Short Communication

Characterization and biotechnological potential of petroleum-degrading bacteria

isolated from oil-contaminated soils
Zhengzhi Zhang
a,b,1
, Lixue Gai
c,d,1
, Zhaowei Hou
d
, Chunyu Yang
b
, Cuiqing Ma
b,
*
, Zhongguo Wang
d
,
Baiping Sun
b
, Xiaofei He
a
, Hongzhi Tang
a
, Ping Xu
a,
**
a
MOE Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, Peoples Republic of China
b
State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, Peoples Republic of China
c
Daqing Petroleum Institute, Daqing 163318, Peoples Republic of China
d
Daqing Oileld Co. Ltd., China National Petroleum Corporation, Daqing 163453, Peoples Republic of China
a r t i c l e i n f o
Article history:
Received 25 January 2010
Received in revised form 28 April 2010
Accepted 21 May 2010
Available online 22 June 2010
Keywords:
Petroleum biodegradation
Microbial consortium
Denaturing gradient gel electrophoresis
Phylogenetic analysis
a b s t r a c t
A collection of 38 bacteria was obtained by enrichment cultivation from oil-contaminated soils of an oil
eld in Daqing, China. Twenty-two strains could utilize diesel oil as the sole source of carbon and energy,
and 11 strains could degrade the total petroleum hydrocarbons (TPHs) of diesel oil by more than 70% in
7 d. Phylogenetically, 19 of the bacteria related to Bacillus species. About 87.5% TPHs of crude oil were
degraded by a consortium of seven strains. Denaturing gradient gel electrophoresis analysis suggested
that ve of the strains persisted throughout the degradation process. The collection of isolated bacteria
might be a useful resource for bioremediation of oil-contaminated soils and biotreatment of oil
wastewater.
2010 Elsevier Ltd. All rights reserved.
1. Introduction
Crude oil is a complex mixture of hydrocarbons and other or-
ganic compounds that can bring up serious environmental prob-
lems when spills occur. Bioremediation is recognized as an
efcient, economic, and versatile alternative to physicochemical
treatment of oil-contaminants. Over the last decade, extensive re-
searches focused on oil bioremediation and crude oil degradation
have been carried out with pure culture or mixed bacterial consor-
tia isolated from oil-contaminated soils (Chaillan et al., 2004;
Mishra et al., 2001). The degradation efciency was limited by sub-
strates and geological, climatological, and ecological factors, when
only a single hydrocarbon degrader was employed (Van Hamme
et al., 2003). Furthermore, since crude oil contains various com-
pounds, a single strain might not be able to degrade all compo-
nents of the crude oil. In general, the combined bacterial
consortium showed better results due to their synergetic effects
(Boopathy, 2000).
The characterization of bacterial populations living in oil-con-
taminated soils and evaluation of their degradation capacities
could potentially serve as guide for improving remediation of such
environments. Culture-dependent and culture-independent ap-
proaches, such as measuring soil respiration and enzyme activities,
microbial counts, and molecular technologies, may provide
information about viable microorganisms presented in such envi-
ronments (Van Hamme et al., 2003). But data from culture-
independent methods reect only the genetic composition and
bacterial distribution of the communities and do not demonstrate
the biochemical capabilities of the populations. Therefore, bacteria
were collected from oil-contaminated soils in Daqing, China, phy-
logenetically characterized and their ability to degrade crude oil
was determined. A consortium of seven bacteria was studied for
its potential bioremediation potential.
2. Methods
2.1. Bacterial isolation and medium
Bacterial strains were isolated by enrichment cultivation from
oil-contaminated soil samples (obtained from the top 20 cm soils)
from an oil eld in Daqing, China. All cultivations were carried out
at 30 C in modied basal salts medium (MBSM) containing 1 g of
NH
4
NO
3
, 0.5 g of KH
2
PO
4
, 5.24 g of K
2
HPO
4
3H
2
O, 0.2 g of
MgSO
4
7H
2
O, 2 ml of 1% CaCl
2
, 200 ll of 1% FeCl
3
, 200 ll of 5%
0960-8524/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2010.05.060
* Corresponding author. Tel.: +86 531 88364003; fax: +86 531 88369463.
