Importance of periphytic biofilms for carbon cycling in paddy fields:A review

Lei ZHOU ,Yonghong WU,* ,Junzhuo LIU ,Pengfei SUN ,Ying XU ,Jan DOLFING ,Robert G.M.SPENCER and Erik JEPPESEN

1State Key Laboratory of Soil and Sustainable Agriculture,Institute of Soil Science,Chinese Academy of Sciences,Nanjing 210008(China)

2Zigui Three Gorges Reservoir Ecosystem,Observation and Research Station of Ministry of Water Resources of the People’s Republic of China,Yichang 443605(China)

3College of Nanjing,University of Chinese Academy of Sciences,Nanjing 211135(China)

4Faculty of Energy and Environment,Northumbria University,Newcastle upon Tyne NE1 8QH(UK)

5Department of Earth,Ocean and Atmospheric Science,Florida State University,Tallahassee FL 32306(USA)

6Department of Ecoscience &Centre for Water Technology(WATEC),Aarhus University,Aarhus DK-8000(Denmark)

7Sino-Danish Centre for Education and Research,Beijing 100049(China)

8Limnology Laboratory,Department of Biological Sciences and Centre for Ecosystem Research and Implementation(EKOSAM),Middle East Technical University,Ankara 06800(Türkiye)

9Institute of Marine Sciences,Middle East Technical University,Mersin 33731(Türkiye)

ABSTRACT Paddy fields play an important role in global carbon(C)cycling and are an important source of methane(CH4)emissions.Insights into the processes influencing the dynamics of soil organic C(SOC)in paddy fields are essential for maintaining global soil C stocks and mitigating climate change.Periphytic biofilms composed of microalgae,bacteria,and other microorganisms are ubiquitous in paddy fields,where they directly mediate the transfer of elements at the soil-water interface.However,their contributions to C turnover and exchange have been largely neglected.Periphytic biofilms affect and participate in soil C dynamics by altering both abiotic(e.g.,pH and redox potential)and biotic conditions(e.g.,microbial community composition and metabolism).This review summarizes the contributions of periphytic biofilms to soil C cycling processes,including carbon dioxide fixation,SOC mineralization,and CH4 emissions.Future research should be focused on:i)the mechanisms underlying periphytic biofilm-induced C fixation and turnover and ii)quantifying the contributions of periphytic biofilms to soil C uptake,stabilization,and sequestration in paddy fields.

Key Words: carbon fixation,carbon mineralization,carbon sequestration,methane emission,microbial aggregates,soil organic carbon

INTRODUCTION

Soils are the largest carbon(C)pool in terrestrial ecosystems,holding over three times more C than the atmosphere,and the roles of soil organic C(SOC)in soil fertility,crop production,climate regulation,and ecosystem stability have been widely recognized (Zhaoet al.,2018;Bossioet al.,2020).Insights into the processes influencing SOC dynamics can help protect and increase SOC stocks,improve the estimates of C-climate feedback,and design agricultural policies for improving soil quality(Liuet al.,2021a).

Paddy fields,the most extensive artificial wetlands on Earth,provide staple foods for nearly half the population of the world but suffer from intensive human impact,especially in rice cultivation(Macleanet al.,2002).Regular and periodic changes between aerobic and anaerobic conditions in paddy soils enhance the accumulation of organic matter(OM),resulting in higher C stocks compared to upland soils (Liu Y Let al.,2019).For example,in four major grain-producing areas in East China,the SOC sequestration efficiency of paddy fields is 39%-127%higher than that of the adjacent dryland counterparts(Chen X Bet al.,2021).Moreover,wetlands are also hotspots of carbon dioxide(CO2) fixation and methane (CH4) emissions (Chen H Yet al.,2021).Monsoon Asia,with 87%of the global paddy soil area and 90%of rice production(FAO,2017),accounts for approximately 25%-36%of the global CH4emissions(Zhanget al.,2020).Hence,elucidating the mechanisms underlying C turnover in paddy fields,including C fixation,transformation,and mineralization,is essential for maintaining global soil C stocks and mitigating climate change(Chen X Bet al.,2021).

