The ISME Journal (2020) 14:288–301 https://doi.org/10.1038/s41396-019-0523-8

ARTICLE

Stable aerobic and anaerobic coexistence in anoxic marine zones

1,2 3 2 2 Emily J. Zakem ● Amala Mahadevan ● Jonathan M. Lauderdale ● Michael J. Follows

Received: 19 March 2019 / Revised: 23 August 2019 / Accepted: 13 September 2019 / Published online: 17 October 2019 © The Author(s) 2019. This article is published with open access

Abstract Mechanistic description of the transition from aerobic to anaerobic is necessary for diagnostic and predictive modeling of fixed nitrogen loss in anoxic marine zones (AMZs). In a metabolic model where diverse - and nitrogen- cycling microbial are described by underlying redox chemical reactions, we predict a transition from strictly aerobic to predominantly anaerobic regimes as the outcome of ecological interactions along an oxygen gradient, obviating the need for prescribed critical oxygen concentrations. Competing aerobic and anaerobic metabolisms can coexist in anoxic conditions whether these metabolisms represent obligate or facultative populations. In the coexistence regime, relative rates of aerobic and anaerobic activity are determined by the ratio of oxygen to electron donor supply. The model simulates key characteristics of AMZs, such as the accumulation of nitrite and the sustainability of anammox at higher oxygen concentrations than denitrification, and articulates how microbial concentrations relate to associated column 1234567890();,: 1234567890();,: transformation rates as a function of redox stoichiometry and energetics. Incorporating the metabolic model into an idealized two-dimensional ocean circulation results in a simulated AMZ, in which a secondary chlorophyll maximum emerges from oxygen-limited grazing, and where vertical mixing and dispersal in the oxycline also contribute to metabolic co-occurrence. The modeling approach is mechanistic yet computationally economical and suitable for global change applications.

