Impact of humic acids on methane emissions from microplastic contaminated paddy soils

Polyvinyl chloride (PVC) microplastics (MP) have been shown to increase methane emissions from paddy soils. Redox-active humic acids (HA) were also found to be enriched in microplastic contaminated soils, and these compounds have the potential to affect greenhouse gas (GHG) emissions. However, the synergistic effect and mechanism of HA and MPs on soil methane emissions is still unclear. This study investigates humic acids inhibitory effect on the methanogenesis process. In order to verify this hypothesis, a long-term anaerobic incubation experiment was conducted using four distinct concentrations of humic acids ranging from 30 mg/L to 240 mg/L (named AHA, BHA, CHA and DHA). The tests were performed on two types of simulated substrates contaminated with microplastics: BA medium supplemented with acetate and soil leachate. BA medium was used to establish a simplified microbiome where the microbial processes can be disentangled more easily, while soil leachate was used to mimic a condition resembling more closely a real paddy field. A total of 36 incubation experiments were performed on serum bottles and a gas chromatograph (GC) was used to periodically measure the methane emissions. After the incubation, the liquid phase was extracted for the volatile fatty acids analysis and the filtered PVC MPs particles were subjected to microbial microscopy and Fourier-transform infrared spectroscopy（FTIR) to identify potential modifications of their chemical properties. The results of GC measurements show that in the BA medium, the BHA group supplemented with 60 mg/L of humic acid had significantly reduced methane emissions compared to the control, while in soil leachate, all four concentrations of HA reduced methane emissions compared to microplastics alone. Microscopy revealed the presence of microorganisms adhering to MPs surface, and FTIR biochemical characterization evidenced an enhanced intensity of peaks associated to -OH and -C＝O functional groups in all HA groups, suggesting that the potential redox reactions were involving these groups. In addition, acetate was found to be accumulated in BHA group supplemented with pure acetate medium. Shotgun sequencing-based metagenomics was used to gain insights on the microbial community composition and for metabolic reconstruction. Notably, with BA medium Methanosarcina sp. upd003, a methanogenic archaeon, displayed a decreased relative abundance in this group compared to the control groups. In contrast, in soil leachate medium, species associated with sulfate reduction, such as Sulfurisoma sp. upd73 and Moraxellaceae sp. upd27, were significantly enriched in the BHA group. Furthermore, a complete sulfate reduction-coupled anaerobic methane oxidation pathway was identified in humic acid-supplemented soil leachate, providing insights on the underlying mechanism leading to the reduction of methane emissions in this substrate. This study thus demonstrates that the substrate has a significant impact on the activity of humic acids, and that humic acids, by acting as electron acceptors, hold broad prospects for methane reduction in microplastic contaminated fields.