De
g
radation of Crude Oil in Contaminated Sediment b
y
P
seudomonas Putida Y3 Strain
Jie He
1,2*
, Xiaoru Fan
1,2,
, Huan Liu
1,2
, Yuan Liu
1,2
and Haifeng Wei
1,2
1
College of Marine Technology and Environment,Dalian Ocean University,Dalian 116023, PR China;
2
Key Laboratory of Nearshore Marine Environmental Science and Technology in Liaoning Province,Dalian Ocean
University,Dalian 116023, PR China.
Keywords: Microbial remediation, Pseudomonas putida Y3 strain, crude oil in sediment, degradation
Abstract: In this study, Pseudomonas putida Y3 strain was used as the test object, and the sediment contamination test
was conducted at different crude oil concentrations (0, 4000, 8000, 12000, 16000 and 20000 mg/kg) in
order to study the remediation effect of microorganisms on crude oil-contaminated sediments. The
degradation rate of crude oil in sediment after the addition of Y3 strain was determined, and the degradation
of crude oil content in contaminated sediment by Y3 strain was analyzed. The results showed that the
growth of Y3 strain was affected by crude oil pollution, and the exogenous bacteria had an adaptive positive
process to the sediment environment. After the microorganisms adjusted themselves, they were able to
adapt to the new environment. The Y3 strain had an effect on the degradation of sediment crude oil. With
the increase of sediment crude oil concentration, the degradation rate constant gradually increased. When
the crude oil concentration was 12000 mg/kg, the rate constant of the experimental group was the highest
(10.584). Studies have shown that Y3 strain has the remedial function for crude oil-contaminated sediments.
1 INTRODUCTION
In recent years, a large amount of petroleum-contam
inated sediment has been produced due to leakage, s
pillage, submergence and other causes in the develo
pment and production activities such as oil explorati
on, transportation, smelting and in the treatment pro
cess of oilfield wastewater. These oil pollutants spre
ad in the environment through various means such a
s volatilization, infiltration into groundwater, and pl
ant absorption, thereby causing serious soil environ
mental pollution problems (Wang et al., 2017; Zhan
g et al., 2018; Daniel and Philip, 2014). The microbi
al remediation of crude oil-contaminated sediment m
ainly relies on microbial metabolism to remove poll
utants. It has the advantages of low cost, convenient
operation and no secondary pollution, which has bee
n rapidly developed in recent years (Xu, 2016; Crisa
fi et al., 2016). The study by Yanfei et al. showed th
at arbuscular mycorrhizal fungi (AMF) can stimulat
e soil microbial activity, improve soil structure and p
lay an active role in petroleum-contaminated soil re
mediation (Lu and Lu, 2015). The study by Mehdi et
al. showed that the paraquat bacteria in Boko Island
had a certain effect on petroleum degradation, whic
h can reach 95% (Mehdi et al., 2014).
In this paper, Pseudomonas putida Y3 strain (Wa
ng et al., 2012) isolated from Panjin beach in Liaoni
ng province was used as the test object. The Y3 strai
n in crude oil-contaminated sediment and the degrad
ation efficiency of sediment contaminated by differe
nt concentration of crude oil were measured to evalu
ate the remediation of oil contaminated sediment by
Y3 strain.
2 MATERIALS AND METHODS
2.1 Sediment
The sediment was collected from the coastal beach
of Panjin, Liaoning Province, dried in a drying oven
at 105°C for 1 hour, crushed, sieved and weighed in
200 g per portion, and placed in a pot for use.
38
He, J., Fan, X., Liu, H., Liu, Y. and Wei, H.
Degradation of Crude Oil in Contaminated Sediment by Pseudomonas Putida Y3 Strain.
In Proceedings of the International Workshop on Environment and Geoscience (IWEG 2018), pages 38-43
ISBN: 978-989-758-342-1
Copyright © 2018 by SCITEPRESS – Science and Technology Publications, Lda. All rights reserved

2.2 Bacterial Strain Used in This Study
The Pseudomonas putida Y3 strain was a laboratory
-preserved strain isolated from Panjin beach of Liao
ning province and cultured in inorganic salt medium
with diesel as the sole carbon source (Wang et al., 2
012).
