Resilience of aerobic methanotrophs in soils; spotlight on the methane sink under agriculture

Abstract Aerobic methanotrophs are a specialized microbial group, catalyzing the oxidation of methane. Disturbance-induced loss of methanotroph diversity/abundance, thus results in the loss of this biological methane sink. Here, we synthesized and conceptualized the resilience of the methanotrophs to sporadic, recurring, and compounded disturbances in soils. The methanotrophs showed remarkable resilience to sporadic disturbances, recovering in activity and population size. However, activity was severely compromised when disturbance persisted or reoccurred at increasing frequency, and was significantly impaired following change in land use. Next, we consolidated the impact of agricultural practices after land conversion on the soil methane sink. The effects of key interventions (tillage, organic matter input, and cover cropping) where much knowledge has been gathered were considered. Pairwise comparisons of these interventions to nontreated agricultural soils indicate that the agriculture-induced impact on the methane sink depends on the cropping system, which can be associated to the physiology of the methanotrophs. The impact of agriculture is more evident in upland soils, where the methanotrophs play a more prominent role than the methanogens in modulating overall methane flux. Although resilient to sporadic disturbances, the methanotrophs are vulnerable to compounded disturbances induced by anthropogenic activities, significantly affecting the methane sink function.


Introduction
Methane is a potent greenhouse gas (GHG), having a 34-fold higher heat r etentiv e ca pacity in a 100-year time fr ame than carbon dioxide (IPCC 2019 ).Atmospheric methane has increased to ∼1857 ppm v in 2018, a 2.6-fold hike since the preindustrial era (IPCC 2019, Saunois et al. 2020 ).The recent trend in methane growth is a cause for concern, exacerbating the impact of climate change (Etminan et al. 2016, Dean et al. 2018 ), and indicates the imbalance of methane sources and sinks whereby the rate of methane production is outpaced by consumption (Saunois et al. 2020 ).Indeed, the net methane flux is a balance of methane production and oxidation, catalyzed by the methanogenic archaea (anaerobic decomposition of organic matter) and methanotrophs, respectiv el y (Conr ad 2009, Kirsc hke et al. 2013, Guerr er o-Cruz et al. 2021 ).P articularl y in well-aerated soils (e.g.forest, upland agricultural soils, and pasture), the methane flux is governed more by the activity of the aerobic methanotrophs than the methanogens (Serr ano-Silv a et al. 2014, Meyer et al. 2017, Ho et al. 2019 ).Hence, disturbances, including a gricultur al pr actices, inflicted upon the methanotrophs will inevitably affect the methane sink function in these soils.Anthropogenic-associated methane emissions, also accounting for a gricultur e-deriv ed methane, contributes up to 65% of the total methane emitted globally (Nazaries et al. 2013 ).
Ne v ertheless, some a gricultur al pr actices ma y ha ve a compar abl y lo w er environmental footprint than others (Lehmann et al. 2020 ).To this end, r egener ativ e a gricultur al pr actices, whic h a pproximate or imitate natural systems are thought to render beneficial effects to soils (see below discussion).While the impact of (r egener ativ e) a gricultur al pr actices on nitr ous oxide fluxes and the associated micr oor ganisms, specificall y in r elation to different (bio-based or mineral) fertilization regimes have been relativ el y well-documented (Cayuela et al. 2014, Yoon et al. 2019, El-Haww ary et al. 2022 ), ho w methane and the aerobic methanotr ophs ar e affected by these interv entions r emain fr a gmented.T his ma y, in part, stem fr om the gener al assumption that a gricultural soils become less important methane sinks after conv ersion fr om pristine envir onments (Le Mer and Roger 2001, Ho and Bodelier 2015, Tate 2015, Kaupper et al. 2020 ).Here, we aim to (i) conceptualize the resilience and response of the methanotr ophs to spor adic (i.e .one-off disturbances , allowing reco very of activity/community composition), recurring, and compounded envir onmental/anthr opogenic disturbances, and (ii) consolidate r esearc h findings on the impact of a gricultur e, with emphasis on r egener ativ e pr actices, on the methane sink function via pairwise comparisons of a gricultur al soils with and without specific interv entions (ma gnitude or % c hange of the ca pacity of the soil to consume methane is documented).Practice-based agricultur al interv entions and the outcomes of these interv entions were documented in a literature survey.We compiled field mana gement pr actices (namel y, nontilla ge, nonc hemical-based fertil-ization, and cov er cr opping; Table S1 , Supporting Information ) lar gel y consider ed to be r egener ativ e (Lehmann et al. 2020, Ne wton et al. 2020 ), and focused on the impact of these practices on the methane flux, and with respect to the methanotroph ecology, when a vailable .T his compilation is not intended to be exhaustiv e, but r ather to ca ptur e the br eadth of the r esults (adv erse to stimulatory effects of the practices on soil methane sink), particularly under upland cropping system.Individual agricultural pr actices wer e consider ed giv en that we cannot unequivocally attribute the response of the methane flux to a specific a gricultur al pr actice wher e m ultiple a ppr oac hes wer e sim ultaneousl y a pplied (i.e.syner gistic effect, suc h as integr ating liv estoc k and cr op farming; Newton et al. 2020 ).

