Bioresource Technology 224 (2017) 327–332

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Biodegradation of crude oil by a defined co-culture of indigenous

bacterial consortium and exogenous Bacillus subtilis
Kaiyun Tao, Xiaoyan Liu ⇑,1, Xueping Chen 1, Xiaoxin Hu, Liya Cao, Xiaoyu Yuan
Laboratory of Environmental Remediation, College of Environmental and Chemical Engineering, Shanghai University, Shanghai 200444, China

h i g h l i g h t s

Crude oil degradation was enhanced by co-cultures with Bacillus subtilis.

Bacillus subtilis ZF3-1 was found to be long-chain n-alkanes degrading strain.

Burkholderiales order was enriched after inoculation of Bacillus subtilis.

a r t i c l e i n f o a b s t r a c t

Article history: The aim of this work was to study biodegradation of crude oil by defined co-cultures of indigenous
Received 12 September 2016 bacterial consortium and exogenous Bacillus subtilis. Through residual oil analysis, it is apparent that
Received in revised form 21 October 2016 the defined co-culture displayed a degradation ratio (85.01%) superior to indigenous bacterial consortium
Accepted 22 October 2016
(71.32%) after 7 days of incubation when ratio of inoculation size of indigenous bacterial consortium
Available online 25 October 2016
and Bacillus subtilis was 2:1. Long-chain n-alkanes could be degraded markedly by Bacillus subtilis.
Result analysis of the bacterial community showed that a decrease in bacterial diversity in the defined
Keywords:
co-culture and the enrichment of Burkholderiales order (98.1%) degrading hydrocarbons. The research
Crude oil
Biodegradation
results revealed that the promising potential of the defined co-culture for application to degradation of
Co-culture of bacteria crude oil.
Bacillus subtili Ó 2016 Elsevier Ltd. All rights reserved.
High throughput sequencing

1. Introduction nity were variable and increased by an exogenously enhanced

Petroleum contamination of environments, which results from
leakage and spillage of crude oil, is always a global concern and
an ongoing threat to human health and natural environments
because of its high incidence and long-term environmental impact
(Lecklin et al., 2011; Li et al., 2015; Lu, 2003; Peterson et al., 2003).
Such a substantial damage to the environment starves for an effi-
cient remediation. Bioremediation, due to its low cost and ability
to convert contaminants to harmless end products, was suggested
for removing crude oil from contaminated sites (Rahman et al.,
2002). Indigenous microorganisms with specific metabolic abilities
played an important role in the bioremediation of crude oil
because they were likely to be better adapted to the environment
requiring treatment (Rahman et al., 2003; Uneo et al., 2007). How-
ever, the metabolic capacities of the indigenous microbial commu-
⇑ Corresponding author.
E-mail address: Lxy999@shu.edu.cn (X. Liu).
1
Equal contributors.
genetic diversity (El Fantroussi and Agathos, 2005).
As an exogenously seeded bacterium, Bacillus subtilis showed
great potential for bioremediation applications. In laboratory tests,
Bacillus subtilis had been successfully used for the degradation of
crude oil and various pollutants, especially n-alkanes (Das and
Mukherjee, 2007). Besides, biosurfactants might be synthesized
by Bacillus subtilis, which can stimulate crude oil degradation by
enhancing the bioavailability of hydrophobic organic compounds
(Nitschke and Pastore, 2006). Moreover, bacterial consortium con-
sisting of Bacillus subtilis and other strains can be defined artifi-
cially and displayed higher ability to completely degrade the
pollutants than individual strains of the consortium due to syner-
gistic interactions among them (Mukherjee and Bordoloi, 2012).
Nevertheless, little is known about how microbes cooperate mutu-
ally and the question of the role of microbial interactions in the
removal of pollutants was thus raised.
Defined bacterial co-culture was often reconstituted by mixing
the individual strains with an equal volume in many studies, but
ratio of inoculation size played an important role in degradation
by defined bacterial consortium (Jin et al., 2012). The bacterial

