Study on the Influence Mechanism of Chitosan on Two Strains of
Petroleum Hydrocarbon Degrading Bacteria
Xiuying Zhao
1a
, Jing Li
1b
, Wu Cui
1c
, Zhiyuan Wu
1d
, Chaocheng Zhao
2e
and Qiyou Liu
2,* f
1
College of Chemical Engineering, China University of Petroleum (East China), Qingdao 266580, China
2
China university of petroleum (east China) state key laboratory of oil pollution control, Qingdao 266580, China
f,*
Keywords: Chitosan, Pseudomonas Aeruginosa, Paenibacillus, Petroleum Hydrocarbon, Environment.
Abstract: With the rapid increase of petroleum use in modern industrialization, the leakage in the process of
exploitation and transportation has caused environmental issue. In order to degrade the pollutants in a green
way, it is very important to study the properties of petroleum hydrocarbon degrading bacteria. The effect of
promising immobilized microbial material, chitosan, on the hydrocarbon degrading bacteria was studied by
plate drilling method. It was found that the acetic acid used to dissolve chitosan had a certain inhibitory
effect on the growth and development of microorganisms. Chitosan had the ability to resist such inhibitory
effect and this ability also depended on the strain itself to a certain extent.
1 INTRODUCTION
1
With the accelerating process of industrialization
and the increasing demand for petroleum and its
ancillary products, petroleum, known as the "blood"
of modern industry, will inevitably have a negative
impact on society from exploitation to use (Dai.
2014). The average annual output of oil in the world
is about 4 billion tons. According to the statistics, 2
kg of pollutants enter the environment every 1 t of
oil produced, so about 800 wt of pollutants enter the
environment every year in the world (Wei. 2020).
At present, there are a lot of remediation
technologies for oil pollution, and bioremediation
technology has great application potential because
of its safety, environmental protection, no secondary
pollution and other advantages. Compared with the
use of free cells, immobilized microorganisms have
several advantages. Chitosan, a new functional
carrier material, is the only basic polysaccharide
found in nature so far. It is the product of chitin
deacetylation. Its chemical name is polyglucosamine
a
https://orcid.org/0000-0002-2369-2391
b
https://orcid.org/0000-0001-5974-6710
c
https://orcid.org/0000-0001-8711-3611
d
https://orcid.org/0000-0002-6122-488X
e
https://orcid.org/0000-0001-7374-8448
f
https://orcid.org/0000-0002-2369-2391
(1-4)-2-amino-b-d-glucose. Its good
biocompatibility, strong adsorption and other
excellent properties have been widely studied in
textile printing and dyeing, food, papermaking,
heavy metal recovery, medical and pharmaceutical,
water treatment and other fields research (Gao et al.
2020; Ou et al. 2020). Chitin waste generated by
shrimp culture can be recycled, which is a profitable
source of income in areas with relatively developed
aquaculture industry (Barreto et al. 2010).
CTS molecular surface is rich in functional
groups but can only dissolve in weak acids. It is
reported that in acidic environment, it can adsorb
organic matter but its -NH
2
protonation into
positively charged -NH
3+
will produce certain
inhibitory effect on microorganisms. Therefore, for
chitosan, the material used by immobilized
microorganisms for degrading petroleum
hydrocarbons, we should not only stay in the study
of pollutants but also pay attention to its effects on
different petroleum degrading microorganisms. In
this paper, the effects of different concentrations of
CTS on different petroleum hydrocarbon degrading
bacteria were compared to provide a theoretical
basis for the petroleum hydrocarbon degrading in
the soil environment.
Zhao, X., Li, J., Cui, W., Wu, Z., Zhao, C. and Liu, Q.
Study on the Influence Mechanism of Chitosan on Two Strains of Petroleum Hydrocarbon Degrading Bacteria.
DOI: 10.5220/0011199400003443
In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 265-268
ISBN: 978-989-758-595-1
Copyright
c
 2022 by SCITEPRESS – Science and Technology Publications, Lda. All rights reserved
265

