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BIOLOGICAL METHOD FOR TREATMENT OF PETROLEUM PRODUCED WATER

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 BIOLOGICAL METHOD FOR TREATMENT OF
PETROLEUM PRODUCED WATER OIL CONTENT IN SUDAN

Mohamed Ahmed Khadam, Muna Ahmed Agab

and Suhair A/Gayoum Saad

Received July. 2008, accepted after revision Feb. 2009

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ABSTRACT
Biodegradation studies of hydrocarbons in untreated produced water from Heglig oil
field in Sudan were undertaken over a period of time using pure indigenous bacterial
cultures. Experiments were performed in 1L glass flasks (Batch cultures) and on a
designed laboratory scale aerobic bioreactor consisting of a mixing and an aeration
tanks (Continuous flow culture). The rate of reduction in petroleum hydrocarbon
fractions, were monitored by means of gas chromatography and change in bacterial
mass. Values of COD, BOD, TDS, and pH were also measured through the experiment
period.
Chromatographic analysis showed that all hydrocarbons content (C4-C34) of 250-300
mg/l in untreated produce water can be reduced to zero value in 36 hours upon
biological treatment only, Also values of COD and BOD were reduced to 95-97 %. No
valuable change in the other parameters.
Simple plantation and germination tests were performed (sorghum and millet) as
confirmation tests for the suitability of treated water to irrigation purposes

Keywords: Biodegradation, Bacterial isolates, hydrocarbons, produced water,

Bioreactor.

1. INTRODUCTION On-shore exploration, like the case in

Sudan, carries potential risks for local
Oil production is a new industry in Sudan
inhabitants and may cause severe
that promotes the economy and makes
damage to the environment upon which
noticeable social development. This
their daily life depends [2]. Also off shore
industry has many environmental impacts
oil production represent a great risk to the
such as air pollution, soil pollution and
marine environment [3].The environmental
hazards associated with waste production.
costs of oil development have been
[1]
extensive. They included destruction of
wildlife and biodiversity, loss of fertile soil

Sudan Engineering Society Journal, March 2009, Volume 55 No.52 23

 BIOLOGICAL TREATMENT OF PETROLEUM PRODUCED WATER IN SUDAN

and degradation of farmland. Oil and gas suspended solids, dissolved solids and
reservoirs have a natural water layer natural low-radioactive elements.
(formation water) that being denser lies
There are many options for treatment of
under the hydrocarbons. To achieve
produced water prior to disposing or re-
maximum oil recovery, in certain stages of
injecting. [13]. Methods are varying greatly
oil field life, additional water is injected in
in efficiency and cost. Biological method is
the reservoirs to help force the oil to the
one of the cheapest and easiest available
surface. Both formation and injected water
options [14].
are eventually produced along with the
hydrocarbons. This produced water is the Both mixed and pure bacterial and fungal
largest volume waste stream in oil cultures have been used successfully in
production operations. Other wastes that the degradation of hydrocarbons [15, 16].
may be generated during this process In Sudan a number of studies were carried
include the residual wastes that remain to isolate and characterise petroleum
after separation of the oil. [4, 5] degradable microorganisms [17, 18].
However, these studies did not investigate
Almost all oilfields produce large
the degradation of produced water
quantities of contaminated water. For
hydrocarbons by these isolates. An
every barrel of oil produced around the
investigation is carried now on the
world approximately 2-10 barrels of water
biodegradation of produce water
is associated [6]. Water production
hydrocarbons by a PhD student in the
quantities continue to increase as the oil
Faculty of Science, University of
and gas fields reach maturity [7]. Since
Khartoum, but no results is published so
great quantities of this water are produced
far, only the proposal of work is available.
in arid areas, the concept of finding
[19]
beneficial uses for produced water arises
in recent years to convert a high-cost Produced water quantities in Sudan oil
liability into an asset. [8] fields increased largely in recent years as
the oil production increased and the old
At the surface, produced water is
field matured. In Sudan Approximately
separated from the oil, treated to remove
1.2 million bb/day of water are produced in
as much oil as possible, and either
GNPOC oil field today. A bioremediation
discharged or injected back into the wells.
project was constructed in Heglig oil field
The general approach for produced water
to treat water coming from central
treatment is de-oiling and de-mineralizing
processing facility (CPF) using large beds
before disposal or utilization.
of reed plants [20, 21]. Another small
Quality of produced water discharges to project was constructed in Adar oil fields
surface or re-injected to wells is controlled to treat about 10 thousands bb/d of water
by rigid environmental regulations in all by hydro cyclones, but water needs further
countries. [9, 10] In Sudan the Ministry of treatment to meet Sudan environmental
Energy and Mining developed national regulations [22]
environmental regulations for petroleum
In this study the biodegradability of
industry [11]. Then more detailed
produced water hydrocarbons by exposing
regulations had been set in 2005 as a part
the hydrocarbons to pure indigenous
of Nivasha peace convention [12].
bacterial cultures is examined.
The composition of produced water is
It is expected that a significant
strongly field-dependent and includes a
degradation of hydrocarbons in produced
variety of inorganic and organic
water by the indigenous bacterial species
compounds. Produced water contains
will help to reduce the problem of
small amounts of emulsified oil, organic
bioaccumulation of these organic
compounds including dissolved
compounds in the environment, and also
hydrocarbons, organic acids, phenols and
the effluent water can be used as useful
traces of chemicals added during
source for irrigation instead of considered
production, inorganic compounds,
as a waste stream.