** Corresponding author. Address: School of Life Sciences and Biotechnology,
Shanghai Jiao Tong University, Shanghai 200240, Peoples Republic of China. Tel.:
+86 21 34206647; fax: +86 21 34206723.
E-mail addresses: macq@sdu.edu.cn (C. Ma), pingxu@sjtu.edu.cn (P. Xu).
1
These authors contributed equally to this work.
Bioresource Technology 101 (2010) 84528456
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NaCl, 5 ml of 1% yeast extract, 200 ll of vitamin mixture, and 5 ml
of metal solution in 1 l of distilled water (Li et al., 2003). Five grams
of soil were inoculated into 100 ml MBSM containing 2% (v/v) die-
sel oil as the sole source of carbon and energy, and incubated with
shaking (200 rpm) for 7 d. Fresh MBSM with 2% diesel oil was inoc-
ulated with 10 ml previous culture and cultivated under the same
conditions. After ve cycles of enrichment, 1 ml of the culture
broth was serially diluted and plated on 1.5% agar MBSM plates,
and incubated for 7 d. Four hundred microliters of diesel oil were
sprayed on the surface of each plate (20 ml MBSM) to obtain a thin
layer. Each colony with a different morphology was characterized
for petroleum hydrocarbon degradation in MBSM supplemented
with 2% diesel oil as the carbon source.
2.2. Characterization of the bacteria
The 16S rRNA gene sequence of each strain was amplied using
Taq DNA polymerase (SolGent Co. Ltd., Korea) under standard reac-
tion conditions with the primers 27F and 1492R (Lane, 1991).
About 800 bp of the PCR products were sequenced with the primer
27F at Shanghai Sangon Biological Engineering Technology and
Services Co. Ltd. (Shanghai, China). The sequences were analyzed
using the BLASTn program (Altschul et al., 1990) of National Center
for Biotechnology Information (NCBI) and aligned with Clustal X
software (Thompson et al., 1997). Phylogenetic trees were con-
structed based on partial 16S rRNA gene sequences (500 bp) using
Neighbor-joining method. The Phylip 3.6 software was used to cal-
culate pairwise distance values (Retief, 2000).
2.3. Biodegradation of diesel oil
Isolates cultured in LuriaBertani medium were washed twice
with 0.85% sodium chloride solution to remove the remaining car-
bon source. The washed cells were transferred into 300 ml Erlen-
meyer asks with stoppers each containing 10 ml of MBSM
supplemented with 200 ll diesel oil that had been left open in
the fume hood to remove volatile hydrocarbons, and cultured with
shaking (180 rpm) for 7 d. After incubation, the cell density was
measured at 600 nm (OD
600
), and the culture broth was extracted
twice with 10 ml of n-hexane each time. All extracts were evapo-
rated to a nal volume of 2 ml with a nitrogen stream. Hydrocar-
bons were quantied by gas chromatographic (GC) system
(Varian 3380, Palo Alto, USA) equipped with an SPB-5 column
(0.32-mm i.d. 30-m length, Supelco). The total area of detected
hydrocarbon peaks was dened as the concentration of TPHs. Per-
centage of degradation was calculated by the following expression:
Percentage of degradation = [(TPHs control TPHs treatment)/
TPHs control] 100. The sterile controls were inoculated with
heat-killed cells autoclaved at 115 C for 10 min. Each test ask
was prepared in triplicate. The correlation of growth and degrada-
tion was analyzed with Statistical Product and Service Solutions
13.0 (SPSS 13.0) software package.
