SPE 109571

Bioremediation of Oil Contaminated Soil at South Santhal CTF, Mehsana, India:

A Case Study
Ajoy Kumar Mandal, Priyangshu Manab Sarma, Manish Dwivedi, Abu Swaleh, Banwari Lal, The Energy and Resources
Institute (TERI); A. Agnihotri, U.S. Hazra, ONGC Corporate, Health, Safety and Environment, New Delhi, India,
and A. Doraiah and S.S. Patidar, ONGC, Mehsana Asset, Mehsana, India

Copyright 2007, Society of Petroleum Engineers

area of land contaminated with oil spills can be
This paper was prepared for presentation at the 2007 SPE Asia Pacific Oil & Gas Conference
and Exhibition held in Jakarta, Indonesia, 30 October–1 November 2007. treated for safe disposal.
This paper was selected for presentation by an SPE Program Committee following review of
information contained in an abstract submitted by the author(s). Contents of the paper, as Results, Observations and Conclusions: In the
presented, have not been reviewed by the Society of Petroleum Engineers and are subject to
correction by the author(s). The material, as presented, does not necessarily reflect any present study, total 1500 tonnes of oil-contaminated
position of the Society of Petroleum Engineers, its officers, or members. Papers presented at
SPE meetings are subject to publication review by Editorial Committees of the Society of soil was undertaken for treatment by bioremediation
Petroleum Engineers. Electronic reproduction, distribution, or storage of any part of this paper
for commercial purposes without the written consent of the Society of Petroleum Engineers is techniques at three sites of South Santhal central
prohibited. Permission to reproduce in print is restricted to an abstract of not more than
300 words; illustrations may not be copied. The abstract must contain conspicuous tank farm area (CTF), Mehsana Asset, ONGC,
acknowledgment of where and by whom the paper was presented. Write Librarian, SPE, P.O.
Box 833836, Richardson, Texas 75083-3836 U.S.A., fax 01-972-952-9435. India. Within 135 days of treatment, the total
petroleum hydrocarbon (TPH) of the oil
Abstract contaminated soil was degraded by 90.98%, 92.08%
Description of the material: The petroleum and 91.47% at Site – I (Well no. 1), Site – II (Well
industry effluents, oily sludge and oil spills cause a no. 205) and Site – III (Well no. 206) respectively.
serious threat to the environment as their constituent Whereas at the control site, without treatment by
compounds have toxic, mutagenic and carcinogenic bioremediation techniques, the degradation of TPH
properties. With the stringent regulatory norms and was negligible. Hence biodegradation technique has
environmental obligations, safe disposal of these been proved to be an effective method for treatment
hazardous wastes is a serious problem. Various of oil contamination.
conventional methods are available for disposal of
the same, but all of them are having one or more Significance of Subject Matter: This study has
disadvantages. Biological methods have been well helped the ONGC authorities to identify an
reviewed and acknowledged for reclamation and environment friendly method for disposal of oily
remediation of environments contaminated with waste. The bioremediation study of oil-
petroleum hydrocarbons. The paper describes a field contaminated soil by applying microbial consortium
case study on the bioremediation of oil is an ongoing investigation and the results are
contaminated soil at an installation of Oil and highly encouraging.
Natural Gas Corporation Ltd. (ONGC), India.
KEYWORDS: Bioremediation, Biodegradation.
Application: A bacterial consortium was developed Oily sludge, Total Petroleum Hydrocarbons
by assemble of four species of bacteria which could
effectively biodegrade different fractions of total INTRODUCTION
petroleum hydrocarbon. Using this bacterial Petroleum industries unavoidably generate
consortium huge quantity of oily sludge and large enormous quantity of oily sludge as well as oil/oily
soaked soil which constitutes a major challenge for
2 SPE 109571

hazardous waste management as well as Therefore, environment friendly technologies are