Key players of aerobic methane oxidation
Disco veries o ver the past two decades have broadened the known diversity of methanotrophs, particularly the anaerobic ones which were found able to couple anaerobic methane oxidation to a suite of electron acceptors, including iron, sulphate , nitrite , and manganese; the ecology , physiology , and potential applications of the anaerobic methanotrophs have recently been reviewed (In 't Zandt et al. 2018, Guerr er o-Cruz et al. 2021 ).On the other hand, the aerobic methanotrophs (henceforth, referred as methanotrophs) oxidize methane to methanol using oxygen as the primary electron acceptor with the enzyme methane monooxygenase (MMO), which can be present as a soluble (sMMO) or membrane-bound particulate (pMMO) form.While the vast majority of methanotrophs harbor the pMMO, the alphaproteobacterial methanotrophs Methylocella and Methyloferula possess only the sMMO (Theisen et al. 2005, Vor obe v et al. 2011 ).In methanotrophs harboring both the pMMO and sMMO, copper regulates the r elativ e expr ession of these enzymes, suppr essing the sMMO, while stimulating the pMMO (Knapp et al. 2007 , Trotsenko andMurrell 2008 ).The pmoA and mmoX gene, r espectiv el y encoding for a subunit of the pMMO and sMMO, are frequently targeted in culture-independent studies to characterize the methanotrophs in complex communities (e.g.Liebner and Svenning 2013, Cai et al. 2016, Wen et al. 2016, Karwautz et al. 2018 ).
In particular, the aerobic rather than the anaerobic methanotr ophs wer e often documented to be the active and k e y methaneoxidizers in many methane-emitting terrestrial environments (Blazewicz et al. 2012, Ho et al. 2013a, Gao et al. 2022, Kaupper et al. 2022 ).Inter estingl y, these methanotr ophs may also foster close interactions with photosynthetic organisms, widening their habitat range to micro-oxic or even anoxic environments (Raghoebarsing et al. 2005, Ho and Bodelier 2015, Milucka et al. 2015, Guerr er o-Cruz et al. 2021 ).It follows that high methane-emitting en vironments (e .g. w astew ater treatment systems, landfill cover, rice paddies, and peatlands) are hotspots for the methanotrophs.Note worthy, methanotr ophs possessing MMO with a low affinity to methane (i.e.high concentration of substrate is required to saturate the MMO) and hence, tend to thrive in methane hotspots, are typicall y r eferr ed to as "low-affinity" methanotr ophs (e.g.Ho et al. 2013a ).Conv ersel y, methanotr ophs oxidizing methane at (circum-) atmospheric methane le v els ar e anticipated to possess the enzyme with a high affinity to methane (henceforth, r eferr ed as "high-affinity" methanotrophs; Knief andDunfield 2005 , Bissett et al. 2012 ).Although r epr esenting a r elativ el y minor fr action of the total bacterial population being members of the r ar e biospher e (Bodelier et al. 2013 ), the "low-affinity" methanotrophs disproportionally contribute to the total soil carbon (i.e.methane-derived carbon 1%-2%; Sultana et al. 2022 ).While the majority of cultur ed methanotr ophs ar e "low-affinity" methane-oxidizers, typically but not exclusively recovered from high methane-emitting en vironments , the "high-affinity" methanotrophs ha ve , for a long time been identified based on their pmoA gene diversity and resisted isolation (Cai et al. 2016, Pr atsc her et al. 2018, Ho et al. 2019, Tveit et al. 2019 ).Traditionally, these "high-affinity" methanotr ophs hav e been cluster ed in specific clades (e.g.upland soil clusters USC-α and USC-γ , r espectiv el y belonging to Alpha-and Gamma-proteobacteria, as well as Jasper Ridge clusters JR1, JR2, and JR3; Knief 2015 ).Recently, a novel methanotroph capable of high-affinity methane oxidation belonging to a genus thought to consist of "lo w-affinity" methanotrophs, Meth ylocapsa gorgona has been isolated in subarctic Norway (Tveit et al. 2019 ), blurring the distinction between "high-" and "low-affinity" methanotrophs on the phylogenetic le v el.Along with this isolate, other members of the same genus, Methylocapsa acidiphila and Methylocapsa aurea have also been shown to grow on atmospheric methane (Tveit et al. 2019 ).Although Methylotuvimicrobium buryatense can oxidize methane at r elativ el y low concentrations, these values ar e still abov e atmospheric le v els ( > 200 ppm v for M. buryatense ), and M. buryatense did not exhibit growth below the threshold methane concentrations (He et al. 2023 ).Ther efor e, with the exception of Methylocapsa species (Tveit et al. 2019 ), the lack of traditional "high-affinity" methanotroph isolates (e.g.members of USC-α, USC-γ , and JR clusters) capable of oxidizing and grow on atmospheric methane makes inter pr etation of their physiological response to disturbances challenging.Much remains unknown of this elusive methanotroph group.Having different affinities to methane may influence methanotroph distribution in the environment, with the "low-affinity" methanotrophs being mor e pr e v alent in envir onments with a high methane availability (% r ange), typicall y acting as a methane biofilter at o xic-ano xic interfaces, while the "high-affinity" ones consume atmospheric methane in well-aerated upland soils (Singh et al. 2010 ).Ho w e v er, it should be noted that the distribution of the "low-affinity" and "high-affinity" methanotrophs is not mutually exclusive, and they ma y co-occur.For instance , "low-affinity" methanotrophs ma y be-come active following a rainfall event in well-aerated upland soils as methane exceeding atmospheric le v els becomes av ailable with incr eased anoxic nic hes r esulting in stim ulated methanogenesis (Shrestha et al. 2012, Ho et al. 2013b ).The different affinities for methane may also determine the response and resilience of the methanotr ophic gr oups to disturbances (see below discussion).

