur most in the presence of unmodified and
calcium montmorillonites; hence, the removal of SARA frac-
tions due to biodegradation was significant and enhanced in the
presence of these two clay samples. However, biodegradation
in the presence of organomontmorillonite and acid-activated
and Cr montmorillonites was hindered. The study indicated that
Cr montmorillonite adsorbed resins most, whereas Zn and K
montmorillonites adsorbed aromatics most after incubation.
Keywords Iatroscan . Biodegradation . Adsorption .
Saturates . Aromatics . Resins . Asphaltenes
Responsible Editor: Philippe Garrigues
U. C. Ugochukwu (*) : M. D. Jones : I. M. Head :
D. A. C. Manning : C. I. Fialips
School of Civil Engineering and Geosciences,
University of Newcastle upon Tyne, Drummond Building,
Newcastle upon Tyne NE1 7RU, UK
e-mail: ugouzochukwu@yahoo.com
Introduction
Environmental pollution by crude oil and its fractions has
become a major concern in the world today as it is the main
cause of ecological and social damage (Burger 1993; Burns et
al. 1993; Shaw 1992). Colloidal supports like clay minerals are
believed to have significant impacts on the environmental fate
of pollutants such as oil spills (Chaerun and Tazaki 2005). In
addition to being able to stimulate microbial growth, some clay
minerals are believed to have the ability to sorb organic, inor-
ganic and microorganisms which might be beneficial in some
environmental remediation processes (van Loosdrecht et al.
1990; Guerin and Boyd 1992; Khanna and Stotzky 1992; Tapp
and Stotzky 1995; Sposito et al. 1999; Murray 2000;
Churchman et al. 2006; Warr et al. 2009). The use of Fourier
transform infrared spectroscopy (FTIR), GC-FID and GC-MS
to assess changes in the oil composition during biodegradation
is limited to hydrocarbons as the changes in resins and
asphaltenes cannot be evaluated. Several studies have shown
that Iatroscan which is essentially thin-layer chromatography–
flame ionization detection (TLC-FID) can be used in the eval-
uation of compositional changes of petroleum arising from
biotic processes. It is indeed a semi-quantitative analytical tool
and does not always produce highly reproducible measure-
ments. However, it is a rapid means of revealing the changes
in the percentage distribution of the saturates, aromatics, resins
and asphaltenes (SARA) fractions of the oil (Pollard et al. 1992;
Napolotano et al. 1998). Consequently, this tool has become
very useful in monitoring the changes in composition of petro-
leum in environmental processes (Pollard et al. 1992;
Napolotano et al. 1998; Stephens et al. 1998). The analysis of
SARA fractions in biodegraded oils in order to estimate extent
of biodegradation has been reported (Goto et al. 1994; Maki et
al. 2001; Jirasripongpun 2002; Ishiyama et al. 2007). However,
the use of Iatroscan for quantifying changes in SARA fractions
 Author's personal copy
of crude oil during biodegradation processes involving solid
supports where adsorption also takes place has not been
reported. Clays in the environment can exist in an unmodified
or modified form such as acid-activated clay, organoclay and
homoionic clays. Acid-activated clay minerals can occur natu-
rally in the environment by the attack of acid on clay minerals
as is the case with the acid mine drainage interacting with clay
minerals (Galan et al. 1999; Dubikova et al. 2002). In the
laboratory, acid-activated clays are prepared by washing or
treating the clay with strong mineral acids such as sulphuric
or hydrochloric acid. During acid activation, the main goal is to
obtain partially dissolved material that is of increased surface
area, porosity and surface acidity (Komadel 2003; Pushpaletha
et al. 2005). Producing organoclays in practice requires the
replacement of the interlayer exchangeable inorganic cations
with organic cations through ion exchange reactions. The re-
sultant organoclay modifies the surface of the original clay
mineral from being hydrophilic to hydrophobic (Hermosin et
al. 1992; Groisman et al. 2004). The homoionic clays are
usually generated by exchanging the interlayer cations of
unmodified clays with excess metallic chloride of the required
metallic ion to produce the desired homoionic clay via cation
exchange reactions (Reddy et al. 2007).
This report is based on laboratory study conducted to
investigate the changes in the SARA fractions due to biodeg-
radation and adsorption in the presence of unmodified and
modified montmorillonites (such as acid-activated, organo-
and homoionic montmorillonites). The following questions
were addressed:
(a) What was the percent distribution of the SARA fractions
in the residual oil after the incubation of the experimental
microcosm containing aqueous clay/oil and oil-degrading
microbes for the test samples in comparison with the
control samples?
(b) How were the losses of the hydrocarbons due to abiotic
processes such as adsorption estimated?
(c) How can the Iatroscan data be used to estimate the
effect of the clays on the biodegradation and adsorption
of the crude oil hydrocarbons and resins?
Materials and methods
Clay mineral used in this study was montmorillonite. Benton-
ite (Berkbent 163) which was the source of montmorillonite
was supplied by Steetley Bentonite & Absorbents Ltd. All the
chemicals were supplied by Sigma-Aldrich. Microbial com-
munities responsible for biodegradation of the crude oil hy-
drocarbons were isolated from beach sediment sample
consisting of fine sand particles collected in a sterilised glass
bottle (Duran) from a site at St. Mary's lighthouse near
Whitley Bay, Newcastle upon Tyne (N 55° 04′ 18″, W 01°
Environ Sci Pollut Res
26′ 59″), UK and were stored at 4 °C in the cold room until
commencement of the experiment. The Bushnell-Haas (BH)
broth as the nutrient source and nutrient agar were supplied by
Sigma-Aldrich. The crude oil was an undergraded North Sea
crude oil originally supplied by British Petroleum.
Characterization of the clay samples
X-ray diffraction
