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Journal of Emerging Trends in Engineering and Applied Sciences (JETEAS) 6(1): 1- 6
© Scholarlink Research Institute Journals, 2015 (ISSN: 2141-7016)
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Journal of Emerging Trends in Engineering and Applied Sciences (JETEAS) 6(1):1- 6 (ISSN: 2141-7016)
Bioremediation of Petroleum Refinery Wastewater Effluent via
Augmented Native Microbes
1
N. M. Musa, S. Abdulsalam, A.D.I. Suleiman and 2Sale, Abdullahi
1
Department of Chemical Engineering,
Abubakar Tafawa Balewa University (ATBU),
PMB 0248, Bauchi, Bauchi State, Nigeria.
2
Microbiology Laboratory,
Department of Biological Science, Abubakar Tafawa Balewa University (ATBU),
PMB 0248, Bauchi, Bauchi State, Nigeria.
Corresponding Author: N. M. Musa
_________________________________________________________________________________________
Abstract
In Nigeria, like other developing countries, industries discharge their wastewater without effective treatment due
to the high cost of existing treatment technologies. Wastewater containing petroleum hydrocarbon is highly
toxic and pose a great danger to the nearby communities. Therefore, there is a need for a more robust and cost
effective treatment technology. In this research, a study was carried out on bioremediation of petroleum refinery
wastewater via augmented native microbes. Four dominant microbial strains isolated from the petroleum
refinery wastewater were identified to be Bacillus subtilis (S1), Micrococcus luteus (S2), Staphylococcus aureus
(S3) and Staphylococcus epidermidis (S4) base on their gram reaction, morphology, microscopic appearance,
and biochemical characteristics. The four strains showed their capability to degrade hydrocarbon as indicated by
their growth in Bushnell-Haas medium. The four native microbes were used to boost the performance of the
indigenous microbes (note that only two out of these four strains were used to boost bioremediation). Five
different treatments (T1-T5) were investigated for 63 days. T1 (Natural attenuation) showed O&G degradation
of 15.23 %. T5 (Aeration + Bioaugmentation with S1 & S2) showed O&G degradation of 66.7 %. From the
result of this study, it has been established that bioaugmentation with native microbes using treatment option
five can be used to developed a robust, cost effective and environmentally friendly process for the treatment of
hydrocarbon contaminated wastewater.
__________________________________________________________________________________________
Keywords: bioremediation, bioaugmentation, effluent, hydrocarbons, native-microbes, wastewater.
pollutants into harmless products. Bacteria and fungi
are often used in the biochemical decomposition of
wastewaters to stable end products. More
microorganisms, or sludge are formed and the portion
of the waste is converted to carbon dioxide, water and
other end products. This is achieved by enhancing the
conditions (pH, nutrients and aeration) of the
indigenous microbes to carry out the bioremediation
process (biostimulation). Alternative to this is
bioaugmentation, where exogenous microbes are
introduced to carry out the process (Hamza et al.,
2012; Long, 2007). In this study, bioaugmentation
using native microbes was used to treat petroleum
refinery wastewater using various treatment options.
The aim of this study is to develop a robust cost
effective process for the treatment of refinery
wastewater using bioremediation technique via
augmented native microbes.
INTRODUCTION
Wastewater released from petroleum refineries is
characterized by the presence of large quantity of
petroleum products, polycyclic and aromatic
hydrocarbons, phenols, metal derivatives, surface
active substances, sulfides, naphthylenic acids and
other chemicals. Due to ineffective purification
systems, the pollutants finds its way into nearby
water bodies and soil with potentially serious
consequences on the ecosystem (Otunkunefor and
Obiukwu, 2005; Bay et al., 2003; Beg et al., 2001).
According to World health organization (WHO), the
mortality rate of water associated diseases exceeds
five million people annually with microbial intestinal
infections accounting for more than 50 % (Cabral,
2010).
Wastewater may be treated by physiochemical or
biological methods, biological treatment is preferred
over physicochemical as the former is cost effective,
efficient and environmentally friendly (Suleimanov,
1995, Hamza, 2012). Bioremediation strategies
involved the use of microbes to transform the harmful
The research objectives are:
i. To develop a bench scale bioreactor for the
treatment of refinery wastewater.