24 Sudan Engineering Society Journal, March 2009, Volume 55 No.52

2. Materials and Methods [23] and Flame Atomic Absorption
Spectrometry [24]. Other chemical
2.1 Samples collection
parameters (nitrate, ammonia, COD,
Produced water samples were brought BOD) were analysed using titration or
from Heglig oil fields (CPF) in different other standard methods.
periods (6th July - 7th December 2008).
Physical tests were carried out in sanitary
Samples were taken from different depths
engineering laboratories in University of
of water ponds constructed for
Khartoum, following the standard water
bioremediation project, using sterile small
tests methods [25].
bucket, and stored in sterile containers of
different sizes. Figures 1 and 2 show 2.3 Isolation Of indigenous Bacterial
ponds and reed bed locations from which Species
samples were collected. Hydrocarbon-utilizing bacteria in produced
water were obtained by plating out low
dilutions (10-1 to 10-4) of samples onto
modified Nutrient agar medium [26]. The
medium has the following composition [in
(g/l)]: NaCl (5), yeast extract (2),
Lablemco meat extract (Oxoid) (1),
Peptone (5), Agar (15), Petroleum water
(instead of distilled water) (1000 ml), and
pH (7.2). The medium was autoclaved at
1.2 kg/cm2 for 15 min. The inoculated
nutrient agar plates were then inverted
and incubated at 37ºC for 48 hours. After
Figure 1: water samples collected from incubation, the bacterial cultures were
storage ponds
stained with methylene blue and observed
under a high-power resolution microscope
(X40).
Cultures were purified by inculcating
separate colonies in modified half strength
nutrient broth and inoculated for 24 hrs in
37ºC then stored at 4ºC to be used for
seeding water samples. Typical
Composition of modified broth media in
(g/liter) is: Peptones 15.0; yeast extract
3.0; sodium chloride 6.0; D(+)glucose 1.0
(also produced water is used instead of
distilled water)
2.4. Oil and grease measurement
Figure 2: Water samples collected from
reed beds Oil is extracted by standard liquid-liquid
extraction method. A measured volume of
2.2 Water chemical and physical
the sample (V=50ml) was introduced into
parameters identification
separating funnel 20ml of hexane and
Chemical and physical tests are carried 10ml of H2 SO4 were added. The layer of
out in water samples to define its main Oil and Hexane was separated in glass
characteristics. Most of chemical elements tubes and centrifuged at 30,000 rpm to
identification tests were made in National remove bacterial cells then transferred
Institute of Researches and geology into a pre-weighed (W1)flask and hexane
research institute laboratories using flame is evaporated in a water bath at 70ºC.
absorption spectrometer. The standard The flask is reweighed (W2) and the Oil
procedures for these tests were taken and grease content is Calculated in ppm
from Atomic Absorption Spectrometry (mg/l) as: (W2 - (W1) * 106 / V.

