4. Discussion
Soil pollution from petroleum is currently considered to be one of the
most severe environmental problems. This type of pollution reduces or
destroys soil fertility, modifies the elemental composition of soil and
water cycles, results in losses in the aesthetic value of ecosystems,
leads to secondary pollution of air and groundwater, and inhibits or
eradicates soil organisms (Koshlaf and Ball 2017, Margesin et al., 2003,
Haritash and Kaushik 2009, Mishra et al., 2001, Hu et al., 2013).
The content of soil carbon, nitrogen, and sulfur increased
significantly. At the same time, the pH decreased significantly
(Fig. 1 ) after petroleum pollution, which corresponds with
former researches (Wang et al., 2010, Anikwe et al., 2017). This is
mainly due to a large amount of carbon, nitrogen, and sulfur in
petroleum, and when sulfur enters the soil, it forms sulfur oxides and
reduces the soil pH. Scanning electron microscope photos visually
describes oil pollution’s adverse effects on soil particle structure
(Fig. S2 ). The determined results of metallomics content in
different soil samples show that petroleum pollution leads to the
accumulation of soil Ca, Cu, Mn, Pb, and Zn (Fig. 2 ). In
addition, the cluster analysis based on the data sets of physical and
chemical properties and metal group content can well distinguish
different samples (Fig. 3 ), which shows that the oil pollution
of different years has a different degree of impact on the soil. The
correlation analysis results show a positive coupling relationship
between a large number of metal elements carried by petroleum.
Previous studies have found that petroleum pollution will lead to shifts
in the soil microbial community. In this study, the microbiome of
oil-contaminated soil in different years was studied. Results showed
that petroleum pollution significantly reduced the soil bacterial and
fungal diversity and shaped the microbial community (Fig. 4 ).
Cheema et al. (2015) found that bacterial diversity in agricultural soil
was significantly higher than in hydrocarbon-contaminated soil.
Moreover, Wang et al. (2018) found that the toxicological effect of oil
pollutants significantly decreased the soil fungal diversity.
In this study, the relative abundance of Proteobacteria and Ascomycota
was increased by petroleum contamination (Fig. 5 ). A previous
study (Zhang et al., 2012) had reported that Proteobacteria was proved
to be the most easily cultivated bacteria in petroleum contaminated
soils. Previous researches (Wu et al., 2019, Zhang et al., 2012) showed
that part of the Proteobacteria can degrade petroleum hydrocarbons or
PAHs in petroleum polluted soil. Kim et al. (2019) enriched
microorganisms producing biosurfactants, which have the potential to
degrade hydrocarbons in oil contaminated soil, and found that
Proteobacteria has the highest relative abundance. Moreover, a previous
study proved that Ascomycota was the most dominant known
petroleum-degrading fungal phyla (Ezekoye et al., 2018, de la
Cruz-Izquierdo et al., 2021). In addition, results also showed that the
relative abundance of Oleibacter and Fusarium was
significantly increased in both PS and OS samples. Oleibacter was
previously proved to be a high performance of HC-degrading bacteria
species (Catania et al., 2015), while Fusarium (Hidayat and
Tachibana 2012, Azin et al., 2018) was also used in petroleum
degradation.
After petroleum hydrocarbon leakage into the soil, it will cause a
series of changes in soil physicochemical properties, microbial
diversity, and composition (Fig. 6 ). This study found that
petroleum hydrocarbons can directly lead to the increase of soil carbon
and nitrogen content, the decrease of bacterial diversity, and the
change of metal group, bacterial and fungal community. The diversity of
the soil fungal community was indirectly reduced by soil metallome.
Carbon and nitrogen were vital factors limiting microbial populations in
petroleum polluted soils. Previous research has proved that soil total
petroleum hydrocarbon, total carbon, and total nitrogen were the most
essential factors influencing the bacterial communities in oil polluted
soils but varied at different contaminating levels (Feng et al., 2020).
It was found that the best optimal Carbon: Nitrogen was between 20:1 and
50:1 for microorganism growth in petroleum polluted soil (Kaufmann et
al., 2004).
The analysis of co-occurrence network may explain the structure and
function of microbial community (Ma et al., 2020; Wagg et al., 2019).
With the change of soil microbial community composition, microbial
co-occurrence network consequentially changes. Our study showed that the
number of nodes, links, and modules decreased in PS and OS samples
compared to Control (Fig. 7 ). Functional cooperative behaviors
have been reported to exists commonly in soil microbial communities
(Cremer et al., 2019). The symbiotic network analysis identifies
microbial taxa that have cooperative or co-dependent relationships,
often representing a population., which usually indicate a mutual
function (Deshpande et al., 2013). The modules have the function of
minimizing the impact of environmental disorder (Kitano, 2004); hence,
the fewer modules in the OS and PS network, the slower communication
speed between modules, and the lower response efficiency to
environmental stimulus.
Otherwise, fewer connections in petroleum-polluted soil samples
represented inhibition in the interactions between soil microorganisms.
This may be due to the decrease of microbial activity in soil caused by
oil pollution (Guo et al., 2012, Labud et al., 2007). What’s more, the
proportion of positive links in PS was lower than that of OS samples.
This indicates that the interaction between soil microorganisms is more
competitive or antagonistic after receiving oil pollution quickly. After
long-term natural recovery, the interaction mode between soil
microorganisms becomes synergy or symbiosis. Meanwhile, the network
invulnerability of Control was highest among all groups, followed by PS
and OS, which means that the soil microbial network becomes less complex
and vulnerable after petroleum contamination.
After long-term oil pollution, more petroleum hydrocarbon-degrading
bacteria may be enriched in the soil. Thus, three bacterial strains with
significant petroleum hydrocarbon degradation efficiency were isolated
from OS samples (Fig. 8 ). Pseudomonas stutzeri (Li et
al., 2020, Gałązka et al., 2012) and Bacillus pumilus (Sheeba et
al., 2017, Patowary et al., 2015) are reported to be potential bacteria
strains in petroleum and polyaromatic hydrocarbons degradation
applications before. Niu et al. (2017) used an agar system and a SynCom
composed of 7 bacterial strains cultured from corn roots to showed that
the absence of Enterobacter cloacae will lead to the whole
collapse of microbial community profiles in the corn seedlings
rhizosphere, proving the significance of microbial interactions and the
presence of crucial taxa in the rhizosphere microbiota. Our study also
found that the SynCom consisting of three bacterial strains could
significantly improve the performance of maize seedlings under
petroleum-pollution stress. This may provide a new strategy for
petroleum-contaminated soil reuse.