2 MANUSCRIPT PREPARATION
2.1 Materials
The soil samples used in this study were obtained
from the soil that was not contaminated by oil in
Dongying, China. All other chemicals were of
analytical grade and commercially available. The
chitosan solution is prepared by dissolving powder
CTS in different concentration of HAC.
2.2 Bacterial Strains and Culture
Media
Mineral salt medium was used for sieve bacteria, the
composition was as follows: 1.0 g of (NH
4
)
2
SO
4
, 1.0
g of K
2
HPO
4
, 1.0 g of KH
2
PO
4
, 0.2 g of
MgSO
4
·7H
2
O, 0.02 g of CaCl
2
, 10.0 g of NaCl, 0.3
g of Crude oil in per liter of deionized water. Beef
extract peptone liquid medium was used for inocula
preparation and maintenance, and the composition
was as follows: 5.0 g of NaCl, 5.0 g of beef extract,
10.0 g of tryptone in per liter of deionized water.
Adjust pH to 7. Sterilization for 20 min at 121 ℃.
After 4 weeks of domestication, absorb 1.0 mL of
the supernatant and placed it in a glycerin tube and
stored in the refrigerator for subsequent use.
2.3 Analytical Techniques
The DNA of strain was extracted from the high
efficient petroleum hydrocarbon degrading bacteria
by using universal primers 27f and 1492r. PCR
amplification of 16S rRNA gene was carried out
according to the corresponding system and
procedure. PCR products were sequenced by Beijing
Qingke Biotechnology Co., Ltd. According to the
results of homology comparison, the genus was
preliminarily identified.
2.4 Experimental Design
The CTS was dissolved in 2.0% and 1.0%HAC to
prepare CTS solution with the concentration of
0.1%, 0.25%, 0.5%, 0.75%, 1.0% and 1.5%. The
solution was mixed uniformly by ultrasound and
sterilized at 105 ℃. Add 200 μL of target bacteria
into 20 mL beef agar medium to be solidified, mix
well, and
make a plate. After the plate was
solidified, three uniform holes were made on the
plate with a 6.5 mm diameter punch as parallel
holes. 50 μL CTS solution with different
concentrations was injected into the holes, and 1.0%
and 2.0% HAC was injected into the holes as
control. The diameter of transparent circle was
measured after 24 h cultivation at 37 ℃.
3 TEST RESULTS AND
DISCUSSIONS
3.1 Experimental Results
Six strains of bacteria were screened from oil
contaminated soil, and the oil reduction rate of each
strain was determined. Two different strains were
selected, one was Pseudomonas aeruginosa (TL-1)
and the other was Paenibacillus (YB-1). The
phylogenetic tree based on 16S rRNA sequence is
shown in Figure 1.
Figure 1: Phylogenetic tree based on 16S rRNA sequence.
ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics
266

3.2 Effect of Chitosan on Bacteria
The transparent circle formed by different
concentrations of CTS dissolved in 1% HAC on the
plate of strain YB-1 and TL-1 is shown in Fig. 3.
For strain TL-1, the inhibition zone was the largest
when the CTS addition was zero (1% HAC solution
control group), and then with the increase of CTS
concentration, the inhibition zone gradually
decreased to the minimum when the CTS
concentration was 1.5%.
Figure 2: Inhibition zone of YB-1 and TL-1 in different concentration of CTS and 1% HAC.
For strain YB-1, 1% HAC alone made the
inhibition zone larger, then with the continuous
addition of CTS, the inhibition zone gradually
decreased to the minimum when the concentration
of CTS was 0.5%. After that, the inhibition zone
increased gradually with the addition of CTS, and
there is a trend that the inhibition zone will be larger
than that formed when 1% HAC solution was
injected alone. We speculate that when the
concentration of CTS was less than 0.5%, the effect
of HAC on the strain was dominant and the addition
of CTS could gradually weaken the effect of HAC
on the strain. But, with the gradual progress of the
reaction, the -NH
2
on the surface of CTS in HAC is
gradually protonated into positively charged -NH
3+
,
which makes CTS become a water-soluble cationic
polyelectrolyte.
Variation of inhibition zone of strain YB-1 with
CTS dissolved in different concentrations of HAC is
as shown in Fig. 3. As for YB-1, the inhibition zone
in CTS dissolved in 2% HAC is higher than that in
1% HAC. It can be concluded that HAC is the main
inhibitor in this concentration range, and with the
increase of CTS, not only does it not have a stronger
antibacterial effect, but it can weaken the inhibitory
effect of HAC on strain TL-1.
Figure 3: Variation of inhibition zone of strain YB-1 with
CTS dissolved in different concentrations of HAC.
Study on the Influence Mechanism of Chitosan on Two Strains of Petroleum Hydrocarbon Degrading Bacteria
267

At the same time, the inhibition zone produced
by two strains at different concentrations of CTS
dissolved in 1% HAC was compared, as shown in
Fig. 4.
Figure 4: the inhibition zone of two strains under different
concentrations of chitosan dissolved in 1% HAC.
When the concentration of CTS was less than
1%, the inhibition zone around strain YB-1 was
significantly smaller than that of strain TL-1. The
reason why we got this result is probably because
YB-1 is a Bacillus. The spores produced by YB-1
can produce certain resistance to the external
environment, so it can resist the influence of HAC
on the strain, and the inhibition zone is much
smaller than that of Gram-negative bacteria TL-1.
So the inhibition of HAC on the strain also depends
on the strain itself. Jing Yingjun (Jing et al. 2006)
also pointed out that different molecular weight, pH
value, metal ions and other external factors of CTS
have different antibacterial effects on different
strains, but the key factor of antibacterial activity of
CTS is the strain itself.
4 CONCLUSIONS
In this paper, plate drilling method is adopted to
study the effect of chitosan material on petroleum
hydrocarbon degrading bacteria. The main
conclusions can be summarized as follows:
(1) Two representative petroleum hydrocarbon
degrading bacteria Pseudomonas Aeruginosa and
Paenibacillus were successfully screened out from
oil-contaminated soil.
(2) The main inhibitory factor on strain was
acetic acid, chitosan can counteract this inhibitory
effect under certain conditions.
(3) The inhibitory effect of HAC and CTS on the
strain also depended on the strain itself.
(4) In terms of the future work, how to prepare
immobilized microbial materials by dissolving
chitosan under appropriate conditions to remove
petroleum pollutants should be carried out.
ACKNOWLEDGEMENTS
This study was supported by the Independent Project
Program of State Key Laboratory of Petroleum
Pollution Control (Grant No. PPC2018014) and the
Fundamental Research Funds for the Central
Universities (No. 16CX06008A)., CNPC Research
Institute of Safety and Environmental Technology.
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