Marine Pollution Bulletin xxx (xxxx) xxxx

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Marine Pollution Bulletin

journal homepage: www.elsevier.com/locate/marpolbul

Quantitative evaluation of in-situ bioremediation of compound pollution of

oil and heavy metal in sediments from the Bohai Sea, China
⁎
Chuanyuan Wanga,b, , Shijie Hec, Yanmei Zoua, Jialin Liua, Ruxiang Zhaod, Xiaonan Yind,
Haijiang Zhangd, Yuanwei Lia
a
Key Laboratory of Coastal Zone Environment Processes and Ecological Remediation, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai
264003, China
b
Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao 266071, China
c
School of Resources and Environmental Engineering, Ludong University, Yantai 264025,China
d
Yantai Oil Spill Response Technical Center of Yantai Maritime Safety Administration, Yantai 264000, China

A R T I C LE I N FO A B S T R A C T

Keywords: Owing to the semi-enclosed environment of the Bohai Sea, the ecological effects caused by an oil spill would be
In-situ bioremediation significant. A typical in- situ bioremediation engineering project for of oil-spilled marine sediments was per-
PAHs formed in the Bohai Sea and a quantitative assessment of the ecological restoration was performed. The bior-
Heavy mental emediation efficiencies of n-alkane and PAHs in the sediment are 32.84 ± 21.66% and 50.42 ± 17.49% after
Sediment
70 days of bioremediation, and 60.99 ± 10.14% and 68.01 ± 18.60% after 210 days, respectively. After
Evaluation
210 days of bioremediation, the degradation rates of two- to three ring PAHs and four-ring PAHs are
84.44 ± 23.03% and 26.62 ± 43.76%, respectively. In addition, the concentrations of the heavy metals first
increased by 6.00% due to oil spill degradation and release, and then decreased by 72.60% with the degradation
of oil caused by bioremediation or vertical migration. According to the continuous tracking monitoring, the
composition of the microbial community in the restored area was similar to that in the control area and the clean
area in Bohai Sea after 210 days of bioremediation. These results may provide some theoretical and scientific
data to understand the degradation mechanism and assessing the ecological remediation efficiency for oil spills
in open sea areas.

1. Introduction
Driven by the development of agriculture and industry, as well as
the population explosion in China, the problem of marine environment
pollution is getting increasingly serious (Wang et al., 2017). The Bohai
Sea (117°32′ E–122°08′ E, 37°07′ N–40°55′ N) is the primary area for
ocean petroleum exploitation in China. Of the four offshore areas of
China, the oil platforms and submarine pipelines in the Bohai Sea are
the most numerous and dense. The expansion of offshore oil and gas
exploitation has increased the risk of oil spill disasters. For example, an
oil spill accident occurred in the Penglai 19-3 oilfield of the central
Bohai Sea on June 11, 2011. The accident resulted in 840 km2 of pol-
luted seawater area, which is worse than fourth-class according to the
“Standards of water quality in the People's Republic of China (GB 3097-
1997)”. The highest oil content in the seawater was 1280 μg/l, which
was 53-times higher than the background value. According to China
Marine Environmental Quality Bulletin, the petroleum hydrocarbons
become the main pollutants in the Bohai Sea after nitrogen and phos-
phorus (SOAC, 2015). Polycyclic aromatic hydrocarbons (PAHs) are an
important class of persistent organic pollutants with carcinogenic,
mutagenic, and degradation-resistant properties that are of particular
concern (Yunker et al., 2002; Lima et al., 2005; Sofowote et al., 2008).
Heavy metals are another kind of prevalent contaminant in the surface
sediments of the Bohai Sea. Unlike petroleum hydrocarbons, heavy
metals cannot be biologically or chemically degraded. In general, sev-
eral kinds of pollutants, such as PAHs and heavy metals, are always
coexistent in the marine environment. The content of heavy metals in
surface sediments in the central Bohai Sea is low (Xu et al., 2019),
which meets the requirements of Class I standard for marine sediment
quality (Table 1; GB 18668-2002). In the typical Bay of Bohai Sea, Cd is
the most dangerous heavy metal followed by Hg, and the other five
heavy metals, Pb, Cu, As, Cr, Zn, are of low ecological hazard (Xu et al.,
2017). At the polluted area of Penglai 19-3 oil spill accident, Pb, Cr, Ni,
Cu and Cd all reach the Class II standard of marine sediment quality,

⁎
Corresponding author at: Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China.
E-mail address: cywang@yic.ac.cn (C. Wang).

https://doi.org/10.1016/j.marpolbul.2019.110787
Received 24 June 2019; Received in revised form 23 November 2019; Accepted 25 November 2019
0025-326X/ © 2019 Published by Elsevier Ltd.