environment management. Apart from this , there is increasingly in demand today for management of
incidence of oil spills in their day to day operations. oily waste [1]. Natural diversity of soil
This also causes enormous environmental pollution. microorganisms is a useful source for detoxification
Some of the environmental impacts due to oil of hazardous waste contamination sites.
contamination can include: physical and chemical Understanding of the microbial diversity in such an
alteration of natural habitats, physical smothering ecosystem leads to formulation of better strategies
effect on the marine life, lethal or sub-lethal toxic of bioremediation. The use of bacteria in breakdown
effects on the marine life, changes in the marine and digestion of oily sludge is a biological process
ecosystem resulting from oil effects on key known as biodegradation [2]. Biodegradation is the
organisms e.g. increased abundance of inter-tidal most ecofriendly and economically viable among all
algae following the death of limpets which normally the available methods of sludge management. These
eat the algae, aquatic birds die due to hypothermia, beneficial microbes break down a wide variety of
drowning , loss in flight , poisoning, etc. Crude oil toxic substrates present in the oily sludge to the
exposure may cause damage to lungs, liver, safest form which does not cause any harm to the
kidneys, intestines and other internal organs of the environment [3].
birds and animals, reproductive impairment in birds,
fish and reptiles. Polycyclic aromatic hydrocarbons
(PAH) may lead to skin erythema (reddening), skin With this rationale, The Energy and Resources
cancer, sinonasal cancer, gastrointestinal cancer, Institute (TERI), New Delhi, India, initiated an
and bladder cancer, Inhalation leads to headache, exhaustive operation of collecting soil samples from
nausea, dizziness, respiratory irritation, BTEX different geo-climatic regions of India followed by a
(Benzene, Toluene, Eethyl benzene & Xylene) series of enrichment and isolation protocol to isolate
present in the oil contamination, cause mutations, and screen hydrocarbon degrading bacterial strains.
cancers, birth defects, endocrine disruptions, still Extensive research at TERI has resulted in the
births, nervous disorders, and liver disease , development of an easy and cost effective
carcinogen, effect on central nervous system, bioremediation process. Providing great
depression , irregular heartbeats. Plants covered by significance to bioremediation in the worldwide
the oil are unable to photosynthesize. Oil effort to tackle oil spills and oily sludge
contaminated soil looses its fertility and effects on contamination, TERI has developed a consortium of
seed germination. The mites and other insects can crude oil/oily sludge degrading bacteria, deriving
not survive in oil contaminated land, leading to a from various bacterial cultures existing in the
major imbalance in the food chain. A large natural environment.
percentage of the oil spill gets emulsified and
solidified along with sea shore, clinging to sand,
rock and stone. The consortium consists of naturally occurring
efficient microbes which exist in normal soil and
Different approaches have been tried since early were isolated from different geoclimatic regions of
times to overcome this problem. Some of them are India. The selected microbes are the most adapted to
known as, land filling, incineration, air spurging, climatic (at temperature ranging from 15°C to
natural remediation (like evaporation of volatile 60°C) and soil conditions to degrade toxic
organic compounds, auto oxidation, photo hydrocarbons. Specially designed nutrients are used
oxidation, etc.), land farming, surfactants, and other during the application of the microbial consortium.
conventional methods like chemical dissociation, This is done to enhance the population of the
dumping in injection wells, surface impoundment, microbial consortium and also to mitigate the initial
waste piles, underground injection wells, etc. toxic shock due to the oil contamination while
However the common drawback in all those application on the oil contaminated soil in the field.
conventional methods is that they are not the The microbial consortium can work in even in high
permanent solution for the environmental pollution saline soil. The microbial consortium consists of
and sometimes they are not cost effective. four different microbes which could degrade alkane,
SPE 109571 3

aromatic, NSO (Nitrogen, Sulphur, Oxygen oily sludge and waste water effluent sludge through
containing compounds) and asphaltene fractions of digestion of harmful chemicals and compounds
crude oil and oily sludge. Bioremediation present in oily sludge and waste water effluent
technology is an ecologically sound, natural sludge into simpler, less toxic or non-toxic
process; oily sludge eating bacteria can increase in substances. Microorganisms, like all living
numbers when an oily sludge (contaminant) is organisms, need nutrients (such as nitrogen,
available. When the contaminant is bioremediated, phosphate, and trace metals), carbon and energy to
the microbial population naturally declines. The survive. These beneficial microbes break down a
residues from the biological treatment are usually wide variety of organic (carbon-containing)
harmless products (carbon dioxide, water and fatty compounds found in oily sludge and waste water
acids). Instead of merely transferring contaminants effluent sludge to obtain energy for their growth.
from one environmental medium to another (e.g., Many species of soil bacteria, for example, use
from water to air or land) bioremediation destroys petroleum hydrocarbons as a food/energy source,
the target chemicals. This technology is usually less transforming them into harmless substances
expensive than other technologies that are often consisting mainly of carbon dioxide, water and fatty
used to clean up hazardous waste. The acids. Bioremediation exploits this natural process
bioremediation process can often be accomplished by promoting the growth of microbes that can
in situ. This eliminates the need to transfer large effectively degrade specific contaminants and
quantities of contaminated waste and the potential convert them to nontoxic by-products. There are
threats to human health and the environment that two basic types of bioremediation: "Biostimulation"
can arise during transportation. The organisms used provides nutrients to the indigenous microbial
in the consortium are not pathogenic and have been populations. This promotes growth and increases
isolated from indigenous soil. metabolic activity that is used to degrade
contaminants. "Bioaugmentation" introduces
specific blends of microorganisms into a
The present paper describes a case study on the
contaminated environment or into a bioreactor to
bioremediation of oil contaminated soil, by
initiate the bioremediation process, which increases
application of the bacterial consortium, at one oil
the population of the fit to handle the
installation of ONGC at South Santhal CTF,
biodegradative process in the contaminated area.
Mehsana, India. Due to an accidental oil spill from
their nearby field, South Santhal CTF had
The present case study was a batch bioaugmentation
accumulated a considerable amount of oil
process carried out at three different sites inside the
contaminated soil, the disposal of which was a
CTF premises. Initially the microbial consortium
serious problem for ONGC. As a case study, the
was selected from the microbial collection of TERI,
said oil contaminated soil was considered for
considering the characteristics of the crude oil of the
disposal by bioremediation technology. After
said location. A feasibility study with the selected
application of the selected microbial consortium, the
microbial consortium was carried out in lab scale at
oil contaminated soil was biodegraded within 135
TERI. After successful laboratory study the said
days time. This has helped the oil installation to find
consortium was produced in bulk and applied on
out a suitable environment friendly method for
the oil contaminated soil at the bioremediation site.
disposal of the oily waste.
In the field study the bioremediation site was
prepared and subsequently the microbial consortium
Statement of Theory and Definitions: was applied along with specially designed nutrients
to enhance the population of the microbial
Bioremediation is a process that uses naturally consortium in the soil under bioremediation. The
occurring microorganisms to transform harmful bioremediation site was tilled and water sprinkling
substances to nontoxic compounds. There are was carried out at regular interval so as to maintain
innumerable strains of microbes under basic aerobic condition as well as moisture content of the
categories of bacteria, yeast or fungi, which degrade bioremediation site. The performance of the
bioremediation job was monitored by analyzing the
4 SPE 109571

