International Biodeterioration & Biodegradation 95 (2014) 33e40

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International Biodeterioration & Biodegradation

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

Degradation of oil and grease from high-strength industrial efuents

using locally isolated aerobic biosurfactant-producing bacteria
Iezzat Emeer Affandi a, Nur Haizarisha Suratman b, Shakilah Abdullah b,
Wan Azlina Ahmad b, Zainul Akmar Zakaria a, *
a
b

Institute of Bioproduct Development, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia
Department of Chemistry, Faculty of Science, Universiti Teknologi Malaysia, 81310 UTM Johor Bahru, Johor, Malaysia

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 17 January 2014
Received in revised form
29 March 2014
Accepted 9 April 2014
Available online 23 May 2014

The potential of oil and grease (O&G)-degrading ability of three local bacterial isolates was evaluated
using wastewaters obtained from food processing, electrical and electronic and oil palm (POME) industries. These bacteria were chosen based on its high bacterial adherence to hydrocarbon (BATH),
culture turbidity and maximum biosurfactant production (BSF) capabilities. From the 16S rRNA analysis,
the food-processing isolate was identied and deposited in GenBank as Serratia marcescens EU555434,
electrical & electronic (Aeromonas hydrophila KF049214) and POME (Bacillus cereus KJ605415). Prior to
evaluation for its O&G degradation ability (effect of contact time, different concentrations of wastewater,
pH and initial organic loading rate), S. marcescens was adapted in used cooking oil while B. cereus in
POME. S. marcescens, with the highest BSF and BATH values, showed maximum oil and grease degradation ability (91%) at pH 7.0 after 12 days of incubation and initial organic loading rate of 1.46
10
1
kg O&G l1 day1. For B. cereus, 100% (v/v) of POME (3012 mg l1 oil and grease) was degraded after 7
days of incubation at 200 rpm, 30  C and pH 6 while A. hydrophila was able to degrade 100% (v/v) of
4.88 mg l1 of O&G from the electronic wastewater, supplemented with tryptone and lactose after only
2 h of incubation at 200 rpm, 30  C at pH 7.0. The role of tryptone and lactose in complete biodegradation
of O&G by A. hydrophila is signicant as neither the addition of tryptone or lactose only resulted in
enhanced O&G degradation, compared to E&E wastewater only. This nding showed the potential of
using local aerobic bacterial isolates as an alternative solution to remove the presence of O&G in various
industrial wastewaters.
2014 Elsevier Ltd. All rights reserved.

Keywords:
Oil
Grease
Bacteria
Biosurfactant
Degradation

1. Introduction
The discharge of oil and grease (O&G) containing wastewater to
the environment increases every year due to rapid urbanization
and industrial development. Major industrial sources of oily
wastewater include petroleum reneries, metal manufacturing and
machining, food processors, electronic and electrical and palm oil
mill efuent (POME). Unlike free or oating oil spilled in the sea,
most of the industrial wastewaters contain oil-in-water emulsions
which can lead to severe problems in the different treatment
stages. The presence of O&G in water treatment units will cause
fouling in process equipment, complication in water discharge requirements and problems in biological treatment stages (Ahmad
et al., 2006).

* Corresponding author.
E-mail address: zainul@ibd.utm.my (Z.A. Zakaria).
http://dx.doi.org/10.1016/j.ibiod.2014.04.009
0964-8305/ 2014 Elsevier Ltd. All rights reserved.

Amongst existing treatment technologies available include

chemical coagulation, gravity separation, parallel-plate coalesces,
gas oatation, cyclone separation, granular media ltration,
microltration and ultraltration. However, very few of these
technologies provide satisfactory solution to meet the increasing
stringent water quality regulations as well as the expensive initial
and operating cost. This makes it imperative to develop effective,
economical and sustainable O&G treatment technologies that can
serve as an alternative to existing treatment systems. One example
of current application of alternative treatment system is the
ponding system which is also the most common treatment system
used by Malaysian palm oil mills to treat POME. One of the most
attractive features for the ponding system is the low capital cost
which can be attributed to the limited requirement for mechanical
mixing, operational control and monitoring (Yacob et al., 2009).
This system consists of a series of specically built ponds including
the anaerobic and facultative ponds incorporated with physico-