Conceptualizing the resilience of the methanotrophic activity and aerobic methanotrophs to sporadic, recurring, and compounded disturbances
The "low-affinity" methanotrophs are remarkably resilient to sporadic or single disturbance events , ha ving been shown to recover following a temper atur e and heat shock up to 45 • C (Ho and Frenzel 2012 ), physical disruption to soil structure (sieving and grinding; Kumar esan et al. 2011 ), incr easing salinity [soil salinity range 0.3-1.0dS m −1 , and up to saltwater salinity le v el (Bissett et al. 2012, Ho et al. 2018 )], and disturbance-induced mortality [soil recolonization following disturbances (Ho et al. 2011, Pan et al. 2014, Kaupper et al. 2020 )], among other anthropogenic-induced disturbances (e.g.contamination of heavy metals, and pollutants such as pharmaceuticals , pesticides , and chemical ad diti ves; see Table S2 , Supporting Information ; Semrau et al. 2010, Deng et al. 2011, Benner et al. 2015 ).Given sufficient recovery time (within days to weeks) and substrate (methane and oxygen) availability, the "lowaffinity" methanotr ophs e v en ov er-compensated for disturbanceinduced activity and diversity loss (Fig. 1 ).Also, relevant factors r estricting micr obial gr owth (i.e .nutrients and space , as a result of disturbance-induced cell die-off) may become available following disturbances .T her efor e, the modified eda phic pr operties may determine the success of the early colonizers, benefiting the fastgr owing methanotr ophs under these favor able conditions (Ho et al. 2017 ).A compositional shift is often detected after disturbance, suggesting the differential response of community members to the disturbance leading to an altered trajectory in community succession ( Table S2 , Supporting Information ; Kumaresan et al. 2011, Andersen et al. 2013, Kaupper et al. 2021a ).In particular, the alpha pr oteobacterial methanotr ophs ( Meth ylosinus and Meth yloc ystis ), which sho w ed habitat pr efer ence for r elativ el y oligotr ophic en vironments (e .g. ombrotrophic peatlands and upland soils), appeared to be generally more resistant to disturbances (Dedysh 2011, Ho et al. 2013a, Knief 2015, 2017 ), while the fast-growing gamma pr oteobacterial methanotr ophs (e.g.Meth ylobacter , Meth ylosarcina , and Methylobacter ) are likely the rapid-responders and early colonizers (Ho et al. 2013a, Pan et al. 2014, Kaupper et al. 2020 ).This suggests adv anta geous ecological tr aits inher ent to some methanotr ophs, likel y r eflecting on their life str ategies, which enabled their persistence and dominance during and after disturbances, r espectiv el y (see r e vie ws Ho et al. 2013a, 2017, Krause et al. 2014 ).
The resilience of the "low-affinity" methanotrophs may be attributable to r elativ el y high methane availability in their habitat, allowing r a pid pr olifer ation among the surviving comm unity members after disturbances, in contrast to the "high-affinity" methanotr ophs, whic h ar e r estricted by substr ate av ailability (atmospheric methane), limiting growth and the population size (Knief and Dunfield 2005, Kolb et al. 2005, Ho et al. 2019 ).Importantl y, the r esilience of the "low-affinity" methanotrophs can also be partly explained by previous exposure to the same disturbance or disturbances, which elicited a similar physiological response, pr ompting r a pid r ecov ery of a comm unity whic h had surviv ed the e v ent (Kr ause et al. 2012, 2017, Baumann and Marsc hner 2013, v an Kruistum et al. 2018 ).It stands to reason that a microbial community primed to a disturbance eliciting a specific physiological r esponse will r espond mor e r a pidl y should the e v ent r eoccur.Although activity r ecov ery can be attributable to prior exposure to a disturbance, results indicate the marginal role of site history in conferring resilience to contemporary disturbances, particularly for the "lo w-affinity" methanotrophs.Regar dless of the community composition, methanotrophs from deep lake sediments recov er ed just as r a pidl y as methanotrophs from a shallow lake and rice pad d y soil from desiccation and heat stress, despite not having prior exposure to the disturbance nor harboring the same community members (Ho et al. 2016 ).Ne v ertheless, prior disturbances likely selected for a reservoir of (seed bank) community members that were resistant or were even favored by the disturbance (Krause et al. 2010, van Kruistum et al. 2018 ).This begs the question whether the resilience of the methanotrophs will be challenged in the face of (intensified) recurring, and compounded disturbances.
To this end, methane uptake rates w ere sho wn to r ecov er after consecutive desiccation-rewetting cycles induced e v ery 2 weeks, but activity was significantly impaired when desiccationr e wetting e v ents intensified (shortened r ecov ery time fr om 2 to 1 week; Ho et al. 2016 ) and the effect increased over stress cycles.This suggests that disturbances may exert a cum ulativ e effect on the soil methane uptake over time, and that the resilience of the "low-affinity" methanotrophs may eventually reach a "tipping point" with recurring disturbances (e.g.increased frequency of desiccation-r e wetting e v ents; Table S2 , Supporting Information ), as demonstrated in other microbial systems (Veraart et al. 2012, König et al. 2018 ).Impaired methane uptake rates were accompanied by a compositional shift in the r ecov er ed methanotr ophic community, favoring members of Methylocystis (Ho et al. 2016 ).Similarly, a step-wise increase in ammonium concentrations from 0.5 to 4.75 g l −1 (in 0.25-0.5 g l −1 incr ements) significantl y impair ed methanotrophic activity or lengthened the la g befor e the onset of activity, but methane uptake could still be detected at the highest a pplication r ate, indicating the emer gence of an ammoniumtoler ant methanotr ophic comm unity with continuous and gr adual exposure to increasing ammonium levels (Qiu et al. 2008, López et al. 2019, Ho et al. 2020 ).Whereas an abrupt ammonium increase elicited a dose-dependent effect on the soil methane uptak e, lik ely favoring the more ammonium-resistant methanotrophs (i.e.able to detoxify products of ammonium oxidation lik e hydro xylamine , nitrate , and nitrite) such as those belonging to gamma pr obacteria (e.g.Meth ylosarcina , Meth ylocaldum , Meth ylococcus , and Methyobacter (Noll et al. 2008, Poret-Peterson et al. 2008, van Dijk et al. 2021 ).These studies demonstrate that intensified and recurring disturbances imposed a cum ulativ e effect on the methanotrophic activity, and profoundly alter the community composition, with consequences for future disturbances.