Soil organic C accumulation is a function of the balance between C input(e.g.,litter,root exudates,and organic amendments)and output(e.g.,mineralization,erosion,and dissolved organic C(DOC)losses)(Basile-Doelschet al.,2020).The contribution of microbial necromass to SOC sequestration has also been widely recognized(Maet al.,2018;Wang Cet al.,2021;Sokolet al.,2022).Additionally,the C input,transformation,and rhizodeposition pathways in paddy soils are distinguished from those in upland soils due to regular flooding,intermittent irrigation,and puddling(Liu Y Let al.,2019;Liuet al.,2021c).Additionally,paddy soils are favorable habitats for the development of periphytic biofilms(Suet al.,2017;Wuet al.,2018),also termed periphyton,which are microbial aggregates composed of algae,bacteria,protozoans,metazoans,epiphytes,and abiotic substances(such as extracellular polymers and minerals)and are ubiquitous on the surfaces of substrates in shallow waters(Battinet al.,2016;Wu,2016).During the seedling,tillering,and jointing stages of rice(approximately 1.5-3 months),periphytic biofilms form at the soil-water interface,and their growth and decay processes can significantly alter the ambient physical and chemical conditions and microenvironments (Fig.1).In one growing season,the periphytic biomass accumulated in one hectare of paddy fields can range from hundreds of kilograms to more than one ton(Reynaud and Roger,1978;Liuet al.,2021c).Moreover,periphytic algal residues are more easily decomposed by microorganisms than plant litter because they are richer in proteins and polysaccharides(Zhouet al.,2019,2021).Periphytic biofilm-derived organic C can further increase the content of biolabile DOC and promote microbial metabolism,ultimately contributing to the emission of CO2or CH4,depending on the redox conditions of paddy fields.Lastly,periphytic biofilms at the soil-water interface not only supplement the SOC pool but also affect the soil microbial community composition and microbial-driven mineralization of SOC.

Fig.1 Photos showing periphytic biofilms at the soil-water interface in paddy fields(a-c)and schematic showing the composition of periphytic biofilms(d)(adapted from Sun et al.,2018).EPS=extracellular polymeric substances.

Compared to the large number of studies focused on the effects of plant input,straw return,fertilization,and conservation tillage on SOC dynamics,knowledge of the contribution of periphytic biofilms to C cycling in paddy fields remains limited.In this review,the role of periphytic biofilms in C fixation and transformation is summarized,and current knowledge gaps are identified.

PERIPHYTIC BIOFILMS IN PADDY FIELDS

Previous studies of natural periphytic biofilms have mainly focused on aquatic shallow ecosystems,such as headwaters,streams,rivers,and shallow lakes.Their results have confirmed that biofilms are hotspots of enzymatic activities,OM cycling,ecosystem respiration,and primary production.Therefore,biofilms form the basis of the local food web(Liboriussen and Jeppesen,2009;Battinet al.,2016).As for paddy ecosystems,the soil-water interface is largely the site where the geochemical processes of C and other nutrients are integrated,and periphytic biofilms are direct mediators of element transfer from the soil to the overlying water(Saikia,2011).The formation of periphytic biofilms often begins with the colonization of heterotrophic bacteria and the production of adhesive extracellular polymeric substances(EPS)as a matrix on solid surfaces,followed by the immigration and adhesion of algae(Wu,2016;Sunet al.,2022b).As the resulting aggregates consist of both autotrophic and heterotrophic microorganisms,the growth of periphytic biofilms involves the consumption of CO2and assimilation of nutrients from the ambient environment.Biofilms have been shown to alter pH and redox conditions and accumulate up to 70 mg nitrogen(N)and 2-25 mg phosphorus(P)per gram of biomass(Crimpet al.,2018;Liuet al.,2021c).They release nutrients back to the soil/water when the microorganisms die and decay(Liuet al.,2021c).Active periphytic biofilms release extracellular enzymes and convert nutrients from organic to inorganic forms(Wuet al.,2018).Conversely,soil properties affect the development of periphytic biofilms.Specifically,SOC content and C-nutrient stoichiometry are reportedly major factors influencing photosynthesis,N interception,and P immobilization by periphytic biofilms(Liu JZet al.,2019;Liuet al.,2021c),suggesting that the interactions between periphyton biofilms and soil/water are important processes that influence biogeochemical cycling at the soilwater interface.Recent reviews on the role and importance of periphytic biofilms in paddy fields have focused on their effects on the transport and transformation of N and P in paddy fields,especially ammonia volatilization,nitrification,and denitrification(Wuet al.,2018;Xuet al.,2020).Although the impact of periphytic biofilms on soil C fixation and transformation has been confirmed(Liuet al.,2021a;Wanget al.,2022),comprehensive knowledge of the interactions between periphytic biofilms and soil C dynamics is lacking.

