Report www.the-innovation.org Contribution of periphytic biofilm of paddy soils to carbon dioxide fixation and methane emissions Sichu Wang,1,2,3 Pengfei Sun,1,2 Guangbin Zhang,1 Neil Gray,4 Jan Dolfing,5 Sofia Esquivel-Elizondo,6 Josep Peñuelas,7 and Yonghong Wu1,2,* *Correspondence: yhwu@issas.ac.cn Received: August 6, 2021; Accepted: November 23, 2021; Published Online: November 26, 2021; https://doi.org/10.1016/j.xinn.2021.100192 ª 2021 The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Graphical abstract Public summary - Field experiments were conducted in tropical, subtropical, and temperate rice paddies - Periphytic biofilm promoted CO2 fixation and CH4 emission - Periphytic biofilm acted as a biotic converter of atmospheric CO2 to CH4 ll www.cell.com/the-innovation Report Contribution of periphytic biofilm of paddy soils to carbon dioxide fixation and methane emissions Sichu Wang,1,2,3 Pengfei Sun,1,2 Guangbin Zhang,1 Neil Gray,4 Jan Dolfing,5 Sofia Esquivel-Elizondo,6 Josep Peñuelas,7 and Yonghong Wu1,2,* 1State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, 71 East Beijing Road, Nanjing 210008, China 2Zigui Three Gorges Reservoir Ecosystem, Observation and Research Station of Ministry of Water Resources of the People’s Republic of China, Shuitianba Zigui, Yichang 443605, China 3College of Advanced Agricultural Science, University of Chinese Academy of Sciences, No. 19(A) Yuquan Road, Shijingshan District, Beijing 100049, China 4School of Natural and Environmental Sciences, Newcastle University, Newcastle upon Tyne NE1 7RU, UK 5Faculty of Energy and Environment, Northumbria University, Newcastle upon Tyne NE1 8QH, UK 6Department €bingen, Germany of Microbiome Science, Max Planck Institute for Developmental Biology, Max-Planck-Ring 5, 72076 Tu 7Consejo Superior de Investigaciones Científicas (CSIC), Global Ecology Unit, Centre for Ecological Research and Forestry Applications (CREAF)–CSIC–Universitat Autonoma de Barcelona (UAB), Bellaterra, 08193 Barcelona, Catalonia, Spain *Correspondence: yhwu@issas.ac.cn Received: August 6, 2021; Accepted: November 23, 2021; Published Online: November 26, 2021; https://doi.org/10.1016/j.xinn.2021.100192 ª 2021 The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Citation: Wang S., Sun P., Zhang G., et al., (2022). Contribution of periphytic biofilm of paddy soils to carbon dioxide fixation and methane emissions. The Innovation 3(1), 100192. Rice paddies are major contributors to anthropogenic greenhouse gas emis- trophs, such as microalgae, dominate the microbial aggregates.16 Oxygen (O2) sions via methane (CH4) flux. The accurate quantification of CH4 emissions released during algal photosynthesis may diffuse into the soil,17 leading to inhibi- from rice paddies remains problematic, in part due to uncertainties and omis- tion of CH4 production18 and facilitating oxidation of CH4 at the interface of soil sions in the contribution of microbial aggregates on the soil surface to carbon and water, preventing CH4 fluxes into floodwater and from there to the atmo- fluxes. Herein, we comprehensively evaluated the contribution of one form of sphere. During the decay phase of PB, heterotrophs replace autotrophs as the microbial aggregates, periphytic biofilm (PB), to carbon dioxide (CO2) and dominant community members.16,19 Heterotrophic respiration and the release CH4 emissions from paddies distributed across three climatic zones, and of labile carbon may lead to greater consumption of O220 resulting in a reductive quantified the pathways that drive net CH4 production as well as CO2 fixation. environment and higher levels of CH4 production. In addition, unlike microalgae in We found that PB accounted for 7.1%–38.5% of CH4 emissions and 7.2%– floodwater, the multispecies aggregates (PB) release extracellular polymeric sub- 12.7% of CO2 fixation in the rice paddies. During their growth phase, PB fixed stances to form a barrier by clogging the pore space at soil-water interface,21 CO2 and increased the redox potential, which promoted aerobic CH4 oxida- limiting carbon transfer to the atmosphere and promoting its reaction with min- tion. During the decay phase, PB degradation reduced redox potential and erals and competing ions in the matrix. To the best of our knowledge, however, increased soil organic carbon availability, which promoted methanogenic mi- none of these hypotheses have been experimentally verified, and the net contribu- crobial community growth and metabolism and increased CH4 emissions. tions of PB to carbon fluxes have not been properly evaluated. Overall, PB acted as a biotic converter of atmospheric CO2 to CH4, and aggra- While a limited number of studies have investigated the effects of periphyton or vated carbon emissions by up to 2,318 kg CO2 equiv ha 1 season 1. Our re- phytoplankton on carbon fluxes in aquatic ecosystems,15,22-24 most have tended sults provide proof-of-concept evidence for the discrimination of the contri- to focus on a single species of algae in rivers and lakes. Rice paddies are much butions of surface microbial aggregates (i.e., PB) from soil microbes, and a shallower and have a lower flow velocity than rivers and lakes, with contrasting profound foundation for the estimation and simulation of carbon fluxes in transport of gases from sediment to floodwater.23 Compared with single micro- a potential novel approach to the mitigation of CH4 emissions by manipu- bial species, PB possesses a more complex microbial composition and struc- lating PB growth. ture.25 This is important as successional