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Journal of Emerging Trends in Engineering and Applied Sciences (JETEAS) 6(1):1- 6 (ISSN: 2141-7016)
samples were left for up to 96 hours (Four days) after
which distinct colonies were formed. Single colonies
were removed from these plates and then subcultured into fresh nutrient agar plates. They were
then incubated at 37oC for 48 hours. The pure
isolated organisms were identified by microscopic
appearance, morphological features, gram reaction,
and biochemical characteristics. The isolates were
identified according to description given in Bergeys
manual of systematic bacteriology to be Bacillus
subtilis
(S1),
Micrococcus
luteus
(S2),
Staphylococcus aureus (S3) and Staphylococcus
epidermidis (S4).
ii.
To determine the hydrocarbon degrading
potential of refinery wastewater in the
bench scale bioreactor developed.
iii. To determine the effect of boosting the
microbial population by adding
enriched
native
microbes
(bioaugmentation) on the rate of
hydrocarbon degradation.
STATEMENT OF THE PROBLEM
In Nigeria, like in other developing countries, the
conventional
cleaning
technologies
(physicochemical) are being used for the remediation
of refinery wastewater, these methods are expensive
and does not lead to complete degradation of
pollutants, therefore, the need for a robust, cost
effective and environmentally friendly cleaning
technology (Hamza et al., 2009; Ojo, 2006).
Determination of Hydrocarbon Degrading
Potentials
The isolates were inoculated in Bushnell-haas
medium and incubated at 37oC, microbial growth was
observed in all the four isolates after three days
indicating their ability to degrade hydrocarbon
compounds (Hamza et al., 2012).
LIMITATION OF THE STUDIES
This study is limited to the development of an
effective bench scale bioremediation technology for
the treatment of refinery wastewater, characterization
of the physicochemical and microbiological
properties of the refinery wastewater, isolation and
identification of native microbes in the wastewater
and bioremediation of refinery wastewater using
augmented native microbes in the bench scale
bioreactor developed.
Preparation of Enriched Native Microbial
Cultures for Bioremediation
Culture media was prepared using manufacturers
standard methods and procedures. For example in a
typical run, 1000 cm3 of distilled water was measured
and 28 g of agar was added to it in a conical flask
after weighing. The mixture was shaken until the agar
was totally dissolved in the distilled water. The
solution was heated on a hot plate then sterilized in
an autoclave at 121oC for 15 minutes; the sterile
media was then placed in a water bath to cool the
media to 47oC before pouring into plates. Same
procedure was done for peptone water but with
different amount of agar (i.e. 15 g in 1000 cm3) and
for macconkey agar (38.5 g in 1000 cm3). Pure
isolates S1 & S2 were then enriched by inoculating
them in all the three sterile media before incubating it
for at 37 oC.
MATERIALS AND METHODS
Sample Collection
The wastewater sample for this research was
collected using sterile containers at the outlet of the
bio-filter of a typical petroleum refining company in
Nigeria. The wastewater sample was then preserved
at low temperature in a deep freezer before
commencement of the experiment.
Wastewater Characterization
The wastewater was analyzed for physicochemical,
organic and heavy metal constituents. Turbidity was
determined with DR\2000 spectrophotometer using
absorphotometric method, COD was determined
using
calorimetric
method
with
DR\2000
spectrophotometer (Long, 2007). BOD was
determined using HACK BOD track instrument
(Anonymous, 2007). O&G was determined using
reflux method with DR\2000 spectrophotometer.
Conductivity was determined using a Wagtech
WEDIST6 conductivity/ TDS meter. Total solids and
suspended solids were determined following the
procedures given in (Anonymous, 1997).
Procedure for Serial Dilution and Pour Plate
Counting Method
Pour plate method was employed as the technique for
recovery of microorganisms from wastewater on
solid media. After autoclaving, the agar was cooled to
45oC before pouring to plates. 0.1 ml of 10-4, 10-6 and
10-8 dilutions were inoculated using pour plate
method. The plates were incubated at room
temperature for 24 h before counting all the colonies
using colony counters. The procedure was repeated
for the same dilution in bushnell-hass media and for
pure isolates S1 & S2 in peptone water.