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I.E. Affandi et al. / International Biodeterioration & Biodegradation 95 (2014) 33e40

chemical and biological treatments, respectively (Tong and Jaafar,

2004; Vijayaraghavan et al., 2007).
Recent study showed that the removal of Chemical Oxygen
Demand (COD) as well as O&G by aerobic oxidation was higher in
anaerobically digested POME as compared to diluted raw POME at
Hydraulic Retention Time (HRT) of 60 h (Vijayaraghavan et al.,
2007). Single culture such as Acinetobacter sp. (KUL8), Bacillus sp.
(KUL39) and Pseudomonas sp. (KLB1) showed higher O&G removal
capacity compared to mixed culture (Bhumibhamon et al., 2002).
Lipase producing bacteria such as Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Serratia marcescens, Pseudomonas
aeruginosa and Staphylococcus aureus has also been reported as a
potent agent for lipid degradation from O&G-containing wastewaters such as those emanating from palm oil mill, dairy, slaughter
house and soap industries (Prasad and Manjunath, 2011). The aim
of this study was to investigate the feasibility of using indigenous
bacteria to remove O&G in specic industrial wastewater namely
POME, Electrical & Electronic, Food Processing Industries wastewater in bench-scale laboratory treatment system.
2. Materials and methods
2.1. Industrial wastewater
The O&G wastewater was obtained from the Wastewater
Treatment Plant section of three different industries located in the
Johor Bahru district, Johor, Malaysia. Each industry has been identied to incorporate O&G in some stages of their operation namely
palm oil mills (mixing pond, POME), electrical and electronic industry, E&E (discharge point) and the food industry (discharge
point). The wastewater samples were collected in clean and sterilized glass containers and the pH of the samples were adjusted to
pH < 2.0 with either 1 M HCl or 1 M NaOH (to prevent abiotic redox
reaction process that might change the chemical speciation in industrial efuents) and transported to the laboratory using refrigerated ice chests in dark condition (APHA, 2005). Each wastewater
sample was then immediately stored at 4  C prior to use. The
average O&G contents in the wastewater was determined to be
between 1886 and 14,510 mg l1 for POME, 1.45e10 mg l1 (E&E)
and 180e79,000 mg l1 (food industry).

absorbance of the aqueous phase was recorded at 600 nm and the

percentage of microbial adhesion to substrate was calculated as
follows:

1

OD600 of aqueous phase after the addition of substrate

100%
OD600 of initial phase before the addition of substrate
(1)

All 25 bacterial isolates from the E&E wastewater were proled

for growth, cell concentration (CFU ml1) and culture turbidity
(OD600) using the following methods: one loopful of respective
24 h-old bacterial colony was inoculated into a series of 250 ml
Erlenmeyer asks containing 25 ml of NB. This was followed by
24 h incubation at 200 rpm and 30  C where bacterial isolates with
the shortest time to reach early stationary phase and OD600 values
of greater than 1.0, were earmarked for subsequent experiments.
From this evaluation, four bacterial isolates denoted as isolate A8,
B1, B6 and B7 were selected for subsequent studies. Bacterial strain
which showed good growth in minimal basal salt medium with
highest cell count was chosen as potential bacteria to degrade O&G
since it has capability to grow well in low minimal medium. The
composition for the minimal basal salt medium is as follows:
(NH4)2SO4 (3.0 g l1, Univar), MgSO4$7H2O (0.5 g l1, Merck),
K2HPO4 (0.5 g l1, BDH), KCl (0.1 g l1, Merck) and yeast extract
(0.1 g l1, Oxoid). One isolate each from respective wastewaters that
shows highest turbidity and fastest growth was identied using the
16S rRNA analysis which was carried out by First BASE Laboratories
Sdn. Bhd., Malaysia and Ktrade Enterprise, Malaysia.
For the food industry wastewater, all 10 isolated strains showed
good growth with OD600 of more than 1.0. One of the isolates, i.e.
isolate A was evaluated for its lipid-degrading ability using Tween
80 peptone agar (peptone e 10 g l1, NaCl e 5 g l1, CaCl2 e 0.1 g l1,
Tween 80 e 5 ml and agar e 18 g l1) where formation of opaque
zone around microbial colonies indicates lipolytic activity. Isolate A
was adapted in 1, 3 and 5% (v/v) of cooking oil at 30  C and 200 rpm
(Certomat, B. Braun) for 24 h while isolates X7 and X10 was adapted
in 100% (v/v) of POME in 2 days. Isolates X10, B1 and A were
maintained in LuriaeBertani glycerol medium and stored at 4  C for
biodegradation study.
2.3. Biosurfactant production and activity

