Chemosphere 169 (2017) 124e130

Contents lists available at ScienceDirect

Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Bioremediation of hydrocarbon degradation in a petroleum-

contaminated soil and microbial population and activity
determination
Manli Wu a, Wei Li a, Warren A. Dick b, Xiqiong Ye a, Kaili Chen a, David Kost b,
Liming Chen b, *
a
School of Environmental and Municipal Engineering, Xi'an University of Architecture and Technology, No.13 Yanta Road, Xi'an, Shaanxi Province 710055,
China
b
School of Environment and Natural Resources, The Ohio State University, Ohio Agricultural Research and Development Center, 1680 Madison Avenue,
Wooster, OH 44691, USA

h i g h l i g h t s

 Biostimulation and bioaugmentation achieved 48% and 58% hydrocarbon degradation.

 Hydrocarbon degradation rate correlated with degrader population increase.
 The degradation rate correlated with degrader activity increase in Biolog assay.
 MPN and Biolog were efficient methods for assessing hydrocarbon degradation rate.

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

Article history: Bioremediation of hydrocarbon degradation in petroleum-polluted soil is carried out by various micro-
Received 10 June 2016 organisms. However, little information is available for the relationships between hydrocarbon degra-
Received in revised form dation rates in petroleum-contaminated soil and microbial population and activity in laboratory assay. In
28 October 2016
a microcosm study, degradation rate and efficiency of total petroleum hydrocarbons (TPH), alkanes, and
Accepted 11 November 2016
polycyclic aromatic hydrocarbons (PAH) in a petroleum-contaminated soil were determined using an
infrared photometer oil content analyzer and a gas chromatography mass spectrometry (GC-MS). Also,
Handling Editor: X. Cao the populations of TPH, alkane, and PAH degraders were enumerated by a modified most probable
number (MPN) procedure, and the hydrocarbon degrading activities of these degraders were determined
Keywords: by the Biolog (MT2) MicroPlates assay. Results showed linear correlations between the TPH and alkane
Bioremediation degradation rates and the population and activity increases of TPH and alkane degraders, but no cor-
Petroleum-contaminated soil relation was observed between the PAH degradation rates and the PAH population and activity increases.
Hydrocarbon degradation efficiency Petroleum hydrocarbon degrading microbial population measured by MPN was significantly correlated
Microbial population
with metabolic activity in the Biolog assay. The results suggest that the MPN procedure and the Biolog
Biolog assay
assay are efficient methods for assessing the rates of TPH and alkane, but not PAH, bioremediation in oil-
contaminated soil in laboratory.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction to have negative effects on human health and the environment.

Petroleum spills often occur by accidents during pumping,
transportation, and refining. Petroleum is a complex mixture
including aliphatic and aromatic hydrocarbons, which are known
* Corresponding author.
E-mail address: chen.280@osu.edu (L. Chen).
Thus, the US Environmental Protection Agency (U.S. EPA, 1986)
considers these hydrocarbons as priority environmental pollutants.
Bioremediation has been considered an efficient, cost-effective,
and environmentally sound method to degrade hydrocarbons in
petroleum-contaminated soils (Salanitro and Dorn, 1997; Liebeg
and Cutright, 1999; Margesin and Schinner, 2001; Zhang et al.,
2008; Chiu et al., 2009; Wu et al., 2013; Kharusi et al., 2016; Ma

http://dx.doi.org/10.1016/j.chemosphere.2016.11.059
0045-6535/© 2016 Elsevier Ltd. All rights reserved.
 M. Wu et al. / Chemosphere 169 (2017) 124e130 125