PERIPHYTIC BIOFILMS AND SOIL C FIXATION

Paddy fields exhibit remarkable C fixation capacities.In the subtropical region of China,the average SOC content of paddy soils has increased by 60%over the past 30 years(Wu,2011).In addition to the photosynthetic C derived from rice,soil autotrophic microbes can fix atmospheric CO2and convert it into microbial biomass,which directly contributes to the SOC pool(Yuanet al.,2012;Geet al.,2013;Jasseyet al.,2022).It has been estimated that the annual C assimilation rate of soil autotrophic microbes in the subtropical region of China is 100-450 kg ha-1,and the global rate for microbial synthesis of organic C is 0.68-4.9 Pg per annum,accounting for approximately 0.9%-4.1%of the total CO2fixed by global terrestrial ecosystem each year (Yuanet al.,2012).Moreover,microbial CO2assimilation processes are predominantly phototrophic,and photosynthetic C fixation mainly occurs in the surface soil(0-1 cm)(Wuet al.,2014),likely by the photoautotrophic microorganisms in periphytic biofilms.Pulse-labeling of soils with and without periphytic biofilms using13CO2has shown that the13C/12C ratios of periphytic biofilms were the highest,followed by those of surface soils with periphytic biofilms,and both were significantly higher than those of soils without periphytic biofilms(Wanget al.,2022).Periphytic biofilms contribute 7.2%-12.7%of CO2fixation in rice paddies,and the assimilated CO2amounts to 78,211,and 118 kg ha-1season-1in the tropical,subtropical,and temperate rice paddies in China,respectively(Table I)(Wanget al.,2022).The majority of CO2fixation by periphytic biofilms occurs in the seedling and tillering stages of rice growth(＜40 d),when the microalgae in periphytic biofilms flourish due to adequate sunlight(Wanget al.,2022).Part of the assimilated C in the surface layer is transported downward to the chemoautotrophic microorganisms in the subsurface layers,facilitating the participation of these organisms in the CO2assimilation processes(Wuet al.,2014).

To date,six autotrophic CO2fixation pathways have been identified:the Calvin cycle(Basshamet al.,1950),the reductive tricarboxylic acid cycle(Evanset al.,1966),the reductive acetyl-coenzyme A(CoA)pathway(Woodet al.,1986),the 3-hydroxypropionate/malyl-CoA cycle (Holo,1989),the 3-hydroxypropionate/4-hydroxybutyrate cycle(Berget al.,2007),and the dicarboxylate/4-hydroxybutyrate cycle(Huberet al.,2008),and the last two are often termed together as the 4-hydroxybutyrate cycle.According to field investigations and incubation experiments based on analyses of marker genes(e.g.,cbbL)and activities of enzymes(e.g.,ribulose 1,5-bisphosphate carboxylase/oxygenase),the Calvin cycle is the most common pathway for autotrophic microbes in paddy soils(Wuet al.,2014;Xiaoet al.,2021).Marker genes associated with the other five autotrophic pathways have also been found in paddy soils.A microcosm experiment conducted by Wanget al.(2022)showed that the growth of periphytic biofilms resulted in the upregulation of acetyl-CoA,malonyl-CoA,and 3-hydroxypropionyl-CoA in the surface soil(0-1 cm),suggesting an important role of the 3-hydroxypropionate/malyl-CoA cycle in periphytic biofilm-induced C fixation.An important characteristic of this pathway is the co-assimilation of many organic compounds,making it suitable for mixotrophic microbes(Zarzycki and Fuchs,2011),which concurs with the characteristics of periphytic biofilms being composed of autotrophs and heterotrophs embedded in a matrix of EPS(Liuet al.,2021b).More field investigations and laboratory experiments are needed to further describe the microbial communities and metabolic pathways involved in periphytic biofilm-induced C fixation and the influencing factors.

