CURRENT MICROBIOLOGY Vol. 43 (2001), pp.

328 335
DOI: 10.1007/s002840010311 Current
Microbiology
An International Journal
Springer-Verlag New York Inc. 2001

In Situ Bioremediation Potential of an Oily Sludge-Degrading

Bacterial Consortium
Sanjeet Mishra,1,2 Jeevan Jyot,1 Ramesh Chander Kuhad,2 Banwari Lal1
1
Tata Energy Research Institute, New Delhi 110 003, India
2
Department of Microbiology, University of Delhi South Campus, New Delhi, India

Received: 20 October 2000 / Accepted: 22 March 2001

Abstract. A field-scale study was conducted in a 4000 m2 plot of land contaminated with an oily sludge
by use of a carrier-based hydrocarbon-degrading bacterial consortium for bioremediation. The land
belonged to an oil refinery. Prior to this study, a feasibility study was conducted to assess the capacity
of the bacterial consortium to degrade oily sludge. The site selected for bioremediation contained
approximately 300 tons of oily sludge. The plot was divided into four blocks, based on the extent of
contamination. Blocks A, B, and C were treated with the bacterial consortium, whereas Block D was
maintained as an untreated control. In Block A, at time zero, i.e., at the beginning of the experiment, the
soil contained as much as 99.2 g/kg of total petroleum hydrocarbon (TPH). The application of a bacterial
consortium (1 kg carrier-based bacterial consortium/10 m2 area) and nutrients degraded 90.2% of the
TPH in 120 days, whereas in block D only 16.8% of the TPH was degraded. This study validates the
large-scale use of a carrier-based bacterial consortium and nutrients for the treatment of land contami-
nated with oily sludge, a hazardous hydrocarbon waste generated by petroleum industry.