The basal spacings of the clay samples were measured by X-
ray diffraction (XRD). The samples were prepared for XRD
measurement by orienting the clays in a glass slide following
a 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 a standard procedure. The XRD was
observed using Cu-Kα generated at 40 kV and 40 mA using
PANalytical X'Pert Pro MPD fitted with an Xcelerator ma-
chine. The data were collected over a range of 2–70° 2θ with
a nominal step size of 0.0167° 2θ and nominal time per step
of 1.00 s. Data were interpreted by reference to X'Pert
accompanying software programme High Score Plus in con-
junction with the ICDD Powder Diffraction File 2 database
(1999) and the Crystallography Open Database (October
2010; www.crystallography.net).
FTIR
The samples were prepared using KBr pellets at sample con-
centration of about 1 %, and the Fourier transform infrared
spectra of the clay samples were recorded on Thermo Nicolet
Nexus 870 fitted with a transmission accessory and equipped
with a DTGS detector. The spectrum of the clay sample was
collected, by collecting 100 scans over a wave number of
400–4,000 cm−1 at 4 cm−1 resolution.
Surface area
The surface area of the clay samples was measured by the
ethylene glycol monoethyl ether (EGME) method following
the method of Carter et al. (1965).
Cation exchange capacity of the clay samples
The cation exchange capacity (CEC) of the clay samples
was determined following the standard ammonium acetate
method (Lewis 1949).
pH
The pH of the clay suspension was measured with a pH
meter.
 Author's personal copy
Environ Sci Pollut Res
Clay mineral modification
The conditions for acid activation of the montmorillonite
clay mineral were as follows: 70 °C, 45 min reaction time
and 3 M HCl. The acid-activated clay mineral samples
prepared (to be used for biodegradation studies) were thor-
oughly washed to ensure that there was no free HCl in the
clay samples by measuring free chloride using Dionex ICS-
1000 ion chromatograph.
At 35 % CEC, the corresponding amount of dodecyldi
methylammonium (DDDMA) bromide was added to mont-
morillonite suspension. The mixture was stirred for 24 h,
and thereafter, the clay suspension was centrifuged, and the
supernatant was rejected, while the produced organomont
morillonite clay was washed repeatedly with de-ionized
water and dried at a temperature of 48 °C and stored in a
desiccator. The homoionic montmorillonites samples were
generated from exchanging the interlayer cations with K+,
Ca2+, Zn2+ and Cr3+ to produce K, Ca, Zn and Cr montmo-
rillonites; 0.5 M solution of the corresponding metal chlo-
ride salt was prepared, and then, 200 mL of each of the
solution was 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 subse-
quently centrifuged. The supernatant was rejected, and the
clay mineral was washed repeatedly until the chloride level
was negligible.
Laboratory biodegradation
Microbial growth
Indigenous microbial cells of Whitley Bay sediments
were isolated and proliferated via several subcultures
prior to use for laboratory biodegradation studies. Micro-
bial growth during culture enrichment was monitored by
measuring absorbance at 600 nm using UV-Visible
spectrophotometer.
Changes of the SARA fractions during biotic and abiotic
processes
The effect of the clay minerals on biodegradation (biotic
process) and adsorption (abiotic process) of the crude oil
SARA fractions was determined by measuring the per-
centage distribution of the SARA fractions after incuba-
tion of the aqueous clay/oil microcosm experiments with
oil-degrading microorganism community for 60 days.
Clay/oil ratio (w/w) of 5:1 was used in all the experi-
ments. Hence, the microcosm consist 250 mg of clay and
50 mg of oil in 10 mL of Bushnell-Haas medium with
the microbial cells. Control experiments to account for
abiotic processes due to volatilization and adsorption in
addition to the positive control were in place (see Table 1
for sample descriptions). Control 2 is the negative control
containing BH medium and oil (no clay and no microbial
cells). It accounts for volatilization and is also used (in
combination with clay controls) to estimate adsorption of
the SARA fractions. Clay controls are a set of controls
containing BH medium, clay and oil (no microbial cells)
and are important in estimating biodegraded and
adsorbed oil. A combination of Iatroscan and gravimetric
analysis was used for the evaluation of biodegraded and
adsorbed oil (see Evaluation of biodegradation and
adsorption section). All experiments were carried out in
triplicates to ensure reproducibility and amenability to
statistical analysis.
Extraction of residual oil (extractable organic matter)
Three stages of extraction with 30 mL of dichloromethane
(DCM) for each stage were employed to extract the residual
oil [extractable organic matter (EOM)] and the 90 mL oil
solution in DCM concentrated to 10 mL solution in a rota-
vaporator. Aliquots of the extract were used for both
Iatroscan and gravimetric analysis. For gravimetric analysis,
empty 10-mL-capacity vial was weighed and noted. Aliquot
(2 mL) of the residual oil extract was transferred to a
weighed 10-mL vial and gently blown down to dryness
using a stream of nitrogen followed by weighing.
Analytical instrumentation
Iatroscan (TLC-FID)
The percentage distribution of the SARA fractions of the
residual oil extract for both test and control samples was
analysed using an Iatroscan MK-5 TLC-FID following the
method of Karlsen and Larter (1991).
Evaluation of biodegradation and adsorption
The quantitation of the SARA fractions was done with
standard oil (North Sea oil) and the SARA fraction distri-
bution generated in percentage. The total weight (in milli-
gram) of residual oil and extractable organic matter and
distribution by weight (in milligram) of SARA fractions in
the residual oil were estimated as follows:
WTS ¼ 5Y ð1Þ
where
WTS =total weight (in milligram) of residual oil (EOM) in
a given sample
5=conversion factor from 2 to 10 mL
 Author's personal copy
Environ Sci Pollut Res