processes and diversity of metabolisms among species dynamically influence soil and water properties, with associated INTRODUCTION impacts on carbon fluxes. Rice paddies are globally important sinks for carbon dioxide (CO2) and sources In this study, we quantified the contribution of PB to CO2 fixation and CH4 emis- of methane (CH4), with high levels of carbon exchange at the soil-atmosphere sion in rice paddies distributed across three climatic zones. We also used incuba- interface.1,2 Annual emissions of CH4 from rice paddies are estimated to be tion and microcosm experiments to explore the underlying mechanisms of PB ef- 20–100 Tg and represent about 5%–20% of annual global anthropogenic emis- fects on CH4 production and aerobic oxidization. The composition and sions.3,4 However, uncertainties in estimates of CO2 fixation and CH4 emission metabolisms of soil methanogenic and methanotrophic communities in the from rice paddies still exist.5 One reason is the underestimation of the contribu- presence of PB were measured using qPCR, high-throughput sequencing, and tion of soil-surface microbial aggregates to these carbon fluxes. For CH4 metabolomics analyses. Our results provide proof-of-concept evidence for the emission estimations and simulations, it is generally assumed that methano- quantization of the contribution of surface microbial aggregates (i.e., PB) to genic substrates are derived only from soil microbes, rice plants, and farmyard CO2 fixation and CH4 emissions and a solid foundation for potential novel ap- manure.6-8 Organic carbon derived from microbial aggregates on the soil surface proaches to CH4 emission mitigation by manipulating PB growth. has been poorly considered. Furthermore, the biogeochemical function of micro- bial aggregates on the soil surface has been neglected due to the challenges of RESULTS distinguishing their contribution from those of the soil microbes.9 CO2 and CH4 fluxes in fields Periphytic biofilm (PB) is a ubiquitous microbial aggregate that develops at the CO2 and CH4 fluxes from rice paddies with and without PB were monitored in soil or sediment surface in aquatic ecosystems (Figure S1). These biofilms situ during the whole rice growing season. CO2 fluxes decreased with time in the comprise a dominant biophase (bacteria, algae, fungi, protozoa, and metazoa), first 10 days and were usually negative from the fifth day after flooding onward along with a minor abiotic phase composed of extracellular polymeric sub- (Figures 1A–1C). Totals of 626, 1,680, and 1,655 kg ha 1 CO2 were fixed in the stances, minerals (iron, Fe; aluminum; and calcium), and nutrients (nitrogen, phos- tropical, subtropical, and temperate experimental paddy fields, respectively, in phorus).10,11 In aquatic ecosystems, PB accounts for 7%–97% of the total primary the presence of PB during the rice season daylight periods. The presence of productivity,12,13 and its biomass in rice paddies may reach 50–620 kg ha 1.14 PB promoted CO2 uptake (Figures 1D–1F): fixation of CO2 by the PB was at Algal biomass in decaying PB is more readily fermented than plant biomass15 79, 211, and 118 kg ha 1 in the tropical, subtropical, and temperate paddies, rep- and, as a result, PB-derived carbon influences the composition of soil organic resenting 12.7%, 12.5%, and 7.2% of the total CO2 fixation. PB tended to fix CO2 carbon (SOC) and provides varied substrates for soil respiration and during the early half of the rice season. methanogenesis. CH4 fluxes increased after flooding and transiently declined to 0 mg m 2 h 1 In addition to affecting CO2 assimilation and substrate supply, PB inevitably during the mid-season aeration period in the subtropical and temperate paddy also affects topsoil and floodwater properties directly associated with CH4 pro- fields and essentially stopped after the 80th day, when the soil was dried for duction and oxidation. During the colonization and growth phase of the PB, auto- rice harvest (Figures 2A–2C). The cumulative CH4 emissions from the tropical, ll The Innovation 3(1): 100192, January 25, 2022 1 Report www.the-innovation.org Figure 1. CO2 flux in the experimental paddy fields CO2 flux in the (A) tropical, (B) subtropical, and (C) temperate experimental paddy fields during a rice growth season with and without periphytic biofilm (PB), and the fluxes induced by PB (D, tropical; E, subtropical; and F, temperate). In (A–C), the error bars indicate the standard deviations (SD) of triplicate results. In (D–F), positive values indicate that gas fluxes in the presence of PB were greater than those in the control (absence of PB), and negative values indicate the reverse. subtropical, and temperate experimental paddy fields with PB were 68.2, 262.4, The presence of PB increased the concentration and changed the composition and 41.2 kg ha 1, respectively. The presence of PB increased CH4 emission (Fig- of the SOC. Effects were most pronounced in the 0–1 cm soil layer, with a ures 2D–2F): the total CH4 emission induced by PB amounted to 4.8, 101, and significant increase in total organic carbon (TOC) concentrations, from 21.5 ± 4.3 kg ha 1 over a single rice growing season across tropical, subtropical, and 1.8 to 24.6 ± 0.9 g kg 1 (Figure 4A). Dissolved organic carbon (DOC) concentra- temperate rice paddies. These represented 7.1%, 38.5%, and 10.4% of the total tions in the soil pore water in the presence of PB gradually increased after flood- CH4 emission of the experimental paddies. During mainly the later half of the ing, from 66.2 ± 8.1 to 81.9 ± 5.6 mg L 