2.3 Experimental Setup
The crude oil was quantitatively stirred in each sedi
ment pot, crushed and sieved so that the crude oil co
ncentration was 0 (CK), 4000, 8000, 12000, 16000 a
nd 20000 mg/kg. To make the crude oil mix well in t
he sediment, 20 g of crude oil was mixed into 980 g
sediment to prepare an initial sediment sample with
a crude oil concentration of 20000 mg/kg. Then a tot
al of 200 g of sediment was filled in each pot, ie the
soil ratio was as shown in Table 1. The sediment we
re poured into pot and mixed evenly. The devices w
ere equilibrated for one month and three concentrati
ons were set for each concentration. In each concent
ration experiment group, 20 mL of Pseudomonas pu
tida Y3 strain with a concentration of 1.0×10
5
cells/
mL were added and distributed homogeneously. The
same concentration group without adding bacteria
was used as a control sample. After adding quantitati
ve Y3 strain to the sediments of crude oil concentrati
on gradient as described above, the contents of Y3 st
rain in sediments were counted using a plate with di
esel as the sole carbon source, and the crude oil cont
ent in sediments were determined by Ultraviolet spe
ctrophotometry (Wang and Hu, 2010) at 30 d, 60 d,
90 d, 120 d, 150 d and 180 d. The rate of oil degrada
tion was calculated. Three parallel experiments were
set up for each experimental group.
Table 1: The ratio of experiment sediment.
Crude oil
concentration
(mg/kg)
0 4000 8000 12000 16000 20000
Initial sediment
sample(g)
0 40 80 120 160 200
Pure sediment(g) 200 160 120 80 40 0
2.4 Measurement Methods
The Pseudomonas putida Y3 strain was counted usi
ng a plate with diesel as the sole carbon source; The
crude oil content in sediment were determined by Ul
traviolet spectrophotometry (GB17378.5-2007) (Wa
ng and Hu, 2010) .
2.5 Calculation Method
Number of Pseudomonas putida Y3 strains: The ave
rage number of colonies on the three plates at the sa
me dilution was calculated according to the followin
g formula.
Colony forming units (cfu) per milliliter = avera
ge number of colonies with three replicates of the sa
me dilution × dilution factor × 5
The rate of oil degradation was calculated to:
%= (C
0
– C
i
) /C
0
×100%
C
0
-------initial petroleum concentration (mg/kg);
C
i
-------residual diesel concentration (mg/kg)
Table 2: The quantity of Pseudomonas putida Y3 strain in different concentrations of oil polluted sediment (cfu/g).
Oil content(mg/kg)
30d 60d 90d 120d 150d 180d
0
CK(×10
2
) 6.50
Aa
3.50
Ba
3.00
Ca
4.90
Da
3.90
Ea
2.00
Fa
+(×10
6
) 21.0
A
b
7.00
B
b
1.70
C
b
3.00
D
b
1.70
C
b
2.60
E
b
4000
CK(×10
2
) 9.00
Aa
2.75
Ba
3.00
Ca
4.10
Da
3.10
Ea
2.60
Fa
+(×10
6
) 19.0
A
b
3.75
B
b
2.40
C
b
2.40
C
b
2.50
D
b
2.50
D
b
8000
CK(×10
2
) 8.00
Aa
2.50
Ba
3.10
Ca
3.50
Da
2.50
Ba
2.50
Ba
+(×10
6
) 19.5
A
b
4.00
B
b
1.50
C
b
1.70
D
b
2.30
E
b
2.50
Fa
12000
CK(×10
2
) 5.50
Aa
2.25
Ba
2.10
Ca
3.30
Da
2.30
Ea
1.90
Fa
+(×10
6
) 17.0
A
b
5.00
B
b
2.40
C
b
1.40
D
b
1.10
E
b
2.20
F
b
16000
CK(×10
2
) 10.0
Aa
2.50
Ba
1.70
Ca
2.70
Da
1.70
Ca
2.40
Ea
+(×10
6
) 13.5
A
b
5.75
B
b
2.50
C
b
1.60
D
b
1.50
E
b
2.40
Fa
20000
CK(×10
2
) 12.0
Aa
1.75
Ba
1.40
Ca
2.40
Da
2.00
Ea
1.90
Fa
+(×10
6
) 12.0
A
b
4.25
B
b
1.12
C
b
1.30
D
b
1.20
A
b
2.30
E
b
CK : no bacteria, control sample; + : add bacteria, experimental sample.