Table 1 Detailed sample descriptions

Sample name Description

BU Test sample containing unmodified montmorillonite, crude oil, Bushnell-Hass medium and microbes
BA Test sample containing acid-activated montmorillonite, crude oil, Bushnell-Hass medium and microbes
BO Test sample containing organomontmorillonite, crude oil, Bushnell-Hass medium and microbes
B-K Test sample containing potassium montmorillonite, crude oil, Bushnell-Haas medium and microbes
B-Ca Test sample containing calcium montmorillonite, crude oil, Bushnell-Haas medium and microbes
B-Zn Test sample containing zinc montmorillonite, crude oil, Bushnell-Haas medium and microbes
B-Cr Test sample containing chromium montmorillonite, crude oil, Bushnell-Haas medium and microbes
CBU Clay control containing unmodified montmorillonite, crude oil and Bushnell-Haas medium. No microbial cells
CBA Clay control containing acid-activated montmorillonite, crude oil and Bushnell-Haas medium. No microbial cells
CBO Clay control containing organomontmorillonite, crude oil and Bushnell-Haas medium. No microbial cells
CB-K Clay control containing potassium montmorillonite, crude oil and Bushnell-Haas medium. No microbial cells
CB-Ca Clay control containing calcium montmorillonite, crude oil and Bushnell-Haas medium. No microbial cells
CB-Zn Clay control containing zinc montmorillonite, crude oil and Bushnell-Haas medium. No microbial cells
CB-Cr Clay control containing chromium montmorillonite, crude oil and Bushnell-Haas medium. No microbial cells
Control 1 Positive control containing crude oil, Bushnell-Haas medium and microbial cells. No clay minerals
Control 2 Control containing Bushnell-Haas medium and crude oil. No clay minerals and no microbial cells

