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.