http://dx.doi.org/10.1016/j.biortech.2016.10.073
0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.
328 K. Tao et al. / Bioresource Technology 224 (2017) 327–332
community shift in response to ratio of inoculation size may be
associated with interactions among bacterial species. Microbial
interactions are likely to be the dominant driver of community
structure (Cordero and Datta, 2016). Co-occurrence patterns of dif-
ferent organisms in a system affected by interactions among them
could reveal important details of a particular community structure
(Fuhrman, 2009). Studying microbial interactions and community
structure in conjunction will help us understand the potential for
functional complementation. Special attention should be paid for
this reason to the changes in microbial community composition
so as to know about synergistic interactions between individual
members in a defined bacterial consortium during bioremediation
of crude oil contamination.
Rapid development of high throughput sequencing methods
has enabled researchers to know about microbial community
structure more conveniently and economically. High-throughput
sequencing of 16S rRNA gene amplicons was widely used to char-
acterize the bacterial composition because of its potential for
reflecting overall community dynamics compared with culture-
dependent techniques and traditional molecular methods, such
as denaturing gradient gel electrophoresis (DGGE) and Sanger
sequencing (Dobson et al., 2011).
Therefore, the objectives of this study were to: (1) investigate
the effect of exogenous Bacillus subtilis on biodegradation of crude
oil by indigenous bacterial consortium from a perspective of ratio
of inoculation size; (2) explore the community composition of
defined bacterial co-culture by high throughput sequencing; (3)
reveal the preliminary mechanisms involved in the mutual bacte-
rial cooperation so that we can apply the defined bacterial co-
culture to bioremediation of petroleum contamination in the
future.
2. Materials and methods
2.1. Chemicals and culture media
Crude oil used in this study was provided by a Shanghai Branch
of China Petroleum Corporation. All other chemicals, analytical
grade or better, were purchased from Sinopharm Chemical Reagent
Co., Ltd., China.
The Luria-Bertani (LB) medium contained 5.0 g of yeast extract,
10.0 g of peptone and 10.0 g of NaCl per liter. The basic mineral salt
medium (MSM) with crude oil as the sole carbon source contained
1.0 g of K2HPO4, 1.0 g of KH2PO4, 1.0 g of (NH4)2SO4, 0.2 g of
MgSO47H2O, 0.02 g of CaCl2, and 1.0 mL of trace element solution
per liter. The trace element solution consisted of 2.5 g of FeSO4-
7H2O, 0.5 g of (NH4)6Mo7O244H2O, 0.3 g of MnSO4H2O and 1 g
of ZnSO47H2O per liter. For solid media, agar (15 g/L) was added
into the solution before autoclaving. The pH was adjusted to 7.0–
7.2 with 1.0 M NaOH before sterilization at 121 °C for 20 min.
2.2. Microorganisms and preparation of inoculum
Indigenous bacterial consortium was enriched from oil-polluted
soil collected from the surface (1–10 cm depth) sediment of
Huangpu-Yangtze River estuary wetland (N31°230 5.400 , E121°30
0
28.300 ) by using the method of restrictive substrate screening
which was described in our previous study (Liu et al., 2011). Bacte-
rial consortium mainly composed of Betaproteobacteria (47.4%) and
Gammaproteobacteria (51.1%) was finally obtained.
Bacillus subtilis named ZF3-1 was once isolated from sludge of
sewage plant. Its 16S rRNA gene sequence was submitted to Gen-
Bank and has been assigned accession numbers KX129926. The
strain was maintained on LB agar slant in 4 °C refrigerator.
Each culture was incubated in LB liquid medium at 150 rpm and
30 °C until the exponential phase was reached. Then cells were
harvested, washed three times with sterile water, and subse-
quently the cell suspensions were adjusted to a optical density of
0.5 at 600 nm using a 752 N UV–vis spectrophotometer (Shanghai