soil samples collected from the bioremediation sites Identification of the bacterial strains: Identification
for total petroleum hydrocarbon (TPH), pH and of the isolated bacterial strains was done by
microbial count. Mehsana being arid zone, sequencing of the 16S rDNA gene with the Microseq
watering of the bioremediation site was a crucial 16S DNA sequencing Kit TM (PE Applied
point in this case study. A special precaution was Biosystems, Inc, USA) (6). The sequences were
taken for watering of the site at regular interval. analyzed with an ABI Prism 310 Genetic Analyzer
Water samples were collected from the bore wells (PE Applied Biosystems, Inc, USA) as per
installed near the bioremediation site, so as to manufacturer's instructions. The sequences of 16S
monitor the presence of oil and grease (if any) in the rDNA genes were subjected to BLAST searches of
ground water during the bioremediation process. the NCBI GeneBank database for identification.

About South Santhal CTF, Mehsana: Degradation of crude oil by bacterial strains:
Situated in the Mehsana District in the Northern Degradation of crude oil by the bacterial isolates was
part of Gujarat State in India. Mehsana is located at monitored in 250 mL flasks in triplicates containing
23.6° N 72.4° E. It has an average elevation of 100 mL MSM with 1 % (w/v) of crude oil (steam
81 meters (265 feet). As the Tropic of Cancer sterilized) as sole carbon source and incubation on a
passes through the district, Mehsana has an rotary shaker (200 rpm) at 37°C. The isolates were
intensely hot or cold climate. The average grown previously in MSM for 24 h with 1 % (w/v)
temperature is 40°C during. summer and 10°C crude oil to a cell density of 108 cells mL-1 and were
during winter. Located in one of the arid zones in inoculated into the medium with 5 % (v/v) as
Gujarat state that face chronic scarcity conditions inoculum. Uninoculated controls were kept to
for want of adequate rains. At Mehsana, ONGC has monitor natural weathering of crude oil. Residual
a big oil exploration station containing crude oil was extracted from the cultures by using
approximately 1250 oil producing wells. South solvents (6). For quantitative analysis, the residual
Santhal CTF is one of the oil installations under crude oil was fractionated by silica gel column (7).
ONGC. The different fractions were analyzed by gas liquid
chromatography (Hewlett Packard 5890 series II)
Description and Application of Equipment and fitted with flame ionization detectors (4). The profile
Processes: of the different fractions of petroleum hydrocarbons
extracted from inoculated flasks was compared with
Isolation of Bacterial strains: The crude oil that of the uninoculated controls to determine the
contaminated soil samples were collected from extent of degradation.
different oil refineries and oil exploration sites of
India. The solvent extractable total petroleum Selection of bacterial consortium: Based on the
hydrocarbon (TPH) in the crude oil contaminated soil efficiency to degrade different fractions of total
samples was estimated (4). For enrichment, 5 g petroleum hydrocarbons (aliphatic, aromatic,
samples of soil were inoculated into 100 mL of asphaltene, NSO compounds) and also based on the
minimal salt medium (MSM) (5) containing steam environmental parameters from where these bacterial
sterilized crude oil (1%, w/v) as carbon source and strains have been isolated, the bacterial consortium
incubated at 37°C on a rotary shaker (200 rpm) for 7 was selected for application on the actual field. A
days. 5 mL of enriched culture was re-inoculated in feasibility study was carried out in the laboratory by
fresh medium under similar conditions and five such checking the efficiency of the said bacterial
cycles were repeated. After five cycles of consortium to degrade crude oil present in the oil
enrichment, 1 mL of culture was diluted up to 108 contaminated soil, to be undertaken for
fold, and 100μL of all dilutions were plated on MSM bioremediation at South Santhal CTF. The study was
agar plates with crude oil (1 % w/v). The bacterial carried out on MSM using crude oil as sole carbon
colonies obtained were further purified on the MSM and energy source as per the methods described
agar plates (with crude oil 1 % w/v). The isolates above.
were routinely sub cultured and frozen stock cultures
were stored in 25 % glycerol at -70°C.
SPE 109571 5