Oil refineries generate huge volumes of oily sludge dur-
ing the refining of crude oil. Oily sludge is carcinogenic
and a potent immunotoxicant [14]. Improper disposal
and handling of oily sludge contaminates soil and may
pose a serious threat to groundwater, and in situ biore-
mediation offers a promising means to reclaim such
contaminated soil [1, 3, 4, 7, 16]. Bioremediation em-
ploys microorganisms capable of degrading toxic con-
taminants for the reclamation of polluted sites [1, 2, 6,
16]. The process treats the contaminants on site (in situ)
instead of merely transporting them elsewhere. Freshly
contaminated sites, such as those used for disposal of
oily sludge, may not harbor microorganisms that have
the ability to utilize hydrocarbons as a carbon source.
Because it is heterogeneous, it is practically impossible
to degrade it by any single microbial species: a mixed
microbial population that has broad substrate specificity
is required to perform this task [12]. Augmenting the
contaminated site with an appropriate bacterial inoculum
is a promising technique to enhance the biodegradation
of hydrocarbons. Moreover, using an indigenous bacte-
Correspondence to: B. Lal; email: banwaril@teri.res.in
rial consortium ensures that the organisms have a higher
tolerance to the toxicity of hydrocarbons and are resistant
to variations in the environment [5, 6]. Many carrier
materials, mostly agricultural byproducts, are used to
transfer the bacterial consortium to the field effectively
[11, 13]. The carrier material provides nutrients, mois-
ture, and physical support for the increased aeration
needed by the microorganisms, and also assists in ex-
tending the survival of the microorganisms until they are
applied in the field. Extended survival of the microor-
ganisms under field conditions is essential for efficient
degradation of the toxic hydrocarbons, especially of the
multi-ringed aromatic hydrocarbons and the recalcitrant
ones, because they are not degraded during the early
stage of the process [10, 15, 16].
The present study aimed to assess the feasibility of
using a bacterial consortium to degrade highly toxic oily
sludge under field conditions and also to assess the
survival of the Acinetobacter baumannii strains, which
are the constituent of the bacterial consortium, during the
course of the treatment. The study was carried out over a
period of 120 days. The bacterial consortium, consisting
of five bacterial strains, was applied as a carrier-based
S. Mishra et al.: Bioremediation by Bacterial Consortium
inoculant to the contaminated site. The site is a part of
the Barauni refinery situated in the state of Bihar in
eastern India.
Materials and Methods
Development of the bacterial consortium. A bacterial consortium
that can degrade oily sludge was developed from a hydrocarbon-
contaminated soil by the enrichment method using a minimal salts
medium [11] in which oily sludge was the sole carbon and energy
source. After five cycles of enrichment, 100 l of the culture after
appropriate dilution was plated on a minimal salt agar medium con-
taining crude oil as the sole carbon and energy source. Out of nine
bacterial isolates that formed the consortium, the five most efficient
isolates were selected for further development of the consortium. The
selected isolates were characterized and identified by biochemical tests
and 16S-rRNA sequencing from MIDI Labs, USA. The isolates were
identified as Acinetobacter baumannii (S19, S26, S30), Burkholderia
cepacia (P20), and Pseudomonas sp. (S24). The consortium was pro-
duced on a large scale in a bioreactor (Bioflow 3000 New Brunswick,
USA) with molasses (0.3% wt/vol) as the sole source of carbon and
energy. The optimal growth conditions consisted of a temperature of
30C, a speed of 250 rpm, aeration 0.75 volume of air/volume of
medium/min, and a pH of 7.0 (adjusted with 1N HCl/1N NaOH), which
were maintained for 15 h; the culture was then harvested and immo-
bilized onto the selected carrier material, namely, steam-sterilized
corncob powder, a biodegradable agricultural residue. A total bacterial
load of 1010 cfu/g of carrier material with a moisture content of 70%
was maintained while immobilizing the bacterial consortium. The
carrier-based culture was dispensed into sterile reusable polythene bags
(4 kg immobilized culture in a 10-kg polybag). The bags were sealed
aseptically and stored at 4C. The shelf-life of the carrier-based culture
during storage at 4C was 3 months [11].
Selection of site and experimental design. A 4000 m2 plot of land
contaminated with oily sludge was selected. The site had been con-
taminated with approximately 300 tons of oily sludge, which was found
to be unevenly distributed in the soil. Based on the degree of contam-
ination, the site was divided into four blocks. Blocks A, B, and C were
treated with the bacterial consortium and nutrients, whereas Block D
was maintained as untreated control. One kilogram of the carrier-based
bacterial consortium and 50 lit of a nutrient mixture (67.5 g KNO3,
8.65 g K2HPO4, 2.5 g MnSO4.2H2O, 0.005 g FeSO4, 0.05 g
CaCl2.2H2O, 0.005 g ZnSO4.7H2O, 0.005 g CuSO4.5H2O, 0.05 g
CoCl2, 0.005 g AlK(SO4)2, 0.005 g H3BO4, and 0.005 g Na2 MoO4) for
every 10 m2 were added to all the blocks except the control block. The
land was thoroughly tilled at 15-day intervals with a tractor fitted with
a harrow.
Analysis of soil and oily sludge. Physical and chemical properties of
soil samples taken at the onset and at the end of the treatment were
analyzed. The samples were collected from four depths, namely, 0 25
cm, 26 50 cm, 5175 cm, and 76 100 cm horizons to check for any
seepage of the constituent petroleum hydrocarbons. The samples were
drawn with a hollow pipe, 4 cm in diameter. Air-dried and pulverized
soil samples were analyzed for organic carbon, nitrogen, available
phosphorus, potassium, moisture level, and pH with standard methods
[9].
Quantification of contamination in soil. To assess the rate at which
the TPH was being degraded, each treatment block was sampled at 10
points. Samples were collected at time zero (just before initiating the
bioremediation), 55 days later, and at the end of the study (120 days
after initiating the process). Total petroleum hydrocarbon from 10 g
329
soil was then consecutively extracted with hexane, methylene chloride,
and chloroform (100 ml each). All three extracts were pooled and dried
at room temperature by evaporation of solvents under a gentle nitrogen
stream in a fume hood. After solvent evaporation, the amount of
residual TPH was determined gravimetrically. After gravimetric quan-
tification, the residual TPH was fractionated into alkane, aromatic,
asphaltene, and NSO (nitrogen, sulfur, and oxygen-containing com-
pounds) fractions on a silica gel column [19]. For this purpose, samples
were dissolved in n-pentane and separated into soluble and insoluble
fractions (asphaltene). The soluble fraction was loaded on a silica gel
column and eluted with different solvents. The alkane fraction was
eluted with 100 ml of hexane followed by the aromatic fraction, which
was eluted with 100 ml of toluene. Finally, the NSO fraction was eluted
with methanol and chloroform (100 ml each). The alkane and aromatic
fractions were analyzed by gas chromatography (GC-FID using a
Hewlett Packard, 5890 Series II analyzer). The alkane fraction was
analyzed by DB5, 30-m long wide-bore column (0.53 mm 1 m film
thickness), while the aromatic fraction was analyzed by 30-m-long DB
5.625 column (0.25 mm I.D, 0.25 m film thickness). The injector and
the detector were maintained at 300C, and the oven temperature was
programmed to rise from 80C to 240C in 5C/min increments and to
hold at 240C for 30 min. Individual compounds present in the alkane
and aromatic fractions were determined by matching the retention time
with authentic standards (Sigma Chemical) and identified in GC as
described earlier [10].
Survival of Acinetobacter baumannii. Acinetobacter baumannii
strains (S19, S30, and S26) were selected as representatives to monitor
the survival of the introduced bacterial consortium during the course of
the study. It was also found that none of the strains of A. baumannii
showed any cross-reactivity with the other bacterial strains in the
consortium or with indigenous microorganisms at the bioremediation
site as observed by the antibiotic resistance pattern and micro-immu-
nodiffusion test. Soil samples (1 g each) were collected before and
immediately after the application of bacterial consortium and sus-
pended in saline water (0.85% NaCl). The suspension, after appropriate
dilution, was plated onto Luria Bertani Agar (LA) containing 50 g/ml
of spectinomycin and ampicillin in one set of petri plates and 25 g/ml
of vancomycin in another set. This enabled us to determine the total
heterotrophic bacterial population and the population of A. baumannii
strains before application of the bacterial consortium at the bioreme-
diation site. Bacterial colonies were picked randomly from selective
Luria agar plates (LA) and subjected to Enterobacteriaceae Repetitive
Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) to ob-
tain DNA fingerprints. DNA fingerprints for standard strains of A.
baumannii were obtained with ERIC-PCR under similar conditions.
The primers, namely ERIC-1R (5-ATG TAA GCT CCT GGG GAT
TCA C-3) and ERIC-2 (5-AAG TAA GTG ACT GGG GTG AGC
G-3), were obtained from Life Technologies, USA. Single isolated
colonies were picked at random from the LA plates, suspended in 50 l
water, and lysed by heating for 10 min at 95C. The cell lysate was
centrifuged at 12,000 rpm and 4C for 3 min, and 2 l of the super-
natant was used in the reaction mixture. The reaction mixture (15 l)
contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2,
0.01% gelatin (wt/vol), 0.2 mM each of dNTPs, 1 M each of primers
ERIC-1R and ERIC-2, and 0.45 units of Taq polymerase (Life Tech-
nologies, USA). The mixture was overlaid with 20 l mineral oil, and
amplification was done on DNA engine PTC-200 (MJ Research, USA).
The amplification protocol included initial denaturation at 95C for 2
min followed by 35 cycles at 92C for 30 s, at 50C for 1 min, 20 s, and
at 68C for 3 min, 20 s. The final extension was done at 68C for 8 min.
The reaction was terminated by using a loading dye (1 l) containing
15% Ficoll, 0.25% bromophenol blue, and 0.25% xylene cyanol. Each
330 CURRENT MICROBIOLOGY Vol. 43 (2001)