As with recurring disturbances, methanotrophic activity is significantly affected by compounded disturbances (i.e.multiple stressors inflicted simultaneously), as would be anticipated during a natural disaster and under anthr opogenic-r elated land-use change such as land conversion for agricultural purposes.Following a peatland forest fire, the potential to oxidize methane was significantly impaired, concomitant to significantly r educed methanotr oph abundance e v en after 7 years postrecovery (Danilova et al. 2015 ).The conversion of pristine to arable lands exacerbates methane emissions (thereafter, see below for effects of specific a gricultur al pr actices on the methane sink function; see Table S1 , Supporting Information ).P articularl y for Figur e 1. T he effect of sporadic (A), recurring (B; i-grey line; ii-orange line), prolonged (B; iii-blue line), and compounded (B; iv-green line) disturbances on the methanotrophic activity (see Table S2 , Supporting Information ).In many instances, the r ecov ery in methane uptake rates is not a reflection of the recovery in the methanotrophic community composition, indicating redundancy among the community members.Given sufficient r ecov ery time under ample substrate (methane and oxygen) av ailability, methanotr ophic activity typically recovers within days/weeks (light gray line; e.g.Pan et al. 2014, Kaupper et al. 2021a ) or e v en ov er-compensate for initial activity loss (dashed light gr a y line; e .g. Ho and Fr enzel 2012 ) likel y attributable to higher nutrient and space availability (derived from disturbance-induced cell lysis and death) after sporadic disturbances (A).In (B), prior exposure to a disturbance may select for a seed bank community resistant to the disturbance for future contingencies .Hence , upon exposure to the same disturbance, activity will fully recover, and may even be less adversely affected (i-grey line; e.g.Krause et al. 2010, Baumann and Marschner 2013, van Kruistum et al. 2018 ).Without allowing a full r ecov ery fr om prior disturbances, the methanotr ophic activity e v entuall y r eac hed a "tipping point", and thereafter, activity no longer r ecov er with intensified r ecurring disturbance (ii-or ange line; Ho et al. 2016Ho et al. , 2020 ) ).Following prolonged disturbances (iii-blue line), methanotrophic activity was profoundly altered, and did not recover to predisturbance levels (e.g.drought; Collet et al. 2015 ).Likewise, compounded disturbances (iv-green line) as expected under land-use change scenarios (i.e.peat mining, deforestation for a gricultur e; Tate 2015 , Meyer et al. 2017, Reumer et al. 2018, Ho et al. 2022 ) significantly impaired the methanotrophic activity (particularly, "high-affinity" methane oxidation), but activity may r eturn r equiring extended r ecov ery time spanning ov er decades (iv-dashed gr een line; e.g.Le vine et al. 2011, McDaniel et al. 2019 ).
well-aerated upland soils, heightened methane emission following land conversion can be attributable to the loss of the methane sink function (Tate 2015, Meyer et al. 2017, Kroeger et al. 2021 , Obr egon Alv ar ez et al. 2023 ), whic h is pr ojected to take up to 80 years to r ecov er after the abandonment of a gricultur e (Le vine et al. 2011, McDaniel et al. 2019 ).Like wise, defor estation of tr opical r ainfor ests for palm oil production significantly lo w ered the capacity of the soil to oxidize methane, but activity gr aduall y r ecover ed ov er decades ( > 30 years) under oil palm a gricultur e (Kaupper et al. 2020, Ho et al. 2022 ).Comparing the methane uptake rates in a pristine, actively mined, and abandoned peatlands under differ ent r estor ation interv entions, activity in the dammed peatland postexcav ation r ecov er ed after > 15 years with the return of Sphagnum , but the community composition was significantl y alter ed, and the network of inter acting micr oor ganisms became less complex and connected (Andersen et al. 2010, Putkinen et al. 2018, Reumer et al. 2018, Kaupper et al. 2021b ).The recovery in activity after peat mining was, thus not reflected in the recovery of the microbial population, resulting in a shift in the trajectory of community succession over time .Nevertheless , community shifts postdisturbance in peatlands may not necessarily be unfavorable with regard to methane emissions, considering that the compar abl y poorl y established methanogenic community may lo w er methane production after r estor ation (Juottonen et al. 2012 ).In contr ast to spor adic disturbances, these examples highlight the vulnerability of the methanotrophs to compounded disturbances, significantly impairing methanotrophic activity, as well as inducing compositional changes to the community.A shift in the methanotrophic composition may alter the collective traits of the methane-oxidizing community, exerting an effect on community functioning (Ho et al. 2013a, Krause et al. 2014, Nijman et al. 2021 ), mor e pr onounced under fluctuating envir onmental conditions.