the different capital (column)/lowercase (line) letters indicated significant differences between groups (p < 0.05); the
same capital (column)/lowercase (line) indicated no significant differences between groups (p > 0.05).
Degradation of Crude Oil in Contaminated Sediment by Pseudomonas Putida Y3 Strain
39

3 RESULTS
3.1 Influence of Crude Oil Pollution on
the Number of Pseudomonas Putida
Y3 Strain in the Sediment
From Table 2, it can be seen that the cell number of
Y3 strain in the experimental group was higher than
that of the control group during the whole experime
nt, and the number was basically stable. This showe
d that the exogenous bacteria have been stabilized an
d balanced in the sediment. This may be due to the f
act that the exogenous bacteria have a certain ability
to adapt to changes in the environment, or due to the
time of September, so the conditions of various asp
ects of sediment are suitable for bacterial growth. At
the end of 180 d, the cell number of Y3 strain in ex
perimental group at the crude oil concentration of 0
mg/kg, 4000 mg/kg and 8000 mg/kg reached the hig
hest value.
Figure 1: The degradation rate of crude oil changes with
the concentration in the polluted sediment (Left: control
group; right: experimental group).
3.2 Degradation of Crude Oil
Contaminated by Pseudomonas
Putida Y3 Strain
3.2.1 Effect of Pseudomonas Putida Y3
Strain on the Degradation Rate of Oil in
Sediment
Figure 2: The degradation rate of crude oil changes with
the time in the polluted sediment (Left: control group;
right: experimental group).
Figure 1 and 2 showed the degradation rate of crude
oil in sediment with concentration and time, respecti
vely, compared with the control group, from the con
centration point of view, with the increase of crude o
il concentration, the degradation rate of crude oil inc
reased first and then decreased. At 150 d, the concen
tration of crude oil in sediment increased from 0 mg/
kg to 8000 mg/kg, and the crude oil degradation rate
increased to 17% - 20%. As the concentration conti
nued to increase, the degradation rate of crude oil de
creased. When the crude oil concentration was 1200
0 mg/kg, the degradation rate of crude oil slightly de
creased. At a concentration of 16000 mg/kg, the cru
de oil degradation rate rapidly decreased to about
6%. This showed that when the crude oil in sedimen
t concentration was 12000 - 6000 mg/kg, it had the g
reatest impact on the Y3 Strain. When the concentrat
ion is 20000 mg/kg, the degradation rate of crude oil
0 30 60 90 120 150 180
0.0
0.5
1.0
1.5
2.0
2.5
3.0
degradation rate of crude oil (%)
Time (d)
0 mg/kg
4000 mg/kg
8000 mg/kg
12000 mg/kg
16000 mg/kg
20000 mg/kg
0 30 60 90 120 150 180
0
3
6
9
12
15
18
21
degradation rate of crude oil (%)
Time (d)
0 m g/kg
4000 mg/kg
8000 mg/kg
12000 mg/kg
16000 mg/kg
20000 mg/kg
0 4000 8000 12000 16000 20000
0
5
10
15
20
degradation rate of crude oil (%)
the concentration of crude oil (mg/kg)
30d
60d
90d
120d
150d
180d
0 4000 8000 12000 16000 20000
0.0
0.5
1.0
1.5
2.0
2.5
3.0
degradation rate of crude oil (%)
the concentration of crude oil (mg/kg)
30d
60d
90d
120d
150d
180d
IWEG 2018 - International Workshop on Environment and Geoscience
40

was reduced to about 4%. It showed that the contam
ination of high-concentration crude oil in sediment h
as a great impact on Y3 strain, resulting in damage t
o the crude oil pollution resistance and elimination f
unction. From a time perspective, the degradation rat
e of crude oil in each concentration group increased
with time, and the speed was even. The degradation
rate of the 8000 mg/kg group was always the highest,
indicating that the Y3 strain had the strongest adapt
ability to this concentration of crude oil and the degr
adation was most effective. The crude oil degradatio
n rate of the experimental group was greater than tha
t of the control group.