Precision Instruments Co. Ltd., Shanghai, China). Finally, bacterial
co-cultures designed by mixing two cultures mentioned above in
different ratios were used as inoculum to obtain an initial count
of 4.5  107 CFU/mL.
2.3. Biodegradation of crude oil and residual oil analysis
For the experiment, 6 groups of microcosms were set up in
50 ml Erlenmeyer flasks containing 20 mL MSM and 1% v/v crude
oil to study the crude oil degradation by microorganisms. Six
experimental groups were set up as follows: CK-control without
inoculum; T1-treatment inoculated with indigenous bacterial
consortium; T2-treatment inoculated with Bacillus subtilis ZF3-1;
T3-treatment inoculated with co-culture of indigenous bacterial
consortium and ZF3-1 (1:1); T4-treatment inoculated with co-
culture of indigenous bacterial consortium and ZF3-1 (1:2); T5-
treatment inoculated with co-culture of indigenous bacterial
consortium and ZF3-1 (2:1). The flasks with inoculum (15% v/v)
added were incubated in a shaker at 150 rpm and 30 °C for 7 days.
After 7 days of incubation, residual oil was subsequently
extracted and analyzed by UV–vis spectrophotometry and gas
chromatography-mass spectrometry (GC–MS), which was used to
determine the degradation ratio of culture samples. Briefly, the
biomass was removed by centrifugation, and the residual oil from
the culture (20 mL medium) was recovered with the addition of an
equal volume n-hexane by vigorously mixing for 3 min on a vortex.
Then the upper organic phase was brought volume to 25 mL in a
volumetric flask after drying by anhydrous Na2SO4. Finally the n-
hexane phase was diluted 50 times and checked for aromatic
fraction content in crude oil by taking absorbance at 225 nm using
UV–vis spectrophotometer (Shanghai Precision Instruments Co.
Ltd., Shanghai, China). The correlation coefficient (r2) of the stan-
dard curve is 0.9992. The organic phase before dilution was exam-
ined directly by GC–MS.
Crude oil fractions dissolved in the n-hexane was analyzed by
an Agilent 7890 Gas Chromatography (GC) and 5975 Mass Spec-
trometer (MS), which was fitted with a manual injector. A HP-5 sil-
ica capillary column (30 m  0.25 mm  0.25 lm) was used with
helium as the carrier gas (1 mL/min). Samples (1 lL) were injected
in the splitless mode. Injector, ion source and detector tempera-
tures were 280, 200 and 250 °C, respectively. The oven tempera-
ture program was set at 35 °C (holding time 5 min) initially and
35–300 °C at 7.5 °C/min (holding time 5 min). The scan mode
was full at m/z 29–400.
2.4. Cell density and surface tension of culture
After 7 days of incubation, the optical density at 600 nm (OD600)
was measured to determine cell density of each culture. The sur-
face tension of culture supernatant was determined with an optical
contact angle measuring device (OCA15EC, Dataphysics, Germany)
as an indirect measure of surfactant concentration.
2.5. High throughput sequencing and data processing
After total DNA was extracted from culture sample, the V4
regions of the bacterial 16S rRNA gene were polymerase chain
reaction (PCR) amplified with the primer pair 520F (50 -barcode+
GCACCTAAYTGGGYDTAAAGNG-30 ) and 802R (50 -TACNVGGGTATC
TAATCC-30 ). The barcode in the forward primer (520F) is a seven-
base sequence unique to each sample. PCR reaction mixtures
 K. Tao et al. / Bioresource Technology 224 (2017) 327–332
contained (lL/25 lL): Q5 high-fidelity DNA polymerase 0.25,
5  reaction buffer 5, 5  high GC Buffer 5, dNTP(10 mM)0.5, for-
ward primer 1 (10 lM), reverse primer 1 (20 lM), DNA templates
1, and H2O 11.25. PCR amplifications were carried for 25 cycles,
with an initial denaturation step of 30 s at 98 °C and 30 cycles of
PCR consisting of 15 s at 98 °C, 30 s at 50 °C, and 30 s at 72 °C,
and a final 5-min extension at 72 °C.
The resulting PCR products were purified with 2% agarose (BIO-
WEST, Spain) using the Axygen DNA Gel Extraction Kit (Axygen,
USA). Quantification of purified PCR products was done using
Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA,
USA). Purified amplicons were pooled in equimolar amounts and
paired-end sequenced (2  300 bp) on an Illumina MiSeq platform
(Personalbio, Shanghai) according to a standard protocol.
Raw FASTQ files were quality-filtered using Quantitative
Insights Into Microbial Ecology (QIIME, version 1.8.0). Sequencing
reads were truncated using a sliding window approach (window
size 10 bp) at an average quality score of P20, and truncated reads
shorter than 150 bp were discarded. Meanwhile reads containing
ambiguous bases were also removed. Overlapping sequences
longer than 10 bp were assembled without a mispaired base. Oper-
ational taxonomic units (OTUs) were clustered under a 97% cutoff
in UCLUST. Chimera detection was performed using the UCHIME
method. MOTHUR program was used to generate the rarefaction
calculation, Shannon diversity index, and Chao1 richness estima-
tor. The Kyoto Encyclopedia of Genes and Genomes (KEGG) path-
ways provided by Phylogenetic Investigation of Communities by
Reconstruction of Unobserved States (PICRUSt) were used to gen-
erate predictions of functional metagenomic (Langille et al., 2013).
2.6. Statistical analysis
All data in this paper are the mean value (±SD) of three indepen-
dent replicates. SPSS 19.0 software was used for statistical analysis.
Statistically significant differences among the experimental treat-
ments were analyzed using one-way analysis of variance (ANOVA)
followed by Duncan’s test at 0.05 probability level.
3. Results and discussion
3.1. Crude oil biodegradation by defined bacterial co-cultures
The degradation experiment was conducted in MSM with 1%
v/v crude oil as the sole carbon source to investigate crude oil
degradation ability of indigenous bacterial consortium with the
addition of exogenous Bacillus subtilis ZF3-1. As shown in Fig. 1,
the ratio of crude oil degradation differed greatly between treat-
ments and the degradation ratio of indigenous bacterial consor-
tium (T1) could reach 71.32%. Although exogenous Bacillus
subtilis ZF3-1 (T2) exhibited limited ability to degrade crude oil,
all the defined bacterial co-cultures with Bacillus subtilis ZF3-1
(T3, T4 and T5) showed a better performance than indigenous bac-
terial consortium (T1) in crude oil degradation, especially group T5
(85.01%). Furthermore, the degradation ratios of saturated fraction
(mainly n-alkanes) and aromatic fraction in crude oil were also
investigated. The degradation ratio of n-alkanes was obviously
higher than that of aromatic fraction which may be due to the
easier degradation of n-alkanes. The degradation behaviors of n-
alkanes were similar to these of crude oil in different treatments.
T5 was also found to have the highest ability of degrading
n-alkanes (94.13%) among all treatments while there was little dif-
ference in degradation of aromatic fraction between different
treatments (about 30%).
In order to further investigate degradation of n-alkanes in T5,
four chosen groups CK, T1, T2 and T5 were analyzed by GC–MS
329
100
crude oil
n-alkanes
a a
90 aromatic fraction
b b a
80 b
c
Degradation ratio(%)
d
70
a a
ab a
30 b
20 c
10
e
0
T1 T2 T3 T4 T5
Treatments
Fig. 1. Degradation ratios of crude-oil, n-alkanes and aromatic fraction after 7 days
of incubation. Different letters up the vertical bars are different significantly
between treatments (P < 0.05).
(Supplementary Fig. S1). C10-C27 n-alkanes and the isoprenoids,
pristane and phytane, in group T5 were found to be eliminated
mostly after 7 days of incubation. Long-chain n-alkanes (C21-C27)
were preferentially metabolized in group T2 while the rest of the
n-alkanes (C10-C20) nearly maintained the same concentrations
except for a slight increase in C11-C13 abundances. The n-alkanes
(C10-C27) in group T1 were degraded in general, but not completely.
Mixed cultures can be superior to pure cultures in the aspect of
complete degradation of the pollutants due to synergistic interac-
