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Characterization of Oil-Degrading Bacteria from Oil-

Contaminated Soil and Activity of their Enzymes
a a ab a
Shaopeng Yan , Qiuyu Wang , Lina Qu & Cong Li
a
Northeast Forest University, College of Life Science, Harbin, P.R. China
b
Daqing normal University, college of life Science, harbin, P.R. China
Published online: 16 Apr 2014.

To cite this article: Shaopeng Yan, Qiuyu Wang, Lina Qu & Cong Li (2013) Characterization of Oil-Degrading Bacteria from
Oil-Contaminated Soil and Activity of their Enzymes, Biotechnology & Biotechnological Equipment, 27:4, 3932-3938, DOI:
10.5504/BBEQ.2013.0050

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 Article HTTP://DX.DOI.ORG/10.5504/BBEQ.2013.0050 A&EB

CHARACTERIZATION OF OIL-DEGRADING BACTERIA FROM OIL-

CONTAMINATED SOIL AND ACTIVITY OF THEIR ENZYMES
Shaopeng Yan1, Qiuyu Wang1, Lina Qu1,2, Cong Li1
1
Northeast Forest University, College of Life Science, Harbin, P.R.China
2
Daqing Normal University, College of Life Science, Harbin, P.R.China
Correspondence to: Qiuyu Wang
E-mail: wqyll@sina.com

ABSTRACT
Ten bacterial strains were isolated from long-term petroleum contaminated soil from the Daqing oilfield. The strains were
preliminarily identified based on 16S rDNA sequence analysis combined with morphological observation, physiological and
biochemical tests, in which strain X2 was identified as Bacillus pumilus, strain Z as Rhizobium sp, strain Y as Microbacterium
Downloaded by [SUNY Health Science Center] at 16:52 03 October 2014

oxydans, strain H as Arthrobacter sp., and strains X1, X3, X4, X5 X6, and X7 as Bacillus spp. The crude-oil degradation ability
and activity of degradation-related enzymes of the strains were also studied, including dehydrogenase, catechol 2,3-dioxygenase,
and lipase. The results showed that strain X6 has the highest oil-degradation rate in both liquid medium and contaminated soil,
which reaches 54.4 % and 51 % in 28 d treatment. The GC-MS analysis showed that all tested strains could totally degrade
chain hydrocarbons with C11~C18, and partially degrade chain hydrocarbons with C19 ~ C24. Most strains showed preference
for growth in alkaline conditions ranging from pH 7.0 to pH 8.0, with strains X2, X6, and Z demonstrating stronger tolerance to
saline and alkaline conditions than the other strains, and hence, displaying better environmental adaptability.