et al., 2016). This method includes bioaugmentation and bio-
stimulation (Llado
et al., 2012; Wu et al., 2016). Bioaugmentation is
a technology of inoculating exogenous microorganisms into the
contaminated soil to degrade hydrocarbons (Wu et al., 2013), and
biostimulation of supplying nutrients to the soil to stimulate the
hydrocarbon degrading capacity of the indigenous microorganisms
(Sanscartier et al., 2009 Chang et al., 2013; Wu et al., 2016).
Some studies have reported the petroleum hydrocarbon
degradation efficiency being associated with the petroleum hy-
drocarbon degrader population (Krutz et al., 2005; Kauppi et al.,
2011; Taccari et al., 2012; Wu et al., 2016). However, information
about relationships between the rate of hydrocarbon degradation
in oil-contaminated soil during bioremediation and hydrocarbon
degrader population and activity as measured in laboratory is
limited (Lors et al., 2010). The most probable number (MPN) is a
popular method for counting microbial populations in laboratory
(Wu et al., 2013), and the Biolog assay is a common method for
identifying microorganisms and measuring the carbon metabolic
activity (Dos Santos et al., 2002; Kadali et al., 2012). These two
methods should be effective and efficient for investigating these
relationships.
We conducted a microcosm study to evaluate the effects of
bioaugmentation and biostimulation in oil-contaminated soil on
hydrocarbon degradation rate and hydrocarbon degrading micro-
bial populations. We also measured the hydrocarbon degrading
activity of microorganisms from the soil by Biolog assay using three
self-defined carbon sources (standard petroleum hydrocarbons, n-
hexadecane, and polycyclic aromatic hydrocarbons). The objectives
were (1) to assess the correlations between hydrocarbon degra-
dation rate in oil-contaminated soil and abundance of hydrocarbon
degrading microbial populations measured by the MPN procedure,
and microbial hydrocarbon degrading activity in the Biolog assay
and (2) to explore relationships between the degrader populations
measured and their activities in the Biolog assay.
2.2. Experiment design for the microcosm study
Detailed information about the experimental design was pre-
viously provided by Wu et al. (2016), but treatments were modified
in this study as presented here in brief. Four treatments included
(1) CK: untreated dried soil; (2) WHC: 20% moisture in soil; (3) BA:
bioaugmentation with a hydrocarbon-degrading consortium
composed of Pseudomonas stutzeri GQ-4 strain KF453954, Pseudo-
monas SZ-2 strain KF453956, and Bacillus SQe2 strain KF453961,
and 20% soil moisture content; (4) BS: biostimulation with
(NH4)2SO4 and KH2PO4 and 20% soil moisture content.
2.3. Total petroleum hydrocarbon (TPH), alkane, and PAH analysis
The TPH was determined as previously described in Wu et al.
(2016). Alkanes and PAHs were separated using the Super Flash
Alumina Neutral columns (SF 15e24 g, 20.8  112 mm, Agilent
Technologies). Alkane and PAH solutions were obtained by eluting
the columns with hexane and dichloromethane, respectively. The
concentrations of alkane and PAH in the solutions were analyzed
using gas chromatography/mass spectrometry (GC-MS) (Agilent
Technologies, Palo Alto, CA).
For alkane and PAH analysis, the separation column was a HP-5-
MS column (30 m  25 mm  0.25 mm; Agilent Technologies), and
helium was carrier gas at flow rates of 1.0 and 1.2 mL min 1,
respectively. Temperature programming for alkane was an initial
5 min at 65  C, and then heating at a rate of 10  C min 1 to 300  C
(holding for 2 min). Programming for PAH was initial 1 min at 50  C,
heating at 15  C min 1 to 170  C (holding for 2 min), followed by
heating at 8  C min 1 to 210  C, and finally at 3  C min 1 to 300  C
(holding for 5 min). For alkanes, the injector and detector tem-
peratures were 300  C and 230  C, respectively. The selected ion
monitoring mode was used, and injection volume was 1 mL in the
splitless mode. For PAH analysis, the multiple reaction monitoring
mode was used, and injection volume was 10 mL.

2.4. Determination of TPH-, alkane- and PAH-degrading microbial

2. Materials and methods populations

2.1. Soil for the microcosm study
Petroleum-contaminated soil was collected in Shaanxi province,
China. The collecting site in the oil field was described by Wu et al.
(2016), but the specific sampling spots were different for these two
studies. Selected chemical and microbiological properties of the soil
for this study are listed in Table 1.
The TPH-, alkane- and PAH-degrading microbial populations in
the microcosm soils were determined by a modified most probable
number (MPN) procedure as described by Wu et al. (2016). For
determination of TPH-degrading microbial population, standard
petroleum hydrocarbons were chosen as the only carbon source.
For determination of alkane- and PAH-degrading microbial pop-
ulations, n-hexadecane and a mixture containing three PAHs were
chosen as the only carbon source, respectively.

Table 1 2.5. Determination of hydrocarbon degrading activity in the Biolog

Selected chemical and microbiological properties of the soil used for the microcosm
assay
study.