tions among members of the associations (Shong et al., 2012).
What is more, the ratio of inoculation size of members in co-
culture may have a marked influence on the degradation
(Chandra et al., 2011). Hence the proportions of indigenous bacte-
rial consortium and exogenous Bacillus subtilis in mixed culture
were adjusted in the present study so that degradation of crude
oil can be enhanced by the well-coordinated co-culture. The opti-
mal ratio of inoculation size in our experimental setup was indige-
nous bacterial consortium: ZF3-1 = 2:1. Such a defined co-culture
performed better than indigenous bacterial consortium in degrada-
tion of crude oil and depletion of n-alkanes. In addition, result
showed that Bacillus subtilis used in the present study depleted
only long-chain (C21-C27) n-alkanes, which is similar to the result
that Bacillus subtilis showed the ability to degrade the long-chain
n-alkanes (>C25) found in research of Gudiña et al. (2013). Consid-
ering these phenomena, synergistic interactions between indige-
nous bacterial consortium and Bacillus subtilis ZF3-1 were
hypothesized to exist in the defined bacterial co-culture.
3.2. Study of bacterial growth
Biodegradation of hydrocarbons depends on the successful sur-
vival of the organisms after their inoculation (Ramos et al., 1991).
The bacterial strains utilized crude oil hydrocarbons as sole source
of carbon and energy, which was evident from the increase in cell
density of culture after incubation. Cell densities of five chosen
groups (T1, T2, T3, T4 and T5) were measured (as shown in
Fig. 2) to monitor bacterial growth after 7 days of incubation.
Group T5 yielded the highest OD600 value of 0.818 among all
groups. The OD600 values of T3 and T4 were significantly lower
than T1 (P < 0.05) while cell density in T2 was below an OD600
value of 0.1. Alone Bacillus subtilis ZF3-1 could not grow well in
MSM which may be due to the non-adaptation of ZF3-1 to crude
oil pollutants. It is notable that the growth of co-culture of indige-
nous bacterial consortium and ZF3-1 was promoted when the ratio
330 K. Tao et al. / Bioresource Technology 224 (2017) 327–332
1.0
0.8
b
Cell density (OD600)
0.6
c c
0.4
0.2 d c
0.0
T1 T2 T3 T4
Treatments
Fig. 2. Cell growth in hydrocarbon biodegradation assays after 7 days of incubation.
Different letters up the vertical bars are different significantly between treatments
(P < 0.05).
of inoculation size of indigenous bacterial consortium and ZF3-1
was 2:1. Such a promotion effect of co-culture may result from
the important role of competitive and cooperative interactions
between organisms in shaping communities (Freilich et al.,
2011). Yet, the consequences of mutual interactions for species
diversity are still poorly understood (Gross, 2008). Therefore, bac-
terial communities were analyzed to further explore the effect of
co-culture on species diversity.
3.3. Study of biosurfactant production
Given biosurfactant-producing ability of Bacillus subtilis, surface
tensions of the six groups (CK, T1, T2, T3, T4 and T5) were mea-
sured (as shown in Fig. 3) to indirectly monitor the biosurfactant
production during the biodegradation of crude oil (Carrillo et al.,
1996). It was reported that a biosurfactant, produced by Bacillus
subtilis using cassava wastewater as substrate, could reduce the
surface tension of medium to 26.6 mN/m (Nitschke and Pastore,
2006). Surprisingly, there was no significant difference in surface
tensions between group CK and T2, which indicated that nearly
no biosurfactant was produced by Bacillus subtilis ZF3-1. In all
cases, biosurfactant production is concurrent with the increase in
cell density (Ron and Rosenberg, 2002). Given low cell density of
T2, limited growth of Bacillus subtilis ZF3-1 due to poor nutrition
of MSM and toxicity of oil may hinder biosurfactant production
by ZF3-1. Besides, carbon-source utilization is an important con-
tributor to biosurfactant production by Bacillus subtilis. Carbohy-