2.2. Isolation and screening of O&G degrading bacteria

The O&G degrading bacteria were isolated from POME, E&E and
food industry wastewaters using the following procedures: 2.5 ml
of each wastewaters were transferred into a series of 250 ml of
Erlenmeyer asks containing 22.5 ml of Nutrient Broth, NB (8 g l1,
Merck) and incubated at 30  C, 200 rpm for 24 h (Certomat, B.
Braun). Then, one loopful of each bacterial culture broth was
inoculated onto Nutrient Agar, NA plates (20 g l1, Merck) followed
by overnight incubation at 30  C (Memmert, USA). The bacterial
colony was then sub-cultured onto fresh NA plates using similar
procedures until a pure culture was obtained.
For POME, the isolated bacteria were further screened for O&G
degradation properties (lipolytic activity) using the Tween 80
peptone agar (Plou et al., 1998). The selected bacterial isolates were
evaluated for its cell surface hydrophobicity properties using the
bacterial adherence to hydrocarbon (BATH) test (Rosenberg et al.,
1980). The BATH tests were carried out using early exponential
phase cells which were rst harvested by centrifugation at
9500 rpm for 5 min (Allegra 25-R Centrifuge, Beckman Coulter)
and resuspended in 0.01 M potassium phosphate buffer (pH 7.0) to
achieve OD600 value of 0.5. Then, 0.5 ml of either palm oil or
parafn oil was added into 5 ml of the cell suspension and vortexed
for 30 s (Thermolyne Mixer II) and let to stand for 15 min. The

The biosurfactant production ability of the bacterial isolate was

evaluated using isolate A by inoculating 5 ml of a 24 h-old culture
broth of isolate A in 500 ml Erlenmeyer ask containing 50 ml
mixture of minimal medium (3 g l1 of (NH4)2SO4, 0.5 g l1 KH2PO4,
0.1 g l1 KCl, 0.5 g l1 MgSO4$7H2O) and 5% v/v of cooking oil. The
mixture was then incubated for 12 h, 30  C and 200 rpm and harvested at 10,000 rpm, 20 min and 4  C. The resulting supernatant
was extracted with chloroform and methanol (2:1 v/v). The extract
was concentrated using rotary evaporator and determined for
biosurfactant activity using the following procedure (Matsumiya
et al., 2007); 30 ml of the concentrated extract was pipetted onto
the center of a series of petri dishes containing a mixture of 50 ml of
distilled water (DW) and 100 ml of cooking oil. One unit of biosurfactant activity (U) is dened as the diameter of the clearing
zone formed after 1 min from the addition of surfactant. The oil
displacement test is an indirect measurement of surface activity of
a biosurfactant sample tested against oil where a larger diameter
represents a higher surface activity of the test solution.
2.4. O&G biodegradation study
The O&G biodegradation study using different industrial
wastewaters was carried out as follows: isolate A (10% v/v) was

I.E. Affandi et al. / International Biodeterioration & Biodegradation 95 (2014) 33e40