Table 1. Composition of oily sludge collected from bioremediation The oily sludge contained 15% solvent-extractable
site at time zero total petroleum hydrocarbons. Aromatics, at 46%, were
Component Content (%)
the largest constituent of the solvent-extractable TPH,
followed by the alkane fraction (37%), asphaltene frac-
Water 11 tion (10%), and NSO (7%). The detailed composition of
Heat-extractable total petroleum hydrocarbons 2 the sludge is given in Table 1.
Solvent-extractable total petroleum hydrocarbon 15
Organic carbon 37 In situ rates of bioremediation. The contamination
Sediments/ash content 35 level of TPH in soil (25 cm horizon) of block A, at the
Composition of the total petroleum hydrocarbon beginning of the study, was found to be 99.2 g/kg of soil.
With application of bacterial consortium and nutrients,
Fractions Content (%) the TPH level was reduced to 16.3 and 9.7 g/kg of soil
Alkane 37 after 55 days and 120 days respectively (Fig. 1). It
Aromatic 46 revealed biodegradation of 83.5% and 90.2% after 55
NSO 7 and 120 days (Table 2).
Asphaltene 10 In block B, at time zero, the level of TPH contam-
ination in 25 cm horizon soil was recorded as 76.1 g/kg
of soil, which decreased to 10.5 and 8.4 g/kg of soil after
PCR product was resolved by electrophoresis on a 2% agarose gel. The 55 days and 120 days respectively owingto biodegrada-
genomic fingerprint thus obtained was compared with the fingerprint of tion of TPH by the bacterial consortium (Fig. 1). The
standard strains of A. baumannii obtained under similar reaction con-
ditions to confirm the survival of the strains at the contaminated site [8].
percentage reduction noted was found to be 86.2% and
88.9% at the end of the respective days. In block C the
level of contamination in soil of 25 cm horizon was
Results found to be 64.5 g/kg of soil. In block C, after the
Soil analysis and composition of oily sludge. The soil treatment by bacterial consortium, the TPH contamina-
at the bioremediation site was loamy and dark brown. tion decreased to 31.1 g/kg and 2.5 g/kg of soil after 55
The content of organic carbon in the soil decreased from days and 120 days respectively (Fig. 1), which revealed
1.9% at time zero to 1.2% at the end of the study. The pH a degradation of 51.7% and 96.1% at 55 and 120 days
was 7.15 at time zero and remained unchanged, whereas respectively. In the untreated block D, the initial con-
the bulk density increased at the end of the experiment. tamination of TPH in soil of 25 cm horizon was found to
The water-holding capacity of the soil increased from be 36.8 g/kg of soil. The contamination decreased to 32.6
59% to 64%. and 30.6 g/kg of soil at the end of 55 days and 120 days