3.2.2 Effect of Pseudomonas Putida Y3
Strain on Crude Oil Content in
Sediment
Figure 3 showed the effect of Pseudomonas putida
Y3 Strain on crude oil concentration in sediment. As
can be seen from the figure, during the experiment,
the crude oil content in sediment of each experiment
al group gradually decreased. The degradation rate, r
eaction order, reaction rate constant and kinetic equa
tion of crude oil in sediment were listed in Table 3.
The greater the reaction rate constant, the higher the
reaction rate, indicating that the Y3 strain has greate
r influence on the degradation of crude oil in sedime
nt. From Table 3, the reaction rate of the experiment
al group at the concentration of 12000 mg/kg was th
e highest, which was 10.584, indicating that the reac
tion rate was the fastest.
4 DISCUSSION
4.1 Effect of Crude Oil Concentration
on the Number of Pseudomonas
Putida Y3 Strain in Sediment
There are a large number of microorganisms that de
pend on organic substances for their existence in sed
iment, such as bacteria and fungi, which have the abi
lity to oxidize and decompose organic matters. After
being contaminated by oil, some microorganisms pr
oduce enzyme systems that decompose pollutants un
der the induction of pollutants, which can degrade p
ollutants and convert them. Microbial remediation re
fers to the use of microorganisms to degrade toxic a
nd harmful crude oil contaminants present in the sed
iment into carbon dioxide and water or to convert th
em into non-hazardous substances. It is an extension
of traditional biological treatment method (Kong, 2
017; Li and Li, 2017; Ren and Huang, 2001).
When cultivating petroleum hydrocarbon degradi
ng bacteria, it is generally believed that the richer th
e energy, the greater the number of bacteria. Howev
er, in this study, the number of Pseudomonas putida
Y3 Strain was the highest in the 4000 mg/kg experi
mental group. After analysis, we mainly considered t
wo factors: on the one hand, dissolved oxygen. The
greater the amount of oil, the more difficult it was fo
r oxygen to enter, thereby affecting the oxygen suppl
y to the Y3 strain and inhibiting the growth of the Y
3 strain. On the other hand, there was an imbalance i
n the nutritional ratio. The large amount of oil in the
sediment resulted in a disproportionate ratio of N an
d P in the soil, thereby inhibiting the growth of the
Y3 strain. Qingxin et al. (Liu and Yi, 2006) also poi
nted out in the research on the growth factors of petr
oleum hydrocarbon decomposing bacteria that the a
mount of degrading bacteria in the experimental gro
up with the oil refueling amount of 1% was higher t
han that in the experimental group with the oil conce
ntration of 2%, verifying that the amount of oil-degr
ading bacteria did not necessarily increase with the i
ncrease of oil concentration.
Table 3: Degradation rate constant and kinetic equation of different concentration of crude oil.
concentration(mg/kg)
reaction
progression
dynamics equation
reaction velocity constant K
/mg/kg∙d
4000 0 y =-2.3425x + 4017.5 R
2
= 0.9196 2.3425
8000 0 y = -9.083x + 7997.1 R
2
=0.9727 9.083
12000 0 y = -10.584x + 11931 R
2
= 0.9637 10.584
16000 0 y = -5.8664x + 15936 R
2
=0.9613 5.8664
20000 0 y =-5.1315x + 19930 R
2
=0.9662 5.1315
Degradation of Crude Oil in Contaminated Sediment by Pseudomonas Putida Y3 Strain
41

Figure 3: The effect of crude oil content in sediment on
the Y3 strain.