Main characteristics Values Biolog (MT2) MicroPlates (Biolog, Hayward, CA) were used to
TPHa (mg kg 1) 20200 ± 170 evaluate the hydrocarbon degrading activity of microorganisms
Alkane (mg kg 1) 15400 ± 130 from the microcosm soil. The carbon sources supplied to the Biolog
Aromatics (mg kg 1) 2180 ± 20 plates were standard petroleum hydrocarbons for TPH-degraders,
pH 8.4 ± 0.3
Total carbon (g kg 1) 765 ± 80
n-hexadecane for alkane-degraders, and PAH solution (0.4 g
Total nitrogen (mg kg 1) 921 ± 40 anthracene, 0.4 g phenanthrene, and 0.2 g pyrene in 1000 mL
Total phosphorus (mg kg 1) 160 ± 20 dichloromethane) for PAH-degraders. Experimental procedures
Total bacterial numbers (cells g 1) 2.3 ± 0.4  107 followed Taha et al. (2015). Degrading activity was expressed as
TPH degraders (MPNb g 1) 1.7 ± 0.4  105
average well color development (AWCD) (Garland and Mills, 1991).
Alkane degraders (MPN g 1) 5.0 ± 0.6  104
PAHc degrader population (MPN g 1) NDd
a
2.6. Statistical analysis
TPH: Total Petroleum Hydrocarbon.
b
MPN: Most Probably Number.
c
Polycyclic aromatic hydrocarbon. All experiments were conducted with three replications. The
d
ND: Not Detected. experimental results are presented as the mean ± one standard
126 M. Wu et al. / Chemosphere 169 (2017) 124e130

deviation (SD). The experimental data were also subjected to degradation in soil.
analysis of variance (ANOVA) by SPSS 19.0 (Statistical Package for Alkanes and PAH are the two fractions of TPH. The changes of
the Social Sciences, China). When the ANOVA generated a signifi- the concentrations of alkanes and PAH during bioremediation of
cant F value (p  0.05) for treatments, the means were compared by the oil-contaminated soil are shown in Fig. 1. During 8 weeks of
the least significant difference (LSD) test. Regression analyses for incubation, alkane concentrations decreased from 15400 ± 130 to
relationships between sets of two variables used Microsoft Excel 12300 ± 420, 12200 ± 360, 7200 ± 280, and 6160 ± 250 mg kg 1 soil
2010. in the CK, WHC, BS, and BA treatments, respectively. Corresponding
concentrations for PAH decreased from 2180 ± 20 to 1900 ± 50,
1720 ± 50, 1630 ± 40, and 1120 ± 40 mg kg 1 soil. These results
3. Results
statistically indicated that the BA treatment was effective for alkane
and PAH degradation and the BS treatment only degraded alkanes
3.1. Degradation of TPH, alkanes, and PAH in microcosms
(P0.05).

After 8 weeks of incubation, TPH degradation in the microcosms

was 58 ± 1% for BA, 48 ± 1% for BS, 21 ± 3% for WHC, and 13 ± 3% for
CK (Fig. 1). Thus, BA treatment was slightly more effective than BS
treatment. Compared to the untreated CK, the moisture only
treatment (WHC) slightly improved, while the BA and BS treat-
ments significantly enhanced TPH biodegradation (P0.05). These
results indicate that inoculating exogenous degrading microor-
ganisms and providing nutrients for the indigenous degraders are
effective methods for bioremediation of petroleum hydrocarbon
CK
20 A A
TPH concentrations (g kg-1)
3.2. Effects of bioremediation on hydrocarbon degrading microbial
populations
The abundances of populations of TPH-, alkane-, and PAH-
degraders in microcosm soils during 8 weeks of incubation are
shown in Fig. 2. For all three types of degraders, the populations
were significantly affected by the treatments (P0.05). For TPH-
and alkane-degraders, the populations were rapidly increased by
the BS and BA treatments during the first three weeks of incubation
and then remained at that level or decreased. For PAH-degraders,
WHC BS BA the populations were rapidly increased by the WHC, BS, and BA
treatments during the first two weeks of incubation and then
maintained at that level or decreased. The BA soils generally had