Fig. 1. Total petroleum hydrocarbon (TPH)

in the contaminated soil of blocks A, B, C,
and D. Soil samples of 10 g each were col-
lected up to a depth of 25 cm from different
treatment blocks, and TPH was extracted
with solvents. Values are means of 12 sam-
ples in each block, and the bars represent
standard deviation.
S. Mishra et al.: Bioremediation by Bacterial Consortium 331

Table 2. In situ biodegradation (%) of various fractions of oily sludge

Biodegradation in 55 days (%) Biodegradation in 120 days (%)

NSO NSO
Block TPH Alkane Aromatic Asphaltane TPH Alkane Aromatic Asphaltane

A 83.5 35.0 36.0 12.5 90.2 36.0 39.7 14.5

B 86.2 36.0 42.0 8.2 88.9 36.0 44.0 8.9
C 51.7 30.9 17.7 3.1 96.1 35.9 44.3 15.9
D 11.4 10.5 0.5 0.4 16.8 11.8 3.6 1.4

Fig. 2. (A) Gas chromatography fingerprinting of a representative alkane fraction of TPH obtained from treatment block B at different times.
Residual TPH was extracted from soil with solvents and was fractionated into alkane, aromatic, NSO, and asphaltene fractions in a silica column.
The alkane fraction was analyzed by GC. Various n-alkane compounds were identified by matching the retention time with authentic standards. (B)
GC fingerprinting of a representative aromatic fraction of TPH obtained from treatment block B at different times. Residual TPH was extracted from
soil with solvents and fractionated into alkane, aromatic, NSO, and asphaltene fractions in a silica column. The aromatic fraction was analyzed by
GC. Various aromatic compounds were identified by matching the retention time with authentic standards. ACN, acenaphthylene; FLO, fluorene;
DBT, di-benzothiophene; PHE, 4H-cyclopenta phenanthrene; CAR, carbazole; PYR, pyrene; CHR, chrysene.