Sudan Engineering Society Journal, March 2009, Volume 55 No.52 25

 BIOLOGICAL TREATMENT OF PETROLEUM PRODUCED WATER IN SUDAN

2.5 Extraction of oil from produced fed at a rate 0.67 ml/min) by a small timing
water for GC tests pump and aerated by air pump of 1L/min
capacity. Continuous mixing of influent
Oil is extracted by standard liquid-liquid
water was accomplished by a mixer of 15
extraction method. A measured volume of
rpm.
the sample (V=50ml) was introduced into
separating funnel, 20ml of hexane was Initial counts of bacteria in samples were
added. The layer of Oil and Hexane was done before seeding and appear in graphs
separated in glass bottles. Bacterial cells in negative time side. Ten ml of the pure
were removed by centrifugation at 30,000 bacterial cultures were introduced into 1L
rpm for 15 min. [26, 27]. flask (batch culture) and other 10 ml
introduced into aerobic reactor (volume
2.6. Gas chromatography of
used for this experiment is 1L) (continuous
Hydrocarbons
flow). The flask was covered with non-
Fresh and degraded hydrocarbons were absorbent cotton wool and placed in a
analyzed by gas chromatography using a position such to allow air passage through
Varian CP-3800 series 11 gas the pores of the cotton wool. The flask
chromatograph, equipped with a single was shaken manually at regular intervals
flame ionization detector (FID) and fitted to allow adequate mixing and
with analog digital converter and a Dell homogeneity of the contents. The
computer. A DB-17 silica capillary column experimental setup was monitored for a
of 15 cm length and an internal diameter period of 48 hrs. At intervals of 3 hrs for
of 0.32 mm wide bore of 1micron film the first 24 hrs and 12 hrs for the
thickness were used. A temperature remaining period, culture samples were
program of 35 - 280oC, increasing at 3.0oC collected and analyzed for microbial load,
per minute for 103.67 min was employed. while the residual hydrocarbon was
Hydrogen with a flow rate of 30 ml/min extracted and analyzed by oil and grease
was used as a carrier gas, while the flow weight method and gas chromatography.
rate of air was 300 ml/min. The detector
temperature was 280oC, while the
injection port temperature was 270oC. A
sample volume of 0.1 µl was injected and
the peaks nC4 – nC34 were recorded.
2.7. Biodegradation and growth studies
Growth and degradation studies over a
time course were carried out using
untreated produced water from Heglig oil
fields as the sole carbon and energy
source. Untreated produced water used
for the study had an initial oil and grease
Figure 3: Batch culture flask
content of 250-300 mg/l. One pure culture
type was used for all experiments.
The experimental work consisted of
designing and constructing a laboratory
scale bioreactor. A high durable
transparent PVC two tanks (15 cm and 10
cm diameter) with 3 L maximum effective
volume were used as the aeration and
mixing tanks respectively. A glass bottle, 4
L capacity, was used as a storage
container. Continuous mixing in aerobic
reactor was performed by means of a
motorized stirring rod and the reactor was Figure 4: Aerobic reactor

26 Sudan Engineering Society Journal, March 2009, Volume 55 No.52

2.8 Germination Tests
Simple germination test were performed to
insure the suitability of treated water to
irrigation purposes. For each test three
types of water were used for irrigation for
comparison: 1- untreated produced water
(petroleum water) 2- treated produced
water (treated with bacteria for 48 hrs
retention time in batch culture) 3- Tap
water. A representative sample of a seed-
lot was obtained by taking sub samples
from seed containers, and then bulk
sample was mixed thoroughly. About 20- Figure 5: Dishes for germination test
30 grams of each seed was taken out to
make a composite sample for the
germination test. [28] Table1: Raw water Characteristics