transferred into 90 ml of food industry wastewater, isolate B1 (10%

v/v) in 90 ml of E&E wastewater and isolate X10 (20% v/v) in 80 ml
of POME in a series of 1 l Erlenmeyer asks followed by shaking at
200 rpm in an orbital shaker (Certomat M, B Braun, Germany) at
30  C for 24 h. POME was sterilized for 15 min at 121  C (200 kPa)
prior to use. The bacterial culture broth was sampled at designated
time intervals (4 h e isolate B1, 7 days e isolate X10 and 12 days e
isolate A) and determined for residual O&G contents. Similar
experimental setup was carried out in the following studies: (1)
effect of various pH of wastewaters (pH 3e11, adjusted using either
1.0 M NaOH or 1.0 M HCl) where samples were analyzed every 2 h
for 4 h (for the E&E wastewater), every 24 h for 7 days (for POME)
and every 24 h for 12 days, for the food industry wastewater; (2)
effect of nutrient supplementation on O&G degradation where
isolate B1 (10% v/v) was cultivated in E&E wastewater (80 ml) that
was added with 1 g l1 of either glucose, succinic acid, lactose,
tryptone, urea, L-alanine, (NH4)2SO4 or NH4NO3, at pH 7 and
monitored for 4 h and (3) effect of initial organic loading rate (OLR)
where isolate A was grown for 12 days in the presence of either
1.08
101 kg O&G l1 day1 or 1.46
101 kg O&G l1 day of
cooking oil while 20% (v/v) of isolate X10 was mixed with 20%, 40%,
60%, 80% and 100% (v/v) of POME and monitored for 7 days. At the
end of the studies, cell concentration (CFU ml1) and O&G concentration were determined. The experiments were carried out in
triplicates.
The O&G content was measured using the oil and grease
determination method as described in APHA2005-5520B (APHA,
2005), with n-hexane used as the oil-extraction solvent. The O&G
content in the suspension was determined for each sample before
and after experiment. All tests were carried out at an ambient
temperature of 25  C. All results were analyzed for standard deviation (s), which measures the condence interval and variation as
described by Eq. (2):

v
u n
uX xi  x2
sx t
n1
i1

(2)

35

4500 SEM) and TEM (Tecnai G2, Philips) at Institute for Medical
Research (IMR), Kuala Lumpur, Malaysia.
3. Results and discussion
3.1. Isolation of microorganisms
For the electric and electronic (E&E) industry wastewater, 25
distinct bacterial colonies were successfully isolated on NB and NA.
Two isolates (isolate X7 and X10) were determined to have the
lipolytic activity after screening with Tween Peptone Agar. One
bacterial isolate, i.e. isolate B1 showed the fastest growth in NB
with OD600 of 1.23 after 2 h of contact time as well as good growth
(1.66
108 CFU ml1 after 24 h) in salt minimal medium (MM). The
isolate is a gram- negative, forming slightly creamy, glistening and
raised colonies when grown for 24 h at 30  C on NA.
From the 10 initially isolated bacteria from the food industry
efuent (FIE), isolate A showed the highest ability to grow in a
nutrient-limiting environment (Minimal Medium) with OD600 of
more than 1.0 after 24 h of incubation compared to other isolates.
Apart from this feature, isolate A was also the only isolate showing
lipolytic activity (distinct formation of opaque zone when grown in
Tween 80 peptone agar) as well as showing good growth when
grown in 5% (v/v) of cooking oil with cell concentration of
108 CFU ml1. Tween, which is the fatty acid esters of polyoxyethylene sorbiton, is the preferred substrate for the detection of
lipolytic microorganisms in agar media (Sierra, 1957) with Tween
80 (the oleic acid monoester of polyoxyethylene sorbitan) being the
most widely used compound. It is based on the precipitation (as
calcium salt) of the fatty acid resulting from the hydrolysis of
Tween (Plou et al., 1998). The ability of microorganism to hydrolyze
oleic acid is a direct indication for its potential use to degrade O&Gcontaining wastewater such as POME which is rich in palmitic acid
and oleic acid contents (Wu et al., 2009). Result from the 16S rRNA
gene sequence analysis is shown in Table 1, hence isolate B1 was
termed as Aeromonas hydrophila (UTM2), isolate A as S. marcescens
(C 19320) and isolate X10 as Bacillus cereus (UTM6). The complete
sequence was deposited in GenBank under Accession number of
KF049214, EU555434 and KJ605415, respectively.