2.4. Biodegradation of crude oil by a bacterial consortium
Strains DQ5, DQ10, DQ11, DQ16, DQ26, DQ62, and DQ85, which
degraded diesel oil and (or) emulsify diesel oil with water ef-
ciently, were stochastically selected to construct a microbial con-
sortium. These seven isolates were cultured separately and
mixed in equal proportions as a consortium. The consortium and
each single isolates were inoculated into 10 ml MBSM supple-
mented with 1 g crude oil as the sole source of carbon and energy
and cultured for 10 d. After incubation, the culture was extracted
sequentially with equal volumes of hexane, methylene chloride,
and chloroform (Mishra et al., 2001). All extracts were pooled
and dried at room temperature by evaporation under a gentle
nitrogen stream, and taken as the residual crude oil.
2.5. Denaturing gradient gel electrophoresis analysis
Genomic DNA samples were extracted and V3 regions of the
16S rRNA genes were amplied with primers corresponding to po-
sition 341534 of the 16S rRNA gene of Escherichia coli (Muyzer
et al., 1993). The PCR products were separated on an 8% polyacryl-
amide gel with a 3050% denaturing gradient. Denaturing gradient
gel electrophoresis (DGGE) was performed with a D-Code instru-
ment (Bio-Rad) for 4 h at 220 V and 60 C. Gels were stained for
three times (15 min each time) with SYBR Green I and imaged
using a GelDoc-It imaging system (Upland, CA, USA). Each bands
of DGGE analysis were recycled and sequenced. The sequences
were aligned with the 16S rRNA genes of each strain by Clustal X
software (Thompson et al., 1997).
2.6. Nucleotide sequence accession numbers
The 16S rRNA gene sequences in this study have been deposited
in the GenBank database under accession numbers GU269267 and
GU377055 to GU377078.
3. Results and discussion
3.1. Degradation of diesel oil
After enrichment cultivation, 38 strains were obtained, and 22
strains could grow in MBSM with 2% diesel oil as the sole carbon
and energy source. The strains degraded between 16.4% and
91.6% of TPHs, and 11 strains degraded more than 70% of TPHs in
diesel oil (Fig. 1A). The biomass of each isolate was proportional
to degradation of TPHs (Fig. 1B). The Pearson correlation coefcient
is 0.881 (p < 0.01), which indicates that the degradation of diesel
oil is positive correlation to growth of the isolates. None of the con-
trols showed growth of biomass and biodegradation of diesel oil.
Gas chromatographymass spectroscopy peaks indicated that the
bacteria, which could grow with diesel oil as the sole source of car-
bon and energy, degraded C12C25 n-alkanes and other hydrocar-
bon fractions (data not shown). Since crude oil is a complex
mixture of hydrocarbons and other compounds, the wide range
of degradation substrates by the strains should be a signicant
advantage in bioremediation of oil-contaminated soils.
3.2. Bacterial population characterization
In the bacterial library, 25 isolates could grow with diesel oil as
the sole source of carbon and energy and (or) emulsify diesel oil
with water efciently. Based on the 16S rRNA genes of the isolates
and BLASTn results, a phylogenetic tree was constructed by the
Neighbor-joining method (Fig. 2). The isolates were grouped in
two main clusters as the c-Proteobacteria and Firmicutes. Five iso-
lates were located in the cluster of c-Proteobacteria, and related
to the genera of Pseudomonas, Enterobacter and Acinetobacter.
Twenty bacteria were grouped in the cluster of Firmicutes, and 19
strains related to the genus of Bacillus; one strain related to the
genus of Exiguobacterium.
The high percentage of isolates related to Bacillus spp. is possibly
because Bacillus strains are easy to be cultivated in the media used,
or favorite the environment conditions of Daqing (latitude 45
north, average annual temperature below 6 C). Psychrotolerance
was observed in Bacillus spp. (Mansilla and Mendoza, 2005), and
Felix and Cooney (1971) also reported that spore-forming bacteria
played an important role in oil biodegradation. Strain D6 closely
Z. Zhang et al. / Bioresource Technology 101 (2010) 84528456 8453
Fig. 1. Degradation of TPHs of diesel oil (A), biomass of isolates and consortium (B). Isolates were cultured in MBSM with 2% (v/v) diesel oil as the sole carbon and energy
source at 30 C for 7 d. Extraction of the un-inoculated control was dened as 100% recovery. Data are represented as the means of triplicate cultures standard derivation.