Please cite this article as: Chuanyuan Wang, et al., Marine Pollution Bulletin, https://doi.org/10.1016/j.marpolbul.2019.110787
C. Wang, et al. Marine Pollution Bulletin xxx (xxxx) xxxx

Table 1
Concentration of heavy metal in sediment (mg/kg dw) and sediment quality guidelines of China for metal.
Item Hg Cd Pb Zn Cu Cr As

a
Standard for sediment quality Class I 0.20 0.50 60.0 150.0 35.0 80.0 20.0
Class II 0.50 1.50 130.0 350.0 100.0 150.0 65.0
Class III 1.00 5.00 250.0 600.0 2000 270.0 93.0
Bohai Seab Nd 0.06–0.22 8.7–32.3 28.7–61.2 6.7–34.6 Nd Nd
Bohai Seac Nd 0.23–0.44 42.8–73.6 Nd 38.1–61.9 89–219.1 20.6–253

Nd: Not available.

a
National Standard of P.R. China (2002).
b
Xu et al. (2019).
c
Li et al. (2019).

which is harmful to the marine ecological environment of Bohai Sea

(Table 1; Li et al., 2019). In the Bohai Sea, due to its semi-closed gulf
properties, the ecological effect caused by oil spill and heavy metal is
significant and cannot be ignored (Li et al., 1994; Hu et al., 2011; Liu
et al., 2017; Li et al., 2019).
Currently, for the emergency treatment of marine oil pollution,
especially sudden oil spill accidents, physical interception, recovery,
and chemical dispersants are used to deal with the marine surface
seawater during the initial stage of an oil spill accident. To reduce the
long-term environmental impact of oil spill, microorganisms play a
crucial role in removing oil contaminants during the middle and late
stage of an oil spill accident (Atlas and Hazen, 2011; Vergeynst et al.,
2018). Biological treatment (bioremediation) is recognized as the most
preferred method for the removal of oil because it is generally cost
effective and environmentally friendly (Braddock et al., 1997; Ghazali
et al., 2004; Zhang et al., 2011; Borah and Yadav, 2017). In contrast,
Fig. 1. Process of launching the remedy agents into the sea during field bior-
microorganisms cannot degrade and destroy heavy metals, but they can
emediation.
alter the migration and transformation of heavy metals in marine en-
vironments by affecting their chemical or physical properties. Because
the marine environment is special, marine construction requires strict results will add to the current understanding of the degradation me-
weather and sea conditions, construction vessels, and maritime opera- chanism, assess the ecological remediation efficiency, and provide
tional experience. It is difficult to spread bioremediation remedy agents theoretical and scientific data for the ecological remediation of marine
(RAs) on contaminated marine sediments. Therefore, currently, most of pollutants.
the bioremediation engineering projects for marine organic pollution
have primarily focused on nearshore areas. In open sea areas, microbial 2. Materials and methods
remediation methods and application efficacies for sediment pollution
are very uncertain (Carriger and Barron, 2011) and require further 2.1. In-situ microbial remediation engineering
study. The primary component of crude oil is organic matter. In addi-
tion, there are approximately 45 kinds of metal elements in crude oil. A screening and resuscitation of indigenous oil-degrading bacteria
Pollution studies have revealed elevated levels of Zn, Cu, Pb, Cr, Ni, and in the Bohai sediments were conducted, which avoided the introduction
V in the sediments and some species of fauna from Niger Delta, Nigeria, of alien species, avoided causing large-scale biological pollution and
indicating inputs from petroleum exploration and exploitation (Horsfall avoided destroying the marine ecology. Then the effect of oil de-
and Spiff, 2002; Howard et al., 2006). Therefore, it is important to gradation due to resuscitated bacteria was evaluated, and the optimal
determine not only the concentration and distribution of petroleum mixing proportion of the different bacteria was obtained. The re-
hydrocarbons but also those of heavy metals in marine sediments after mediated region covered an area of 0.76 km2, in which 370 tons of RAs
such in situ microbial remediation project work. were uniformly thrown onto the ocean floor containing oil-polluted
In this study, a typical oil spill pollution area in the central Bohai sediments via 5 kg degradable mesh bags. The strains used were
Sea is selected and in situ bioremediation engineering of oil-soaked Acinetobacter calcium acetate, Pseudomonas putida and Salfobacillus. Oil-
sediments is performed. The primary engineering work covers the re- degrading bacteria were adsorbed on powdery zeolite/granular zeolites
lease, transshipment, shipment, marine delivery of microbial remedia- surfaces and then wrapped with poly-γ glutamic acid. The physical
tion products, and residual pollutant tracking and monitoring (Fig. 1). parameters of the flow field, wind field and effective wave in the de-
A profound knowledge of the biodegradation potential of spilled oil in signated remediation sea area and its surrounding sea area were mon-
deep seas is thus essential for an ecological risk assessment, as well as itored and tracked to provide reference for the design of the area and
for developing contingency plans for oil spill responses after an oil spill monitor the speed of fungicide delivery. The construction of microbial
accident. To the best of our knowledge, there have been few assess- remediation products for warehousing, transshipment, shipment and
ments performed on the effectiveness assessment of in situ microbial delivery were performed at sea. For more detailed information on this
remediation of residual oil and heavy metals in marine sediments in the in-situ bioremediation engineering of oil-soaked marine sediments refer
Bohai Sea. The specific objectives of this study were to 1) quantitatively to Zhao et al. (2018).
evaluate the potential for biodegradation of residual oil in marine se-
diments in deep sea areas according to in situ bioremediation en-
2.2. Study area and sampling methods
gineering methods mentioned above, and 2) determine the relationship
between heavy metals distribution and oil spill biodegradation. The
A background investigation of the repaired sea area was completed