2.5. Electron microscopy analysis

The morphology of isolate A grown in a mixture of 0.5 mL of
cooking oil in 5 mL phosphate buffer was analyzed using the
Scanning Electron Microscope (SEM) and Transmission Electron
Microscope (TEM). Sample preparation for SEM was prepared using
modied procedure from that described by Zakaria et al. (2007) as
follows: the bacterial cell pellets were obtained by centrifugation at
9500 rpm, 4  C for 30 min. The cell pellets were xed in 2.5% (v/v)
glutaraldehyde (in phosphate buffer, pH 7.4) for 1e2 h and washed
twice with deionized water. It was then post-xed using 2% (v/v)
osmium tetroxide (Fluka, Switzerland) for 1 h and again washed
with deionized water. The cell pellets were subsequently dehydrated using increasing concentration of ethanol (10%, 30%, 50%,
70%, and 100% v/v; 5 min each) and left to dry overnight at 70e
80  C in a desiccator.
For the TEM analysis, the ethanol-dehydrated cells were
embedded in 50% and 100% (v/v) of epoxy resin in acetone (Q rc)
for 15 min each. Then, the cell pellet was inltrated a second time
with fresh 100% (v/v) epoxy resin and cured at 60  C overnight.
Specimens of 90 nm thickness were sectioned from the embedded
blocks using a Leica UltraCut UCT ultramictrotome and mounted on
200-mesh copper TEM grids. The specimens were stained with
uranyl acetate and post-stained with lead-citrate for 5 min each
(Southam et al., 2001; Chaerun et al., 2004). All samples were
maintained in a desiccator prior to viewing under SEM (Hitachi S-

3.2. Characterization of microorganisms

3.2.1. Bacterial adherence to hydrocarbon
The bacterial adherence to hydrocarbon (BATH) was carried out
to determine the degree of cells hydrophobicity toward
Table 1
Identication of isolate B1, X10 and A by 16S rRNA gene sequence analysis.
Isolate

Species as close relatives

Accession no.

Percent
similarity

B1

Aeromonas
Aeromonas
Aeromonas
Aeromonas

NR_042155.1
NR_074841.1
NR_043638.1
NR_037013.2

99%
99%
99%
99%

X10

Bacillus
Bacillus
Bacillus
Bacillus
Bacillus
Bacillus

cereus strain
cereus strain
cereus strain
cereus strain
sp. LT3
cereus strain

KF494192.1
FJ493043.1
KF376341.1
KF494193.1
FJ932655.1
FJ932655.1

99%
99%
99%
99%
99%
99%

Serratia
Serratia
Serratia
Serratia
Serratia
Serratia

marcescens strain
marcescens strain
marcescens strain
nematophila strain
marcescens
marcescens strain

AB244453
AB244433
AB244291
EU036987
EF415649
AB270613

99%
99%
99%
99%
99%
99%

hydrophila sub sp. dhakensi

hydrophila sub sp. hydrophila
hydrophila
jandaei
B234
2Y-2
YLB-P5
B236
XZM002

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I.E. Affandi et al. / International Biodeterioration & Biodegradation 95 (2014) 33e40