Selection and preparation of bioremediation revival and growth. The nutrient formulation was
sites: At South Santhal CTF, Mehsana, dissolved in water and the liquid nutrient solution
bioremediation job was undertaken for a total was spread uniformly to the bioremediation site
quantity of 1500 metric tonnes of low oil containing with the help of water sprinkler. After application of
oil contaminated soil. Three separate sites located in the microbial consortium, mixing of oil
nearby area of the CTF were selected for contaminated soil and microbes were done by tilling
undertaking the bioremediation job with the said of bioremediation sites site with a tractor attached
1500 metric tones of oil contaminated soil. The sites with cultivator. In the control site (Site- IV),
were identified as Site – I (near well no. 1), Site – II microbial consortium was not added, however rest
(near well no. 205) and Site – III (near well no. 206) of the other activities like tilling, watering etc. were
respectively. One control site of 5 meter x 5 meter carried out in the same manner as the experimental
area was also prepared near the bioremediation site bioremediation sites.
– I, where no treatment was given. This site was
identified as Site – IV (near well no. 1). This was Tilling: Tilling of all the bioremediation sites was
done to compare the natural degradation of the oily done at a regular interval of once in a week. This
sludge under the similar climatic situations. All the was done with the help of a tractor attached with
three sites were within half kilometer radius, hence cultivator. Regular tilling of the bioremediation site
the control site was kept only in one area. The was done to maintain aeration for the microbial
bioremediation sites were prepared by removing consortium applied on the oil contaminated soil at
stones, debris and other non-biodegradable the bioremediation sites.
materials from the site. The sites were leveled using
JCB backhoe loader before spreading of oil Watering: Regular watering of the bioremediation
contaminated soil. The bottom of sites was layed sites was done at every fourth day using tap water
down by high density polyethylene (HDPE) liner of collected in a water tanker and by sprinkling the
500 micron thickness to avoid the leaching of oil same with the help of water sprinkler attached to the
contamination to the underground and prevent water tanker. Mehsana being arid zone and also the
ground water contamination. The sites were job being carried out in summer season, watering of
barricaded and labeled for proper identification. The the bioremediation sites was required to be done
oil contaminated soil was transferred to the more frequently.
bioremediation sites using Dumper Truck and the
same was spread uniformly using JCB backhoe Sampling: Samples were collected from the
loader in all the three bioremediation sites as well as bioremediation sites at 0, 15, 45, 75 and 135 days
the control site. The height of the oil contaminated after application of the microbial consortium to the
soil was maintained to maximum 20 cm. in all the bioremediation site. Each bioremediation sites were
sites. divided in four equal blocks, which were further
divided in four sub-blocks. Equal quantity of
Application of microbial consortium on oil samples was collected randomly from each sub-
contaminated soil: The microbial consortium was block i.e. total 16 samples were collected from one
produced in 100 liter bioreactor at TERI. The site. Samples were collected using a hollow
microbial consortium culture was immobilized with stainless steel pipeline of 3 inch diameter and 50
a suitable carrier material, packed in sterilized cm. in length and by inserting the same vertically
polybags (packing size 5 kg) and transported to the on the bioremediation site from the surface till the
respective sites for its application on oil bottom of the site in one particular point. This was
contaminated soil. The microbial consortium was done to collect uniform samples from each depth of
applied on oil contaminated soil by manual the bioremediation site. The samples were collected
spreading at regular intervals of one month up to in sterile plastic containers. The sixteen samples
three applications. Previously selected nutrient were mixed uniformly to get a homogenized
formulation was also sprayed on oil contaminated composite mixture which was considered as the
soil along with the microbial consortium. This was representative sample from the site. Mixing was
done to help the microbial consortium for their done in a large container by hand with hand gloves.
6 SPE 109571