Y=weight of oil residue (in milligram) in 2 mL aliquot Weight of whole oil adsorbed, as follows:
(see Laboratory biodegradation section)
Wads ¼ WTS ðfor control 2Þ
Fw ¼ WTS X ð2Þ
 WTS ðfor clay control sampleÞ ð5Þ
where
Fw =weight (in milligram) of a given SARA fraction in Percentage of whole oil adsorbed
the oil residue
X=percent of fraction as measured by Iatroscan ¼ ½Wads =WTS ðfor control 2Þ  100 ð6Þ
Weight (in milligram) of whole oil biodegraded, as follows:
Percentage biodegradation of a given SARA fraction, as
Wob ¼ WTS ðfor clay control sampleÞ follows:
2 3
 WTS ðfor test sampleÞ ð3Þ
Fbio ¼ 4ðFw of control sample  Fw of test sampleÞ5100
Percentage of whole oil biodegraded, as follows: =Fw of control sample
Tob% ¼ ½Wob =WTS ðfor control sample clayÞ  100 ð4Þ ð7Þ

Table 2 Basal spacing of 001

reflections and selected FTIR Sample XRD FTIR
absorption bands of the clay
samples d spacing (Å) Absorption band (cm−1)

Ethylene glycolated Air dried Heat treated OH stretch C–H Carbonate

BU 17.1 12.5 10.6 3,623 – 1,430

BO 16.8 14.2 13.2 3,623 2861, 2935 1,430
BA 16.8 14.8 10.1 3,623 – –
B-K 17 12.6 10.0 3,623 – 1,430
BU, BO, BA, B-K, B-Ca, B-Zn,
B-Cr represent unmodified, B-Ca 17 14.4 10.2 3,623 – 1,430
organo-, acid-activated, potassi- B-Zn 17 14.5 10.2 3,623 – –
um, calcium, zinc and chromium B-Cr 17 14.6 10.3 3,623 – –
montmorillonites, respectively
 Author's personal copy
Environ Sci Pollut Res

Table 3 EGME surface area, pH and cation exchange capacity (CEC) organomontmorillonite is due to the C–H symmetrical and
of the clay samples
asymmetrical stretch of the DDDMA moiety of the
Sample pH Surface area (m2/g) CEC (mEq/100 g) organoclay, respectively. The acid-activated, zinc and Cr mont
morillonites did not show absorption band at 1,430 cm−1
BU 9.0 645 83.3 (which is normally assigned to carbonates such as calcite) as
BA 4.1 722 – the carbonates must have been digested during the acid acti-
BO 9.2 471 – vation process and cation exchange reactions.
B-K 7.8 455 76.4
B-Ca 7.6 598 79.1
Surface area, CEC and pH of the clay samples
B-Zn 5.9 525 73.8
B-Cr 4.8 489 83
The EGME surface area, pH and cation exchange capacity
of the clay samples are shown in Table 3.
Percentage adsorption of a given SARA fraction, as On acid activation, the clay samples tend to increase in
follows: surface area and acidity (Table 3). The EGME surface area
of organo- and K montmorillonites (B-K) is low on account
2 3
of reduced ability of the EGME molecule to access the
Fads ¼ 4ðFw of control 2  Fw of clay control sampleÞ5 100 hydrophobic surfaces of these clay samples.
=ðFw of control 2Þ
ð8Þ Changes in percent composition of the SARA fractions