hydrocarbon. The BATH index is important to evaluate the afnity

of a particular bacterial strain to carry out any hydrocarbon
degradation process. In this study, B. cereus (UTM6) showed the
highest BATH capacity with a value of 84.51% followed by
A. hydrophila (UTM2) (22.86%). The higher hydrocarbon afnity for
B. cereus (UTM6) can be attributed to its original location of isolation, i.e. POME which contains high concentration of O&G
compared to A. hydrophila (UTM2) which was isolated from the E&E
wastewater with lower O&G concentration. Being originally
adapted to live in high O&G concentration environment, B. cereus
(UTM6) is anticipated to have cell surface properties that would
allow migration of hydrocarbon into the inner bacterial cells region
where degradation process would take place (Chang and Su, 2002).
The use of oil-adapted cells was successful in increasing BATH value
for bacterial cells as demonstrated by S. marcescens (C 19320)
where oil-adapted cells of S. marcescens (C 19320) showed a BATH
value of 81% compared to the non- adapted cells (44% only). The
formation of distinct white layer at the interface between oil and
aqueous layers clearly indicate the afnity of S. marcescens (C
19320) toward oil-phase, hence its high BATH value. By introducing
an environmental isolate such as S. marcescens (C 19320) into an
oil-rich solution, it is anticipated that the fatty acid component on
the cell membrane would be increased. This condition proceeds
due to the ability of the bacterial cells to change its membrane
uidity by isomerization of the fatty acids contents from cis to
trans-unsaturated fatty acids. The secretion of biosurfactant of the
cell surface directly assisted the adhesion of isolates onto the oil
droplets.
3.2.2. Production of biosurfactant
Due to its highest BATH value, S. marcescens (C 19320) was
further evaluated for its biosurfactant activity using cooking oil as
substrate. Formation of smaller oil droplets at the early stationary
phase of growth indicates initial point for the secretion of biosurfactant (data not shown). Control set (not inoculated with bacteria) showed oil droplets with a much bigger diameter and
numbering much less. The crude biosurfactant was also evaluated
using the oil displacement test where the results clearly indicate a
much superior biosurfactant activity for the crude biosurfactant of
S. marcescens C 19320 with a value of 380  42.4 U ml1 compared
to the commercial surfactant, Tween 80 (79  0.93 U ml1). Control
set (consisted of a mixture of cooking oil-water) did not show any
biosurfactant activity. Higher biosurfactant activity would result in a
larger and faster rate of clearing zone formation. Biosurfactant
excreted by the bacteria assisted in the emulsication and encapsulation of the oil in smaller droplets by reducing the surface tension
of the oil (Kosaric, 2001). This actually, increases the rate of organic
substrate dissolution, hence its utilization by the isolate
(Papanikolaou and Aggelis, 2011). High biosurfactant activity is
desirable in oil and grease biodegradation process as high biosurfactant activity would result in higher surface area of hydrophobic water-insoluble substrates followed by increasing the
bioavailability of hydrophobic compounds, hence increasing the
overall oil and grease degradation waste (Ron and Rosenberg, 2002).
SEM analysis of S. marcescens (C 19320) (after a 24 h cultivation
in the presence of oil droplets) showed marked changes in the cell
morphology, formation of macropores and cell disintegration
resulting in the formation of exopolymer matrix that would lead to
interconnection of cells into intricate network of coherent mass
(Fig. 1). The exopolymer matrices shown in Fig. 1b (arrow) can be
attributed to the excretion of biosurfactant by the cells as a mean
for cells anchorage to oil droplets as well as to allow degradation
process to take place. From the TEM micrographs, translucent
globules of oil droplets (100e200 nm) were clearly discernible at
the inner region S. marcescens (C 19320) grown in the presence of

oil droplets (Fig. 1a). None of these globules are present in bacterial
cells grown without the presence of oil droplet (Fig. 2b).
3.3. Biodegradation of oil and grease
3.3.1. Effect of contact time and concentration
A. hydrophila (UTM2) showed the ability to carry out complete
degradation of O&G (initial concentration of 5.186 mg l1) from E&E
wastewater after 4 h of incubation at 30  C. High cell concentrations
of more than 108 CFU ml1 maintained throughout the biodegradation study indicate good adaptability of the cells to grow in an oil
environment (Fig. 3). An interesting point to note is that even
though A. hydrophila (UTM2) was isolated from a low O&G concentration environment (1.5e10 mg l1 in E&E wastewater), it
shows high adaptation ability to survive and utilize O&G in
wastewater containing O&G indicating its potential to be applied
for biodegradation process.
For the food industry wastewater, S. marcescens (C 19320)
showed maximum O&G removal capacity of 89% after 12 days of
incubation at pH 7.0 and 5% (v/v) of cooking oil (Fig. 4). The oil
removal increased gradually with increasing cell concentrations.
After 2 days of incubation, 39% of oil was degraded, followed by 72%
(4 days), 80% (6 days), 84% (8 days), 85% (10 days) and 89% (12
days). High cell concentration of around 108 CFU ml1 was also
maintained throughout the study. The complexity of compounds
present in cooking oil induces the excretion of either lipase or
biosurfactant to facilitate the uptake process of the complex
nutrient into the cell (Dumore and Mukhopadhyay, 2012).
For POME, B. cereus (UTM6) showed the ability to carry out
higher O&G degradation with increasing POME concentration.
More than 90% of O&G were removed from all concentrations of
POME used (20e100% v/v) with complete removal achieved for
20%, 80% and 100% (v/v) of POME after 6 days of contact time at
200 rpm and 30  C. A point to note is the ability of B. cereus (UTM6)
to degrade O&G even at the highest concentration used, i.e. 100% (v/
v) POME (correspond to 14,510 mg l1) compared to both 20% (v/v)
and 80%(v/v) of POME. As shown in Fig. 5, the cell concentrations
also remained high throughout the experiment with a value of
3.25
108 CFU ml1 (at 100% O&G removal) and
1.88
109 CFU ml1 for 40% (v/v) of POME (lowest O&G removal at
90.53%). Even though this study has clearly demonstrated the role
of B. cereus (UTM6) in carrying out the O&G degradation process by
using pre-sterilized POME, more studies need to be carried out
using ltered (to remove suspended solids) but non-sterilized
POME to evaluate potential role of indigenous microbes in O&G
degradation. This would give better indications on the feasibility of
using B. cereus (UTM6) for eld application. This is because in nonsterile POME, there is always the possibility of indigenous microorganisms in POME to proliferate and participate in the O&G
degradation process as well as consuming the nutrients present
(Salihu and Alam, 2012). This situation may or may not work to the
advantage of B. cereus (UTM6) in any attempts to evaluate its O&G
degrading ability at a bigger scale (open treatment systems/eld
application) where the effect of other environmental factors such as
uctuation in pH, temperature, possible introduction of other pollutants to the aqueous environment would be much higher.
Similar condition was observed for the O&G degradation from
the food industry wastewater with highest O&G removal of 90%
was obtained at the highest organic loading rate used, i.e.
146 mg O&G l1 day1. This is followed by 85% and 82% removal for
OLR
of
1.20