Approximately 1 Kg. of the composite sample was Characterization of oil contaminated soil (oil
taken to laboratory for necessary analysis for content and its various fractions):
monitoring of the bioremediation job. Oil content in the oil contaminated soil were
measured in terms of total petroleum hydrocarbon
The bore well water samples were collected from (TPH) which was extracted from a known quantity
each bore wells installed at the nearby area of the of oil contaminated soil by solvent extraction
bioremediation site. The samples were collected in method using various solvents like hexane,
sterile plastic bottles. The bore wells were flushed methylene chloride and chloroform consecutively.
thoroughly before collecting the samples. The extracts were pooled and dried at room
temperature by evaporation of solvents under gentle
Equipments : The following equipments were nitrogen stream in a fume hood. The amount of
utilized for the bioremediation treatment operation TPH recovered from the oily sludge was quantified
in the field: by gravimetric method. The sediments/ash content
a. JCB Backhoe Loader – used for loading oil in the residual oily sludge was measured by heating
contaminate soil to the dumper truck for the sludge, after TPH extraction, at 600°C for five
transportation to the bioremediation site hours using a crucible and subsequent cooling to
from the storage location. Also used for room temperature. The amount of ash recovered
spreading of oil contaminated soil at the from the residual sludge was quantified
bioremediation site. gravimetrically (3).
b. Dumper Truck – used for transportation of
the oil contaminated soil. The TPH extracted from the oil contaminated soil
c. Light Commercial Vehicle (LCV) – used for was further fractionated for various fractions like
transportation of the microbial consortium alkane, aromatic, NSO and asphaltene fractions. A
from laboratory to the bioremediation site. known quantity of TPH was dissolved in n-pentane
d. Bioreactor assembly – used for bulk and separated into soluble and insoluble fractions.
production of the microbial consortium. The insoluble fraction (asphaltene) was quantified.
e. Mixer / Blender – used for immobilization The soluble fraction was further loaded on silica gel
of the microbial consortium. column and eluted with different solvents (7). The
f. Sealer – used for sealing of the packing bags alkane fraction was eluted with hexane, followed by
containing microbial consortium. the aromatic fraction that was eluted with benzene.
g. Tractor with cultivator – used for tilling of The NSO fraction was eluted with chloroform and
the soil at the bioremediation site. methanol. Alkane and aromatic fractions were
h. Water Tanker with sprinkler – used for concentrated by evaporation of solvents and then
spraying of sweet water to the soil at the 0.2 μl of each was analyzed by gas chromatography
bioremediation site. (GC Hewlett Packard, 5890 Series II) to identify all
i. Bore wells – used for collection of ground the compounds present in the alkane and aromatic
water for regular monitoring. fractions by matching the retention time with
j. Small hollow pipe – used for collection of authentic standards (3).
soil samples from the bioremediation site.
Determination of moisture content: Moisture
Monitoring of bioremediation process: The content of the oil contaminated soil was determined
bioremediation process was monitored by analyzing by the standard method IS – 2720 – P2.
the soil samples from time to time for the following
parameters: Determination of microbial count: The oil
a. Oil content and its various fractions. contaminated soil samples collected from the
b. Moisture content bioremediation site were monitored for Total
c. Microbial count Bacterial Count (TBC) by standard spread plate
d. pH method with serial dilutions of the samples.
The bore well water samples were analyzed for pH Standard Luria Bertini agar plate (Himedia catalog
and oil & grease only. no. M 557) was used for determining TBC. (8)
SPE 109571 7

Determination of pH: sampling locations were purified and preserved as

To 20 gm. soil sample 50 ml. water was added. The frozen stock cultures in 25 % glycerol at –70 ΟC
same was kept on a shaker at 370C for overnight for further experiments. All the bacterial strains
incubation. The same was allowed for settling at were given an accession number. All the bacterial
room temperature. The liquid solution layer was strains were identified by the method mentioned
gently decanted to one 100 ml. beaker. The pH of above. It was found that out of 324 isolates only 110
the solution was measured using standard pH meter different species of bacterial strains were obtained
(Orion Expandable Ion Analyzer model no. EA – (Table 1).
940). The pH was determined by following the
operating manual of the pH meter. The pH meter Degradation of crude oil by selected bacterial
was calibrated before checking the pH. The pH of strains. All the bacterial strains that have been
the ground water samples was measured directly by isolated were analyzed for its efficiency to degrade
the above mentioned pH meter. the total petroleum hydrocarbon (TPH) and its
different fractions of the crude oil and oily sludge. A
Determination of oil and grease in ground water representative data of one site (Indian Oil Corporation
sample: Oil and grease in the ground water samples Ltd., Gujarat refinery, India) is presented below in
were determined as per the standard method IS Figures 1 and 2.
3025 (P 39) : 1991.
Selection of bacterial consortium: From the
Biodegradation of TPH in the oil contaminated bacterial collections of TERI, a specific bacterial
soil: The oil contaminated soil samples were consortium that could degrade different fraction of
collected from the bioremediation site at zero day oil contaminated soil of South Santhal CTF was
i.e. before initiation of the bioremediation job and developed in minimal salt medium, with oil
after regular interval of one month till the contaminated soil of South Santhal CTF as the sole
completion of the bioremediation job. The TPH and source of carbon and energy. This indigenously
its various fractions were analyzed for every developed bacterial consortium consisted of four
samples. The decrease in the TPH content and its bacterial isolates. These four bacterial isolates could
fractions with time and the percent biodegradation degrade alkane, aromatic, NSO and asphaltene
was calculated. Simultaneously, biodegradation of fractions of the crude oil. The optimized consortium
alkane and aromatic fractions was assessed by was used for the field biodegradation studies at
quantitative measurement of the peaks from the GC South Santhal CTF, Mehsana.
chromatogram with the help of standard calibration
curve of each compound of alkane and aromatic Bioremediation of oil contaminated soil at South
fractions (3). Santhal CTF, Mehsana:

Bioremediation site: The detailed descriptions of

Presentation of Data and Results: the sites selected for the bioremediation job at South
Santhal CTF are given in Table – 2. The sites were
Isolation and identification of hydrocarbon prepared as per the method mentioned earlier.
degrading bacterial strains: Culturable bacterial
strains were isolated by enrichment culture Composition of oil contaminated soil:
technique from the total petroleum hydrocarbon The oil contaminated soil undertaken for
contaminated soil samples. The enrichment of the bioremediation at South Santhal CTF contained
soil samples was done with crude oil as sole source 7.42% solvent extractable total petroleum
of carbon and energy. A total of 324 culturable hydrocarbon (TPH), 18.73 % moisture content and
bacterial strains were isolated from crude oily 73.85 % residual soil. TPH extracted from the oil
sludge and oil contaminated soil samples collected contaminated soil contained 65% alkane fraction,
from 15 oil installations located at different 24% aromatic fraction, 11% fractions of heavy
geoclimatic regions of India (Table 1). The hydrocarbons like asphaltene, resins etc.
culturable bacterial strains isolated from all the
8 SPE 109571

Biodegradation : After complete application of the in the control site where no treatment was given.
microbial consortium to the bioremediation sites, Hence bioremediation technology using the
the solvent extractable TPH in the oil contaminated microbial consortium can be a very good
soil in all the three sites was found to be degraded to environment friendly process for disposal of
more than 90% in 135 days compared to the initial hydrocarbon contaminations.
day TPH. The details of TPH content and percent
degradation of TPH in all the bioremediation sites Acknowledgements:
are described in Table – 3 and Figure - 3. Figure – The authors are thankful to Dr. R. K. Pachauri,
4, 5 & 6 below describes the chromatograms of GC Director-General of TERI, for providing the
analysis indicating the biodegradation of the alkane infrastructure to carry out the present study. The
and aromatic fraction of the oil contaminated soil at authors also thank the management of ONGC for
the bioremediation Sites – I, II and III respectively. providing the opportunities for field study at their
In the control site it was found that there was only oil installations at South Santhal , Mehsana Asset.
14% degradation of the TPH in the same period. The authors are grateful to various officials of
This indicates that the bioremediation process by ONGC installations for their kind co-operation and
using the microbial consortium is an efficient support during the period of study. A special thanks
process for degradation of oil contamination. to Ms. Anju Singh and the lab attendants of
Microbial Biotechnology Area of TERI for helping
pH and microbial count of the soil samples: The in the analysis of the samples. Authors acknowledge
soil samples collected from time to time were the secretarial help in typing the manuscript by Ms.
checked for pH and total bacterial count. It was Neena Mata and Ms. Jyoti Gupta.
found that throughout the bioremediation treatment
process, pH of the samples were within 7.5 to 8.5 in Nomenclature:
all the sites. The microbial counts were maintained
in the range of 107 to 108 CFU per gram of soil TPH = Total Petroleum Hydrocarbon concentration
sample in Site – I, II and III respectively. However, in the oil contaminated soil (mass of oil / mass of
in the control site the microbial count was in the dry soil) (%).
range of 104 to 105 CFU per gram of soil sample PAH = Polycyclic Aromatic Hydrocarbons
(Table – 4). concentration present in the oil contaminated soil
(mass of oil / mass of dry soil) (%).
Ground water quality: Ground water samples
collected from the bore wells near the BTEX = Benzene, Toluene, Eethyl benzene &
bioremediation sites were analyzed for pH and oil Xylene present in the oil contaminated soil (mass of
and grease. It was found that pH in all the ground oil / mass of dry soil) (%).
water samples varied from 7.5 to 8.5. Whereas, in NSO = Nitrogen, Sulphur, Oxygen containing
all the samples the oil and grease was found to be compound present in the oil contaminated soil
nil. This indicated that there was no leaching of oil (mass of oil / mass of dry soil) (%).
to the underground water.
CTF = Central Tank farm Area of the oil
Conclusions: installation.
In South Santhal CTF, Mehsana Asset, of ONGC, MSM = Minimal salt medium without the carbon
total 1500 tonnes of oil contaminated soil was source. Used for isolation and enrichment of
biodegraded using a specially designed microbial specific microbial strain.
consortium. The bioremediation job was carried out RPM = Revolutions Per Minute, measures the
in three separate sites and also in one control site rotational speed of the motor's spindle of the rotary
where no treatment was given. The microbial shaker.
consortium could degrade more than 90% of the
TPH present in the oil contaminated soil in 135 days mL = milliliter, a metric unit of volume equal to
period in the experimental sites. Whereas there was one thousandth of a liter.
only 14% degradation of the oil contaminated soil
SPE 109571 9