1, while DOC concentrations in soil without rice season, fluxes of CH4 were lower in the presence of PB than in its absence. PB were significantly lower, fluctuating between 48.6 ± 5.9 and 58.8 ± 4.6 mg L 1 (Figure 4B). In the presence of PB, concentrations of 491 different soil organic molecules increased (Figure 4C), with lipids and organic acids accounting for CH4 production and oxidation by PB 45% and 13% of those increased organic molecules, respectively (Figure S6A), To test whether PB itself produces CH4, we incubated PB in serum bottles and while the concentrations of 214 soil organic molecules, mostly heterocyclic com- measured CH4 concentrations in the headspace over time (Figure 3A). We found pounds (Figure S6B), reduced. that under anaerobic conditions CH4 emission was not detected in the presence To test whether the increased SOC derives from the CO2 fixed by PB, we of PB, even with an additional supply of acetate, which is a methanogenic sub- labeled CO2 in the incubation chamber containing soil columns with and without strate (CH3COOH / CH4 + CO2);26 in contrast, CH4 emission was observed PB (Figure S3B). The 13CO2 (enrichment >98 atom%) was injected into the sealed from the anaerobic soil, and the presence of PB increased CH4 flux in the chamber, then the 13C enrichment of PB and surface soil was determined after initial 2 days. incubation. The atomic proportion of 13C/12C in the PB reached 2.41% ± To test whether PB is capable of CH4 oxidation, we incubated different 0.002% (Figure 4D), while the soil TOC was more 13C enriched in the presence amounts of PB in Woods Hole medium with a high initial concentration of CH4 of PB (1.10% ± 0.01%) than when PB was absent (1.08% ± 0.004%). These results (10,000 ppm/6,530 mg L 1). The CH4 was consumed over time in all bottles suggest that PB fixed CO2 from the atmosphere and released the carbon to the (Figure 3B), including in the control without PB. Nevertheless, by day 4, the deple- surface soil. tion of CH4 in the incubations that had received the largest amounts of PB amend- ment was significantly greater (p < 0.05) than in the absence of PB. Consistent Redox potentials with this evidence of biologically mediated CH4 removal, in separate experiments Redox potentials (Ehs) of surface soil and floodwater were profiled during the we detected higher concentrations of dissolved O2 in floodwater in the presence growth phase (day 10 after flooding) and the decay phase (day 40 after flooding) of PB (Figure S2). of PB. At day 10, Ehs of the floodwater and at the 1.5-cm soil depth were similar, but the Eh decreased sharply between the depths of 1.5 and 3.5 cm (Figure 5A). Soil organic carbon The presence of PB significantly (p < 0.01) increased the Eh of floodwater and Flooded soil was the main CH4 source in rice paddy. To evaluate the effect of surface soil ( 1.5 to 1.5 cm depth) from 237 to 257 mV. At depths >3.5 cm, PB on soil properties, soil columns were incubated for 40 days in microcosms un- the Eh in soil declined to 30 mV, both with and without PB. At day 40 after flood- der flooding (Figure S3A). The PB grew rapidly in the first 10 days (growth phase), ing there was no change in Eh of the floodwater and surface soil (0–1.5 cm layer) reached a maximum biomass, and then began autogenic sloughing between day where PB was absent, and in soil >3.5 cm deep, the Eh decreased to 158 mV 10 and day 20, and then autogenic sloughing became the dominant process (Figure 5B). In the presence of PB, there was a steeper decrease in Eh of the sur- (decay phase) from day 20 onward,10 as indicated by the F0 in Figure S4A and face soil, from 248 mV at the water surface to 200 mV in soil >3.5 cm deep. observed in Figure S5. Regardless of the presence of low-Eh microsites likely present throughout the 2 The Innovation 3(1): 100192, January 25, 2022 www.cell.com/the-innovation Report Figure 2. CH4 flux in the experimental paddy fields CH4 flux in the (A) tropical, (B) subtropical, and (C) temperate experimental paddy fields during a rice growth season with and without PB, and the fluxes induced by PB (D, tropical; E, subtropical; and F, temperate). In (A–C), the error bars indicate the SD of triplicate results. In (D–F), positive values indicate that gas fluxes in the presence of PB were greater than in the control (absence of PB), and negative values indicate the reverse. soil column, this low Eh in bulk soil below 3.5 cm is consistent with the potential anotrophs showed no significant differences (p > 0.05) between microcosms onset of methanogenesis.27 with or without PB. Based on mcrA and pmoA gene sequence analyses, there were similar Microbial composition and metabolism methanogen and methanotroph genera found in the soils regardless of PB The size and composition of the communities of methanogens and aerobic presence or absence, but the relative abundances in the relevant sequence li- methanotrophs in the soil microcosms were influenced by the presence of PB. braries significantly differed (p < 0.05). More specifically, the presence of PB Regardless of the presence or absence of PB, the mcrA and pmoA gene abun- tended to increase the relative abundance of Methanosarcina in both the sur- dances (indicators of methanogen and methanotroph, respectively) both ap- face and the deep soil, and led to decreases in Methanobacterium in surface peared to be higher in the surface (0–1 cm) soil layer than the abundances soil (0–1 cm layer) and Methanocorpusculum in the deep soil (5–20 cm layer) at deep soil layers (5–20 cm) (Figures 6A and 6B). This