Anthropogenic activity affecting soil methane sinks; spotlight on agricultural practices
Agriculture expansion and intensification to meet the global food, feed, and biofuel demands pose a threat to soil processes worldwide, including methane consumption.Although land conversion to a gricultur e may adv ersel y impact soil ecosystem function, specific a gricultur al mana gement pr actices ma y lea ve a less se v er e imprint.To this end, r egener ativ e farming has been perceiv ed as a gricultur al mana gement a ppr oac hes, whic h hav e a r elativ el y lo w er environmental impact on soil ecosystem functions than conv entional a gricultur e, at times, e v en pur ported to r e v erse the impact of conv entional a gricultur e (e.g.carbon stock accum ulation).Consider ed "sustainable land management practices" by the Inter gov ernmental P anel on Climate Change (IPCC), r egener ativ e a gricultur e has been her alded as an effectiv e str ategy for continuous sustainable crop production (IPCC 2019 ).Yet, the concept lacks a clear definition or has been defined differently by users, albeit the widespread usage of the term.Agricultur al pr actices, whic h ar e fr equentl y associated with r egener ativ e farming include reducing/eliminating tillage, use of cover crops including green manure, and integrated farming ( Table S1 , Supporting Information ; Newton et al. 2020 ).Other exclusionary measures include no or minimum synthetic fertilizer input or replacing these with bio-based or organic residues ( Table S1 , Supporting Information ; Lehmann et al. 2020 ).The impact of these a gricultur al pr actices particularl y on eda phic par ameters, crop yield, as well as carbon dioxide and nitrous oxide emissions in relation to (in)organic fertilization have been r elativ el y well-documented in recent work (see discussion below).Although methane turnover in wetland rice cultivation is well-studied (e.g.Krüger et al. 2001, Kim ur a et al. 2004, Shrestha et al. 2011, Lee et al. 2014, Li et al. 2021 ), the impact of a gricultur e on the methane sink and the associated methanotrophs in upland soils remain fr a gmented.In particular, the r esponse of the methanotr ophic community composition and abundances are pertinent to explain variation in the response of the methane sink to diverse agricultur al pr actices (Shr estha et al. 2012, Judd et al. 2016 ).

The impact of agricultural practices on the methane sink
Her e, we elabor ate on the effects of specific a gricultur al pr actices (i.e .nontillage , exclusion of chemical N fertilization or incorporation of bio-based residues , co ver cropping) on the methane sink function, with emphasis on upland soils ( Table S1 , Supporting Information ; Lehmann et al. 2020, Newton et al. 2020 ).Because of the wide range of organic or bio-based residues used in case studies r ele v ant at the local-or r egional-scale (e.g. oil palm kernel and husks, diverse aboveground crop residues; Kaniapan et al. 2021, Shinde et al. 2022 ), we focused on compost and bioc har, whic h can be deriv ed fr om v arious waste str eams, as well as manure or digestate, a commonly applied bio-based fertilizer.
The effects of tillage on soil methane emissions are contradictory, having been documented to significantl y stim ulate (e.g.Yeboah et al. 2016 ) or lo w er (e.g.Tian et al. 2013 ) methane uptake in a gricultur al soils (Fig. 2 ; Table S1 , Supporting Information ).T his inconsistency ma y stem from the different types of cropping systems (wetland or well-aerated upland agriculture), exhibiting starkl y differ ent methane flux rates, in turn determining the magnitude and direction of fluxes (i.e.methane source or sink), and the response of the predominant indigenous methanotrophs ("lowaffinity" or "high-affinity") pr esent.Similarl y, the pr ocesses governing methane flux is different in the two cropping systems, with methanogenesis and anaerobic methane oxidation becoming important in the wetland soils.Howe v er, a gener al tr end emer ged when comparing the effects of nontillage and conventional tillage in wetland and upland a gricultur al soils independentl y, showing ov er all lo w er methane emission under nontillage in pad d y fields (which may depend on the rice growing stage; Li et al. 2011 ), and having no a ppar ent effects or lo w ered methane emission in upland a gricultur al soils (see r e vie w; Maucieri et al. 2021 ; Fig. 2 ; Table S1 , Supporting Information ).Compar ativ el y lo w er methane emissions under nontillage in rice paddies are consistent with previous work (Huang et al. 2018 ).Rice paddies ar e commonl y tilled between rice plants to r emov e weeds during the rice growing season.Tilla ge r esults in the aer ation of soil and the oxidation of reduced electron acceptors, thereby providing thermodynamically favor able electr on acceptors for micr obial r espir ation and suppressing methanogenesis (Brune et al. 2000, Liesack et al. 2000 ).Mor eov er, tilla ge also disrupts the methane-oxygen counter gradient, which forms on the soil surface-o verla ying floodwater interface (upper 1-3 mm, based on electr ode measur ements of substrate depth profiles), where the methanotrophs thrive .Here , the contribution of the methanotrophs to the net methane flux, typically determined using specific inhibitors, exhibited substantial methane consumption potentially up to 90% of total methane pr oduced (Liesac k et al. 2000 , Kajan and Fr enzel 2006 , Reim et Figur e 2. T he impact of selected a gricultur al pr actices on methane emissions in well-aerated upland soils, comparing the effects of the treatments to agricultural soils without treatments (see Table S1 , Supporting Information ).The arrow indicates the direction of the c hange (incr ease or decr ease); the ma gnitude of the c hange (%) is giv en in Table S1 ( Supporting Information ).Dashed outline indicates that the effect of an intervention has yet to be unambiguously resolved (e.g.potentially lo w er methane emissions follo wing compost addition into upland a gricultur al soils).A dash indicates that the interv ention imposed marginal or no change to