Biotechnol. & Biotechnol. Eq. 2013, 27(4), 3932-3938 through special substrate enrichment (18, 28). Therefore,
Keywords: crude-oil contamination, oil-degrading bacteria, bioremediation of oil contaminated soil has broad prospects
16S rDNA, GC-MS analysis, activity of degradation related because of its low cost, no secondary pollution, processing in
enzyme situ and so on (1, 2, 3, 15, 17).
At present, microbial remediation of oil contaminated soil
Introduction has been carried out and widely reported in the world (7, 16, 23,
Crude oil is mainly composed of alkanes, cycloalkanes and 26), which provides a reference and guidance for development
aromatic alkanes, which constitute about 50 % to 80 % of the oil and practical application of bioremediation technology. In this
content. These constituents are called petroleum hydrocarbons research, 10 natural bacterial strains were isolated from oil
for short (9). More than 230 hydrocarbons have been identified contaminated soil from the Daqing oilfield in northern China,
in oil (6). As an important energy source, oil has played an and identified by 16S rDNA sequence analysis combined with
indispensable role in industrial production. Therefore, the 20th morphological observation, physiological and biochemical
century was named the “oil century” (22). Daqing is the largest tests. The optimum growth condition, crude oil biodegradation
oilfield in China, whose crude oil is paraffin-based, with a high and degradation-related enzymes of these strains were also
wax content (20 % to 30 %), high freezing point (25 °C ~30 °C), investigated. The purpose was to select and provide new
high viscosity (the ground viscosity in 35 cP) and a low sulfur strains with fast growth and high degradation ability for
content (0.1 %). Over the years, the Daqing oil field has provided bioremediation of the contaminated soil of the Daqing oilfield.
large amounts of crude oil for the country, which has made great
contributions to Chinese industrial development. However, the Materials and Methods
oil exploration and transportation has contaminated the soil of Soil samples
the Daqing area in various degrees. A numbers of alkali spots
Petroleum-contaminated soil samples were collected from
have formed, which limit the vegetation cover and crop growth.
spots around the oil wells of the Daqing oilfield constructed in
Moreover, petroleum hydrocarbons are absorbed by plant roots
1962. The samples were sealed separately in a sterile bag and
and accumulated. From there, they could potentially get into
saved under -20 °C.
human’s body through the food chain and pose a threat to human
health (5, 10). Crude oil
It is well known that a lot of soil bacteria and fungi can Crude oil from the Daqing oil administration bureau was
utilize petroleum hydrocarbons as a carbon source. At the black solid at room temperature. Petroleum ether was used
same time, some aboriginal microbes have gradually adapted to dissolve the oil to a 10  % concentration (100  g·L-1). The
to the long-term oil contaminated soil and developed a resulting solution was stored in brown reagent bottles away
superior community which can make use of oil contaminants from direct sun light after sterilization.
3932 Biotechnol. & Biotechnol. Eq. 27/2013/4
 Medium The isolated strains were inoculated in inorganic salt
Mineral salt medium: NaCl 10 g·L-1, MgSO4 0.5 g·L-1, NH4Cl medium adjusted to pH in 6, 7, 8, and 9, with 0.1 mol·L-1 HCl
0.5 g·L-1, CaCl2 0.2 g·L-1, K2HPO4 1.0 g·L-1, KH2PO4 0.5 g·L-1, or NaOH, and were cultivated on a shaker for 3 days at 30 °C,
KCl 0.1  g·L-1, FeCl3.6H2O 0.03  g·L-1, pH  7.0; enrichment 160  r·min-1. Then, the absorbance of the culture broth was
medium: 1 % inorganic salt medium with 1 % crude oil; LB measured at 258  nm in order to determine the optimum pH
solid medium: yeast extract 5  g·L-1, peptone 10  g·L-1, NaCl range for strain growth.
10 g·L-1, agar 15 g·L-1 ~ 20 g·L-1, pH 7.4 ~ 7.6. The culture The strains were inoculated in medium with 1  %, 3  %,
media were sterilized at 121 °C for 20 min. 5 %, and 7 % NaCl, and cultured at 30 °C, 160 r·min-1 for 7
days. The OD258 of the culture medium was measured in a UV
Isolation of oil-degrading bacterial strains
spectrophotometer to determine the optimal salt concentration
One gram of oil-contaminated soil sample was added in a
for strain growth.
flask containing 100 mL of sterile water as 10-2 diluent, which
was then shaken for 30 min at 160 r·min-1 at 30 °C. And then Crude-oil degradation ability of the strains