Isolation and Identification of Bacteria
Following the aseptic technique, the standard dilution
methods for recovery of bacteria from the wastewater
was used. The nutrient agar plates were inoculated
with 0.1 ml of diluted sample and then incubated at
37oC. Growth was observed after 48 hours and the
Bioremediation Studies
Five plastic buckets (20 L) each were used for this
experiment. Four of these plastic buckets, each
containing 13.3 L of wastewater were aerated while
the fifth bucket containing the same quantity of
wastewater (13.3 L) was left un-aerated (figure 1);
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Journal of Emerging Trends in Engineering and Applied Sciences (JETEAS) 6(1):1- 6 (ISSN: 2141-7016)
Treatment 1 (T1), the first plastic bucket (un-aerated)
was left as collected to monitor natural degradation
(control). Treatment 2 (T2), the second bucket
contains the same sample as the first but was aerated
at 1.2 Lair/min. Treatment 3 & 4 (T3 & T4), 50 ml
each of enriched pure isolate S1 & S2 were added to
plastic buckets 3 & 4 respectively and the reactors
were aerated at 1.2 Lair/min. Treatment 5 (T5),
combination of 25 ml each of enriched pure isolates
S1 & S2 were added to plastic bucket 5. Effect of
aeration, potentials of adding pure isolates S1 & S2
separately and the use of consortium of bacteria were
compared with natural attenuation.
CONCLUSION
The following conclusions were drawn from the
results obtained:
i. Bioremediation of petroleum refinery wastewater
was effective to an appreciable extent via
augmented native microbes (67 % O&G removal
in 63 days) in the bench scale bioreactor rig
developed.
ii. A full scale treatment technology can be sized
using treatment option 5 (T5).
iii. Four microorganisms namely bacillus subtilis,
micrococcus luteus, staphylococcus aureus and
staphylococcus epidermidis were identified in
the petroleum refinery wastewater and can be
enriched to boost the rate of biodegradation.
(remember only two were used for boosting)
RESULTS AND DISCUSSION
Wastewater Characterization
The results of physicochemical characterization
(Table 1), showed that the refinery discharge its
wastewater without treating as required by the
international
standards
for
discharge
of
environmental pollution. Therefore the need for a
more robust treatment process.
REFERENCE
Adekunle A. A., Adekunle, I. M. and Igba U. T.
2012. Assessing and Forecasting the Impact of
Bioremediation Product Derived From Nigeria Local
Raw Materials on Electrical Conductivity of Soils
Contaminated with Petroleum Products. Journal of
applied hytotechnology 2 (1): 57-66.
Substrate Reduction
COD and BOD tests are commonly used to determine
the amount of organic pollutants found in water,
making COD and BOD a useful measure of water
quality (Okerentugba and Ezeronye, 2003, Hamza et
al., 2009). Biodegradation potentials of five different
treatments (T1 – T5) were measured in a bench scale
bioreactor by monitoring O & G contents. Fig 2 – 4
show results of bioremediation of a typical Petroleum
refinery wastewater.
Adekunle, I.M. 2011. Bioremediation of soils
contaminated with Nigerian petroleum products using
composted municipal wastes. Bioremediation
Journal. 15(4): 230-241
Bay S., Jones B.H., Schiff K. and Washburg L. 2003.
Water quality impacts of storm-water discharges to
santa monica Bay. Marine environmental research
56: 205-223
This result (fig. 2 -4) showed Treatment 1 (natural
attenuation), which is a very slow process for
biodegradation can be boosted by augmentation with
strain of bacteria or consortium of bacteria. In T1
(Treatment 1), only 15.23 %, COD reduction of 11.93
% and BOD reduction of 10.11 % compared to T5
(Treatment 5) boosted with a consortium of bacillus
subtilis and micrococcus luteus which showed O&G
decrease of 67 %, COD decrease of 51.27 % and
BOD reduction of 62.79 %. Micrococcus and
Bacillus species were reported to be hydrocarbon
degraders (Long, 2007; Anonymous, 2007, 1997). T2
(Treatment 2) showed that injecting air can boost
biodegradation process compared to natural
attenuation. T2 showed 21.52 % O&G decrease,
COD decrease of 30.97 % and BOD decrease of
30.97 %. Table 2 & 3 showed the summary of results
obtained after 63 days and parameters used for the
bioremediation studies.
Beg M. U., Al-Muzaini S., Saeed T., Job P.G., Beg
K.R., Al-Bahloul M., Al-Matrouk K., Al-Obaid T.
and Kurian A. 2001. Chemical contamination and
toxicity of sediment from a costal area receiving
industrial effluents in kuwait. –Archives of
environmental contamination and toxicology 41:289297
Cabral J.P., 2010. Water Microbiology. Bacteria
Pathogens and water. Int. J. Environ Res. Pub.
Health, 7:3657-3703
Hach Company, 2007. DR/2000 Procedure manual.
8th edition, U.S.A
Hamza, U. D., I.A Mohammed, I. M., Ibrahim, S.
2009. Kinetics of Biological Reduction of Chemical
Oxygen Demand from Petroleum Refinery
Wastewater. Researcher, 1(2).