2
C. Wang, et al.
Fig. 2. Study area and the location of sampling sites in the Bohai Sea.
before the bioremediation engineering project was applied. Prior to
conducting the ecological bioremediation, the surface sediment
(0–5 cm) was collected in August 2015 using an Ekman style stainless
steel grab sampler (0.1 M3). The location of the sampling station is
shown in Fig. 2. BH21 is non-oil and gas acquisition area and was used
as the control station without bioremediation. In this study, only some
representative stations were located near oil platforms (the PL4, PL5,
PL6, PL11 and PL17 stations), and these were selected for quantitative
evaluation of in-situ bioremediation based on a remediated region of
0.76 km2. After 70 and 210 days of bioremediation, the surface sedi-
ment samples in the same stations near oil platforms and control area
were collected from the polluted area, and the oil content was de-
termined to evaluate the ecological bioremediation efficiency.
2.3. Analytical procedures
2.3.1. Extraction and fractionation
Sediment samples were freeze-dried, extracted, and analyzed after
the biodegradation experiment. Detailed information on the sample
treatment and analysis had also been reported in previous studies (Hu
et al., 2011; Wang et al., 2011, 2017). Briefly, extraction was performed
using the Soxhlet method with dichloromethane and methanol (93:7) as
the solvent mixture for 72 h. The aliphatic hydrocarbons, aromatic
hydrocarbons and resins were separated by column chromatography
using activated alumina and silica gel, and gradient solvents as eluent:
ligarine, ligarine/dichloromethane (3:9, v/v) and MeOH, respectively.
Recoveries of crude oil groups varied from 89.54% to 93.75%. In ad-
dition, the recovery of crude oil components increased by 4.16% and
was affected by the addition of Tween-20.
2.3.2. Instrumental analysis of the oil spill
The instrumental analysis was performed on a Hewlett-Packard
6890 gas chromatograph interfaced with a Hewlett-Packard 5973 mass-
selective detector made by Agilent (USA). Helium was used as the
carrier gas. n-Alkane and PAHs were identified using the GC–MS in full
scan mode. Sample extracts were injected in a splitless mode onto a H-5
fused capillary column (30 m × 0.2 mm i.d) coated with 0.25 μm thick.
Individual n-alkanes were identified based on the retention time of the
3
Marine Pollution Bulletin xxx (xxxx) xxxx
authentic standards (nC10-40, Sigma). Alternately, individual PAHs were
quantified based on the retention time and the m/z ratio of an authentic
PAH mixed standard (Sigma). The relative abundance was calculated
from the respective mass chromatogram peak areas. Five surrogate
standards (naphthalene-d8, phenanthrene-d10, dibenzothiophene-d10,
fluoranthene-d10, and pyrene-d10) were added to all the samples to
monitor the matrix effects. The average recoveries of surrogate stan-
dards varied from 81.6% to 92%. For the detailed analytical procedure,
conditions, and quality control refer to previous studies (Hu et al.,
2011; Wang et al., 2017).
2.3.3. Instrumental analysis of heavy metals
The samples were freeze-dried, crushed, and then sieved through a
100-mesh sieve. Approximately 0.1 g dried surface sediment samples
were digested in 5 mL aqua regia (HF: HNO3: HClO4 = 5:2:1) in Toflon
cans using closed microwave digestion. They were then heated at
180 °C until the samples turned grey. Then 4 mL HNO3 was added into
the cooled samples. The residues were diluted with deionized water to
50 mL to determine the concentrations of the heavy metals. The con-
tents of the heavy metals in the sample solutions were determined using
inductively coupled plasma mass spectrometry (ICP-MS; erkin Elmer
NexION 300Q, USA). The heavy metal concentrations were obtained
using an average of three repeated measurements for each sample.
2.4. Data analysis
In this study, the average measurement value of the triplicate
samples found using GC-Ms and ICP-MS was used to develop figure and
table to show the regulation of data change. To evaluate the precision of
the analysis data, three replicates of each sample were tested every five
samples. Repeat values of the sample were presented as the mean ±
standard deviation (SD). The relative standard deviation (RSD) of the
replicates was 5.0%. For this sample, the average value was used for the
convenience of drawing.
The single factor index method was used to classify the water
quality by comparing the measured data with a standard, and to select
the worst type of water quality as the evaluation result (Liu et al.,
2017). This method was also used to evaluate the pollution status of
C. Wang, et al. Marine Pollution Bulletin xxx (xxxx) xxxx