pattern implies that (Figure 6C). For the methanotrophs, the presence of PB increased the relative CH4 production and its aerobic oxidation were generally more prevalent in abundance of Methylocaldum from 13.7% to 77.7% in the surface soil. In the the surface soil. However, the presence of PB enhanced the size of the metha- deep soil, PB increased the relative abundance of Methylocystis, but decreased nogen community in the surface soil (p < 0.05), which increased from (6.25 ± that of Methylomonas (Figure 6D). The methanogen and methanotroph genera 0.68) 3 108 to (8.97 ± 0.78) 3 108 gene sequences g 1 soil (Figure 6A). In found in PB were consistent with those in soil. Methanobacterium and Methyl- contrast, pmoA gene abundances were higher (p < 0.05) in soil without PB (Fig- ocystis were the most abundant methanogen and methanotroph genera, ure 6B), indicating that the proliferation of aerobic methanotrophs was inhibited respectively, in PB, but were in proportions of only 3.8%–12.2% and 3.8%– in the presence of PB. The community size of methanogens and aerobic meth- 23.8% in soil. Figure 3. Methanogenesis and CH4 oxidation ca- pacity of PB (A) Methane emissions by PB and soil under anaerobic incubation. (B) Consumption of CH4 in the headspace of the bottles with different amounts of PB. Error bars are SD of three replicates. ll The Innovation 3(1): 100192, January 25, 2022 3 Report www.the-innovation.org Figure 4. Effects of PB on SOC in microcosms (A) Concentration of TOC in different soil layers after in- cubation. (B) Temporal changes in concentration of DOC in soil pore water after flooding. (C) Changes in soil concentrations of organic molecules in the pres- ence of PB. The horizontal axis represents the loga- rithm of the fold changes in the concentration of organic molecules calculated as average concentra- tion in the PB treatment/average concentration in control. Light and dark blue circles indicate increases and decreases in concentrations of organic mole- cules, respectively, and gray circles indicate organic molecules with no change in concentration (p > 0.05). (D) Proportion of 13C/12C within PB biomass (light blue circles), soil when PB was absent (orange circles), and soil in the presence of PB (dark blue cir- cles) after pulse labeling by 13CO2. *, p < 0.05 and **, p < 0.01. Fixation of CO2 was accompanied by release of O2, and might promote CH4 oxidation. Unex- pectedly, PB promoted CH4 emission from soil (Figures 2 and 3A), possibly due to the effects of PB on the soil properties and soil microbial composition directly associated with CH4 pro- duction, e.g., availability of methanogenic substrates, soil Eh, and activity of methano- gens.22,27,33-35 Fixed carbon was released to the soil as meth- anogenic substrates accompanied by the growth The main methanogenic pathways in paddy soil are aceticlastic methanogen- and decay of PB: over the growth and decay phases of PB, the CO2 that was fixed esis (CH3COOH / CH4 + CO2) and hydrogenotrophic methanogenesis (4H2 + in its biomass was gradually released into the surface soil and pore water as ex- CO2 / CH4 + 2H2O).28 We analyzed key metabolites involved in methanogenesis udations and debris, as indicated by the higher levels of TOC and DOC (Figures 4A via metabolomics (Figure S7 and Table S1) and found that concentrations of and 4B). For instance, microbiomes in PB release organic compounds, such as coenzyme F420, which is a key electron transporter involved in two steps of hydro- complex organic acids and monosaccharides, into the extracellular environ- genotrophic methanogenesis from methenyltetrahydromethanopterin to methyl- ment;36-39 biopolymers within the PB debris, such as cellulose, were likely depoly- tetrahydromethanopterin;29 F420H2; and their precursor, the coenzyme F420-1, merized to form sugar monomers and lipids, which were converted to glycerol were upregulated in the presence of PB, leading to an acceleration of methano- and long-chain fatty acids,26,40 as indicated by the greater concentrations of lipids genesis via the hydrogenotrophic pathway. In addition, the upregulation of the and organic acids (Figure S6) and diversity of other organic molecules in soils metabolites acetyl-coenzyme A, malonyl-coenzyme A, and 3-hydroxypropionyl- with PB (Figure 4C). coenzyme A in the 3-hydroxypropionate (3HP) bicycle metabolic pathway30 Variations in Eh reflected the dynamics of the release and depletion of elec- also suggested a more active carbon fixation in the PB group (consistent with trons and terminal electron acceptors such as O2 and Fe3+.20,41 Photosynthetic Figure 3). microalgae were the dominant community during the growth phase of PB,16 and O2 released by microalgae diffused into floodwater and surface soil and DISCUSSION increased the Eh (Figure 5A). As PB began autogenic sloughing, the exudations Indigenous capacity of periphytic biofilm to produce and oxidize CH4 and minimal debris were released to the soil and degraded by soil microbes, lead- The incubation experiments indicated that PB has little indigenous capacity to ing to a depletion of soil O2 and of other electron acceptors.20 When O2 released produce CH4, but may promote aerobic CH4 oxidation. However, based on the by PB was in excess of that consumed during degradation of PB debris, the Eh sequence analyses, both mcrA and pmoA genes were present in PB (Figure S8), decreased in the near-surface soil during the decay phase of PB (Figure 5B). indicating that methanogens and methanotrophs coexist in PB. As methanogens This methanogenic potential