The changes in percent composition of the SARA fractions as

Results/discussion
Characterization of the clay samples
XRD and FTIR
Unmodified and potassium montmorillonites have almost
identical d spacing (001) on air drying (12.5 and 12.6 Å,
respectively), whereas di- and trivalent cations in the
interlayer of montmorillonite caused the d spacing on air
drying to shift to between 14.4 and 14.6 Å as a result of
increased hydration energy which favours attraction of two
water layers (Table 2). The broad IR absorption band observed
at 3,623 cm−1 in the spectra for organomontmorillonite and
acid-activated and homoionic montmorillonites corresponds
to OH stretching of AlAlOH which is typical of montmoril-
lonites. The absorption bands at 2,861 and 2,935 cm−1 for the
60
50
percentage
revealed from the Iatroscan analysis are shown in Figs. 1 and 2.
The result of the gravimetric analysis of the EOM and
saturates/aromatics and saturates/resin analysis is presented
in Table 4.
The distribution pattern of SARA fractions in the residual
oil for experiments containing acid-activated montmorillon-
ite (BA), organomontmorillonite (BO) and control 1 is sim-
ilar (Fig. 1). The percentage composition of aromatics is
higher in these samples than that in their control samples
(Fig. 1). This indicates that the removal of saturates was
higher in these samples than the removal of aromatics and is
most probably due to biotic process of biodegradation as
saturates are relatively easier to be broken down by microbes.
The EOM and saturates/resins ratio (Table 4) indicate that
there have been changes in the experiments containing BA,
BO, unmodified montmorillonite (BU) and control 1 that
could be attributed to biodegradation. However, the pattern
of percentage distribution of the SARA fractions with sample

40
30 saturates
20
aromatics
10
0 resins
asphaltenes

sample

Fig. 1 Percentage distribution of SARA fractions after incubation in (without cells) for acid-activated, organo- and unmodified montmoril-
the presence of acid-activated, organo- and unmodified montmorillon- lonites, respectively (see Laboratory biodegradation section). Controls
ites. BA, BO and BU represent acid-activated, organo- and unmodified 1 and 2 are described in Laboratory biodegradation section
montmorillonites, respectively. CBA, CBO and CBU are clay controls
 Author's personal copy
Environ Sci Pollut Res

Fig. 2 Percentage distribution 70

of SARA fractions after 60
incubation in the presence of K,

percentage
50
Zn, Cr and Ca
40 saturates
montmorillonites. B-K, B-Zn,
B-Cr and B-Ca represent K, Zn, 30 aromatics
Cr and Ca montmorillonites, 20 resins
respectively. CB-K, CB-Zn, 10
CB-Cr and CB-Ca are the clay asphaltenes
0
controls (controls without cells) B-K CB-K B-Zn CB-Zn B-Cr CB-Cr B-Ca CB-Ca
sample