petroleum hydrocarbons in the sediments in this study. The single 0d 70d 210d BR-70d BR-210d

Residual rate of n-alkane(%)

factor pollution index (PIi) is used to characterize the pollution level of
(a)

Biodegradation rate (%)

a single pollutant. The equation used to calculate PIi is as follows (Li 120 100
et al., 2016; Liu et al., 2017): 100 80
PIi = Ci/ Si, 80 60
40
where PIi is Standard Index of term i pollutants; Ci is the measured 60
20
concentration (average value) of term i pollutants; Si, is the evaluation 40 0
criterion of term i pollutants. The contamination degrees were classified
20 -20
as low degree (PIi < 1), moderate degree (1 < PIi < 3), high degree
(3 < PIi < 6) and heavy high degree (PIi > 6). 0 -40
BH-21 PL5 PL6 PL17 PL11
3. Results and discussion Station ID

3.1. Environmental quality assessment of petroleum hydrocarbons pollution 0d 70d 210d BR-70d BR-210d
(b)

Residual rate of PAHs(%)

Biodegradation rate (%)

According to previous studies on the core sediment (0–60 cm in 120 100
depth) in the central of Bohai Sea (Liu et al., 2017), the content of total 100 80
petroleum hydrocarbons (TPHs) in sediments below 40 cm (i.e., before
the 1930s) was low (20.49 × 10−6). Hence, this level was used as the 80 60
background value in the study area due to the weaker influence of 60 40
human activities on the Bohai Sea. The assessment of petroleum hy- 40 20
drocarbon concentration and pollution in the surface sediments without
ecological remediation is shown in Fig. 3. The oil pollution level in the
20 0
sediment increased gradually with a decrease in the distance from non- 0 -20
oil fields to near oil platforms. The pollution levels of the non-oilfield BH-21 PL-5 PL-6 PL-17 PL-11
stations (BH21 = 0.61, BH28 = 0.91) showed no pollution or low Station ID
pollution levels. The pollution levels of the near-platform stations (BH-
8 = 3.98, BH-9 = 5.03) showed high pollution levels. The PI of the oil Fig. 4. Residual rate and biodegradation rate (BR) of n-alkane (a) and PAHs (b)
platform stations (PL5, PL6, PL11, and PL17) ranged from 23.77 to with the bioremedition time.
Note: 0d: Before bioremediation; 7d: Residual rate 70 days after bioremedia-
41.97, indicating heavy pollution levels.
tion; 210 days: Residual rate 210 days after bioremediation; BR-70d:
Biodegradation rate 70 days after bioremediation; BR-210d: Biodegradation
3.2. Distribution of n-alkane during the bioremediation rate 210 days after bioremediation.

The most abundant fraction in crude oil is alkanes, which are more
biodegradation, the bioremediation efficiency of n-alkane in the sedi-
likely to be utilized by microbes and easier to degrade (Nikolopoulou
ment were 32.84 ± 21.66% and 60.99 ± 10.14% after bioremedia-
et al., 2013; Wang et al., 2016). The oily sediment samples showed a
tion for 70 days and 210 days, respectively. Compared to long-chain n-
typical non-biodegraded profile, having a full suite of n-alkanes. How-
alkanes, compounds with low- to middle-molecular weight are gen-
ever, the samples (70 days and 210 days after bioremediation) showed
erally transformed and biodegraded at higher rates by the microbes.
significantly lower abundance of n-alkanes. The residual rate of total n-
Thus, the low molecular weight to high molecular weight n-alkane ratio
alkanes (T-ALK) in the sediment from the bioremediation area varied
(LMW/HMW) decreased greatly (Fig. 5a). Since the disappearance of
from 33.87% to 80.36% (avg. 62.11%, 70 days) and from 29.15% to
light components occurred primarily during the initial stage of de-
70.12% (avg. 39.00%, 210 days), respectively (Fig. 4a). However, the
gradation, the change in the LMW/HMW ratio further decreased, with
residual rate of n-alkane of the control station (BH21) without bior-
little magnitude after 210 days. In addition, the degree of oil biode-
emediation was very little. In the biodegraded oil residue, there was a
gradation was also assessed using the Pr/n-C17 (Didyk and Simoneit,
significant increase in the relative abundance of unresolved complex
1989; Lemkau et al., 2010). According to the already established pat-
mixtures (UCM). According to the analysis of hydrocarbon
tern, the higher Pr/n-C17 ratio for biodegraded oils (BR-70d, BR-210d)
than their non-biodegraded counterparts (0d) was observed (Fig. 5b).
Oil Platform
1000 45 Compared with the background survey, it was found that the con-
TPH（ppm）

900 TPH 40 centrations of oil spilled pollutants displayed a significant reduction

800 PI 35 trend. The stations nearest to the drilling platform were PL5 and PL6,
700 30 and it was these areas that displayed the best quantitative evaluation
600 25
500 Near Oil for spilled-oil bioremediation. In addition, the concentration of spilled
PI

400 20 oil decreased further with an increase in the degradation time.

Platform 15
300 Non oilfield
200 10
100 5 3.3. Evolution of the chemical composition of PAHs during the
0 0 bioremediation

It is well known that PAHs pose serious threats to coastal habitats

Station ID due to their more toxic effects (Pongpiachan et al., 2018). A total of 16
PAHs have been listed as toxic pollutants by the US Environmental
Fig. 3. Concentrations and pollution index of the petroleum hydrocarbons in Protection Agency (EPA) (Perelo, 2010). Detailed knowledge of PAHs
the surface sediments. biodegradation can provide some useful information for further un-
Note: TPH means total petroleum hydrocarbons; PI means the single factor derstanding their environmental fate and potential eco-toxicological
pollution index. effect. In this study, the total concentration of 16 USEPA priority PAHs

4
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2.00 evaporation, but the loss of other PAHs was most likely due to biode-
0d gradation. The results further supported that the degree of the PAHs
BR-70d degradation decreased with an increasing in the number of aromatic
LMW/HMW ALK