A
18 AB A A A A A the greatest populations of TPH- and alkane-degrading
BCA A
16 C B A A
B B B B
14 B CK WHC BS BA
12 B
C C C C
C TPH degraders
10 C 8.0
8 C
7.5
Log10(MPN)

D D D
6 7.0
6.5
Alkane concentrations (g kg-1)

16 6.0
A A
A 5.5
14 B A A
B B A A A 5.0
B
12 A A
B A A A
C C 7.0 Alkane degraders
10
B B 6.5
Log10(MPN)

C B B
8 D B 6.0
B B
C 5.5
6 C C 5.0
4.5
2.2 A 4.0
PAH concentrations (g kg-1)

A A
2.0 A A A A
A 2.2 PAH degraders
1.8 A A B B B B
A 2.0
Log10(MPN)

1.6 B A
B 1.8
B C C
1.4 B 1.6
B
1.2 C
1.4
D
1.0 C C D 1.2
0 1 2 3 4 5 6 7 8 1.0
0 1 2 3 4 5 6 7 8
Time (weeks)
Time (weeks)
Fig. 1. Degradation of TPH, alkanes, and PAH by bioremediation in oil-contaminated
soil. Errors bars represent standard deviation. Different letters in the same week Fig. 2. Effects of bioremediation on TPH-, alkane-, and PAH-degrading microbial
represent a significant difference at P 0.05. populations in oil-contaminated soil. Errors bars represent standard deviation.
 M. Wu et al. / Chemosphere 169 (2017) 124e130 127

microorganisms, which were respectively 1.2  108 and decreased afterwards. This suggests that soil microorganisms have
7.3  106 cells g 1 soil in the third week after incubation. The BS the strongest bioactivities in the third week of incubation.
treatment also significantly enhanced TPH- and alkane-degrading
microbial populations compared to the CK treatment (P0.05),
3.4. Relationship between hydrocarbon degradation rate and
and the greatest populations of TPH- and alkane-degrading mi-
microbial population increase in oil-contaminated soil
croorganisms for the BS treatment were respectively 1.6  107 and
6.5  105 cells g 1 soil in the third week after incubation. Among
The relationship between hydrocarbon degradation rate and the
the three types of degraders in the microcosm soils of different
population increase of TPH-, alkane-, and PAH-degraders is shown
treatments, TPH-degrading populations were greatest, alkane-
in Fig. 4. There were linear correlations for TPH degradation rate
degrading populations were intermediate, and the PAH-degrading
populations were lowest.

3.3. Degrading activities of petroleum hydrocarbon degraders in the

Biolog plates

The average of well color development (AWCD) in the Biolog

plates is an indicator of carbon metabolism by the microbial com-
munity. In this study, TPH-, alkane-, and PAH-degrading activities
by microorganisms from the oil-contaminated soil were assessed
using the Biolog plates with three self-defined carbon sources
(standard petroleum hydrocarbons, n-hexadecane, and PAH). As
shown in Fig. 3, the AWCD value was greatest when standard pe-
troleum hydrocarbons were the sole carbon source, and smallest
when PAH were the sole carbon source. This indicated that the soil
microorganisms utilized alkanes more than PAH as the carbon
source substrate.
The AWCD value was generally highest in the BA soil, and then
followed by the BS soil (Fig. 3). Statistical results indicated that
bioaugmentation and biostimulation could significantly enhance
hydrocarbon degrading activity of microorganisms in the biore-
mediation soil (P0.05). For the BA and BS treatments, the AWCD
value increased during the first three weeks of incubation and then

CK WHC BS BA

TPH utilization
AWCD (590nm)

1.0
0.8
0.6
0.4
0.2

0.7 Alkane utilizatio n

AWCD (590nm)

0.6
0.5
0.4
0.3
0.2
0.1

0.21
AWCD (590nm)

PAH utilization
0.18
0.15
0.12
0.09
0.06
0 1 2 3 4 5 6 7 8

Time (weeks)
Fig. 3. Degrading activities of petroleum hydrocarbon degraders in the Biolog (MT2) Fig. 4. Relationship between hydrocarbon degradation rate in oil-contaminated soil
MicroPlates. Errors bars represent standard deviation. and the population increase of TPH-, alkane-, and PAH-degraders.
128 M. Wu et al. / Chemosphere 169 (2017) 124e130

versus TPH degrader population increase and for alkane degrada-

tion rate versus alkane degrading population increase during the
first three weeks of incubation (Fig. 4A and B). The correlation
equations may be useful for predicting TPH and alkane degradation
in oil-contaminated soil incubated in laboratory at room temper-
ature from increases in the abundance of degrading microbial
population measured by the MPN procedure. There was no corre-
lation between hydrocarbon degradation rate and microbial pop-
ulation when microbial population was not increased or decreased
after 3 weeks of incubation. No correlation was observed for PAH
degradation rate and PAH degrader population at any time (Fig. 4C).

