Pyrene effects on methanotroph community and methane oxidation rate, tested by dose–response experiment and resistance and resilience experiment

Abstract

Methanotrophs are an important group of bacteria that can metabolize methane. Polycyclic aromatic hydrocarbons (PAHs) are widespread contaminants and present in all ecosystems. We hypothesize that PAHs may affect methanotrophs and methane oxidation. In this study, we assessed dose–response curves for the inhibition of methane oxidation and methanotrophs diversity by pyrene, and resistance and resilience of soil methane oxidation rate and methanotrophs composition in response to pyrene contamination. Methanotrophic bacterial diversity was determined by terminal restriction fragment length polymorphism analysis of the pmoA gene, while methanotrophs composition was assessed by cloning and sequencing of the pmoA gene. Sequences with 98% or more identity were considered as the same operational taxonomic unit (OTU). The pyrene concentration at which methane oxidation decreased by 50%, as compared to the non-spiked control soil (EC50), was determined. Both EC50 concentration and 500 mg kg−1of pyrene were applied as disturbances in the resistance and resilience experiment. Resistance and resilience were determined 1 and 40 days, respectively, after spiking pyrene. Methane oxidation rate decreased with increasing pyrene concentrations and the EC50 value was 22.0 mg kg−1. Methanotrophic bacterial community diversity decreased in 200 and 500 mg kg−1 pyrene treatments, and methanotroph community structure shifts occurred in 100, 200, and 500 mg kg−1 pyrene treatments. Methane oxidation was neither resistant nor resilient to pyrene disturbance. However, methane oxidation of soil with 22.0 mg kg−1 pyrene disturbance recovered to some extent after 40 days incubation. There were five OTUs identified in the control samples, but the number of OTUs increased 1 day after the addition of 22 mg kg−1 of pyrene. It suggests that a low level of disturbance may increase diversity. Forty days after 500 mg kg−1 of pyrene disturbance, only one OTU belonging to Methylocaldum was detected. The resilience of Methylocaldum to a high level of pyrene could be due to the high genomic GC content, which reduces the frequency of insertion by pyrene into the DNA duplex. In addition, we found that the number of OTUs decreased in all treatments after the 40-day incubation. Methane oxidation activity was more sensitive to pyrene than the methanotroph community structure, but could recover under a low level of pyrene. Significant decrease in diversity and shift in species composition occurred only after severe perturbation. A low level of disturbance could increase biodiversity, while a high level of disturbance could decrease it.