1.50 BR-210d rings.

3.4. Relationship between the heavy metal distribution and oil spill
(a) bioremediation
1.00
Heavy metals such as Cu, Zn, Ni, Cr, Fe, and Mn are essential mi-
0.50 cronutrients for various organisms including microorganisms, plants
and animals. The concentrations of heavy metals changed significantly
after remediation. From the variety of heavy metal concentrations, it
0.00 can be seen that the concentration of heavy metals increased from
600.13 mg/kg on a dry wt. basis (dw) to 635.70 mg/kg dw in the
BH21 PL17 PL5 PL6 PL11
bioremediation project after 70 days. Then a sharp decrease occurred
Station ID from 635.70 mg/kg dw to 174.15 mg/kg dw after 210 days of in-situ
bioremediation work. However, the Cu and Pb concentration showed a
1.50 continuous decrease with a relatively stable change (Fig. 7). Compared
0d with the environmental background value of the Bohai sediment (Li
BR-70d et al., 1994), the content of Pb, Cu and Cd in the area was higher than
BR-210d that of its background value prior to the bioremediation project. Among
1.00 them, concentrations of Pb were approximately three to five times the
Pr/n-C17

background value, and concentrations of Cu and Cr were two to three

(b)
0.50
0.00
BH21 PL17 PL5 PL6 PL11
Station ID
Fig. 5. Variation of LMW/HMW ALK (a) and Pr/n-C17 (b) with the bior-
emediation time.
Note: 0d: Before bioremediation; BR-70d: 70 days after bioremediation; BR-
210d: 210 days after bioremediation.
(T-PAHs) in the oil spill pollution area ranged from 93.11 to 273.42 ng/
g dw with a mean concentration of 155.28 ng/g dw. After bior-
emediation, the residual rate of T-PAHs in the sediment from the
bioremediation area varied from 24.38% to 63.11% (avg. 49.58%,
70 days) and from 12.69% to 54.69% (avg. 31.99%, 210 days), re-
spectively (Fig. 4b). However, the residual rate of PAHs without the
bioremediation from the control station (BH21) was little.
Major compositional change in the PAHs observed in this study was
consistent with previous reports. The rate of biodegradation of the
different aromatic hydrocarbon classes decreased with an increasing
number of aromatic rings and with an increasing number of alkyl
substituents (Wang and Fingas, 2003; Huang et al., 2004; Haritash and
Kaushik, 2009; Chen et al., 2013). For some 16 PAHs detected in the
oily sediment 70 days after bioremediation, the degradation rates of
two- to three-ring PAHs and four-ring PAHs were 65.04 ± 32.39 ng/g
on a dry wt. basis (dw) and 43.21 ± 30.01 ng/g dw, respectively. For
the oily sediment 210 days after bioremediation, the degradation rates
of two- to three-ring PAHs and four-ring PAHs were 84.44 ± 23.03 ng/
g dw and 26.62 ± 43.76 ng/g dw, respectively. However, some of the
more recalcitrant compounds of 5–6 ring PAHs were largely un-
degraded after bioremediation (Fig. 6). In contrast, some five to six ring
PAHs increased after bioremediation, which may have been attributed
to the atmospheric deposition of PAHs or new marine water pollution.
Even though two-ring PAHs (e.g., naphthalene) dominated in the crude