TBC = Total bacterial count in the oil contaminated

soil (CFU / gm. of soil). (CFU = colony forming
unit)
HDPE = High Density Polyethylene plastic, has a
very high seam strength and is also resistant to
many chemicals and ultra violet light. For this
reason it is often used to make pipes used in the
hydrocarbons industry.
References:

1. ATLAS R. M.: “Microbial hydrocarbon

degradation - biodegradation of oil spills”,
[J] .Chem. Tech. Biotechnol. (1991), 52:
149-156.
2. HEAD, I.M.: “Bioremediation: towards a
credible technology”, Microbiology (1998),
144 : 3 : 599.
3. Mishra, S. et al.: “Evaluation of inoculum
addition to stimulate in situ bioremediation
of oily sludge contaminated soil” Appl.
Environ. Microbiol (2001), 67:1675.
4. Mishra, S. et al.: “In situ bioremediation
potential of an oily sludge degrading
bacterial consortium”. Current
Microbiology (2001), 43: 328.
5. Lal, B. et al.: “Mineralization of [14C]
octacosane by Acinetobacter calcoaceticus,”
Canadian Journal of Microbiology (1996), 42
: 1225.
6. Sarma, P.M. et al.: “Assessment of
intraspecies diversity among strains of
Acinetobacter baumannii isolated from sites
contaminated with petroleum
hydrocarbons”. Canadian journal of
Microbiology (2004), 50: 405.
7. Walker, J D. et. al.: “Microbial petroleum
degradation:application of computerized
mass spectroscopy”. Can. J. Microbiol
(1975) 21:1760.
8. Mishra, S. et al.: “Crude oil degradation
efficiency of a recombinant lux tagged
Acinetobacter baumannii strain and its
survival in crude oil contaminated soil
microcosm”. FEMS Microbiology Letters
(2004), 235: 323.
10 SPE 109571

Table 1 Total petroleum hydrocarbon degrading bacterial strains isolated from sampling sites situated in different geoclimatic regions
in India.

Isolation sites (Regional location in Geographical location Tempe- Total number of Total number of
India) (Latitude & Longitude) rature culturable species among the
Latitude Longitude range (0C) bacterial strains bacterial strains
isolated
IOCL, Mathura refinery (Northern ) 27026′ N 77043′ E 10 – 40 30 14
IOCL, Barauni refinery (Eastern) 25028′ N 85059′ E 19 – 35 16 8
IOCL, Haldia refinery (Eastern ) 22000′ N 88005′ E 15 – 35 26 6
IOCL, Gujarat refinery (Western ) 22016′ N 73014′ E 20 – 38 30 9
IOCL, Panipat refinery (Northern) 29023′ N 76058′ E 15 – 40 20 5
ONGC oil well, Jorhat (North Eastern) 26040′ N 95035′ E 10 – 35 11 4
Oil well of Oil India Ltd., Duliajan, 27015′ N 95015′ E 10 – 35 26 12
(North Eastern)
IOCL, Digboi refinery (North Eastern) 27015′ N 95015′ E 12 – 35 33 11
IOCL, Guwahati refinery (North 26009′ N 91046′ E 15 – 35 22 10
Eastern)
BPCL, Mumbai refinery, (Western) 18056′ N 72051′ E 24 – 35 18 7
HPCL, Visakhapatnam (South Eastern) 17041′ N 83017′ E 21 – 42 19 6
CRL, Cochin refinery (Southern ) 9055′ N 76014′ E 19 – 37 12 6
BRPL, Bongaigoan refinery (North 22016′ N 73014′E 18 – 32 13 6
Eastern )
Vadinar refinery (Western) 23044′ N 72039′ E 15 – 45 16 3
Reliance refinery, Jamnagar (Western) 22026′ N 70026′ E 15 – 45 32 3
Total 324 110