4.2 Degradation of Pseudomonas Putida
Y3 Strain on Different Concentratio
ns of Contaminated Crude Oil
The physiological processes of microbial metabolis
m of crude oil pollutants are generally accomplished
through intracellular metabolism of absorbents that
contact and adsorb crude oil, secrete extracellular en
zymes and generate crude oil contaminants (Ren and
Huang, 2001). The key to the degradation of crude
oil is the oxidation of crude oil by oxidase (Liu et al.,
2009). Fungi and bacteria complete the oxidative m
etabolism of crude oil contaminants through the acti
on of extracellular enzymes and intracellular enzyme
s. The microbial metabolic pathways of crude oil hy
drocarbon compounds are crucial for elucidating the
mechanism by which microorganisms degrade organ
ic pollutants. Studies have shown that the key step in
the degradation of crude hydrocarbons by bacteria a
nd fungi is the oxidation of substrates by oxidases. I
n addition, microbial uptake of crude oil hydrocarbo
ns and transport process is also an important part of t
he study of microbial degradation mechanism. Gas c
hromatography analysis shows that Pseudomonas ba
cteria selectively transports crude oil hydrocarbons f
rom the extracellular to the intracellular and has a ce
rtain selectivity in composition (Liu et al., 2008). In
the natural environment, the process of microorganis
ms degrading hydrocarbon organic pollutants in cru
de oil is very complicated, involving the interactions
between microorganisms, substrates, and microorga
nisms and substrates.
From the comparison of the results of the experi
ments with and without the addition of bacteria, the
degradation rate of crude oil is sorted by size: experi
mental group with bacteria > control group. Althoug
h bacterial contamination in sediment may have som
e influence on the experiment, Pseudomonas putida
Y3 Strain still played a leading role in the degradatio
n of crude oil. The activity of Y3 strain improved th
e physicochemical properties of sediment, thereby pr
omoting the degradation of crude oil in sediment, w
hich is similar to the study of Qiang et al. (Ma, 200
8). The petroleum degradation rate was inversely pro
portional to the sediment crude oil concentration, wh
ich was consistent with the reports of Routani et al.
(Routani, 1985). In other words, high oil concentrati
on would inhibit the growth of oil-degrading bacteri
a, and even lead to a large number of deaths, resultin
g in a significant drop in oil removal rate.
0 30 60 90 120 150 180
3400
3600
3800
4000
4200
Crude oil content (mg/kg)
Time (d)
experimental group
control group
0 30 60 90 120 150 180
6000
6400
6800
7200
7600
8000
8400
Crude oil content (mg/kg)
Time (d)
experimental group
control group
0 30 60 90 120 150 180
10000
10400
10800
11200
11600
12000
Crude oil content (mg/kg)
Time (d)
experimental group
control group
0 30 60 90 120 150 180
15000
15200
15400
15600
15800
16000
Crude oil content (mg/kg)
Time (d)
experimental group
control group
0 306090120150180
19000
19200
19400
19600
19800
20000
Crude oil content (mg/kg)
Time (d)
experimental group
control group
IWEG 2018 - International Workshop on Environment and Geoscience
42

5 CONCLUSIONS
The growth of Pseudomonas putida Y3 Strain was af
fected by crude oil pollution. With the increase of cr
ude oil concentration, the number of Y3 strain gradu
ally decreased. Exogenous bacteria can adapt to the
sediment environment. When the sediment environ
ment changed, the number of exogenous bacteria wo
uld be affected. However, after the adjustment of the
microbes themselves, they were generally able to ad
apt to the new environment.
The Y3 strain had an effect on the crude oil degr
adation of sediments. With the increase of crude oil
concentration, the reaction rate constant increased gr
adually. When the concentration was 12000 mg/kg, t
he rate constant of the experimental group was the hi
ghest (10.584).
ACKNOWLEDGEMENT
We acknowledge the Natural Science Foundation of
Liaoning (No.2015020616), the Wetland Degradatio
n and Ecological Restoration Program of Panjin Pin
k Beach (PHL-XZ-2017013-002).
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