the solution was also diluted 5 times with sterile water to Crude oil hydrocarbon degradation in liquid medium.
10-7 diluent concentration, and then 0.1  mL was loaded onto Each strain was cultured in LB liquid medium containing 1 %
LB solid medium plates and incubated for 1 or 2 days at crude oil at 30 °C, 160 r·min-1. The degradation rate of crude
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30 °C. Microbial colonies with different color and form were oil hydrocarbons was measured by the weighting method every
transferred with an inoculation loop onto solid LB medium week for a month, and non-inoculated liquid medium was used
again for separation and purification. The purified strains were as the control.
precultivated 3 to 4 times on enrichment medium, and were Crude oil hydrocarbon degradation in contaminated
used as the petroleum-degrading bacteria for further study. soil. Ten grams of fresh soil from the Daqing oilfield were
The selected oil-degrading bacteria with superior growth were oven dried after sieving and sterilized at 121 °C for 1 h. The
propagated on test tube slants and stored at 4 °C. sterilized soil was mixed with treated oil solution so as to
Morphological observation, physiological and biochemical obtain soil with a 1 % concentration of crude oil. The soil was
tests inoculated with 1 % inoculum, and then cultured at 30 °C for
The strains were plated on LB solid medium and cultured for 1 or a month. The oil degradation rate in the soil was measured by
2 days at 30 °C. The morphological characteristics of the colonies the weighting method every week, and non-inoculated soil was
were observed, e.g. colony color, form and size, elevation, gloss, used as the control.
viscosity, medium color (4). Gram staining (4) and capsule GC-MS analysis of residual oil components after
staining (13) were also performed. Seven physiological and biodegradation. The liquid medium after biodegradation
biochemical assays were carried out (24), including Methyl red treatment was used as the sample, and non-inoculated medium
(MR) test, V.P. test (Vogex-Proskauer), oxidase test, catalase without crude oil solution was used as a control. After 28 days
test, produced H2S, indole test, starch hydrolase test . of biodegradation, the medium was mixed with petroleum
ether and centrifuged, the residual oil components for both the
Molecular identification samples and the controls were measured by Agilent GC-MSD
Total bacterial DNA was extracted with UNIX-10 (6890N-5973) with pole temperature kept at 80 °C for 4 min,
bacterial DNA extraction kit (Shanghai Biotechnology then increased at a rate of 5 °C·min-1 to 250 °C and maintained
Company). The universal 16S rDNA primers (19) 27F at 250 °C for 20 min .
5’-AGAGTTTGATCATGGCTCAG-3 ‘and 1492 R Activity of degradation-related enzymes. The activity of
5’-TACGGTTACCTTGTTACGACTT-3’ were used for the some degradation-related enzymes of the strains was studied.
PCR reaction. After that, the amplified products were separated Dehydrogenase activity was measured spectrophotometrically
by electrophoresis in a 1  % agarose gel. The obtained 16S (11, 20). Catechol 2,3-dioxygenase activity was measured by
rDNA fragments of all strains were packaged and sent to the nebulization (25). Lipase was measured by the alkali titration
Beijing Genomics Institute for sequencing. The sequencing method (8).
results were analyzed by BLAST online comparison (http://
www.ncbi.nlm.nih.gov) for identification of the strains (21). Statistical analysis
Pearson correlation analysis between crude oil degradation
Optimization of strain growth
rate and degragation related enzymes of the strains was done
The strain growth at different pH values and salt concentrations
using the Statistical Package for the Social Sciences statistical
was evaluated by single-factor analysis. There were three
software (SPSS 16.0).
replications for each treatment. The strains were cultivated in
LB liquid medium to the logarithmic phase; then the cells were
Results and Discussion
collected by centrifugation and washed three times with sterile
water to remove residual culture medium. The cell density was Strain isolation and identification
measured in a UV-1800 UV-Vis spectrophotometer (Shimadzu, In this study, 54 strains were isolated from 7 soil samples from
CORP). the Daqing oilfield. Ten oil-degrading bacteria were selected
Biotechnol. & Biotechnol. Eq. 27/2013/4 3933
 TABLE 1
Morphological features, physiological and biochemical characteristics of the strains