Biomass Growth and Conductivity Increase
In figure 5, the biomass growth was measured by
turbidity/optical density increase (Leah and Colwell,
1990). In figure 6, the rate of conductivity increased
with decrease in O&G (Adekunle, 2011; Adekunle et
al., 2012).
Hamza, U.D., Mohammed, I. M., Sale, A. 2012.
Potentials of bacterial Isolates in bioremediation of
petroleum refinery wastewater. Journal of applied
hytotechnology 1(3):131-138
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Journal of Emerging Trends in Engineering and Applied Sciences (JETEAS) 6(1):1- 6 (ISSN: 2141-7016)
Long, H. (2007); Wastewater treatment methods and
disposal, 3441, Empire road U.S.A., 2007.
www.water.me.vccs.edu/courses/ENV149/treatment.
htm (accessed 12th December, 2013)
Table 2: Summary of results
Initial
Value
Treatment 1
Treatment 2
Treatment 3
Treatment 4
Treatment 5
Standard
Values
Noweco Laboratory, 1997. Norwalk Wastewater
Equipment Company, Inc. 220 republic street
Norwalk,
Ohio
U.S.A.
44857.
http://www.norweco.com/html/lab/whatTest.htm
(accessed January 2014)
Leahy J.G., Colwell, R. 1990. Microbial degradation
of hydrocarbons in the environment. American
Society of Microbiology. Microbiological Reviews,
54(3):305-315.
COD
(mg/L)
285.00
BOD
(mg/L)
155.00
Turbidity
(NTU)
47.00
17.80
14.80
10.50
12.00
7.00
10.00
251.00
197.33
129.67
138.67
151.68
250.00
139.33
107.00
67.33
78.67
57.67
30
100.00
59.67
73.00
94.00
83.00
145.33
4-10.00
Table 3: Summary of Results and Isolates Identified
Ojo, O.A., 2006. Petroleum Hydrocarbon Utilization
by Native Bacterial Population from a Wastewater
Canal Southwest Nigeria. African Journal of
biotechnology, 5(4): 333-337.
Okerentugba P.O. and Ezeronye, O. U. 2003.
Petroleum Degrading Potentials of Single and Mixed
Microbial Cultures Isolated from Rivers and Refinery
Effluent
in Nigeria.
African
Journal
of
Biotechnology, 2 (9): 288-292.
Otunkunefor T. V., Obiukwu, C. 2005. Impact of
refinery effluent on the physicochemical properties of
a water body in Niger Delta. Applied ecology and
environmental research 3: 61-72
Suleimanov, A.Y. 1995. Conditions of waste fluid
accumulation at petrochemical and processing
enterprise prevention of their harm to water bodies,
Meditsina Truda Promyswe Nnaia Ekologila. 12:3136
APPENDIX
Table 1: Physicochemical Characteristics of KRPC
Wastewater before Treatment
Parameter
pH
Conductivity (µs.cm-1)
Turbidity (NTU)
TDS (mgL-1)
TSS (mgL-1)
Organics
BOD (mgL-1)
COD (mgL-1)
O&G (mgL-1)
Inorganic
Phosphate
Total-N (mgL-1)
Total-C (mgL-1)
Heavy Metals
Fe (mgL-1)
Cu (mgL-1)
Zn (mgL-1)
Cr (mgL-1)
O & G
(mg/L)
21.00
Value
6.55
450
47
224
47
155
285
21
7.36
28.68
49.25
0.93
0.5
0.08
0.075
4
S/N
1
Parameter
Volume of bioreactor
Value obtained
20 L
2
Working volume or Effective
volume
2/3 of Total volume (13.3
L)
3
4
Aeration flow rate
Geometry of bioreactor
1.2 L/h (0.02L/min)
Cylindrical
5
Continuous monitoring
63 days
6
Isolates
microbes)
Bacillus subtilis
Micrococcus luteus
Staphylococcus aureus
Staphylococcus
epidermidis
7
The best treatment option
(indigenous
T5 (67 % O&G removal)
Journal of Emerging Trends in Engineering and Applied Sciences (JETEAS) 6(1):1- 6 (ISSN: 2141-7016)
Figure 1: Schematic representation of a bench-scale bioreactor rig.
Figure 2: Rate of COD Reduction
Figure 3: Oil and Grease Removal
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Journal of Emerging Trends in Engineering and Applied Sciences (JETEAS) 6(1):1- 6 (ISSN: 2141-7016)
Figure 4: Rate of BOD Reduction
Fig. 5: Rate of Turbidity increase
Fig. 6: Rate of Conductivity increase
6