oil, the much higher solubility and volatility than heavier PAHs caused
much more profound losses (Liu et al., 2012; Yin et al., 2015; Duan
et al., 2018). The loss of naphthalene may have been caused by
times. After 210 days of in-situ bioremediation work, the concentra-
tions of Pb, Cr, Cu, Ni, V, and Cr were still higher than the background
values, but they all were within the national standard.
Generally, bioremediation involves adsorption of the contaminants
on organic matter or reduction of the metal bioavailability by micro-
organisms. Heavy metals cannot be degraded by microorganisms, but
microorganisms have a certain resistance and detoxification effect on
heavy metals, and they can adsorb and transform heavy metals (Jacob
et al., 2018). With the exception of organic matter, there are approxi-
mately 45 kinds of metal elements in crude oil (Luo et al., 2003). In the
Bozhong oilfield in the Bohai Sea, the average concentrations of Ni, V,
Cr, Cu, and Pb were 5.15 mg/Kg, 0.59 mg/kg, 1.71 mg/kg, 0.99 mg/kg,
and 0.29 mg/kg, respectively (Li et al., 2010). The primary form of
heavy metals in petroleum form complexes with organic matter
(Barančíková and Makovníková, 2003). With the addition of petroleum
hydrocarbon degrading bacteria, the contents of some organic matter
and light hydrocarbon components in crude oil are degraded (Wang
et al., 2011; Zhao et al., 2018). This process leads to a release of heavy
metals from crude oil or other organic matter, leading to an increase in
heavy metal (Ni, V, Cr, Cd) concentrations (Fu, 2014). In this study, as
time went on to 70 to 210 days after bioremediation work, the de-
graded components in the oil were degraded completely. In addition,
some bacteria began to play a role in the physical precipitation and bio-
deposition of the heavy metals, resulting in a reduction of heavy metal
(Ni, V, Cr, Cd) concentration. This decreasing trend in heavy metal
concentration may have been due to physical precipitation, chemi-
sorption, bio-deposition, and other processes (Dong et al., 2013; Jacob
et al., 2018). For example, the use of microbial biomass is a potential
alternative for the uptake of heavy metals from different polluted media
(Arivalagan et al., 2014; Dhanarani et al., 2016).
Because the depth of sampling was approximately 5 cm, the vertical
migration after heavy metal activation may have be one of the reasons
for the reduction in heavy metals. Pb and Cu have strong vertical mi-
gration ability, and the vertical migration of other heavy metals is re-
latively stable. This could also explain why the content of Pb and Cu in
the sediments had been decreasing during the remediation process, in
spite of degradation and release of crude oil. Based on the analysis, the
final content of the heavy metals was lower than the concentration of
heavy metals in the sediments prior to the restoration. Some important
bacterial strains such as Bacillus and Pseudomonas sp. have been widely
used for the removal of heavy metals from wastewater and soil because
of their high metal binding affinities (Bachate et al., 2013; Ullah et al.,
2015).