3.5. Relationship between hydrocarbon degradation rate in oil-

contaminated soil and hydrocarbon degrading activity increase in
the Biolog plates

The relationship between hydrocarbon degradation rate in oil-

contaminated soil and TPH-, alkane-, and PAH-degrading activity
increase in the Biolog plates is shown in Fig. 5. There were linear
correlations for TPH and alkane degradation rates in oil-
contaminated soil during the first three weeks of incubation
versus the respective TPH and alkane degrading activities in the
Biolog plates (Fig. 5A and B). It is possible to use these equations to
predict TPH and alkane degradations in oil-contaminated soil
incubated in laboratory at room temperature from increases in
degrading microbial activity measured in the Biolog plates. There
was no correlation between hydrocarbon degradation rate and
microbial activity when microbial activity was not increased or
decreased after 3 weeks of incubation. No correlation was observed
for PAH degradation rate and PAH degrading activity at any time
(Fig. 5C).

3.6. Relationship between degrading microbial populations

measured by the MPN procedure and hydrocarbon degrading
activities in the Biolog plates

The relationship between degrading microbial populations by

the MPN procedure and degrading microbial activities in the Biolog
plates is shown in Fig. 6. Linear correlations existed between TPH-,
alkane-, and PAH-degrading microbial populations and corre-
sponding metabolic activities. These equations describing these
relationships may be useful for predicting TPH-, alkane-, and PAH-
degrader populations in oil-contaminated soil incubated in labo-
ratory at room temperature from the corresponding degrading
microbial activity in the Biolog plates.