respectively. This revealed a biodegradation of 11.4%
and 16.8% at the end of the study by the indigenous
bacterial population. Biodegradation of various individ-
ual fractions of TPH in the different blocks is shown in
Table 2. GC analysis of alkane fraction and aromatic
fraction, obtained after silica gel fractionation, of a rep-
resentative soil sample collected from block B is shown
in Fig. 2A and 2B.
Survival of Acinetobacter baumannii. The population
of A. baumannii strains was stable even after 120 days
following the application of the bacterial consortium,
332
Fig. 2. Continued.
indicating very high survivability of the introduced strain
(Fig. 3). A. baumannii could not be detected in the soil at
time zero (before application of the bacterial consortium)
and also in block D, the control block. DNA fingerprints
of the A. baumannii strains were found to be similar,
whereas the antibiotic resistance patterns were different.
Bacterial strains can be differentiated by genomic DNA
fingerprints based on ERIC-PCR obtained after gel elec-
trophoresis, as some of the standard strains are shown in
Fig. 4. During the screening, genomic DNA fingerprints
of isolates obtained from different treatment plots were
compared with those of A. baumannii S30; in many cases
they matched exactly. The strains of similar bacterial
species could be differentiated from others by using
ERIC-PCR-based DNA fingerprints, as shown in Fig. 5.
Discussion
The present study was carried out to assess the capability
of a bacterial consortium to biodegrade oily sludge, a
hazardous hydrocarbon waste generated by the petro-
CURRENT MICROBIOLOGY Vol. 43 (2001)
leum industry. The bacterial consortium was found to
adapt to the local soil environment. This was also be-
cause the consortium was developed from bacterial iso-
lates obtained from hydrocarbon-contaminated soil. Dib-
ble and Bartha [5] reported that environmental factors
play a vital role in the bioremediation of soil contami-
nated with oily sludge, and the results obtained in this
study corroborate this view. The initial population of
hydrocarbon-degrading bacteria at the site was found to
be 103 cfu/g of soil, but Acinetobacter strains were not
detected in this indigenous population. It has been re-
ported previously that bioremediation is negligible if the
population of hydrocarbon-degrading microorganisms is
less than 105 cfu/g in soil [7]. This suggested that a
bacterial consortium needs to be added. A carrier-based
formulation made it easy to transfer the microorganisms
from the laboratory to the field. In a previous study, the
survival of the consortium was studied with different
carrier materials. Corncob powder (agricultural byprod-
uct) was found to be the best for the purpose; a survival
of 73.3% was recorded during storage over 3 months
S. Mishra et al.: Bioremediation by Bacterial Consortium 333

Fig. 3. Survival of A. baumannii strains in the

contaminated soil during the bioremediation
study. Soil samples of 1 g each were sus-
pended in normal saline (0.85% NaCl) and,
after proper dilution, were plated onto Luria
agar plates containing 50 g/ml of spectino-
mycin and ampicillin in one set of Petri
plates and 25 g/ml vancomycin in another.
Values shown here are means of 12 samples
from each block, and bars represent standard
deviation.

Fig. 4. ERIC-PCR fingerprint of known bacterial strains on 2% agarose gel. Bacterial colonies were heat lysed, and whole genomic DNA was
subjected to ERIC-PCR to get the fingerprint. Lane 1, 100-bp marker; Lane 2, A. baumannii S30; Lane 3, A. baumannii S19; Lane 4, Acinetobacter
sp. B15; Lane 5, A. calcoaceticus BD413; Lane 6, Escherichia coli JM109; Lane 7, negative control.

