Research paperDetection of functional microorganisms in benzene [a] pyrene-contaminated soils using DNA-SIP technology
Graphical Abstract
Introduction
Polycyclic aromatic hydrocarbons (PAHs) are recalcitrant compounds and occur widely in various environmental compartments including soils, surface waters and groundwaters (Aranda, 2016). In particular, the toxicity and persistence of PAHs increase with increasing ring number and thus the high molecular weight (HMW) PAHs are of special concern. Benzo [a] pyrene (BaP), a HMW-PAH with a five-ring structure, has been allocated to the first class of ‘human carcinogens’ and is frequently detected in soils (Juhasz and Naidu, 2000).
Bioremediation is considered as an effective method to clean up HMW-PAH-contamination in environments, according to the biodegradation ability of microorganisms (Doyle et al., 2008). Until now, the reported degraders that can metabolize HMW-PAHs (such as pyrene and BaP) include Rhodococcus, Nocardia, Mycobacterium, Gordona (Kastner et al., 1994), and Paracoccus aminovorans HPD-2 (Mao et al., 2008). According to these isolates, PAH-ring hydroxylating dioxygenases (PAH-RHD) catalyze the first step of the PAH degradation pathway, comprising nah, pah, arh and phn genes in Gram-negative (GN) bacteria and nid, nir, phd and nar genes in Gram-positive (GP) bacteria (Song et al., 2015). Thus, PAH-RHD have been widely used as biomarkers of soil PAH degradation potential (Jurelevicius et al., 2012). However, most microorganisms in nature still cannot be cultured (> 99%) and direct-cultivation underestimates the microbial diversity of functional microorganisms (Song et al., 2015). Though high-throughput sequencing can reveal the structure of complex microbial communities and reflect the environmental function, it cannot accurately show the metabolic capacities of microorganisms (Gutierrez, 2011). DNA-based stable isotope probing (DNA-SIP) technology is a powerful technique that distinguishes microorganisms and links identity to function (Dumont and Murrell, 2005).
Previous work found significantly different abilities of four different types soils to degrade PAH-pyrene, the fastest pyrene dissipation was observed in paddy soil (anthrosols), and the least reduction occurred in red soil (ferric acrisols), and the pyrene dissipation in mixture of red soil and paddy soil was higher than that in red soil (Ren et al., 2016, Wang et al., 2018). Moreover, the microbial community composition in mixture differed from the two soils (Wang et al., 2018), and the microbial community largely determine the function of PAH removal in soil (Wang et al., 2018). However, the analysis of enriched microorganisms alone is not sufficient to link the microorganisms and function. In this study, 13C-BaP was employed as a HMW-PAH to explore and compare the different active BaP-degrading bacteria in paddy soil and red soil, and investigate the active degraders in mixture of the two soils with DNA-SIP technique.
Section snippets
Soils and reagents
The paddy and red soils, ferric acrisols and anthrosols according to the FAO soil classification system, were sampled to a depth of 15 cm at field experiment stations of the Chinese Academy of Sciences located at Changshu, Jiangsu province and Yingtan, Jiangxi province, respectively. Both soils are considered to be uncontaminated, because of the concentrations of 16 PAHs < 200 μg kg−1, and the initial concentrations of benzo [a] pyrene (BaP) were 2.93 and 9.90 μg kg−1 in the red and paddy
Soil residual BaP dynamics
The BaP biodegradation potential results from the microcosm culture experiments are shown in Fig. 1. The initial concentrations of soil BaP were ~10 mg kg−1. After 14 d the mean concentrations of BaP were 6.9 and 7.5 mg kg−1 in the paddy soil and paddy-red soil mixture, respectively, and the removal efficiencies reached 29.5% and 25.3%. However, there was no noteworthy decline BaP in the red soil in which the residual BaP was 9.8 mg kg−1 after 14 d of incubation, slightly lower than the initial
Discussion
Microorganisms play essential roles in the biodegradation of organic pollutants in contaminated ecosystems, especially microorganisms that use pollutants (such as PAHs) as carbon sources (Thomas et al., 2019). Microbes do not usually live in isolation but tend to live together in ecosystems (Ratzke et al., 2020). Based on the DNA-SIP technology, numerous bacteria have been identified as PAH degraders such as Methylibium and Legionella in PAH-contaminated groundwater (Zhang et al., 2012), and
Conclusions
Here, DNA-SIP technology was used to identify the active BaP degraders in the soil microbial community of R, P and PR. Significantly different bacterial species were labeled in ‘heavy’ DNA fractions. Only one OTU830 (Ramlibacter) belonging to the Gammaproteobacteria was labeled as a BaP degrader in R, and the BaP removal rate was much lower in R than in P or PR. The community of P (paddy soil) showed the highest BaP removal efficiency (29.5%), and the highest diversity of active BaP degrading
CRediT authorship contribution statement
Beibei Wang designed the experiment, analyzed the data, created the figures, and wrote the preliminary version of the manuscript. Ying Teng conceived the research topic, revised the manuscript, and is the corresponding author. Huaiying Yao advised and helped with the ultracentrifugation of DNA and qPCR, revised the manuscript. Peter Christie revised the language and monitored for scientific accuracy. All authors interpreted the data and contributed to the manuscript.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was funded by the Outstanding Youth Fund of Jiangsu Province, China (No. BK20150049) and the National Key Research and Development Project of China (No. 2019YFC1803700). We thank Juan Wang and Yaying Li for help with the ultracentrifugation in the DNA-SIP experiment at Ningbo Urban Environment Observation and Research Station, Chinese Academy of Sciences.
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