Introduction microbes utilize inorganic nitrogen species, producing nitrogen gas (N2) and the potent greenhouse gas nitrous Oxygen reaches low concentrations in aquatic environ- oxide (N2O) [1, 4–6]. The interpretation and prediction of ments where aerobic consume oxygen quickly how these rates of fixed N loss change with warming- relative to the rate of its supply [1–3]. As oxygen con- induced deoxygenation require appropriate descriptions of centrations decline, the viable biological the transition from aerobic to anaerobic activity [7–9]. generally consists of smaller organisms, and only micro- Observations suggest that this transition is not a sharp one. organisms can efficiently utilize oxygen at the lowest Rather, aerobic and anaerobic metabolisms seem to co-occur (nanomolar or lower) concentrations [4, 5]. When oxygen in AMZs [10–16]. Metagenomics and metatranscriptomics is sufficiently depleted, metabolically diverse micro- suggest widespread aerobic metabolic potential and activity organisms use alternative electron acceptors for anaerobic throughout AMZs [13, 17]. The consumption of oxygen respiration. In pelagic anoxic marine zones (AMZs), produced by at the secondary chlorophyll maximum indicates that aerobic metabolism is active in anoxic or nearly anoxic conditions [18, 19]. This co-occurring aerobic and anaerobic activity must be accounted for when Supplementary information The online version of this article (https:// doi.org/10.1038/s41396-019-0523-8) contains supplementary predicting N loss as a function of organic matter respiration material, which is available to authorized users. [17], since neglecting the portion of organic matter that is oxidized aerobically within AMZs overestimates N loss. * Emily J. Zakem Explanations for co-occurrences typically invoke varia- [email protected] tions in ambient oxygen concentrations in time or space, such 1 Department of Biological Sciences, University of Southern as from lateral intrusions of oxygen from equatorial jets or California, Los Angeles, CA, USA anoxic niches inside particles [17, 20–25]. Mixing and par- 2 Department of Earth, Atmospheric and Planetary Sciences, ticle sinking can also supply immigrant cells that are adapted Massachusetts Institute of Technology, Cambridge, MA, USA to different environments and that continue to metabolize 3 Woods Hole Oceanographic Institution, Woods Hole, MA, USA upon their arrival [26–28]. However, as intervals in time and Stable aerobic and anaerobic coexistence in anoxic marine zones 289 space separating sustainable aerobic and anaerobic activity Here, we explore the coexistences and competitive exclu- become small, stable coexistence best describes community sions among the diverse O2- and N-cycling metabolisms that activity. Stable coexistence is supported by evidence that emerge when metabolisms are related more directly to che- aerobic respiration is viable at low (nanomolar) oxygen mical potential. Ideally, a model of concentrations [18, 29], and that community metabolism would anticipate biogeochemical function by estimating the responds to rapid (as short as hourly) fluctuations in oxygen activity of from first principles—as a func- availability [30]. tion of underlying chemical and physical constraints— The energy that drives most metabolisms in pelagic AMZs allowing for universal applicability. We work towards this originates directly or indirectly from in the ideal by constructing a microbial model where sunlit layer. Therefore, we can consider the supply of two metabolic functional types are described by underlying redox substrates—oxygen and organic matter—as the dominant chemistry, by theoretically and empirically determined effi- control on AMZ formation [31, 32]. To understand the che- ciencies, and by parameterizations of resource uptake mical transformations in AMZs that impact the climate system, grounded in physiology. Unlike models that rely on we must consider a diversity of N-cycling metabolisms. Two imposed oxygen inhibition concentrations and other AMZ- anaerobic metabolisms are responsible for the bulk of fixed N specific measured parameters [38, 39], this allows for a self- fi À À loss in the ocean: heterotrophic denitri cation (NO3 or NO2 consistent model that has no information about AMZ meta- + organic matter→N2O + N2) and chemoautotrophic anaero- bolic as input, and can then be compared with À + þ→ À + bic ammonia oxidation (anammox; NO2 NH4 NO3 observations. Moreover, resolving interactions of metabolic À À N2)[6]. Since NO2 (aswellasNO3 ) accumulates in AMZs, functional types obviates the need for prescribed oxygen we can assume that heterotrophic denitrification is limited by thresholds and allows for the emergence of stable coexistence þ organic matter and that anammox is limited by NH4 . Thus, of competing metabolisms. À fi fi when NO2 is abundant, we may describe the transition from Speci cally, we use Resource Ratio Theory [40]to rst aerobic to anaerobic activity as the outcome of two competi- develop and demonstrate a theory for sustained coexistence tions: the of denitrification against aerobic het- with an idealized example of aerobic and anaerobic hetero- erotrophy for organic matter, and the competition of anammox trophic populations competing for organic matter. We con- þ against aerobic ammonia oxidation for NH4 . The control of sider this competition as either actual competitive oxygen on these two outcomes seems to be distinct. Incubation interactions between two distinct obligate aerobic and experiments have demonstrated that anammox tolerates higher anaerobic populations or an internal cellular process of a oxygen concentrations than denitrification [30]. This is con- facultatively anaerobic population that switches its electron sistent with observations in the Bay of Bengal, where ana- acceptor to maximize its growth rate in a dynamic envir- mmox, but not denitrification, was measured at intermediate onment. Second, we develop a redox-based parameterization oxygen concentrations (10–200 nM) [33]. of a set of diverse microbial metabolisms crucial to AMZ O2 However, understanding the transitions of this complex and N cycling, and examine the outcome of their interactions network of metabolisms along an oxygen gradient in a simplified system (a virtual chemostat). Finally, since remains incomplete. Though observations and model physical processes as well as biological processes shape À estimates of NO2 production and consumption rates AMZs, we incorporate the metabolic model into a two- À – reveal excessive NO2 supply [16, 34 36], reasons for the dimensional (2D) circulation that captures the essential differing rates remain unclear. Accumulation to con- physical features giving rise to AMZ formation. The model, centrations beyond those limiting any metabolism distin- expanded to include phytoplankton and func- À guishes this secondary NO2 maximum from the primary tional types, represents a fully dynamic microbial ecosystem À NO2 maximum, which may constitute the limiting sub- spanning oxic to anoxic states. In the idealized AMZ, À sistence concentration for aerobic NO2 oxidizing micro- coexistence is sustained in the oxycline from vertical mixing organisms [37]. Also, why is anammox a viable as well as at an emergent secondary chlorophyll maximum. metabolism at higher oxygen concentrations than deni- trification [30]? One explanation for inhibition of anae- robic metabolism is the oxygen sensitivity of specific Materials and methods enzymes [30]. An alternative perspective is that microbial community function forms, and is formed by, the che- Theory: stable coexistence despite competition mical potential of the environment, and that specific enzymatic machinery has consequently evolved over time Metabolic model in response to this chemical potential. Thus, chemical potential may explain the energetic favorability of meta- We develop a theoretical model of two metabolisms. Each bolism at a more fundamental level. requires two resources: electron donor S and electron acceptor 290 E. J. Zakem et al.