methane emission.Abbreviations: i.c., inconclusive (insufficient studies to derive conclusion).Graphic of the crop is reproduced from Brenzinger et al. ( 2021Brenzinger et al. ( ). al. 2012 , Pr aja pati and Jacinthe 2014 ).Hence, a gricultur al pr actices, whic h destr oy this micr ohabitat will ine vitabl y affect the role of the methanotrophs as a methane biofilter in rice paddies, requiring time (days to weeks; Ho et al. 2011 ) for the gradient and methanotroph population to re-establish.In contrast to wetland a gricultur e, tilla ge in well-aerated upland soils may act to r elie v e gas exchange limitation and promote methane uptake.When both nontilled and conv entionall y tilled upland agricultural soils act as methane sinks, atmospheric methane uptake can be lo w er in the nontilled than tilled site (Plaza-Bonilla et al. 2014 ), albeit the stimulatory effect of tillage could not be unambiguously confirmed in the presence of other confounding factors (Maucieri et al. 2021 ).Rele v ant local soil physico-chemical par ameters, whic h may confound tillage-induced effects are moisture and temperature (Boeckx andCleemput 1996 , Hiltbrunner et al. 2012 ).Lo w er soil methane uptake in nontilled soils had been attributed to lo w er in situ temper atur e and high soil moisture in a field study, covering seasonal variation over a year (Tian et al. 2013 ), with lo w er temper atur e limiting biological activity including methane oxidation, whereas the high moisture content is thought to restrict gas (methane and oxygen) diffusion into the soil.While nontillage minimizes soil erosion and degradation, this intervention exerts different effects on soil methane emission, depending on the cropping system.
Another r ele v ant a gricultur al pr actice that r egener ates or ganic matter in soil is the exclusion and/or replacement of inorganic fertilizers with bio-based/organic residues (e .g. manure , as well as compost and biochar from diverse waste streams; Jenkinson 1991 ).Ho w e v er, the incor por ation of bio-based or ganic r esidues, particularl y manur e , ma y still ha v e undesir able side effects, in-cluding heightened methane emission via stimulation of the indigenous soil methanogens and/or the addition of r esidue-deriv ed methanogens into the soil (Gattinger et al. 2007, Radl et al. 2007, Thangarajan et al. 2013,Ho et al. 2015 ).Manure-induced increase in methane emissions typically occur in rice paddies, while gener all y imposing little effect in upland a gricultur al soils (Fig. 2 ).Supplementing rice pad d y soil with fresh manure promoted the soil-borne methanogens in flooded rice paddies, leading to significantly higher methane production (e.g.Kim et al. 2018 ), but can be remedied with the application of manure ad diti ves to the manure to suppress methane production, besides odor control (ammonia volatilization; Zhu 2000 ).Other bio-based residues show promising methane mitigation or crop growth-promoting capabilities; when locally sourced materials from diverse waste streams (e.g. a gricultur e, industry, and household) were applied to representativ e a gricultur al (sandy loam and clay) soils, some bio-based r esidues (e.g.nitr ogen-ric h se wa ge sludge and aquatic plant material) significantly increased crop (wheat) yield at the expense of having a higher global warming potential (GWP), mainly driven by nitrous oxide emissions (Ho et al. 2015(Ho et al. , 2017 ) ).In the same study, the incor por ation of compost in upland a gricultur al soils imposed compar abl y lo w er GWP than in the soils without any residue addition, and only marginally affected the soil bacterial community composition, including the methanotrophs, and fungal abundance (Ho et al. 2017, Brenzinger et al. 2018 ), in addition to promoting plant beneficial microbes (Bonanomi et al. 2018 ).Specific compost suppressed methane emission in well-aerated upland soils in the short-term ( < 2 months) by significantly stimulating the a ppar ent cell-specific methane uptake rates, offsetting up to 16% of the total carbon dioxide emitted (Ho et al. 2015, 2019, Brenzinger et al. 2018 ).Presumably, compost-derived r ar e earth metals (e.g.La and Ce) and other elements (e.g.copper and calcium) at minute concentrations ( μg g soil −1 range; El-Ramady 2011 ) may have stimulated methanol dehydrogenase (catal yzes the conv ersion of methanol to formaldehyde) and/or the pMMO (in the case for copper) of some methanotrophs (Ho et al. 2013c, Zheng et al. 2018 ); Agegnehu et al. 2016, Vekeman et al. 2016, Krause et al. 2017 ).While methanotrophs may possess a copper sequestration mechanism by releasing methanobactin, a c halkphor e with a high affinity for copper, and thus ov ercome copper limitation, a scav enging mec hanism for the r ar e earth elements is as y et unkno wn in methanotrophs (Pol et al. 2014, DiSpirito et al. 2016 ).In contr ast, compost induced significantl y higher methane emission in wetland a gricultur al soils, considering high methane production under water-logged conditions.Despite having gener all y compar able physico-c hemical pr operties (e.g.stable C fraction, or absence/minimal labile carbon), matur e compost deriv ed fr om differ ent waste str eams may differ entiall y influence methane production and oxidation, affecting the ov er all flux (Br enzinger et al. 2018, v an den Ber gh et al. 2023 ).Hence, nuances in mature compost (e.g.presence of heavy metal contaminants or r ar e earth elements) may impose a strong effect on the soil methanotrophic community and activity.Although having no a ppar ent effects on cr op yield in these studies, compost amendment may thus reduce methane emissions and benefit other aspects of soil function (e.g.long-term carbon accumulation in soil; Ryals et al. 2015 ).Evidently, no improvement in crop yield was a trade-off for lo w er GWP, but the carbon dioxide offset by increased methane uptake suggests that cr op pr oductivity can be impr ov ed considering compost addition complemented with other N-rich soil ad diti v es (Br enzinger et al. 2021 ) at optimal combinations to minimize ov er all GHG emissions.