is also consistent with the observed reduction of are strict anaerobes,18 methanogenesis was suppressed by photosynthetic the electron acceptor Fe3+ at this depth in the PB group soil at day 40 (Figure S9), microalgae due to O2 release in the PB environment. Therefore, PB worked as which similarly implies a more reducing environment in the presence of PB. a CH4 sink through both the production of O2 and the provision of a habitat for Following the typical model of a thermodynamically controlled redox succession aerobic methanotrophs. in saturated soil,20 the reduction of Fe3+ would be predicted to proceed prior to the onset of methanogenesis as a function of depth. Accordingly, it appears that the The underlying reasons that PB promoted CH4 emission and CO2 fixation bulk-phase solid Fe had been reduced. The promotion of CO2 fixation by PB may be attributed to autotrophs present in Changes in size and composition of microbial communities are closely related PB. Following fertilizer application and flooding, PB colonizes rapidly on the soil to their environments, and Eh and substrate composition and availability are ma- surface due to favorable light and nutrient conditions.19 Microalgae and other jor drivers of this change.42,43 For instance, since methanogens are strict anaer- autotrophs in PB fix CO2 from the atmosphere during photosynthesis;16,17 the obes,18 a higher SOC availability, as well as lower Eh, promoted an increase in the temporal fluctuation in CO2 flux was consistent with variations in daily weather methanogen community size in the surface soil with PB (Figure 6A). The majority patterns (temperature and illumination).31 After the early stages, PB decayed of methanogens, e.g., genera Methanocorpusculum, Methanosarcina, and as the canopy cover of the developing rice increased, as a result of reduced light Methanocella, produce CH4 through the hydrogenotrophic pathway,44 but Meth- and nutrient availability.19 The light dependence indicated that microbial CO2 anosarcina has a broader substrate spectrum,40 which could also utilize acetate fixation in rice paddies is phototrophic rather than chemotrophic.32 Microbial to produce CH4.43,45 Evidence for the shift toward Methanosarcina in the soil of CO2 fixation plays a significant role in the terrestrial carbon sink. The rate of mi- the PB group (Figure 6C) suggested a promotion of aceticlastic methanogenesis. crobial phototrophic CO2 fixation was significantly higher in rice paddies than in Aerobic methanotrophs obligately require an aerobic environment,46 and upland soils,32 which might be attributed to the CO2 fixation of algae in PB (7.2%– accordingly, we found that their abundance was lower (relative to the control) 12.7% of the total CO2 sink in this study). in the presence of PB, due to the lower Eh in the surface soil. In the surface 4 The Innovation 3(1): 100192, January 25, 2022 www.cell.com/the-innovation Report Figure 5. Effect of PB on redox potential (Eh, mV) of floodwater and surface soil Eh profiles in the micro- cosms with and without PB during (A) the growth phase (day 10 after flooding) and (B) the decay phase (day 40 after flooding) of PB. Error bars representing SD (n = 3) are mostly smaller than the data symbols and hence not observable. rapidly, and the surface soil was relatively oxidized.27 PB increased Eh of the surface soil and floodwater; however, degradation of the PB exudates and debris gradually enhanced SOC availability, leading to the increase in CH4 produc- tion. The PB growth phase coincided with the time of high CH4 production, when most of the CH4 was emitted quickly through ebullition, so there was less or a negligible chance for CH4 oxidation on the soil surface by PB.26 During the decay phase, accumulation and degradation of PB debris and exudates were maximal and contributed to a higher abundance of methano- gens, a lower abundance of methanotrophs, and, as a result, the greatest levels of CH4 emis- sion. Although PB works as an indigenous CH4 sink, its contribution to CH4 production plays a soil, limitation of O2 imposed by the PB selected for an increased relative abun- more prominent role. This is the reason that PB promoted CH4 emission in dance of the genus Methylocaldum (Figure 6D) against a backdrop of declining the field. community size of methanotrophs (Figure 6B), which is perhaps because Previous studies in mangrove reported a decrease in CO2 fluxes by biofilms Methylocaldum is adapted to surviving periods of stress.47 Methylocaldum is a due to photosynthetic CO2 consumption at the sediment surface,49 which is potential thermophile, but additionally shows a capability for adapting to drastic consistent with the results in rice paddies. However, different from in rice paddies, temperature gradients often found in transiently warm environments such as the CH4 flux was also reduced by biofilm on the mangrove soil surface. This paddies.47,48 This property probably gives it an advantage over the genus Meth- might be attributed to the different biofilm biomass, microbial composition, tidal ylocystis, as it possesses survival strategies in response to the stress of limited O2 cycle (flooding and drainage), and flux measurement intervals compared with rice availability. Furthermore, as a type I methanotroph, Methylocaldum is thought to paddies. grow quickly under favorable conditions.45,46 Taken together, our results show that the presence of PB affected CH4 emis- Contribution of periphytic biofilm to carbon flux sion through the regulation of microbial composition and