drate was regared as a kind of ideal carbon source and oil was
once reported to inhibit biosurfactant production (Fox and Bala,
2000; Joshi et al., 2008). Our further investigation of the effect of
different carbon sources on biosurfactant production by ZF3-1
revealed that ZF3-1 could reduce the surface tension to about
29.21 mN/m using beef extract and oil really inhibited biosurfac-
tant production by ZF3-1 (Supplementary Fig. S2).
In addition, the surface tension was often reduced to values
below 30 mN/m as a result of biosurfactant synthesis (Shavandi
et al., 2011). In the present study, the surface tensions of T1 and
T5 dropped dramatically to 57.50 and 54.48 mN/m respectively
when compared with the surface tensions of CK (72.05 mN/m),
but were still higher than 30 mN/m. Besides, there was no signifi-
cant difference in surface tensions between group T1 and T5, which
indicated that the addition of ZF3-1 did not lead to the enhanced
biosurfactant production of indigenous bacterial consortium. As
for groups T3 and T4, the surface tensions of meida were also
75
a a
a
70
Surface tension (mN/m)
65
b b
60
c
55
T5
50
CK T1 T2 T3 T4 T5
Treatments
Fig. 3. Surface tension changes in hydrocarbon biodegradation assays after 7 days
of incubation. Different letters up the vertical bars are different significantly
between treatments (P < 0.05).
decreased, but still higher than the surface tension of T1. These
results illustrated that biosurfactant production during biodegra-
dation of crude oil was limited and there was no apparent relation-
ship between enhanced biodegradation of crude oil and bacterial
biosurfactant production in the defined co-culture. The bacterial
community structures and functions in the defined co-culture
should be figured out necessarily.
3.4. Changes in bacterial taxonomy composition in co-culture
High throughput sequencing technique was applied to further
analyze bacterial communities in media of groups T1 and T5.
Group T1, inoculation with indigenous bacterial consortium, is
named ‘MCI’. Group T5, inoculation with coculture with exogenous
Bacillus subtilis, is named ‘MCE’. A total of 77 311 and 69 458 high
quality sequences were obtained from MCI and MCE respectively.
All rarefaction curves tended to approach the saturation plateau
(Supplementary Fig. S3). Up to 281 and 168 OTUs were then recov-
ered from MCI and MCE respectively. Moreover, both the Shannon
diversity index and Chao1 richness estimator of MCE were obvi-
ously lower than these of MCI (shown in Fig. 4a), which indicated
a decrease in the diversity of indigenous bacterial community after
co-culture with exogenous Bacillus subtilis. The taxonomic assign-
ments are summarized in Fig. 4b. The bacterial OTUs in MCI were
mainly assigned to 7 different orders, among which Xanthomon-
adales (48.9%) was the most abundant order, followed by
Burkholderiales (47.1%) and Enterobacteriales (2.2%). However, the
proportion of Burkholderiales in MCE showed a high variation,
increasing sharply to 98.1%, which made it become predominant
in the bacterial community. In addition, the genus Bacillus did
not appear in MCI, but existed in MCE.
The shift in composition of the indigenous bacterial community
caused by exogenously introduced strain has been demonstrated
in several studies (Boon et al., 2000; Yang et al., 2016). However,
Xiong et al. (2013) reported that inoculation of exogenous microor-
ganisms in the pollutant-contaminated soil had negligible impact
on the bacterial community structure. In our studies, the co-
culture system of exogenous Bacillus subtilis and indigenous bacte-
rial community with specific metabolic capacity was investigated
in liquid medium. The results indicated the dramatic changes in
bacterial community structure, which suggest that the structure
of the bacterial community was dynamic, but not static after inoc-
ulation of exogenous microorganisms. Indeed, Burkholderiales
 K. Tao et al. / Bioresource Technology 224 (2017) 327–332 331