In addition to manure and compost, biochar application gained attention in the past decade , ha ving been proposed as a carbon stor a ge str ategy in soils (Lehmann et al. 2006 ), and was projected to ac hie v e carbon neutr ality in a gr o-systems (rice, wheat, and corn production systems) when applied in combination with other climate-smart a gricultur al pr actices (intermittent dr aina ge in rice production and reduced N-fertilization input; Xia et al. 2023 ).Although the effects of biochar amendments alongside conventional fertilizers on the edaphic properties have been welldocumented (i.e .impro ved water and nutrient retention, cation exc hange ca pacity, soil por osity, and a ggr egation leading to higher cr op gr owth and yield; Liang et al. 2006, Mau and Utami 2014, Agegnehu et al. 2016, Bamminger et al. 2018, Rasa et al. 2018 ), the effects of biochar on GHG fluxes remain contentious.Biochar amendment can suppress or stimulate fertilizer-associated nitrous oxide emission (Yanai et al. 2007, Spokas et al. 2009, Cayuela et al. 2014, Harter et al. 2014, Shen et al. 2014, Agegnehu et al. 2016, Bamminger et al. 2018, Bor char d et al. 2019 ).Similarly, what little is known on the effects of biochar on methane turnover is based on case studies, showing both a stimulation on methane production (e.g.Wang et al. 2012 ) and enhanced methane uptake (e.g.Karhu et al. 2011, Syed et al. 2016, Kubaczy ński et al. 2022 ; Table S1 , Supporting Information ), as well as having no or marginal effects on methane emission (e.g.Bamminger et al. 2018 ).Like the effects of tilla ge, the a ppar ent contr asting effects of bioc har on the methane flux may stem from the cropping system, as well as the variable application rate in different studies (9-240 t ha −1 ;  Spokas et al. 2009 , Karhu et al. 2011 , Bamminger et al. 2018 , Zhao  et al. 2021 , Kubaczy ński et al. 2022 , Xia et al. 2023 ) and the delayed detectable effect over time (e.g.significant effects of biochar amendment detected only after 1 year; Major et al. 2010 ).Incorporation of biochar to wetland rice agricultural soils increased the methane sink strength or decreased the methane source when compared to amendments in upland a gricultur al soil, whic h sho w ed marginal effects (Jeffery et al. 2016, Bamminger et al. 2018, Zhao et al. 2021 ).On the other hand, a recent study sho w ed significant stimulation of methane uptake in upland agricultural soils concomitant to increased methanotroph abundance over at least 5 years after biochar addition (Kubaczy ński et al. 2022 ).Mor eov er, bioc har a ppear ed to hav e a stabilizing effect, r educing the v ariability in methane fluxes (Karhu et al. 2011 ).Regardless of the feedstock (exception, biosolids) for biochar production, the pyrolysis temper atur e a ppears to be r ele v ant in determining the effect of the final product on soil methane emission, with biochar under gone high pyr ol ysis temper atur e exceeding 600 • C significantl y incr eased the methane sink function after incor por ation into soils (Jeffery et al. 2016 ).Bioc har deriv ed fr om high pyr olysis ( > 600 • C) contains less labile material (Bruun et al. 2011 ) and hence, less substr ate av ailability for micr oor ganisms (r esistant to degradation), including the methanogens .Likewise , high por osity in bioc har incr eases aer ation, potentiall y suppr essing methane pr oduction, or pr omotes methane oxidation (Karhu et al. 2011, Joseph et al. 2021 ).It thus a ppears that bioc har modifies the edaphic properties, in turn, affecting microbially mediated soil pr ocesses; the dir ect effect of biochar, as well as other amendments, on methanotroph metabolism remains to be determined.