Seeds of millet and sorghum were planted Constituent Heglig produced

in suitable three plastic pots, for each Water
plant, filled with a well mixed agricultural Color Brown
clay soil. Then pots irrigated in regular Odour Mineral oil
intervals and growth noticed daily. In pH-value 8.9 –9.5
germination test three samples of 100 Total Dissolved Solids 1000-1210
seeds were randomly selected, excluding mg/l
Oil & Grease mg/l 250-300
damaged seeds. Three Petri dishes were
Turbidity 100-103 NTU
prepared by covering their bottoms with 2
BOD mg/l 100-130
filter papers with a layer of non-absorbent COD mg/l 560-600
cotton wool in between to form a pad in Cadmium (Cd) mg/l ND
each dish. These pads were moistened Chromium mg/l 0.033
with described water samples.100 seeds Copper (Cu) mg/l 0.014
samples were spread evenly on the filter Mercury mg/l ND
paper with suitable distances between Nickel (Ni) mg/l 0.043
seeds. Dishes were placed in room Ammonia (NH3) mg/l 1.12
temperature (39ºC). Germinated seeds Nitrate (NO2) mg/l 0.782
were counted 24 hrs after sawing. Fluoride mg/l ND
Iron (Fe3) mg/l 0.34
3. RESULTS Zinc (Zn) mg/l 0.009
Raw produced water used for the Manganese (Mn) mg/l 0.064
experiment was found to have physical
and chemical characteristics as shown in Table 2: Treated water Characteristics
(Table1). Treated water was tested for the
main contaminants and found to have Constituent Higlig Treated
significant reduction in them except TDS Water
(Table 2). Color Colorless
Odour Odourless
When water samples initially tested under pH-value 8.5-8.7
microscope, it was found to be very rich Total Dissolved Solids 1000-1200
with different undefined species of mg/l
microorganisms.A large number of cells Oil & Grease mg/l ND
was easily noticed to grow in the first 16 Turbidity 5 NTU
hrs of incubation. Cells chosen for this BOD mg/l 08
study is motile gram –ve small rods COD mg/l 20
organisms.

Sudan Engineering Society Journal, March 2009, Volume 55 No.52 27

 BIOLOGICAL TREATMENT OF PETROLEUM PRODUCED WATER IN SUDAN

A very rapid increase in bacterial counts

was noticed in both continuous and batch
cultures (Charts 1 and 2). Reduction in oil
and grease was very clear through first
hours of experiment, but as degradation
increased, remaining weight couldn’t be
measured because of the limit of digital
balance accuracy (0.0001 mg) (Chart 3).
Gas chromatographs of inlet and outlet
after 36hrs retention time showed a Figure 8: Browning of plant leaves
complete degradation of all peaks (nC4 -
nC34) in both batch and continuous
cultures (Charts 4, 5 and 6).
Germination test showed that growth in
pots irrigated by biodegradable water was
of better quality in length and intensity for
both crops compared to raw and tap water
(Figures 6 and 7). In dishes 100% growth
was noticed in all dishes after 24 hrs, but
when allowing seeds to grow, a very clear
toxic effect was noticed in raw water
dishes as typically described by Connor
[29] as leaves of plant show browning of
tips going inwards (Figures 8, 9 and 10) Figure 9: Toxic effects (Raw water)
while a very healthy growth noticed in
biodegradable water dishes.

Figure10: Healthy growth (treated water)

Bactria growth curve in
Continous Flow Culture

Figure 6: Millet growth (pot irrigated with 1.00E+12

treated water in the middle) 1.00E+11

1.00E+10

1.00E+09

1.00E+08
C ount (C FC )

1.00E+07

1.00E+06

1.00E+05

1.00E+04

1.00E+03 Chart 1
1.00E+02

1.00E+01

1.00E+00
Figure 7: Sorghum growth (pot irrigated -10.00 0.00 10.00 20.00 30.00 40.00 50.00
Time (hrs)
with treated after in the middle)

28 Sudan Engineering Society Journal, March 2009, Volume 55 No.52

 1.00E+12 4. CONCLUSION
1.00E+11
It can be concluded from the aforementioned
1.00E+10 that:
1.00E+09 Chart 2 1. The toxicity of produced water due to
high hydrocarbon concentrations, can be
C o u n t (C F C )