metabolism, availability The global CH4 emission factor of flooded rice fields was estimated to be of methanogenic substrates, and soil Eh. During its growth phase, PB grew 1.19 kg ha 1 day 1 (4.96 mg m 2 h 1),50 and the seasonal average CH4 flux in Figure 6. Effects of PB on the size and composition of methanogen and aerobic methanotroph communities in the surface and deep layer of paddy soil microcosms after incu- bation Gene abundances of (A) mcrA, quantifying the methanogens, and (B) pmoA, quantifying the methanotrophs, and genus-level compositions of (C) methanogenic and (D) methanotrophic communities in the soil. ll The Innovation 3(1): 100192, January 25, 2022 5 Report China is 9.02 mg m 2 h 1.51 In this study, the seasonal CH4 fluxes from tropical, 2. Runkle, B.R.K., Suvocarev, K., Reba, M.L., et al. (2019). Methane emission reductions from the www.the-innovation.org subtropical, and temperate experimental paddy fields were 4.12, 9.42, and alternate wetting and drying of rice fields detected using the eddy covariance method. Environ. Sci. Technol. 53, 671–681. 1.48 mg m 2 h 1 on average in the presence of PB, respectively, and 3.82, 3. Intergovernmental Panel on Climate Change (1997). Methane emissions from rice cultiva- 5.57, and 1.32 mg m 2 h 1 from fields without PB, which are in the same range tion: flooded rice fields. In Revised 1996 IPCC Guidelines for National Greenhouse Gas as other studies. Inventories: Reference Manual https://www.ipcc-nggip.iges.or.jp/public/gl/guidelin/ We quantified the contribution of PB to paddy carbon flux by comparing the ch4ref5.pdf. fluxes induced by PB with the total flux from fields with PB. In the “with PB” field, 4. Intergovernmental Panel on Climate Change (2013). Climate change 2013: the physical sci- the paddy was managed based on local practice, and PB grew normally without ence basis. In Working Group I Contribution to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change https://www.ipcc.ch/site/assets/uploads/ intervention, which is suitable for being a reference. The contributions of PB to the 2018/02/WG1AR5_all_final.pdf. total CO2 sink and CH4 efflux are estimated as 7.2%–12.7% and 7.1%–38.5%, 5. Bridgham, S.D., Cadillo-Quiroz, H., Keller, J.K., and Zhuang, Q. (2013). Methane emissions respectively, in the experimental paddy fields. Variations in the contribution of from wetlands: biogeochemical, microbial, and modeling perspectives from local to global PB to carbon flux were found among the three climatic zones. scales. Glob. Change Biol. 19, 1325–1346. Soil TOC content (Table S2), DOC content (Figure S4B), and PB biomass (Fig- 6. Yan, X., Akiyama, H., Yagi, K., and Akimoto, H. (2009). Global estimations of the inventory and ure S4A) were measured in the three experimental paddy fields. The PB biomass, mitigation potential of methane emissions from rice cultivation conducted using the 2006 Intergovernmental Panel on Climate Change Guidelines. Glob. Biogeochem. Cy. 23, GB2002. as well as TOC and DOC contents, was highest in the subtropical field compared 7. Huang, Y., Sass, R.L., and Fisher, J.F.M. (1998). A semi-empirical model of methane emission with tropical and temperate, indicating a stronger CO2 uptake of the subtropical from flooded rice paddy soils. Glob. Change Biol 4, 247–268. PB. The increase in CH4 emission induced by PB is mainly attributed to the 8. Fumoto, T., Kobayashi, K., Li, C., et al. (2008). Revising a process-based biogeochemistry organic input from PB. The highest PB biomass led to maximum DOC accumu- model (DNDC) to simulate methane emission from rice paddy fields under various residue lation and methanogen abundance and, as a result, the most active CH4 emission management and fertilizer regimes. Glob. Change Biol 14, 382–402. induced by PB. Further studies are still needed to investigate the factors driving 9. Oertel, C., Matschullat, J., Zurba, K., et al. (2016). Greenhouse gas emissions from soils—a review. Geochemistry 76, 327–352. PB biomass (e.g., soil and climate properties) and quantitatively explain the 10. Wu, Y. (2016). Periphyton: Functions and Application in Environmental Remediation changes in CH4 and CO2 flux. (Elsevier). Despite the variations in contribution of PB to carbon flux, microbial aggregates 11. Zhu, N., Wu, Y., Tang, J., et al. (2018). A new concept of promoting nitrate reduction in surface on the soil surface of tropical, subtropical, and temperate rice paddies all waters: simultaneous supplement of denitrifiers, electron donor pool, and electron media- increased CH4 emissions through CO2 fixation, carbon release, and associated tors. Environ. Sci. Technol. 52, 8617–8626. increases in labile SOC availability. PB functioned as a biotic converter that trans- 12. Azim, M.E. (2009). Photosynthetic periphyton and surfaces. In Encyclopedia of Inland Waters, G.E. Likens, ed. (Academic Press), pp. 184–191. ferred atmospheric CO2 into CH4. The amount of CO2 fixed by PB was 2.1–57.3 13. Flipo, N., Rabouille, C., Poulin, M., et al. (2007). Primary production in headwater streams of times that of induced CH4 emissions, indicating a net flow of carbon from the at- the Seine basin: the Grand Morin river case study. Sci. Total Environ. 375, 98–109. mosphere to the rice paddies. However, CH4 is a potent greenhouse gas, with a 14. Kimura, M., Murase, J., and Lu, Y. (2004). Carbon cycling in rice field ecosystems in the 25-fold greater greenhouse warming potential than CO2 on a 100-year time context of input, decomposition and translocation of organic materials and the fates of their horizon.52 The presence of PB reduced greenhouse gas emissions by 11.8 kg end products (CO2 and CH4). Soil Biol. Biochem. 