Besides no-tillage and incorporation of organic amendments into soils, r egener ativ e farming includes cov er cr opping to minimize nitrogen loss via leaching and/or (de)nitrification in the presence of the main crops (intercropping) and during fallow after harv est (P a ppa et al. 2011, Gabriel et al. 2012, Sanz-Cobena et al. 2014 ).Cov er cr ops (e.g.legumes such as vetch and peas) may also be incor por ated into the soil as gr een manur e, ther eby r etaining accumulated N (i.e .ha ving r elativ el y slo w er miner alization r ates; Baggs et al. 2000, Kim et al. 2012 ) in the field for the next cropping season.Also, depending on the selection of cov er cr ops (mixtur es or monocr op), substr ate utilization pr ofile assessed using a Biolog ECO plate analysis of soils amended with cover crop mixtures significantl y incr eased, indicating a r elativ el y higher micr obial functional (metabolic) diversity when compared to soils that receive r esidues fr om monocr op (Dr ost et al. 2020 ).Species-specific effects of cover crops on carbon dioxide and nitrous oxide emissions have been documented, showing varied results (higher, lo w er, or comparable emission rates in fields without cover crops) for both intercropping and as green manure (Baggs et al. 2000, Pappa et al. 2011, Sanz-Cobena et al. 2014 ).Ho w e v er, the effects of cov er cr opping and gr een manur e a pplication on soil methane uptake are less kno wn.Regar dless of the choice of cover crops (barley, r a pe, and v etc h), an upland a gricultur al soil planted to maize remained a methane sink, albeit having v etc h as a cover crop turned the soil into a weak but not significant methane source during fallow (Sanz-Cobena et al. 2014 ).Like for carbon dioxide and nitrous oxide emissions (Sanz-Cobena et al. 2014, Drost et al. 2020 ), it appears that the C:N ratio of the cover crop is relevant when determining methane emissions.To this end, the choice of a cover crop as gr een manur e in rice a gricultur e w as sho wn to exert a strong effect on methane emission, with v etc h possessing a lo w er C:N ratio r esulting in significantl y lo w er methane emission than rye (higher C:N), prompting the authors to suggest that the extraneous carbon (compar ativ el y higher total C and labile C fr actions) av ailability in rye upon incor por ation into soil stim ulated methanogenesis (Kim et al. 2012 ).Besides inducing a lo w er methane emission, v etc h also significantly increased crop yield (total biomass and grain yield).Hence, a tailored selection of cover crops, also as gr een manur e, for specific main cr ops and cr opping systems ar e r equir ed to r educe methane emissions, while incr easing yield.Evidentl y, futur e studies to explore the impact of cover cropping on methanotr ophs ar e warr anted.

Conclusion and perspective
The methanotrophs are evidently affected by disturbances, but ma y still reco ver from sporadic events.Upon disturbance recurrence, ho w ever, methanotrophic activity was impaired, and required decades to recover following compounded disturbances associated to change in land use and natural disasters.Accumulating evidence indicates that the methane-oxidizing community is comprised of both methanotrophs and nonmethanotrophs, each play r ele v ant r oles, enabling and e v en exerting syner gistic effects on community functioning (e.g.Stock et al. 2013, Ho et al. 2014, Benner et al. 2015, Veraart et al. 2018 ).Giv en the r ele v ance of the nonmethanotrophs in modulating methanotrophic activity, future work could focus on interkingdom interaction in response to disturbances (incor por ating soil micro-and macro-organisms e .g. viruses , protists , soil isopods; Murase and Frenzel 2008, Kuiper et al. 2013, Heffner et al. 2023a , b ), and possibly, to establish earlywarning indicators of a collapsing interaction network, leading to impair ed comm unity function.Mor eov er, inter action-induced r elease of (volatile) organic compounds can significantly influence the methanotrophs (Veraart et al. 2018 ), as well as the selection of beneficial micr oor ganisms essential for cr op pr otection (e.g.disease suppr essiv e soils; Carrión et al. 2019, Weisskopf et al. 2021 ).
Although evidence suggests the transition to specific agricultur al pr actices (e.g.nontilla ge, or ganic fertilization, and cover cropping) ma y fa vor or do not exert an adverse impact on the methanotr ophs, a ppl ying suc h pr actices alone may not be suffi-cient to ac hie v e food security for a gr owing human population.To this end, ecological intensification is gener all y thought to enhance soil ecosystem services by complementing and/or replacing conv entional a gricultur al a ppr oac hes to boost cr op yields (Tittonell 2014 , Kleijn et al. 2019, MacLaren et al. 2022 ).Central to ecological intensification is the enhancement of belowground (micr o)or ganism inter action, whic h facilitates the usa ge of r esources mor e efficientl y.For instance, a gricultur al pr actices (e.g.low and sparse fertilization; Pandey et al. 2019 ) that favor dissimilatory nitr ate r eduction to ammonium ov er denitrification to r etain N in soil (e.g.Putz et al. 2018, Yoon et al. 2019 ).Also, while the impact of specific a gricultur al pr actices on methane emissions and by extension, other parameters determining the multifunctionality of soils (e.g.physico-chemical characteristics, other GHG, microbial diversity) have been documented, the trade-off when applying multiple practices concurrently in conjunction with the individual pr actices, potentiall y yielding ad diti v e, syner gistic, anta gonistic, and/or net neural effects needs further probing (Lehmann et al. 2020, Xiao et al. 2021 ).
Emerging soil "modifiers," such as nano-and microplastics are r elativ el y persistent compounds, that not only alter soil c har acteristics, affecting gas diffusivity and the emissions/consumption of GHG, but also significantly affect the soil microbial (plastispher e;Rohrbac h et al. 2022, Zhu et al. 2022 ) and inv ertebr ate (e.g.earthworms and soil isopods; Lahive et al. 2022, Hink et al. 2023 ) communities .In addition, nanoplastics ma y accumulate in plants (Gong et al. 2021 ), and modify plant c har acteristics (e.g.c hange in root anatomy; Elena Pradas del Real et al. 2022 ), potentially affecting crop yield.Although the application of specific organic compounds such as biochar as soil ad diti ves has generally been well-r eceiv ed as a strategy to sequester carbon and immobilize heavy metals in soils (Gong et al. 2022 ), the environmental impact of long-term accumulation of the immobilized heavy metal remains unclear.The ambiguity of the long-term impact of these compounds (e .g. nanoplastics , microplastics , and biochar) in soils necessitates thorough environmental assessments.Summarized, r egener ativ e a gricultur al pr actices can str engthen the methane sink and favor the methanotrophs, depending on the cropping system, but further work is needed to shed light on the mechanistic understanding of the outcomes of these a gricultur al pr actices.