Table – 2 : Bioremediation site details

Particula Location at South Area of the Dimension of site with Quantity of Quantity of Microbial
rs Santhal CTF, bioremediati oil contaminated soil oilcontaminated soil consortium applied on
Mehsana on site (L x B x H) undertaken for the site
bioremediation
Site – I Near well no. 1 at 4785 m2 87 m x 55 m x 0.2 m 1100 Metric Tonnes 3000 kgs.
Scrapyard
Site – II Near well no. 205 800 m2 40 m x 20 m x 0.2 m 250 Metric Tonnes 750 kgs.
Site – III Near well no. 206 880 m2 40 m x 22 m x 0.2 m 250 Metric Tonnes 750 kgs.
Site – IV Near well no. 1 at 25 m2 5 m x 5 m x 0.2 m 5 Metric Tonnes Nil
Scrapyard

Table 3. Biodegradation of TPH in oil contaminated soil at the bioremediation sites of South Santhal CTF, Mehsana Asset.
Time of Site I (Well no. 1) Site II (Well no. 205) Site III (Well no. 206) Site IV (Control site)
treatment % % biodegradation % % biodegradation % % biodegradation % % biodegradation
TPH TPH TPH TPH
Zero day 7.87 --- 7.32 --- 6.92 --- 7.57 ---

After 15 days 5.12 34.94 5.27 28.01 4.29 38.01 7.28 3.83
After 40 days 4.21 46.51 3.98 45.63 3.14 54.62 7.00 7.53
After 75 days 2.04 74.08 2.41 67.08 1.95 71.82 6.78 10.44
After 135 days 0.71 90.98 0.58 92.08 0.59 91.47 6.48 14.40
SPE 109571 11

Table 4. pH and total bacterial count (TBC) of oil contaminated soil samples from the bioremediation sites of South Santhal CTF,
Mehsana Asset.
Time of Site I (Well no. 1) Site II (Well no. 205) Site III (Well no. 206) Site IV (Control site)
treatment by pH TBC pH TBC pH TBC pH TBC
microbial (CFU/gm. soil) (CFU/gm. soil) (CFU/gm. soil) (CFU/gm. soil)
consortium
Zero day before 7.81 1.2 X 104 8.03 3.1 X 104 7.89 2.6 X 104 7.94 1.8 X 104
application
Zero day after 7.72 3.7 X 109 8.01 2.6 X 109 7.86 3.2 X 109 7.88 2.1 X 104
application
After 15 days 7.68 5.9 X 108 7.89 7.1 X 108 8.12 6.2 X 108 8.11 3.8 X 104
After 40 days 7.78 3.9 X 108 8.05 4.2 X 108 8.06 4.6 X 108 8.16 6.4 X 104
After 75 days 7.86 1.9 X 108 7.92 2.1 X 108 7.98 2.6 X 108 8.02 8.3 X 104
After 135 days 7.76 5.3 X 107 7.96 6.1 X 107 8.21 5.8 X 107 7.98 1.1 X 105

100

90

80

70

60
Degradation (%)

50

40

30

20

10

0
TERI 7001

TERI 7002

TERI 7003

TERI 7004

TERI 7005

TERI 7006

TERI 7007

TERI 7008

TERI 7009

TERI 7010

TERI 7011

TERI 7012

TERI 7013

TERI 7014

TERI 7015

TERI 7016

TERI 7017

TERI 7018

TERI 7019

TERI 7020

TERI 7021

TERI 7022

TERI 7023

TERI 7024

TERI 7025

TERI 7026

TERI 7027

TERI 7028

TERI 7029

TERI 7030

Bacterial strains

Figure 1. Degradation of TPH by the culturable bacterial strains isolated from crude oily sludge and oil contaminated soil of Gujarat
Refinery, India.
12 SPE 109571

Figure 2. GC chromatogram indicating the degradation of Aliphatic (left) and Aromatic (right) fractions of the TPH by different
bacterial strains isolated from crude oily sludge and oil contaminated soil of Gujarat Refinery, India.

9
7.87

7.57
7.32

8
7.28
7.00
6.92

6.78
6.48

6
5.27
5.12
% TPH --->

5
4.29
4.21

3.98

4
3.14
2.41

3
2.04

1.95

2
0.71

0.59
0.58

0
Site I Site II Site III Site IV

Zero day After 15 days After 40 days

After 75 days After 135 days

Figure 3 . Biodegradation of TPH of the oily sludge at CPF Gandhar, ONGC Ankleshwar.
SPE 109571 13

Figure 4 : GC chromatogram indicating the biodegradation of Alkane (left) and Aromatic (right) fractions of the TPH of the oil
contaminated soil at Site – I of South Santhal CTF, Mehsana.

Figure 5 : GC chromatogram indicating the biodegradation of Alkane (left) and Aromatic (right) fractions of the TPH of the oil
contaminated soil at Site – II of South Santhal CTF, Mehsana.

Figure 6 : GC chromatogram indicating the biodegradation of Alkane (left) and Aromatic (right) fractions of the TPH of the oil
contaminated soil at Site – III of South Santhal CTF, Mehsana.