101
kg O&G l1 day1
and
1.08
101 kg O&G l1 day1, respectively (Fig. 6). Moreover, the
initial OLR can also be related to HRT, thus a good balance between
these two parameters has to be obtained for good degradation
operation (Khemkhao et al., 2011).

I.E. Affandi et al. / International Biodeterioration & Biodegradation 95 (2014) 33e40

37

Fig. 1. SEM micrographs of (a) Serratia marcescens (C 19320) without oil (control) and (b) S. marcescens (C 19320) in the presence of oil.

Fig. 2. TEM micrographs of S. marcescens (C 19320) growing (a) in the presence of oil and (b) without oil droplets; arrow indicates the presence of translucent globules of oil
droplets.

3.3.2. Effect of pH
pH plays a very important role in the O&G biodegradation
process. This was clearly demonstrated in this study where complete O&G degradation from POME was determined to occur at
initial pH of 6.0, 6.5 and 7.0 whereas insignicant O&G degradation
(less than 10%) was recorded at initial pH values of less than 5.5 i.e.
pH 5.5 and 5.0, indicated by the absent of surviving cells (Fig. 7).
This situation can be directly correlated with the amount of cell
concentration present in the POME where for complete O&G
degradation, CFU values of more than 108 CFU ml1 were always
present whereas for the insignicant O&G degradation (less than
10%), the cell concentrations were less than 105 CFU ml1 Ahmad

et al. (2009) suggested that minimum cell concentration of

105 CFU ml1 is required for efcient bacterial-based bioremediation process. The presence of high concentrations of oil in POME
resulted in very low water activity as well as low pH that directly
affect bacterial survival (Nwuche and Ogbonna, 2011).
Similar condition was also recorded for the O&G biodegradation
from the E&E wastewater by A. hydrophila (UTM2) with 100%
degradation achieved at initial pH 7.0 (Fig. 8). Increasing or
decreasing the initial pH of the E&E wastewater resulted in uctuation of the O&G degradation values between 90% (pH 11) to 75%
(pH 3). Nevertheless, this high O&G degradation values can be

Fig. 3. Biotic and abiotic degradation of O&G in electric and electronic wastewater by
Aeromonas hydrophila (UTM2).

Fig. 4. Percentage removal of O&G and bacterial survival during biodegradation of food
industry efuent at different time intervals.

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I.E. Affandi et al. / International Biodeterioration & Biodegradation 95 (2014) 33e40

Fig. 6. Effect of initial OLR on the removal of oil and grease for S. marcescens (C 19320)
during biodegradation of food industry efuent.