TABLE IEffects of periphytic biofilms(PBs)on soil pH,redox potential(Eh),and SOC/DOC/DOMa) contents and their contributions to CO2 fixation and CH4 emission in paddy fields

PERIPHYTIC BIOFILMS ALTER SOIL C COMPONENTS

The C sources of periphytic biofilms are mainly dissolved OM(DOM)and photosynthetic C,which enter higher trophic levels through predation and are transferred as C-rich nutrient components(Saikia,2011;Wu,2016).Hence,the growth and decay of periphytic biofilms at the soil-water interface can alter SOC characteristicsviathe consumption and production of organic compounds(Fig.2).Specifically,SOC has been shown to be a major regulatory factor of photosynthesis and biomass accumulation at experimental and regional scales (Liuet al.,2021c).At high SOC and DOC levels,periphytic biofilms are inhabited and shaped by speciesrich algal and bacterial communities and upregulate the expression of many photosynthesis-associated pathways(Liuet al.,2021c).The fate of photosynthetic C generated in periphytic biofilms varies and includes mineralization and release into the atmosphere as CO2/CH4and incorporation into SOC pools,especially biolabile soil C components such as microbial biomass C and DOC (Geet al.,2013;Wuet al.,2014;Xiaoet al.,2021).Accordingly,elevated DOC concentrations in soil pore water and increased surface SOC contents have been observed during the growth and decay of periphytic biofilms(Table I)relative to soils without periphytic biofilms(Liuet al.,2021a;Wanget al.,2022).Increased DOC may result from the release of soluble organic substrates,residues,and their decomposition products(Geet al.,2013;Xiaoet al.,2021).The interplay is complex,as not only the DOC level,but also the DOM level and composition in the soil affect the interactions occurring in periphytic biofilms(Liuet al.,2021a).

Fig.2 Schematic showing the role of periphytic biofilms in soil C dynamics in paddy fields.Periphytic biofilms act as biotic converters who transfer atmospheric CO2 into CH4 by incorporating photosynthetic C into the soil organic C(SOC)pool.The growth and decay of PBs at the soil-water interface alter soil C characteristics via SOC utilization and production,causing changes in pH and redox potential(Eh).Periphytic biofilm-derived labile C(e.g.,dissolved organic C)is processed by soil microorganisms and transformed into CO2/CH4 (aerobic/anaerobic mineralization),microbial biomass C(anabolism),and extracellular C(e.g.,metabolites,enzymes,and aggregation agents).Microbial necromass and metabolites are the precursors of stable C.Extracellular C derived from PBs or soil microorganisms may also influence both labile and stable C.Enzymes may catalyze the depolymerization of soil macromolecular constituents(A),whereas other extracellular substances may promote the aggregation and protection of SOC(B).

High levels of humic-like substances,which are closely related to soil texture,moisture,and complex formation,augment the biomass and N and P contents of periphytic biofilms(Liuet al.,2021c).As microbial aggregates are rich in EPS(Sunet al.,2022b),the growth of periphytic biofilms typically results in the release of organic compounds,including enzymes,complex organic acids,monosaccharides,and proteins,into the surrounding environment (Fig.2)(Kalscheuret al.,2012).The biopolymers(e.g.,cellulose)in periphytic biofilm debris may be depolymerized to form sugar monomers and lipids,which are then transformed into glycerol and long-chain fatty acids (Thaueret al.,2008;Malyanet al.,2016).Specifically,the growth and decay of periphytic biofilms release humic-and tryptophan-like materials into soils and increase the abundances of carbohydrate-,lignin-,lipid-,protein-,and aminosaccharide-like compounds(Table I)(Liuet al.,2021a;Wanget al.,2022).Most of these compounds are considered biolabile substrates for microbes and can act as hotspots for microbial metabolic activities (Zhouet al.,2020).Accordingly,the presence of periphytic biofilms promotes the metabolism of lipids,carbohydrates,and amino acid compounds,including glycerophospholipids,glycine,pyruvate,serine,and threonine,resulting in the upregulation of acetyl-CoA (Wanget al.,2022).These metabolic changes may be intimately associated with the microbial decomposition of periphytic biofilm residues and further coupled with nutrient cycles,including N immobilization and P fraction changes,emphasizing the important role of periphytic biofilms in biogeochemical cycles in paddy fields(Wuet al.,2018).