4. Discussion

The C: N: P ratio in the petroleum-polluted soil was

100:0.12:0.02 (Table 1). For bioremediation operations, US EPA
recommended a ratio of 100:10:1 in soil for appropriate nutrients
to stimulate microorganism growth (U.S. EPA, 2002). This study and
our previous study (Wu et al., 2016) indicate that biostimulation
(i.e., addition of nutrients N and P) strategies can enhance biore-
mediation of the petroleum-contaminated soil collected in north-
Fig. 5. Relationship between hydrocarbon degradation rate in oil-contaminated soil
ern Shaanxi province, China. These two studies respectively and TPH-, alkane-, and PAH-degrading activity increase in the Biolog (MT2)
achieved 48% and 60% degradation of TPH. Also, before bioreme- MicroPlates.
diation, this petroleum-contaminated soil had low populations of
TPH-, alkane- and PAH-degrading microorganisms (Table 1). Thus,
inoculating suitable petroleum hydrocarbon degraders can increase stutzeri GQe4 strain KF453954, Pseudomonas SZe2 strain
these populations in the oil-polluted soil, leading to more effective KF453956, and Bacillus SQe2 strain KF453961) achieved 58%
TPH degradation. In our previous study, bioaugmentation only with degradation of TPH.
Acinetobacter SZ-1 strain KF453955 degraded 34% of TPH after six Biolog (MT2) MicroPlates are useful tools for the screening and
weeks of incubation (Wu et al., 2016). In this study, bio- evaluation of bacterial strains growing on selected carbon sources
augmentation with a TPH-degrading consortium (Pseudomonas (Pierce et al., 2014). This method has been successfully used as a
 M. Wu et al. / Chemosphere 169 (2017) 124e130
Fig. 6. Relationship between degrading microbial population by the MPN procedure
and degrading microbial activity in the Biolog (MT2) MicroPlates.
rapid means of identifying bacteria that are able to metabolize
hydrocarbons (Lu et al., 2008; Kadali et al., 2012). However, infor-
mation about using Biolog assay for evaluating the degrading ac-
tivities of soil microorganisms during bioremediation was limited.
In our study, Biolog (MT2) MicroPlates with standard petroleum
hydrocarbons, n-hexadecane, or PAHs as the carbon source were
used for determining the hydrocarbon metabolic activities of
129
microorganisms from the microcosm soil. The purpose of this
experiment was to evaluate the relationship between hydrocarbon
degrading rate in petroleum-contaminated soil and hydrocarbon
degrading activity in the Biolog plates and to find an assay
describing bioremediation potential that is easier to perform than
the MPN assay. The linear correlation between the degrading mi-
crobial populations and the metabolic activities (Fig. 4) indicates
the Biolog assay can be a substitute to predict hydrocarbon
degradation in oil-contaminated soil incubated in laboratory at
room temperature. Also, the Biolog assay is more cost-effective and
convenient than the MPN assay.
There are reports that TPH degrading microbial population size
is associated with TPH degradation (Miya and Firestone, 2001;
Krutz et al., 2005; Wu et al., 2013). Wu et al. (2016) reported that
inoculating hydrocarbon degrader Acinetobacter SZ-1 strain
KF453955 enhanced biodegradation of TPH in oil-polluted soil. In
this study, TPH-degrading microbial populations were greater in BA
soil than in BS soil, and TPH degradation efficiency was 58% for BA
treatment and 48% for BS treatment, which suggested the existence
of a positive relationship between hydrocarbon degrader popula-
tion and hydrocarbon degradation efficiency.
In the first 3 weeks of incubation, because TPH and alkane de-
graders grew rapidly, TPH and alkanes were greatly consumed and
degraded. In this stage, the TPH and alkane degrading microbial
population increases were linearly correlated to TPH and alkane
degradation rates. However, after 3 weeks of incubation, the
degrading microbial populations did not increase and even began
to decrease. This led to changes in TPH and alkane usage patterns.
Therefore, there was no correlation between hydrocarbon degra-
dation rate and microbial population when microbial population
was not increased or decreased after 3 weeks of incubation. Infor-
mation about the relationship between hydrocarbon degradation
and microbial population is limited. Lors et al. (2010) reported
changes of bacterial community during bioremediation of PAHs in a
coal tar contaminated soil. Daghio et al. (2015) described hydro-
carbon degrading microbial communities in bench scale aerobic
biobarriers for gasoline-contaminated groundwater treatment.
Hou et al. (2015) reported that petroleum degradation was posi-