BU is similar to that of the clay control sample (CBU) (Fig. 1), to their control samples, this does not imply that there have
indicating that percentage distribution of SARA fractions not been changes due to biodegradation. The EOM and
alone is not enough for the assessment of biodegradation. saturates/resins ratio indicate that there have been changes
The combined use of percentage distribution of SARA frac- in the presence of these clay samples that are most likely due
tions, EOM and saturates/resins ratio gives a better indication to biodegradation (Table 4). Adsorption which is estimated
of changes due to biodegradation (Fig. 1; Table 4). in this study by comparing EOM of clay controls and control 2
The percentage distribution of saturates and aromatics is in addition to saturates/aromatics ratios for the clay controls
almost the same for sample B-Cr (Fig. 2), indicating that indicates that CB-Cr, CB-Zn and CB-K adsorbed more oil
there was a slight removal of saturates after incubation. This than the other control samples (Table 4; Figs. 1, 2 and 4). It is
removal of saturates was most probably due to biodegrada- therefore expected that the corresponding test samples would
tion. The other test samples (homoionic montmorillonites) remove some oil via adsorption.
have the same pattern of SARA distribution in comparison
with their controls, making it necessary to look at the EOM Biodegradation and adsorption of oil and SARA fractions
and saturates/resin ratio (Table 4) for a better assessment of
changes that are likely due to biodegradation. Though the The biodegradation and adsorption of the oil and its SARA
pattern of distribution of SARA fractions in the presence of fractions are shown in Figs. 3 and 4.
homoionic montmorillonites such as B-K, zinc montmoril- Biodegradation of saturates, aromatics, resins and whole
lonite (B-Zn) and calcium montmorillonite (B-Ca) is similar oil appear to have been hindered in samples BA, BO and B-
Cr, in comparison with the positive control, control 1
(Table 4; Figs. 3, 6 and 7). It is suggested that samples BA
Table 4 EOM and ratio analysis of saturates, aromatics and resins. and B-Cr being more acidic than the other samples may
Values are reported as mean have hindered biodegradation by having toxic effect on the
microbes. Sample BO may have hindered biodegradation as
Sample Saturates/aromatics Saturates/resin EOM
ratio ratio (mg) a result of the ability of organomontmorillonite to render the
hydrocarbons unavailable (by hydrophobic interaction) for
BA 0.9 2.9 19.4 biodegradation. The high surface area of BA (Table 3) could
CBA 1.5 5.7 30.4 have been a useful factor for providing a huge support for
BO 1.0 2.5 20.5 the microbes, but the toxic effect of the acidic surface seems
CBO 1.4 3.9 32.3 to outweigh the supposed benefit of the high surface area;
BU 1.4 2.8 8.5 hence, the general hindrance of biodegradation was ob-
CBU 1.5 5.3 24.8 served. There has been neither enhancement nor hindrance
Control 0.9 2.6 18.7 of biodegradation in the presence of B-K and B-Zn as the
1 percentage biodegradation of the oil, and its fractions was
Control 1.2 4.9 35.6
comparable with control 1 (Fig. 3). This may be due to
2
B-K 1.2 1.8 9.7 adsorption of the oil on these two clay mineral samples
CB-K 1.8 4.0 17.6 (potassium and zinc montmorillonites), reducing the avail-
B-Zn 1.4 2.9 11.6 ability of the oil for biodegradation (Fig. 4). The percentage
CB-Zn 1.8 5.2 21.3 removal of saturates and aromatic fractions by biodegrada-
B-Cr 1.0 2.3 12.3 tion in the presence of unmodified and calcium montmoril-
CB-Cr 1.6 6.4 19.1
lonite was in the range of 70–76 % (Fig. 3). Also, the
B-Ca 1.2 2.0 6.8
percentage removal of whole oil and resins via biodegrada-
CB-Ca 1.4 4.6 25.3
tion in the presence of these two clay mineral samples was
in the ranges of 65–70 and 42–50 %, respectively, which
 Author's personal copy
Environ Sci Pollut Res

100

% biodegraded
80
60
whole oil
40
20 saturates

0 aromatics
resins

Sample

Fig. 3 Biodegradation of oil, saturates, aromatics and resins supported calcium montmorillonites, respectively. Control 1 control containing
on clay mineral. BA, BO, BU, B-K, B-Zn, B-Cr and B-Ca represent microbes, oil and BH medium, but no clay
acid-activated, organo-, unmodified, potassium, zinc, chromium and

Fig. 4 Adsorption of oil, 80

saturates, aromatics and resins % adsorbed 60
supported on clay mineral whole oil
40
saturates
20
aromatics
0
BA BO BU B-K B-Zn B-Cr B-Ca resins
Sample

Standard (rod 2) (2,1)

Acquired 05 August 2010 15:00:45 defproj,nrg_ch06.CeG_Iatroscan 08-05 1456,2,1,1
Response

0.21

0.14
75 0.33

70

0.43
65

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55
Retention time

Fig. 5 TLC-FID chromatogram for standard oil (North Sea oil)

Sample 2 (rod 5) (5,1)

defproj,nrg_ch06.CeG_Iatroscan 08-05 1456,5,1,1
Acquired 05 August 2010 15:03:05
Response