36, 1399–1416. CO2 equiv ha 1 season 1 in the temperate experimental paddy, and increased 15. West, W.E., Coloso, J.J., and Jones, S.E. (2012). Effects of algal and terrestrial carbon on methane production rates and methanogen community structure in a temperate lake sedi- by 40.8 and 2,318.0 kg CO2 equiv ha 1 season 1 in the tropical and subtropical ment. Freshw. Biol 57, 949–955. experimental paddies, respectively. 16. Wu, Y., Liu, J., Lu, H., et al. (2016). Periphyton: an important regulator in optimizing soil phos- Worldwide, the role of PB in CH4 emissions has not been recognized. PB is phorus bioavailability in paddy fields. Environ. Sci. Pollut. R. 23, 21377–21384. ubiquitously distributed across tropical, subtropical, and temperate rice paddies, 17. Mori, T., Miyagawa, Y., Onoda, Y., and Kayaba, Y. (2018). Flow-velocity-dependent effects of and the assimilated CO2 by PB reached 79, 211, and 118 kg ha 1 season 1, turbid water on periphyton structure and function in flowing water. Aquat. Sci. 80, 6. respectively. The decay phase of PB (days 20–40 after flooding) exactly over- 18. Angel, R., Claus, P., and Conrad, R. (2012). Methanogenic archaea are globally ubiquitous in aerated soils and become active under wet anoxic conditions. ISME J. 6, 847–862. lapped with the period when the paddy soils showed maximum CH4 emission 19. Li, J.Y., Deng, K.Y., Cai, S.J., et al. (2020). Periphyton has the potential to increase phosphorus potentials, and therefore, the PB exudations and debris provided abundant use efficiency in paddy fields. Sci. Total Environ. 720, 137711. organic carbon for CH4 production. This suggests the possibility of developing 20. Arndt, S., Jørgensen, B.B., LaRowe, D.E., et al. (2013). Quantifying the degradation of organic mitigative strategies for greenhouse gas emissions through PB regulation. matter in marine sediments: a review and synthesis. Earth-sci. Rev. 123, 53–86. Decay of PB is due to the low availability of light and nutrients at the tillering 21. Leopold, A., Marchand, C., Deborde, J., et al. (2013). Influence of mangrove zonation on CO2 stage of rice.19 If its light and nutrient capture ability are improved, the lifespan fluxes at the sediment–air interface (New Caledonia). Geoderma 202-203, 62–70. 22. Liang, X., Zhang, X., Sun, Q., et al. (2015). The role of filamentous algae Spirogyra spp. in of PB is expected to be prolonged, which on one hand helps the rice paddy to methane production and emissions in streams. Aquat. Sci. 78, 227–239. fix more CO2 and, on the other hand, staggers the times of PB carbon input 23. Bogard, M.J., del Giorgio, P.A., Boutet, L., et al. (2014). Oxic water column methanogenesis as and maximum CH4 emission potentials, hence enhancing the carbon sink of a major component of aquatic CH4 fluxes. Nat. Commun. 5, 5350. terrestrial ecosystems and achieving sustainable development and carbon 24. Iguchi, H., Umeda, R., Taga, H., et al. (2019). Community composition and methane oxidation neutrality.53,54 activity of methanotrophs associated with duckweeds in a fresh water lake. J. Biosci. Bioeng. 128, 450–455. 25. Tang, J., Zhu, N., Zhu, Y., et al. (2018). Sustainable pollutant removal by periphytic biofilm via Conclusions microbial composition shifts induced by uneven distribution of CeO2 nanoparticles. This study provides proof-of-concept evidence for the discrimination of the Bioresour. Technol 248, 75–81. contributions of surface microbial aggregates (i.e., PB) from those of soil mi- 26. Malyan, S.K., Bhatia, A., Kumar, A., et al. (2016). Methane production, oxidation and mitiga- crobes to CO2 and CH4 emissions throughout the rice growing season. Microbial tion: a mechanistic understanding and comprehensive evaluation of influencing factors. aggregates (i.e., PB) on soil surfaces increased CH4 emission through CO2 fixa- Sci. Total Environ. 572, 874–896. tion by autotrophs and were associated with increases in availability of labile 27. Wang, Z.P., DeLaune, R.D., Patrick, W.H., and Masscheleyn, P.H. (1993). Soil redox and pH effects on methane production in a flooded rice soil. Soil Sci. Soc. Am. J. 57, SOC and changes in Eh and the soil microbiome. Thus, PB functioned as a biotic 382–385. converter of atmospheric CO2 into CH4. Overall, microbial aggregates at the soil 28. Fenchel, T., King, G.M., and Blackburn, T.H. (2012). Chapter 1 – bacterial metabolism. In surface play a pivotal role in carbon biogeochemistry of rice paddies, confirming Bacterial Biogeochemistry, Third Edition, T. Fenchel, G.M. King, and T.H. Blackburn, eds. that they should be accounted for in the development and optimization of predic- (Academic Press), pp. 1–34. tive models of global carbon fluxes. 29. Allen, K.D., and White, R.H. (2018). Identification of the radical SAM enzymes involved in the biosynthesis of methanopterin and coenzyme F420 in methanogens. Method. Enzymol. 606, 461–483. MATERIAL AND METHODS 30. Herter, S., Farfsing, J., Gad’On, N., et al. (2001). Autotrophic CO2 fixation by Chloroflexus aur- Material and methods are provided in the supplemental information. antiacus: study of glyoxylate formation and assimilation via the 3-hydroxypropionate cycle. J. Bacteriol. 183, 4305–4316. REFERENCES 31. Yan, Z., Shen, T., Li, W., et al. (2021). Contribution of microalgae to carbon sequestration in a 1. Saito, M., Miyata, A., Nagai, H., and Yamada, T. (2005). Seasonal variation of carbon dioxide natural karst wetland aquatic ecosystem: an in-situ mesocosm study. Sci. Total Environ. exchange in rice paddy field in Japan. Agr. For. Meteorol. 