tively correlated with specific petroleum degrading
microorganisms.
For predicting hydrocarbon degradation, microbial respiration
as indicated by the amount of evolved CO2 is a popular method
(Liebeg and Cutright, 1999; Aspray et al., 2008; Kharusi et al., 2016).
Palmroth et al. (2006) used the Biolog plates for determining mi-
crobial activity from hydrocarbon-polluted soil. There is little
research on the relationship between hydrocarbon degradation
rate in oil-contaminated soil and hydrocarbon degrading activity in
the Biolog plate. Our result suggests that the Biolog assay can be
used to predict TPH- and alkane degradation rates in oil-
contaminated soil incubated in laboratory at room temperature
when hydrocarbon degrading activity increases.
There was no correlation between PAH degradation rate and
PAH degrader population and activity increase in this study. This
indicates that the three PAH compounds used in the MPN and
Biolog plates do not represent all the PAH in the contaminated soil
for microbial degradation and different microorganisms degrade
different PAHs. Guo et al. (2016) extracted soil with solution before
bioremediation to assess potential degradation of PAH. They re-
ported correlations between the rapidly desorbing fractions of PAH
extracted by solutions before bioremediation and the amounts of
PAH degraded by bioaugmentation in soil.
5. Conclusions
Bioaugmentation with a petroleum-degrading consortium
130 M. Wu et al. / Chemosphere 169 (2017) 124e130
composed of Pseudomonas stutzeri GQ-4 strain KF453954, Pseudo-
monas SZ-2 strain KF453956, and Bacillus SQe2 strain KF453961
and biostimulation with nutrients nitrogen and phosphorus
enhanced TPH and alkane degradation in the oil-polluted soil
incubated in laboratory at room temperature. Linear correlations
were observed for TPH and alkane degradation rates in oil-
contaminated soil and TPH and alkane degrader population in-
creases by the MPN procedure, and TPH and alkane degrading ac-
tivities in the Biolog (MT2) microplates during the first three weeks
of incubation. There are linear correlations between the degrading
microbial populations and the hydrocarbon degrading activities in
the Biolog plates. The MPN and the Biolog plate procedures are
efficient methods for assessing the rates of TPH and alkane, but not
PAH, bioremediation in oil-contaminated soil incubated in
laboratory.
Acknowledgments
This research was supported by the National Natural Science
Foundation of China (No. 21577109), Program for Innovative
Research Team in Shaanxi (PIRT) (Grant No. 2013KCT-13), Project on
the foundation of Natural Science Foundation of Shaanxi Province
(2015JM5163), Key Laboratory Project of Shaanxi Provincial Edu-
cation Department (13JS048), and state and federal funds appro-
priated to The Ohio State University and The Ohio Agricultural
Research and Development Center, Wooster, OH, USA.
References
Aspray, T., Gluszek, A., Carvalho, D., 2008. Effect of nitrogen amendment on respi-
ration and respiratory quotient (RQ) in three hydrocarbon contaminated soils of
different type. Chemosphere 72, 947e951.
Chang, W., Akbari, A., Snelgrove, J., Frigon, D., Ghoshal, S., 2013. Biodegradation of
petroleum hydrocarbons in contaminated clayey soils from a sub-arctic site: the
role of aggregate size and microstructure. Chemosphere 91, 1620e1626.
Chiu, S., Gao, T., Chan, C.S., Ho, C.K., 2009. Removal of spilled petroleum in industrial
soils by spent compost of mushroom Pleurotus pulmonarius. Chemosphere 75,
837e842.
Daghio, M., Tatangelo, V., Franzetti, A., Gandolfi, I., Papacchini, M., Careghini, A.,
Sezenna, E., Saponaro, S., Bestetti, G., 2015. Hydrocarbon degrading microbial
communities in bench scale aerobic biobarriers for gasoline contaminated
groundwater treatment. Chemosphere 130, 34e39.
Dos Santos, L.F., Defrenne, L., Krebs-Brown, A., 2002. Comparison of three microbial
assay procedures for measuring toxicity of chemical compounds: ToxAlert®10,
Cell Sense and Biolog MT2 microplates. Anal. Chim. Acta 456, 41e47.
Garland, J.L., Mills, A.L., 1991. Classification and characterization of heterotrophic
microbial communities on the basis of patterns of community-level sole-car-
bon-source utilization. Appl. Environ. Microbiol. 57, 2351e2359.
Guo, M., Gong, Z., Allinson, G., Tai, P., Miao, R., Li, X., Jia, C., Zhuang, J., 2016. Vari-
ations in the bioavailability of polycyclic aromatic hydrocarbons in industrial
and agricultural soils after bioremediation. Chemosphere 144, 1513e1520.
Hou, J., Liu, W., Wang, B., Wang, Q., Luo, Y., Franks, A.E., 2015. PGPR enhanced