Strain
X1 X2 X3 X4 X5 X6 X7 H Y Z
Characteristics
Morphological features
Colony form circular or irregular circular irregular irregular circular circular circular circular circular
Colony color white buff white white white white white buff buff white
Cell shape rod rod rod rod rod rod rod round rod rod
Physiological and biochemical characteristics
Gram staining + + - + + + + - + -
Capsule staining + + + + + + + - + +
Catalase + + + - + + + + - -
*Oxidase ++ - + - - - - - - ++
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Indole + + + + + + + + + +
M.R + + - + - + + - - +
V.P. + + + + - + + - + +
H2S production - + - - - + + - - -
Starch hydrolase + - - - + + + - - -
Note: “+” is positive, “-” is negative. *Oxidase test: “++” is positive, “+” is slow-positive, “-” is negative.

TABLE 2
Bacterial identification based on 16S rDNA sequencing data

No. of strain Total length (bp) GenBank accession No. Identification result Similarity (%)
X1 1442 EU584539.1 Bacillus sp. 99
X2 1450 EU594558.1 Bacillus pumilus 99
X3 1428 EU915719.1 Bacillus sp. 99
X4 1449 GQ199726.1 Bacillus sp. 99
X5 1435 EU584552.1 Bacillus sp. 100
X6 1452 EU236733.1 Bacillus sp. 100
X7 1448 EU863836.1 Bacillus sp. 99
H 1412 GU257944.1 Arthrobacter sp. 99
Y 1417 EU714369.1 Microbacterium oxydans 99
Z 1368 EU556969.3 Rhizobium sp. 100

according to their colony form and growth on solid LB medium the other strains due to their fast growth and large colony size
with crude oil. The strains were designated as X1, X2, X3, X4, (Fig. 1).
X5, X6, X7, H, Y, Z. Several physiological and biochemical parameters were
Morphological observation, physiological and biochemical assayed. All 10 strains were indole-positive, whereas only
tests, combined with 16S rDNA sequence analysis were used strains Z and X1 were oxidase-positive. Almost all of the
for identification of the bacterial strains. All the 10 isolates strains showed positive capsule staining, except for strain H.
were found to be rod-shaped, with round and opaque colonies. The results are shown in Table 1.
Strains X2, X3, X6, X7, H, and Y formed circular colonies, The 16S rDNA PCR amplification product of the strains is
and strains X1, X4, and X5 formed irregular colonies. The a fragment about 1.5 Kb in size (Fig. 2). All the fragments were
colony size of strain Z was much smaller than that of the others highly similar to known bacterial strains according to the 16S
because of its slow growth. On solid LB medium, strains X1, rDNA sequence comparison. Based on these results, together
X3, X4, and X5, grew better and formed larger colonies than with the morphological, physiological and biochemical

3934 Biotechnol. & Biotechnol. Eq. 27/2013/4

 characteristics, strain X2 was identified as Bacillus pumilus; In the medium with 7  % salt concentration, strains X3, X5,
strain Z, as Rhizobium sp.; strain Y, as Microbacterium and X7 could hardly grow. Strain Z showed the best growth at
oxydans; strain H, as Arthrobacter sp.; strains X1, X3, X4, all the tested salt concentrations. The growth of strain X5 was
X5, X6, and X7 as Bacillus spp. (Table  2). However, the always the poorest, even at 1 % salt concentration.
colony morphology and some physiological and biochemical The fact that some of the strains demonstrated an ability to
properties of these Bacillus spp. strains were quite similar. grow at pH values up to 9 and salt concentration up to 7 %,
Therefore, further identification to the species level is needed. such as strain Z, X6 and X2, could be considered a result of
natural selection and evolution. Notably, strain Z, which was
identified as Rhizobium sp., showed extremely high adaptation
compared to the other strains under all the tested saline and
alkaline conditions.
Crude-oil degradation ability of the tested strains
Crude-oil hydrocarbon degradation in liquid medium.
A great difference was observed in the oil degradation rate
among the studied strains, especially the 7 Bacillus sp. strains
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(Fig. 4A). The mean oil degradation rate of the strains in liquid