135, 93–109. 768, 144387. 6 The Innovation 3(1): 100192, January 25, 2022 www.cell.com/the-innovation Report 32. Ge, T., Wu, X., Chen, X., et al. (2013). Microbial phototrophic fixation of atmospheric CO2 in 48. Medvedkova, K.A., Khmelenina, V.N., Suzina, N.E., and Trotsenko, Y.A. (2009). Antioxidant China subtropical upland and paddy soils. Geochim. Cosmochim. Ac. 113, 70–78. systems of moderately thermophilic methanotrophs Methylocaldum szegediense and 33. Said-Pullicino, D., Miniotti, E.F., Sodano, M., et al. (2015). Linking dissolved organic carbon Methylococcus capsulatus. Microbiology 78, 670–677. cycling to organic carbon fluxes in rice paddies under different water management prac- 49. Jacotot, A., Marchand, C., and Allenbach, M. (2019). Biofilm and temperature controls on tices. Plant Soil 401, 273–290. greenhouse gas (CO2 and CH4) emissions from a Rhizophora mangrove soil (New 34. Hatala, J.A., Detto, M., and Baldocchi, D.D. (2012). Gross ecosystem photosynthesis causes Caledonia). Sci. Total Environ. 650, 1019–1028. a diurnal pattern in methane emission from rice. Geophys. Res. Lett. 39, L06409. 50. Wang, J., Akiyama, H., Yagi, K., and Yan, X. (2018). Controlling variables and emission factors 35. Penning, H., and Conrad, R. (2007). Quantification of carbon flow from stable isotope frac- of methane from global rice fields. Atmos. Chem. Phys. 18, 10419–10431. tionation in rice field soils with different organic matter content. Org. Geochem. 38, 51. Sun, M., Zhang, H., Dong, J., et al. (2018). A comparison of CH4 emissions from coastal and 2058–2069. inland rice paddy soils in China. Catena 170, 365–373. 36. Klein, T.A., Ahmad, S., and Whitney, J.C. (2020). Contact-dependent interbacterial antago- 52. Jiang, Y., Liao, P., van Gestel, N., et al. (2018). Lime application lowers the global warming nism mediated by protein secretion machines. Trends Microbiol. 28, 387–400. potential of a double rice cropping system. Geoderma 325, 1–8. 37. Spaepen, S., Vanderleyden, J., and Remans, R. (2007). Indole-3-acetic acid in microbial and 53. Chen, J.M. (2021). Carbon neutrality: toward a sustainable future. The Innovation 2, 100127. microorganism-plant signaling. FEMS Microbiol. Rev. 31, 425–448. https://doi.org/10.1016/j.xinn.2021.100127. 38. Yu, C., Harrold, D.R., Claypool, J.T., et al. (2017). Nitrogen amendment of green waste im- 54. Cheng, H. (2020). Future earth and sustainable developments. The Innovation 1, 100055. pacts microbial community, enzyme secretion and potential for lignocellulose decomposi- https://doi.org/10.1016/j.xinn.2020.100055. tion. Process. Biochem. 52, 214–222. 39. Kalscheur, K.N., Rojas, M., Peterson, C.G., et al. (2012). Algal exudates and stream organic matter influence the structure and function of denitrifying bacterial communities. Microb. ACKNOWLEDGMENTS Ecol. 64, 881–892. This work was supported by the National Natural Science Foundation of China 40. Thauer, R.K., Kaster, A.K., Seedorf, H., et al. (2008). Methanogenic archaea: ecologically rele- (41825021, 41961144010, and 31772396), the Natural Science Foundation of Jiangsu Prov- vant differences in energy conservation. Nat. Rev. Microbiol. 6, 579–591. ince (BZ2019015 and BE2020731), and the Original Innovation Project of the Chinese Acad- 41. Ali, M.A., Sattar, M.A., Islam, M.N., and Inubushi, K. (2014). Integrated effects of organic, inor- emy of Sciences (ZDBS-LY-DQC024). ganic and biological amendments on methane emission, soil quality and rice productivity in irrigated paddy ecosystem of Bangladesh: field study of two consecutive rice growing sea- sons. Plant Soil 378, 239–252. AUTHOR CONTRIBUTIONS 42. Singh, A., Singh, R.S., Upadhyay, S.N., et al. (2012). Community structure of methanogenic S.W. and Y.W. designed the study. J.D., G.Z., N.G., and J.P. improved the experimental de- archaea and methane production associated with compost-treated tropical rice-field soil. signs. S.W. performed the experiments with help from P.S. S.W., Y.W., S.E.-E., and J.P. wrote FEMS Microbiol. Ecol. 82, 118–134. 43. Wang, C., Shen, J., Liu, J., et al. (2019). Microbial mechanisms in the reduction of CH4 emis- the paper with input from all coauthors. sion from double rice cropping system amended by biochar: a four-year study. Soil Biol. Biochem. 135, 251–263. DECLARATION OF INTERESTS 44. Alpana, S., Vishwakarma, P., Adhya, T.K., et al. (2017). Molecular ecological perspective of methanogenic archaeal community in rice agroecosystem. Sci. Total Environ. 596-597, The authors declare no competing interests. 136–146. 45. Kong, D., Li, S., Jin, Y., et al. (2019). Linking methane emissions to methanogenic and meth- LEAD CONTACT WEBSITE anotrophic communities under different fertilization strategies in rice paddies. Geoderma 347, 233–243. The lead contact website for this article is at http://sourcedb.issas.cas.cn/yw/rc/fas/ 46. Gao, D., Sheng, R., Whiteley, A.S., et al. (2020). Effect of phosphorus amendments on rice rhi- 201501/t20150107_4295452.html. zospheric methanogens and methanotrophs in a phosphorus deficient soil. Geoderma 368, 114312. 47. Sherry, A., Osborne, K.A., Sidgwick, F.R., et al. (2016). A temperate river estuary is a sink for SUPPLEMENTAL INFORMATION methanotrophs adapted to extremes of pH, temperature and salinity. Environ. Microbiol. Supplemental information can be found online at https://doi.org/10.1016/j.xinn.2021. Rep. 8, 122–131. 100192. ll The Innovation 3(1): 100192, January 25, 2022 7