(a) (b)
3.0 300 100
Xanthomonadales
Shannon diversity index
90 Enterobacteriales
Chao1 richness estimator
SBla14
2.5 250 Burkholderiales
80
Rhizobiales
70 Lactobacillales

Chao1 richness estimator

Relative abundance (%)

Shannon diversity index

2.0 200 Bacillales

60 Nostocales
Flavobacteriales
1.5 150 50 Actinomycetales

40
1.0 100
30

20
0.5 50
10

0.0 0 0
MCI MCE
MCI MCE --
Treatments Treatments

Fig. 4. Bacterial community analysis of the defined co-culture. a) The Shannon diversity index and Chao1 richness estimate in the defined co-culture. b) Order distribution
level of defined bacterial co-culture.

order seemed to be enriched in the co-culture system, while others then Burkholderiales order degrading hydrocarbons enriched, thus
tended to be less abundant. This order is known to include a vari- enhancing crude oil degradation.
ety of bacteria capable of degrading hydrocarbons, which may
contribute to the efficient crude oil degradation by the defined 3.5. Predicted bacterial functions in coculture
co-culture (Kasai et al., 2005; Yuste et al., 2000). The proportion
of the genus Bacillus in bacterial community of MCE was relatively PICRUSt was employed to predict the presence and relative
small (nearly 0.1%), which may be related to utilization of long- abundance of gene families in different microbiomes MCI and
chain n-alkanes by strain ZF3-1. Once long-chain n-alkanes were MCE. Overall 41 KEGG orthologies (KOs) were represented in the
exhausted, there may be no source of carbon and energy for its data set (Supplementary Fig. S4). Both MCI and MCE microbiomes
growth, and the rest of the hydrocarbons were sequentially biode- mainly exhibited functions included metabolism, genetic informa-
graded by other bacterial genera, as reported in the research of tion processing, environmental information processing and some
Horowitz et al. (1975). other unclassified functions.
Each member in a bacterial co-culture has a specific role and Within the metabolism category (Fig. 5), an obvious increased
may need to depend on the presence of other members to degrade relative abundance of metagenomic sequences grouped into the
complex mixtures of hydrocarbons in crude oil by synergistic category of ‘‘xenobiotic biodegradation and metabolism” in KEGG
interactions. One of the possible mechanisms in which hydrocar- was observed in MCE microbiomes, suggesting that MCE micro-
bon degraders benefit from synergistic interactions is that one spe- biome has an enhanced potential for degradation when compared
cies are able to degrade some compounds that others are able to with MCI microbiome. Metabolism functional prediction further
only partially (Alexander, 1999). We hereby hypothesize that proved that the defined co-culture of indigenous bacterial consor-
long-chain n-alkanes might be degraded by Bacillus subtilis ZF3-1 tium and Bacillus subtilis ZF3-1 (2:1) is superior to indigenous bac-
at an early stage of crude oil biodegradation of co-culture system, terial consortium in the aspect of complete degradation of crude oil.

MCE
MCI
Amino Acid Metabolism
Biosynthesis of Other Secondary Metabolites
Carbohydrate Metabolism
Energy Metabolism
Enzyme Families
KEGG class

Glycan Biosynthesis and Metabolism

Lipid Metabolism
Metabolism of Cofactors and Vitamins
Metabolism of Other Amino Acids
Metabolism of Terpenoids and Polyketides
Nucleotide Metabolism
Xenobiotics Biodegradation and Metabolism

0.00 0.05 0.10 0.15

The relative abundance

Fig. 5. Metabolism functional predictions for co-culture with Bacillus subtilis.

332 K. Tao et al. / Bioresource Technology 224 (2017) 327–332
4. Conclusions
Co-cultures of indigenous bacterial consortium and exogenous
Bacillus subtilis displayed enhanced ability in degrading crude oil.
When ratio of inoculation size of indigenous bacterial consortium
and Bacillus subtilis was 2:1, crude oil especially n-alkanes was
nearly degraded completely. Enhanced crude oil degradation may
be explained by the predominance of Burkholderiales order
(98.1%) in the defined co-culture with long-chain n-alkanes-
degrading Bacillus subtilis. This work demonstrates the promising
potential of the defined co-culture for bioremediation applications.
Further research should aim at practical application of the defined
co-culture in petroleum-contaminated soil.
Ethical statement
Conflict of interest: The authors declare that they have no con-
flict of interest.
Acknowledgements
The work was funded by the National Natural Science Founda-
tion of China (Nos. 41373097, 21677093), Program for Innovative
Research Team in University (No. IRT13078).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biortech.2016.10.
073.
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