Fig. 2. Neighbor-joining tree based on partial 16S rRNA gene sequences (500 bp) of the isolates and related species found by BLASTn search. Bootstrap analysis was performed
with 1000 repetition and only values higher than 50% are shown. Bar = 0.1 nucleotide substitution per site, and (T) = Type strain.
8454 Z. Zhang et al. / Bioresource Technology 101 (2010) 84528456
related to Exiguobacterium, which was previously reported to be
prevalent in permafrost and has adapted to the long-term freezing
conditions (Rodrigues and Tiedje, 2007). Members of the bacterial
library adapted to harsh environment, which might be of great
use for biotreatment of oil-contaminants under such circumstances.
The isolates belonging to c-Proteobacteria include bacteria re-
lated to Acinetobacter and Enterobacter, and some bacteria in these
genera have been observed to degrade petroleumcompounds (Ver-
ma et al., 2006; Molina et al., 2009). The isolates, DQ2 and DQ8 re-
lated to Pseudomonas aeruginosa. The genus Pseudomonas is known
to include species that show a high efciency to degrade polycyclic
aromatic hydrocarbons and alkanes (Molina et al., 2009).
3.3. Biodegradation of crude oil by a bacterial consortium
Seven isolates, Bacillus sp. DQ5, Bacillus sp. DQ10, Enterobacter
sp. DQ11, Bacillus sp. DQ16, Bacillus sp. DQ26, Bacillus sp. DQ62,
and Bacillus sp. DQ85, which degraded diesel oil and (or) emulsify
diesel oil with water efciently (see Supplementary material,
Fig. S1), were stochastically selected to form a microbial consor-
tium. TPHs of diesel oil were degraded about 95.8% during a 7-d
incubation (Fig. 1A), and the OD
600
of the culture reached 3.5
(Fig. 1B). After incubation for 10 d, about 87.5% of TPHs of crude
oil were degraded (from 1.0 to 0.125 g) (Fig. 3A), while only about
1064% TPHs of crude oil were degraded by the single strains
(Fig. 3B). There was no signicant degradation of TPHs of diesel
oil and crude oil by sterilized cells. The stability of microbial con-
sortium is also an important property in bioremediation process.
DGGE analysis was carried out to determine changes in the compo-
sition of the consortium during the degradation process. Seven
bands were observed at day one, and no signicant change in the
banding pattern over the 10-d degradation period was observed.
Only in the end, two bands, corresponding to strains DQ5 and
DQ85, disappeared. The densities of most bands were the same, ex-
cept that the intensity of the band corresponding to strain DQ11 in-
creased during the incubation process (Fig. 3C). In general, a
microbial consortium can degrade hydrocarbons from oil-contam-
inated sites more efciently than pure isolated microorganisms
mainly due to synergetic effects (Jacques et al., 2008). Efcient deg-
radation of oil-contaminants and synergetic effects of consortia
were also observed by Jacques et al. (2008) and Molina et al. (2009).
4. Conclusion
A constructed microbial consortium composed of seven bacte-
ria, which were isolated from oil-contaminated soils, was more
efcient in crude oil degradation than individual strains and could
be potentially be useful for bioremediation efforts.
Acknowledgements
This work was partially supported from a research project of
Daqing Oileld Co. Ltd. China National Petroleum Corporation
(Contract Number: DQYT-1201002-2007-JS-9128). The authors
also acknowledge partial nancial support by the Chinese National
Program for High Technology Research and Development (Project
Numbers: 2007AA061101 and 2007AA10Z401).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.biortech.2010.05.060.
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