phytoremediation of petroleum contaminated soil and rhizosphere microbial
community response. Chemosphere 138, 592e598.
Kadali, K.K., Simons, K.L., Skuza, P.P., Moore, R.B., Ball, A.S., 2012. A complementary
approach to identifying and assessing the remediation potential of hydro-
carbonoclastic bacteria. J. Microbiol. Methods 88, 348e355.
Kauppi, S., Sinkkonen, A., Romantschuk, M., 2011. Enhancing bioremediation of
diesel-fuel-contaminated soil in a boreal climate: comparison of biostimulation
and bioaugmentation. Int. Biodeter. Biodegr 65, 359e368.
Kharusi, S.A., Abed, R.M.M., Dobretsov, S., 2016. EDTA addition enhances bacterial
respiration activities and hydrocarbon degradation in bioaugmented and non-
bioaugmented oil-contaminated desert soils. Chemosphere 147, 279e286.
Krutz, L.J., Beyrouty, C.A., Gentry, T.J., Wolf, D.C., Reynolds, C.M., 2005. Selective
enrichment of a pyrene degrader population and enhanced pyrene degradation
in Bermuda grass rhizosphere. Biol. Fert. Soils 41, 359e364.
Liebeg, E.W., Cutright, T.J., 1999. The investigation of enhanced bioremediation
through the addition of macro and micro nutrients in a PAH contaminated soil.
Int. Biodeter. Biodegr 44, 55e64.
Llado
, S., Solanas, A.M., de Lapuente, J., Borra s, M., Vin
~ as, M., 2012. A diversified
approach to evaluate biostimulation and bioaugmentation strategies for heavy-
oil-contaminated soil. Sci. Total Environ. 435e436, 262e269.
Lors, C., Ryngaert, A., Pe
rie
, F., Diels, L., Damidot, D., 2010. Evolution of bacterial
community during bioremediation of PAHs in a coal tar contaminated soil.
Chemosphere 81, 1263e1271.
Lu, W., Wang, H., Huang, C., Reichardt, W., 2008. Aromatic compound degradation
by iron reducing bacteria isolated from irrigated tropical paddy soils. J. Environ.
Sci. 20, 1487e1493.
Ma, J., Yang, Y., Dai, X., Chen, Y., Deng, H., Zhou, H., Guo, S., Yan, G., 2016. Effects of
adding bulking agent, inorganic nutrient and microbial inocula on biopile
treatment for oil-field drilling waste. Chemosphere 150, 17e23.
Margesin, R., Schinner, F., 2001. Bioremediation (natural attenuation and bio-
stimulation) of diesel-oil-contaminated soil in an alpine glacier skiing area.
Appl. Environ. Microbiol. 67, 3127e3133.
Miya, R.K., Firestone, M.K., 2001. Enhanced phenanthrene biodegradation in soil by
slender oat root exudates and root debris. J. Environ. Qual. 30, 1911e1918.
Palmroth, M.R.T., Koskinen, P.E.P., Pichtel, J., Vaajasaari, K., Joutti, A., Tuhkanen, T.A.,
Puhakka, J.A., 2006. Field-scale assessment of phytotreatment of soil contami-
nated with weathered hydrocarbons and heavy metals. J. Soils Sediments 6 (3),
128e136.
Pierce, M.L., Ward, J.E., Dobbs, F.C., 2014. False positives in biolog EcoPlates™ and
MT2 MicroPlates™ caused by calcium. J. Microbiol. Methods 97, 20e24.
Salanitro, J.P., Dorn, P.B., 1997. Crude oil hydrocarbon bioremediation and soil eco-
toxicity assessment. Environ. Sci. Technol. 31, 1769e1776.
Sanscartier, D., Laing, T., Reimer, K., Zeeb, B., 2009. Bioremediation of weathered
petroleum hydrocarbon soil contamination in the Canadian High Arctic: labo-
ratory and field studies. Chemosphere 77, 1121e1126.
Taccari, M., Milanovic, V., Comitini, F., Casucci, C., Ciani, M., 2012. Effects of bio-
stimulation and bioaugmentation on diesel removal and bacterial community.
Int. Biodeter. Biodegr 66, 39e46.
Taha, M., Kadali, K.K., AL-Hothaly, K., Smith, A.T., Ball, A.S., Adetutu, E.M., 2015. An
effective microplate method (Biolog MT2) for screening native lignocellulosic-
straw-degrading bacteria. Ann. Microbiol. 65, 2053e2064.
U.S. Environmental Protection Agency, 1986. In: Test Method for Evaluating Solid
Waste, SW-846, third ed., 1A. U.S. EPA, Washington, DC.
U.S. EPA Environmental Protection Agency, 2002. Application, Performance, and
Costs of Biotreatment Technologies for Contaminated Soils. EPA/600/R-03/037,
Battelle Contract No. 68-C-00e185.
Wu, M., Chen, L., Tian, Y., Ding, Y., Dick, W.A., 2013. Degradation of polycyclic aro-
matic hydrocarbons by microbial consortia enriched from three soils using two
different culture media. Environ. Pollut. 178, 152e158.
Wu, M., Dick, W.A., Li, W., Wang, X., Yang, Q., Wang, T., Xu, L., Zhang, M., Chen, L.,
2016. Bioaugmentation and biostimulation of hydrocarbon degradation and the
microbial community in a petroleum-contaminated soil. Int. Biodeter. Biodegr
107, 158e164.
Zhang, K., Hua, X., Han, H., Wang, J., Miao, C., Xu, Y., Huang, Z., Zhang, H., Yang, J.,
Jin, W., Liu, Y., Liu, Z., 2008. Enhanced bioaugmentation of petroleum- and salt-
contaminated soil using wheat straw. Chemosphere 73, 1387e1392.