Composed of complex mucopolysaccharide matrixes with embedded microorganisms,periphytic biofilms act as barriers by clogging the pore space at the soil-water interface(Leopoldet al.,2013),thereby reducing the movement of some minerals and ions(Sunet al.,2021,2022a).Proteinand humic-rich periphytic biofilm residues can sorb onto mineral surfaces (e.g.,iron and aluminum oxides),form new organo-mineral associations,and be incorporated into soil aggregates,promoting the stabilization of soil OM(SOM),which is vital for global C sequestration (Kopittkeet al.,2018).Specifically,photosynthetic C has been found in humins(Xiaoet al.,2021),which are thought to be strongly associated with soil minerals and stable to microbial degradation(Gautamet al.,2021).Other fates of periphytic biofilm-derived C include biomass(cellular components),CO2,excreted metabolites,and microbial necromass,which is an important precursor of stable SOC(Fig.2)(Leopoldet al.,2013;Mitchellet al.,2020).The C isotope labeling technique,together with high-resolution microscopy and high-throughput sequencing,can be utilized for further investigation of periphytic biofilms to better understand the turnover of periphytic biofilm-derived C and its potential role in the stabilization and sequestration of soil C,as well as the associated microbial community composition and activity.

PERIPHYTIC BIOFILMS AND SOIL C MINERALIZATION

The mineralization of SOC is an important SOC output process in which microorganisms use organic molecules and transform C and nutrients into CO2/CH4and soluble inorganic forms (Qiuet al.,2018;Mitchellet al.,2020;Weigleinet al.,2022).Microbial biomass,SOC stability,and electron acceptor availability mainly determine the intensity and rate of SOC mineralization in paddy soils(Liet al.,2021).The growth and decay of periphytic biofilms at the soil-water interface can influence SOM mineralization by altering both abiotic(e.g.,pH and redox potential(Eh))and biotic factors(e.g.,microbial composition and metabolism),leading to changes in CO2and CH4emissions from paddy fields(Fig.2)(Qiuet al.,2018).The presence of periphytic biofilms enhances CH4emissions and contributes 7.1%-38.5%of the total CH4efflux in the tropical,subtropical,and temperate experimental paddy fields (Table I) (Wanget al.,2022).This enhancement is ascribed to the increases in biolabile substrate availability and microbial metabolic activities,as demonstrated by the increased methanogen community in the surface soil (0-1 cm) (Wanget al.,2022).Moreover,periphytic biofilms release exoenzymes that catalyze the hydrolysis of organic compounds,such as phosphate esters,which become available for microbial utilization (Fig.2)(Ellwoodet al.,2012;Wuet al.,2018).

During the colonization and growth phases of periphytic biofilms(seedling and tillering stages of rice growth),microalgae dominate the microbial aggregates and increase oxygen(O2)content and Eh due to photosynthesis,which enhances microbial mineralization by providing electron acceptors(Liet al.,2021)and inhibits CH4production by facilitating the oxidation of CH4at the soil-water interface(Wanget al.,2022).In addition,periphytic biofilms form a physical barrier at the water-soil interface by releasing extracellular polymers,blocking soil pores,and limiting gas transfer to the atmosphere(Leopoldet al.,2015;Jacototet al.,2019).However,as the growth phase of periphytic biofilms coincides with CH4emission bursts through ebullition from paddy fields,periphytic biofilms lack the capacity to fully prevent such emissions by oxidizing all CH4,although CH4oxidation by periphytic biofilmsper sehas been observed in microcosmic experiments(Wanget al.,2022).

Heterotrophs dominate the microbial aggregates when periphytic biofilms decay (Liet al.,2020).Heterotrophic respiration and microbial decomposition of biolabile DOC derived from periphytic biofilm exudates and debris can lead to greater consumption of O2and other electron acceptors(e.g.,ferric ion and nitrate ion)and thus decrease soil Eh (Arndtet al.,2013).This substrate-rich reductive environment can further promote methanogen community activities,especially aceticlastic methanogenesis by the genusMethanosarcinawith a broader substrate spectrum than other methanogens,and reduce the abundance of aerobic methanotroph communities (Thaueret al.,2008;Malyanet al.,2016).Consequently,the presence of periphytic biofilms has been shown to result in higher levels of CH4production and emission.Overall,periphytic biofilms act as biotic converters that transform atmospheric CO2into CH4by incorporating photosynthetic C into biolabile SOC(Fig.2)with an extent varying spatially and temporally among different climatic zones(Wanget al.,2022).Further scientific studies at the molecular level are needed to illustrate how climatic factors and soil properties influence the contribution of periphytic biofilms to C flux by altering biomass or microbial community characteristics.