Fig. 5. Effect of different POME concentrations on O&G removal and CFU ml1 by
Bacillus cereus (UTM6): (a) 20%, (b) 40%, (c) 60%, (d) 80% and (e) 100%.

attributed to the alteration of the O&G structures due to the low/

high pH values that would allow abiotic degradation process to
occur (Leahy and Colwell, 1990). Verstraete et al. (1976) reported
the increase in the degradation rate of gasoline in soil when the pH

Fig. 7. Effect of pH on O&G degradation and cell concentration of B. cereus (UTM6): (a)
pH 6, (b) pH 6.5, and (c) pH 7.

I.E. Affandi et al. / International Biodeterioration & Biodegradation 95 (2014) 33e40

39

resulted in gradual reduction in O&G removal capacity as follows:

pH 7.0e77%, pH 6.0e72% and pH 5.0e71% (Fig. 9). Together with the
high concentration of lipid in the food industry wastewater and the
fact that optimum pH for lipase production and activity is 8.0, this
should directly explain pH 8.0 being the optimum pH for O&G
degradation by S. marcescens (C 19320). A point to note is the optimum pH of 8.0 that may prove advantageous for the survival of
bacterial cells during real application where a low pH (due to the
accumulation of high VFA concentration) has been reported to
inhibit bacterial growth (Stafford, 1982) resulting in drastic reduction of O&G removal level and possible reactor failure. Thus, the
concentration of volatile fatty acids (VFA) should also be used as
one of the parameters to be monitored during bioreactor operation
(Buyukkamaci and Filibeli, 2004).
Fig. 8. Effect of pH for oil and grease degradation in electrical and electronic
wastewater.

Fig. 9. Effect of pH on the removal of oil and grease for S. marcescens (C 19320) during
biodegradation of synthetic food industry efuent.

3.3.3. Effect of nutrient supplementation

The percentage of O&G degradation by A. hydrophila (UTM2) in
the presence of carbon and nitrogen supplementation is shown in
Fig. 10. The addition of either 1 g l1 of lactose, tryptone, (NH4)2SO4
or succinic acid into E&E wastewater resulted in complete removal
of O&G with high concentration of surviving cell between 107 and
109 CFU ml1. Other carbon and nitrogen sources supplementation
resulted in high O&G degradation, i.e. 91% (NH4NO3), 96% (Lalanine), 94% (urea) and 88% (glucose). Control experiments
(without the addition of bacterial cells) showed minimal role of
abiotic degradation with values ranging from 1.33 to 12.96%.
Various studies reported the effect of carbon supplementation toward the production of biosurfactant by bacteria which ultimately
increased the biodegradation process (Nitschke et al., 2005; Gouda
et al., 2007; Abouseoud et al., 2007). However, Gouda et al. (2007)
reported that the addition of glucose as a co-substrate strongly
inhibits the degradation of kerosene by Gordonia sp. DM. Similar
situation was reported by Leahy and Colwell (1990) where the
supplementation of nutrient would limit the microbial degradation
of hydrocarbons.
4. Conclusion

was increased from 4.5 to 7.4. However, the degradation value

dropped signicantly when the pH increased to pH 8.5.
pH 8.0 was determined as the optimum pH for O&G degradation
of food industry wastewater by S. marcescens (C 19320) with
highest O&G removal of 91%. However, decrease in pH values

This study demonstrates the potential application of a bacterialbased O&G removal system as a cost-effective and environmentalfriendly process. The ability of the bacterial isolates to breakdown
O&G is advantageous as this would limit the need to supplement
the system with the normally expensive complex or synthetic

Fig. 10. Percentage of O&G degradation by Aeromonas hydrophila (UTM2) in the presence of carbon and nitrogen supplementation.

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I.E. Affandi et al. / International Biodeterioration & Biodegradation 95 (2014) 33e40

growth medium. Nevertheless, more studies need to be carried out

such as evaluation of the continuous/pilot-scale before any attempts on introduction of the system to the industry.
Acknowledgments
The authors would like to thank the Ministry of Higher Education (MOHE), Malaysia for the MyMaster scholarship award to
Iezzat Emeer Affandi and Nur Haizarisha Suratman. Our sincere
gratitude also to Universiti Teknologi Malaysia for the Research
University Grant (03H84, 02H84) and Universiti Tun Hussien Onn
(UTHM) for the SLAI scheme to Shakilah Abdullah.
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