Anaerobic oxidation of methane in paddy soil
by Lichao Fan
Date of Examination:2020-09-30
Date of issue:2020-11-18
Advisor:Prof. Dr. Michaela Dippold
Referee:Prof. Dr. Michaela Dippold
Referee:Prof. Dr. Volker Thiel
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Abstract
English
The anaerobic oxidation of methane (AOM) is a globally important CH4 sink that offsets potential CH4 emission into the atmosphere. AOM is estimated to consume up to 90% of CH4 produced in marine sediments before it reaches atmosphere, but it is an underappreciated CH4 sink in terrestrial ecosystems. This calls for the study of the specific mechanisms of terrestrial AOM and the estimation of the ecological relevance for CH4 sink, especially in ecosystems exposed to long-term anaerobic conditions such as rice paddies. Flooded paddy soils are the hotspot area of methanogenesis along with a high availability of alternative electron acceptors (AEAs) needed by methanotrophic microorganisms to oxidize CH4 anaerobically. However, the role of AEAs and the intensity of the AOM process in reducing the CH4 fluxes from rice paddies remain unclear. Moreover, it remains unclear how AEAs from different fertilization modes affect anaerobic microbial interactions, and whether a preferred AOM pathway exists in these interactions. Current studies on AOM are largely based on microcosm incubations with headspace CH4 injection and shaking. However, shaking introduces mechanical disturbances but the lack of shaking may lead to a systematical underestimation of CH4 oxidation due to the relatively low solubility of CH4. To address these and the above challenges, four research aspects were investigated in this: (i) utility of the silicone tube approach for CH4 oxidation studies, (ii) the occurrence of AOM under shaking and steady conditions with silicone tubes, (iii) role of AEAs and fertilization for AOM, (iv) active AOM pathways and functioning of the microbial community network in paddy soils. These aspects were investigated by tracing the 13C-label from CH4 into CO2, soil organic matter, total microbial biomass and phospholipid fatty acids (PLFA) in fertilized (manure, biochar, NPK) paddy soils amended with alternative electron acceptors (NO3-, Fe3+, SO42-, humic-acids) to quantify CH4 oxidation, and identify microbial groups by 16S rRNA sequencing analyses. Our results implied the injection of CH4 belowground via porous silicone tubes to compensate for the poor solubility of CH4 and replace the common shaking method. During a 29-day incubation of soil slurry, the highest net CH4 oxidation rate was 1.6 µg C-CO2 g-1 dry soil h-1 after injecting 13CH4 into the slurry through a silicone tube without shaking. This was 1.5-2.5 times faster than the respective CH4 oxidation after headspace injection without shaking. Furthermore, it was found that CH4 oxidation rates were similar between silicone tube injection without shaking and headspace injection with shaking. Consequently, the silicone tube approach can substitute the common shaking method. As the silicone tube approach maintains the gas concentration gradients, it can more realistically reflect natural soil conditions. Secondly, by 13C enrichment of CO2 after 13CH4 injection we clearly confirmed the hypothesized occurrence of AOM in paddy soil during a 59-day anaerobic incubation. The cumulative AOM reached 0.16-0.24 μg C-CO2 g-1 dry soil without shaking, but it was 33-80% lower with shaking. Unexpectedly, the effect of silicone tubes on AOM was insignificant either with or without shaking, suggesting that the main limiting factor for AOM was not the CH4 concentration in water (slurry) but the availability of AEAs. Without shaking, the methanogenesis control (no CH4 addition) revealed a steady increase of CH4 in the headspace/tube, whereas the CH4 concentration in jars with shaking was constantly low during 59 days. This suggests that shaking inhibited methanogenesis, possibly by disturbing the AOM-related microorganisms which were co-localized to the substrates (i.e. CH4 and AEAs). Added NO3- was the most effective electron acceptor during 84 days of anaerobic incubation. The highest AOM rate was 0.80 ng C g-1 dry soil h-1 under pig manure fertilization followed by the control and NPK, while AOM was the lowest under biochar application. The role of Fe3+ in AOM remained unclear. SO42- inhibited AOM but strongly stimulated the production of unlabeled CO2, indicating intensive sulfate-induced decomposition of native organic matter. Added humic acids were the second most effective electron acceptor for AOM, but increased methanogenesis by 5-6 times in all fertilization treatments. We demonstrated for the first time that organic electron acceptors are among the key AOM drivers and are crucial in paddy soils. Finally, we determined AOM pathways by tracing 13C incorporation from 13CH4 into total microbial biomass and PLFA, and related these pathways to the microbial community’s network. The co-occurrence network revealed a set of major and minor AOM pathways with synergistic relations between complementary anaerobic microbial groups. A set of comparative analyses confirmed that NO3--driven AOM was the major AOM pathway. It co-existed with minor pathways involving NO2- reduction by NC10 bacteria, putative reduction of humic acids and Fe3+ by Geobacter species, and SO42- reduction by sulfate-reducing bacteria linked with anaerobic methanotrophs. In a broader ecological view, AOM is ubiquitous in paddy soils but still is an underappreciated CH4 sink. NO3--induced AOM together with manure fertilization has the potential to recycle ~3.9 Tg C-CH4 annually before the produced CH4 released to the atomsphere, which was equivalent to roughly ~10–20% of the global net CH4 emissions from rice paddies. Consequently, the application of suitable organic and mineral fertilization strategies can provide an effective control on the CH4 sink under anaerobic conditions in submerged agricultural ecosystems.
Keywords: Anaerobic oxidation of methane; paddy soil; PLFA; soil microbial community; Methane turnover