medium gradually increased with time, and for three of them
exceeded 50  % for the 28  d processing period: 54.4  % for
strain X6, 52.3 % for strain X2, and 50 % for strain X5. The
slowest oil-degrading rate at all the tested points in time was
that of strain X7.
Crude-oil hydrocarbon degradation in contaminated
soil. The results showed that the oil degradation rate of strain
X6 was also the highest in contaminated soil and reached 51 %
for the 28 d period. The oil degradation rate of strains X1, X2,
X3, X5, Y, H, and Z, ranged from 40 % to 50 %. Strains X4 and
Fig. 1. Morphology of the strains. X7 displayed the lowest degradation rate: 39.1 % and 33.4 %,
respectively (Fig. 4B).
GC-MS analysis of residual oil components after
biodegradation. Strains X2, X5, and X6, which were
identified as Bacillus spp., were found to have higher oil
degradation ability than the others, both in liquid medium and
in contaminated soil. These strains, together with Bacillus
sp. X7, which showed the lowest degradation efficiency, as
well as strains from the other bacterial species (strains H, Y,
and Z) were selected for the residual oil component analysis
Fig. 2. Electrophoresis of 16S rDNA PCR products of the studied strains. M: before and after biodegradation, using GC-MS. The results
Marker; CKL blank control.
revealed 25 types of saturated hydrocarbons in the crude oil
Growth conditions before biodegradation processing: short-chain hydrocarbons
with C11~C21 carbons accounted for 52.69 % and long-chain
As shown in Fig.  3A, strain X3 reached its maximum
hydrocarbons with C22~C33 carbons accounted for 47.31 %,
absorbance value at pH = 6. The highest absorbance for strains
this also includes two kinds of branched-chain alkanes with 12
X1, X2, X5, X6, X7, H. and Z was at pH = 7; and for strains
and 15 carbon atoms on the main chain (Fig. 4C).
X4 and Y, at pH = 8 . At pH = 9, strains X2 and X4 still showed
high absorbance values, indicating that these two strains During the 28-day biodegradation experiment, all the
are well adapted to high-alkaline conditions. Strain Z was studied strains showed different ability to degrade different
observed to grow much better than the other strains at the same crude-oil components, degradation rate being higher in liquid
pH values, even in the most alkaline conditions. Almost all medium than in contaminated soil. Only strain X6, however,
of the strains, except strain X3, showed good growth at pH 7 showed the highest degradation rate (over 50  %) in both
to pH 8, indicating that these strains have adapted to grow in kinds of culture medium. This suggests that strain X6 could
a more alkaline soil environment. This is in good agreement be a promising target for further research, such as field tests
with the fact that the soil of the Daqing oilfield is normally and creation of new genetically engineered bacteria for oil
with pH 7.6 to pH 8.6 and less than 5 % salinity (27). biodegradation.
At 3 % salt concentration of the medium, strains X2, X4, The shorter chain hydrocarbons in crude oil could be
X6, X7, H, Y, and Z, reached an absorption peak (Fig.  3B). degraded more rapidly than the longer ones. Almost no
Biotechnol. & Biotechnol. Eq. 27/2013/4 3935
 A B
Fig. 3. Effect of pH (A) and salt concentration (B) on the growth of strains. OD values of strain Z (left-hand side Y axis); OD values of the remaining strains
(right-hand side Y axis).
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A B

C D
Fig. 4. Degradation of single-strain petroleum hydrocarbons in liquid medium (A), and in contaminated soil (B). Crude-oil component before biodegradation
processing (C), and residual oil component degraded by strain X6 (D). GC-MS analysis