In addition to the microbial mineralization of SOC derived from exudates and debris of periphytic biofilms,biolabile inputs may stimulate the microbial decomposition of native,soil-borne organic C(the C priming effect),leading to the mineralization of native refractory C into gaseous or soluble forms that are then lost from the soil system(Kuzyakovet al.,2000;Mitchellet al.,2020).Algal debris reportedly has a positive priming effect on sediment SOM mineralization and induces CO2and CH4emissions.Furthermore,the intensity of the priming effect is related to soil properties and the quantity of added substrates(Wang Y Ret al.,2021;Yanget al.,2022).Decomposition byproducts,such as low-molecular-weight acids released as DOC,can destabilize native mineral-associated SOM(Kaiser and Kalbitz,2012).These results indicate that periphytic biofilminduced CO2/CH4emissions may also come partly from native stable soil C.However,knowledge of the priming effect of periphytic biofilms on SOM mineralization is still limited.The compound-specific stable isotope technique can be applied to discriminate the C sources (newly added or native SOC) of gases and explore how periphytic biofilm debris affects the priming effects of SOM mineralization by altering soil N availability,nutrient stoichiometry,microbial co-metabolism,etc.In addition to soil and microbial properties,SOM stabilization mechanisms(e.g.,chemical recalcitrance and physicochemical protection)play a vital role in regulating the intensity of priming effects by altering the microbial accessibility of soil C sources (Chenet al.,2019;Liuet al.,2022).To maximize the direct and indirect C sequestration effects of periphytic biofilms in paddy fields,it is necessary to reduce CO2/CH4emissions and stimulate the role of periphytic biofilms in increasing the stock of stable SOC.Therefore,the contribution of periphytic biofilm-derived C input to stable SOC(stabilization)versusgreenhouse gas losses(mineralization)and the underlying mechanisms should be explored comprehensively and quantitatively to determine and manipulate the net C sequestration effects of periphytic biofilms in paddy fields.

CONCLUSIONS AND OUTLOOK

This review highlights that periphytic biofilms play an important role in soil C dynamics,including C fixation,SOC transformation,and mineralization,and act as biotic converters that transfer atmospheric CO2to CH4.The importance of periphytic biofilms at the soil-water interface in soil C cycles is emphasized by outlining that they affect and participate in soil C dynamics by altering soil physicochemical properties and microbial community activities.However,in-depth studies are needed to elucidate the mechanisms underlying CO2fixation and C transformation induced by periphytic biofilms and their responses to changes in environmental conditions,such as climatic factors and soil properties.Furthermore,gathering quantitative,spatially explicit information about the contributions of periphytic biofilms to soil C uptake,stabilization,mineralization,and sequestration is essential for a better understanding of soil C dynamics,maintenance of soil C stock,and mitigation of greenhouse gas emissions from paddy fields.For instance,more knowledge is needed on how to regulate the community composition and structure of periphytic biofilms and how to manipulate the growth of periphytic biofilms with agricultural management techniques(e.g.,irrigation and fertilization)to maximize C fixation and minimize CH4emissions in paddy fields.Promoting C sequestration in paddy fields can increase C sinks in terrestrial ecosystems and help achieve sustainable development and C neutrality.

ACKNOWLEDGEMENTS

We acknowledge the financial support from the National Natural Science Foundation of China(Nos.41825021 and 42207447),the National Key Research and Development Program of China (No.2021YFD17008),the Provincial Natural Science Foundation of Jiangsu,China(No.BK20220004),the Postdoctoral Science Foundation of China(Nos.BX2021325 and 2022M723242),and the State Key Laboratory of Lake Science and Environment Foundation,China (No.2022SKL008).EJ was supported by the TÜBITAK program BIDEB2232 of Türkiye(No.118C250).We thank Ms.Anne Mette Poulsen for the language editing.