1.00E+08
reduced using indigenous bacterial
1.00E+07
species which could result in reducing
1.00E+06 environmental pollution that affect both
1.00E+05
land and vegetation.
1.00E+04
2. Good quality of treated water is obtained
1.00E+03 in the laboratory scale which can be used
-10.00 0.00 10.00 20.00 30.00 40.00 50.00
Time (hrs) successfully for germination of plants and
crops.
Chart 1 and 2: Bacterial Counts in
Continuous Flow Culture, Bacterial Counts 3. TDS were not affected by this treatment.
in Batch Culture4.
Biodegradation in Batch flow

350.00

300.00
Chart 3: Batch flow Biodegradation
Oil concentration mg/l

250.00

200.00

150.00

100.00

50.00

0.00
0.00 3.00 6.00 9.00 12.00 15.00

time (hrs)

Chart 4: Raw Water Chromatograph

Sudan Engineering Society Journal, March 2009, Volume 55 No.52 29

 BIOLOGICAL TREATMENT OF PETROLEUM PRODUCED WATER IN SUDAN

Chart 5: Treated Water in Batch Culture Chart 6: Treated Water in Continuous

(36 hrs)
Culture (36 hrs)

5. RECOMMENDATIONS 4. Veil J. A; Markus G. Puder, Deborah

Elcock, Robert J. Redweik, Jr. (2004)
1. Further research is essential to study
‘A White Paper Describing
the biodegradability potentials for all Produced Water from Production of
other isolated species and the synergy Crude Oil’ Prepared for: U.S.
between them, if any. Department of Energy National Energy
2. Bacterial growth methods (suspended, Technology Laboratory.
attached or hybrid) must be studied 5. Stephenson M.T. (1992) ‘Survey of
carefully and mathematically modelled Produced Water Studies’
to optimize the bioreactor design. "Proceedings of the 1992 International
Produced Water Symposium, held, in
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1. Yager TH. R. (2001) ‘The mineral 6. Verbeek P.; Davies S.; (2008)
industry of Sudan’ U.S. geological ‘Produced water Treatment and
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26.2-26.4. Symposium on Produced water
2. International Finance Corporation Management Stavanger.
(IFC) and World Bank Group (WBG) 7. Sumi L. (2005) ‘Produced Water
‘Environmental, Health, and Safety from Oil and Gas Production’ Paper
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Development’ April 30, 2007 gas summit, New Mexico.
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environment. com /index.html ‘Convention For The Protection Of
The Marine Environment Of The

30 Sudan Engineering Society Journal, March 2009, Volume 55 No.52

 North-East Atlantic’ Text as Capable of Degrading Crude Oil
amended on 24 July 1998, updated 14 Under Aerobic Conditions’ PhD
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Industry’ Khartoum, Sudan Ming L.B; Sabbar A. (Oct. 2006)
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 BIOLOGICAL TREATMENT OF PETROLEUM PRODUCED WATER IN SUDAN

refinery effluents in Nigeria’ African 29. Connor J. A.; Newell C. J.’(2006)

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The Authors:
Dr. Mohammed Ahmed Khadam. Professor Muna Ahmed Agab.
Dr. Khadam has BSc in Civil Prof. Muna has BSc and MSc in food
Engineering from the University of microbiology from the University Of
Khartoum, and MSc in Sanitary Khartoum. She did her PhD in Belfast
Engineering from Delft University in (United Kingdom). She was the head of
biotechnology section in Shambat
Netherlands. He did his PhD in
Agriculture Institute and now she is the
University of Khartoum (Environmental technical administrator in Sudanese
Engineering ).He was the head of civil Standard Metrology Organisation and a
engineering department and now he is lecturer in MSc courses (Environmental
the Deputy Dean for Academic Affairs Engineering, University of Khartoum)
and the head of research board of the
Faculty of Engineering, University of
Khartoum.

Eng. Suhair A/Gayoum

(Corresponding Author)
She has BSc and MSc in Civil Engineering from
the University of Khartoum. Now she is a PhD
student in Environmental engineering section in
U of K and a lecturer in Sudan University for
Science and Technology, – Faculty of Water and
Environmental Engineering.
([email protected])

32 Sudan Engineering Society Journal, March 2009, Volume 55 No.52

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