[11]. Corncob powder, being a good porous material,
may have enhanced the rate of degradation by providing
air pockets in the soil, which facilitate growth and expose
a greater surface area to microbial action. Various soil
parameters affect the bioremediation process and are
indicators of bacterial activity. Organic carbon decreased
from 1.9% to 1.2%, indicating that the TPH content of
the soil had been lowered. Similarly the water-holding
capacity of the soil increased to 64%, showing a reduc-
tion in the oil content of the soil.
334
Fig. 5. ERIC-PCR fingerprint of unknown bacterial isolates on 2% agarose gel. Bacterial colonies were isolated from soil at the bioremediation site.
Pure colonies were picked, heat lysed, and whole genomic DNA was subjected to ERIC-PCR to get the fingerprint. Lane 1, HindIII marker; Lanes
214, fingerprints of different isolates obtained from selected plates containing 25 g/ml of Vancomycin; Lane 15, standard bacterial strain of A.
baumannii S30.
In situ bioremediation is a cheap and easy method to
reduce oily sludge contamination. This approach does
not require the soil to be moved from its site, thus
reducing any chance of secondary contamination. Using
microbial inoculants has been a common practice, since
it enhances the rate of biodegradation [6, 10]. Many
agricultural residues have also been found to stimulate
bacterial activity and degradation [12, 13]. The degrada-
tion was greater (55 60%) during the first half (0 55
days) of the study (Table 2), probably because the alkane
fraction and the lower fractions of aromatic compounds
were degraded in the first 55 days. Once the concentra-
tion of the lower fractions is lowered, the bacterial ac-
tivity shifts to degrading the higher fractions. Extensive
tilling and watering ensured the maintenance of the re-
quired moisture content and facilitated aeration for faster
degradation. In the control block D, the degradation was
minimal, which shows that the indigenous population
can biodegrade the oily sludge, but the process is very
slow.
Survival of the microorganisms in the soil after their
application is a deciding factor in the rate of degradation
of hydrocarbons [15]. Recent developments in molecular
techniques, especially the polymerase chain reaction, are
being widely used to assess the survival of the introduced
strains in the field [8, 17, 18, 20]. Repetitive DNA
sequences have been characterized primarily from Esch-
erichia coli and Salmonella typhimurium. These se-
quences, referred to as enterobacterial repetitive inter-
genic consensus sequence (ERIC) or intergenic repeat
units (IRUs), are 126-bp elements containing a highly
conserved central inverted repeat and are located in ex-
tragenic regions [18]. The analysis of this element by
CURRENT MICROBIOLOGY Vol. 43 (2001)
PCR-synthesized oligonucleotide has provided unique
DNA fingerprints. It is reported that ERIC probes hy-
bridized preferentially to genomic DNA from Gram-
negative enteric bacteria [8]. In the present study, sur-
vival of the microorganisms was tracked with the ERIC-
PCR-based genomic DNA fingerprinting method. The
strains of Acinetobacter baumannii were found to be
stable at the end of 120 days at the site. This might be
due to the maintenance of proper soil conditions, includ-
ing moisture level, nutrients, and aeration, and also be-
cause the constituent microorganisms in the consortium
had been isolated from the hydrocarbon-contaminated
site.
The present study clearly demonstrates that, if suit-
ably developed, application of a carrier-based indigenous
bacterial consortium can be used to remediate soil con-
taminated with oily sludge. Maintenance of proper soil
conditions is an essential aspect to be looked into and
needs to be studied in further detail when taking up such
studies because soil conditions influence the survival of
the microorganisms.
ACKNOWLEDGMENTS
The authors are thankful to Dr R.K. Pachauri, Director, TERI, for
providing the infrastructure to carry out the present study. Thanks are
in order to the management of the Barauni Refinery, where the study
was carried out, and the Department of Biotechnology, Govt. of India,
for their financial assistance. The technical assistance provided by Mr.
Vinod Kumar is appreciated. The authors thank Mr. Yateen Joshi for
critically editing and Mr. G. Gopalakrishnan for typing the manuscript.
The authors also wish to thank the Council of Scientific and Industrial
Research for providing a fellowship during the work.
S. Mishra et al.: Bioremediation by Bacterial Consortium
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