hydrocarbon constituents less than 19 carbons remained after Activity of oil-degradation related enzymes
28 days, indicating that all tested strains can totally degrade Considerable differences in dehydrogenase activity and
chain hydrocarbons with C11~C18, and partially degrade catechol 2,3-dioxygenase activity were observed among the
chain hydrocarbons with C19~C24. No strains were found to 10 strains. Moreover, the activity of these enzymes was much
degrade hydrocarbon components longer than C24 (Table 3). higher in the strains induced by crude oil than in the non-
By comparison with the control, it could be suggested that induced ones (Table  4), indicating that the dehydrogenase
petroleum hydrocarbon components shorter than C15 can of the strains may be inductively expressed in the presence
naturally volatilize in liquid culture. In the future, it is necessary of crude oil. Strain X1 showed the highest dehydrogenase
to identify and study the other groups of microorganisms that activity, which reached 371  mg·L-1·h-1 (non-induced cells)
can act on long-chain hydrocarbons, such as soil fungi and and 501 mg·L-1·h-1 (oil-induced cells). Strain X4 exhibited the
actinomycetes, in the Daqing area.
3936 Biotechnol. & Biotechnol. Eq. 27/2013/4
 TABLE 3
Components of petroleum hydrocarbons after biodegradation processing

Oil components
Strain
Totally degraded Partially degraded Non-degraded
X2 C15–C19 C20–C24 C25–C33
X5 C15–C21 C22, C23 C24–C33
X6 C15–C18 C19–C24 C25–C33
X7 C15–C19 C20–C23 C24–C33
Y C15–C19 C20–C23 C24–C33
H C15–C20 C21–C24 C25–C33
Z C15, C17 C16, C18–C24 C25–C33

TABLE 4
Activity of degradation related enzymes of the strains
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Strain
X1 X2 X3 X4 X5 X6 X7 Y H Z
Enzyme activity
DH (no-induced) (L-1·h-1) 371 133 47 9 87 78 3 48 21 11
DH (oil-induced) (L-1·h-1) 501 172 123 108 159 168 10 59 137 78
C23Oase (no-induced) (OD375) 0.452 0.391 0.338 0.441 0.483 0.203 0.420 0.330 0.384 0.313
C23Oase (oil-induced) (OD375) 1.146 2.026 0.566 4.738 1.014 0.704 1.608 0.654 0.876 1.564
Lipase (U·mL-1) 38 49 118 62 100 50 78 103 53 135
DH: dehydrogenase; C23Oase: 2,3-dioxygenase

highest catechol 2,3-dioxygenase activity: 0.4408 (OD375) and Arthrobacter sp., based on morphological, physiological
for non-induced cells and 4.738 (OD375) for oil-induced and biochemical characteristics, and 16S rDNA sequence
cells (Table  4). The Pearson correlation analysis revealed analysis. Rhizobium sp. strain Z showed the fastest growth and
some positive correlations between crude oil degradation rate highest adaptability in saline-alkaline environment, possibly
and dehydrogenase activity of the strains, with a correlation as a result of long-term natural selection and evolution. GC-
coefficient of 0.568 and 0.637 for non-induced strains and MS data revealed C11 to C33 chain hydrocarbons in the
oil-induced strains, respectively. These results suggest that crude oil. In the 28-day biodegradation experiment, the tested
bacterial dehydrogenase could possibly play a role in the bacterial strains showed better degradation ability towards
biodegradation of oil-contaminated soil. On the other hand, C15~C19 chain hydrocarbons, but not for chain hydrocarbons
there was a negative correlation (although non-significant) longer than C24. The enzyme activity assays indicated that
between the oil degradation rate of the strains and their lipase the dehydrogenase in the studied strains could be inductively
activities with a correlation coefficient of -0.443, implying expressed in the presence of crude oil. Basillus sp. strain X6
that, to a certain extent, crude oil might be considered as a showed the highest biodegradation rate (over 50  %), which
restraining factor for lipase expression in the studied strains. No makes it a promising target for further research.
clear correlation was found between the crude oil degradation
rate and the catechol 2,3-dioxygenase activity of the strains, Acknowledgements
although it can be much efficiently induced by crude oil. The This research was supported by Project “948” of the Chinese
reason for this might be that the crude-oil samples used in National Forestry Bureau (2008-4-34).
our study contained chain hydrocarbons, but not polycyclic
aromatic hydrocarbons, which can be degraded by some oil-
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