75
0.13
0.20
0.33
70

0.44
65

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55
Retention time

Fig. 6 TLC-FID chromatogram for sample organomontmorillonite

 Author's personal copy
Environ Sci Pollut Res

Sample 1 (rod 3) (3,1) defproj,nrg_ch06.CeG_Iatroscan 08-06 1528,3,1,1

Acquired 06 August 2010 15:32:49
Response

80

75

0.13
0.20
70

0.33

0.43
65

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55
Retention time

Fig. 7 TLC-FID chromatogram for sample acid-activated montmorillonite

were relatively high (Fig. 3). Extensive biodegradation in
the presence of these two clay mineral samples is also
evidenced by the chromatograms and relatively low EOM
values (Table 4; Figs. 8 and 9). There is a significant
difference between BU and control 1 and between B-Ca
and control 1, indicating that unmodified and calcium mont-
morillonites enhanced the biodegradation of the oil. These
samples may have enhanced biodegradation as a result of
high surface area (Table 3) and ‘local bridging effect’ which
leads to the accumulation and effective delivery of the
nutrients to the microbial cells, hence the improved extent
of biodegradation (Bright and Fletcher 1983; Fletcher and
Marshall 1982; Warr et al. 2009).
Adsorption of the crude oil and its fractions by clay
minerals is significant with B-K, B-Zn and B-Cr (Fig. 4).
The extent of hydration of the exchangeable cation of the
montmorillonite is a function of the cation type, and this, in
turn, determines the accessibility of the hydrophobic silox-
ane surface. Exposure of the hydrophobic siloxane surface
would occur if the interlayer of the montmorillonite is
occupied by large and monovalent cations such as K+
(Jaynes and Boyd 1991). Potassium montmorillonite is
therefore able to attract the crude oil aromatics into the
hydrophobic siloxane surface where pi–cation interaction
will eventually take place, making potassium montmorillon-
ite possesses good adsorptive capacity for the hydrocarbons,
especially the aromatic fraction of the crude oil. It is not yet
understood in this study why Cr and Zn montmorillonites
showed high adsorptive capacity for the oil. Acid-activated
clay minerals would have protons in the interlayer of the
montmorillonites, reducing the hydrophobicity of the surface
and consequently reducing adsorption. The organic phase of
the organoclay actually interacts with the crude oil aromatic
fraction, but this interaction appears not to have much to do

Sample 4 (rod 9) (9,1) defproj,nrg_ch06.CeG_Iatroscan 08-09 1555,9,1,1

Acquired 09 August 2010 16:05:02
Response

75

70 0.12

0.20
0.33
0.44
65

60

0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55
Retention time

Fig. 8 TLC-FID chromatogram for sample unmodified montmorillonite

 Author's personal copy
Environ Sci Pollut Res
Sample 2 (rod 6) (6,1)
Acquired 07 August 2010 16:11:16
Response
75
70
0.14
0.22
65
0.00 0.05 0.10 0.15 0.20 0.25
Fig. 9 TLC-FID chromatogram for sample calcium montmorillonite
with pi–cation interaction; hence, the weak hydrophobic in-
teraction was readily overcome by the solvent (DCM) used for
extraction. Unmodified and Ca montmorillonites adsorb hy-
drocarbons, but to a lesser degree due to their reduced ability
to expose the interlayer siloxane surface.
Selected TLC-FID chromatograms are presented in Figs.
5, 6, 7, 8 and 9.
Conclusion
This study reveals that the changes in the compositions of
the SARA fractions of the crude oil due to biodegradation
and adsorption are dependent on the form of clay mineral
mediating the processes. The SARA compositions in the
presence of organomontmorillonite and acid-activated
montmorillonite after incubation follow the same pattern in
which the aromatic fraction is higher than the other frac-
tions. The positive control experiment (without solid sup-
port) after incubation also has the same pattern in which the
aromatic fraction is the most abundant fraction. However,
the SARA compositions in the presence of homoionic mont-
morillonites after incubation follow a different pattern in
which saturates fraction is higher than all other fractions
except for chromium montmorillonite where both saturates
and aromatics occur at equal abundance. Zinc and potassium
montmorillonites appear to remove more of the aromatic
fraction by adsorption than any other clay mineral sample,
whereas chromium montmorillonite seemed to remove more
of the resins by adsorption than any other clay mineral
sample. Generally, adsorption of the oil occurred most in
the presence of zinc and potassium montmorillonites. Re-
moval of the oil and its fractions, especially the saturates and
aromatics due to biodegradation, appeared to be most ex-
tensive and enhanced in the presence of unmodified and
calcium montmorillonites. However, removal of the oil
defproj,nrg_ch06.CeG_Iatroscan 08-07 1602,6,1,1
0.34
0.45
0.30 0.35 0.40 0.45 0.50 0.55
Retention time
due to biodegradation in the presence of Cr montmorillonite,
organomontmorillonite and acid-activated montmorillonite
was hindered.
The use of Iatroscan in this study for the evaluation of the
compositional changes of SARA fractions due to biodegra-
dation and adsorption on solid supports such as clay min-
erals appears to be successful.
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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