5
C. Wang, et al.
Fig. 6. Relative degradation efficiency of PAHs with different numbers of rings with the bioremedition time.
Fig. 7. Biodegradation rate of heavy metals and PAHs with bioremediation
time.
3.5. Assessment of the current bio-environmental remediation
After 70 days of ecological restoration, the variation range in the
total bacterial count in sediments was 5.76 × 107–2.31 × 109 (CFU/g),
with an average value of 1.33 × 109 CFU/g, which was significantly
higher than that of 5.06 × 106 CFU/g prior to restoration. After
210 days, Bacillus spp. and Haimonas spp. were still detected in the
microbial remediation area, but the proportions of strains in each sta-
tion were greatly reduced. According to the results of high-throughput
microbial analysis of the two tracking monitoring, the oil pollution
remediation of sediments achieved good results for the microbial
community and for ecological restoration. The composition of the mi-
crobial community in the restored area was similar to that in the control
area and the clean area. However, in terms of the microbial diversity
and abundance, the microbial diversity and abundance of most stations
in the restored area surpassed that in the unrestored area, and the
microbial community structure was effectively restored.
Author contributions section
Chuanyuan Wang: Data curation, Writing- Original draft
6
Marine Pollution Bulletin xxx (xxxx) xxxx
preparation. Shijie He: Data analysis and Editing. Yanmei Zou:
Experiment. Jialin Liu: Data analysis. Ruxiang Zhao, Xiaonan Yin,
and Haijiang Zhang: Reviewing and Revising. Yuanwei Li: Data
analysis.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
We thank LetPub (www.letpub.com) for its linguistic assistance
during the preparation of this manuscript. This study was co-supported
by Shandong Province Key Research and Development Program of
China (Public Welfare Project) (2019GSF109081); Key Projects in the
Yantai Science & Technology Pillar Program (2018ZHGY079), Project
of on-site sediment microbial remediation of public area of central
Bohai Sea, North China Sea Branch of State Oceanic Administration
(QDZC20150420-002), and Inter cooperation, CAS, Chinese-foreign
cooperation in key projects (133337KYSB20160002).
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