X μ −1 à . An equation for the growth rate i (t ) of a population of oxygen, O2 [43, Table 2]. If DIN supply is reduced, the associated with each metabolism i can be formulated from the anaerobic population would also become limited by its fi V −1 −1 Àà Àà speci crateofuptake (mol resource mol biomass t )of electron acceptor at NO3 (or NO2 ). each required resource and the yield y (mol biomass mol resource−1) associated with each resource. Using Leibig’s The threshold for stable aerobic and anaerobic coexistence Law of the minimum, the limiting growth rate is Following previous work [40, 44, 45], we derive an μ ¼ min½ŠyS VS ðSiÞ; yX VX ðXiÞ ; ð1Þ i i i i i expression that determines whether the simulated aerobic where uptake is a function of the resource concentration and heterotroph competitively excludes the anaerobic hetero- may be represented with a saturating (Michaelis–Menten) troph or whether the two coexist as a function of the relative form or as limited by diffusive supply, for example. The supply rates of organic matter (OMin) and oxygen (O2in)ina two metabolisms may also be considered as occurring chemostat where growth rate μi equals dilution rate D.In (“competing”) within one facultative population that can Appendix 1, we provide the details of the derivation, which use multiple electron acceptors. considers the condition allowing both aerobically and We can anticipate the outcome of a competitive interac- anaerobically sustained biomass in the steady state balances tion between the two populations by comparing the for organic matter and oxygen (Eqs. (T3) and (T4) in population-specific subsistence concentrations of that Table 1). We call this threshold ϕ: resource [40]. Here we derive and compare the expressions Dð À ÃÞ O2in O2 À1 for the subsistence concentrations of organic matter for ϕ ¼ à r ; ð3Þ DðOMin À OM Þ populations carrying out aerobic and anaerobic heterotrophy. O We describe organic matter uptake with a Michaelis–Menten where r is the ratio of oxygen to organic matter demand of À1 form with specific maximum uptake rate VmaxOM and half- the aerobic heterotrophic metabolism: r ¼ yOM y (mol O O O2 2 saturation constant KOM, and then combine the organic utilized per mol OM utilized; Eq. (A11)). If ϕ > 1, more matter-limited growth rate (yOMVOM) with a steady oxygen is supplied than is required to aerobically consume state balance in which growth rate equals the specific loss all of the organic matter supplied, and the anaerobic rate L (t−1), which may represent mortality, maintenance metabolism can be competitively excluded. If ϕ = 1, oxygen costs, consumption by grazers, or viral lysis. Solving for the and organic matter are supplied in the exact ratio demanded organic matter concentration OM* gives by the aerobic metabolism. If ϕ < 1, more organic matter K L is supplied than can be processed aerobically, and the à OMi i OMi ¼ : ð2Þ excess organic matter can be metabolized anaerobically. yOM VmaxOM À Li i i Thus, ϕ = 1 is the threshold below which aerobic and The kinetic parameters for organic matter uptake are anaerobic metabolism can stably coexist. The aerobic uncertain and should vary with substrate and with metabolism is never competitively excluded (as long as population, but if we assume that uptake kinetics for the oxygen remains an energetically favorable electron same substrate may be optimized similarly for different acceptor). organisms adapted to similar conditions (which must be the In an AMZ, where organic matter supplies nearly all case for a facultatively aerobic population), and if we chemical energy, ϕ can broadly delineate the locations of assume that specific loss rates are also similar (such as if the anaerobic activity. However, the incoming and outgoing populations are subject to the same rate), the fluxes of oxygen and organic matter cannot be easily subsistence organic matter concentrations differ predomi- decomposed as in the chemostat (where the incoming flux y F ¼ D ϕ nantly by the yield OMi in the denominator. Free energies of O2in O2in), and so we use an approximate form of . reactions predict that O2 is a superior electron acceptor to When it is feasible for both oxygen and organic matter À NO3 across a wide range of activities [41, 42], and thus that substrates to be depleted to low concentrations relative to y fl F ¼ the aerobic organic matter yield ( OMO ) is greater than the supply, one can neglect the outgoing uxes (i.e., O2out y y D à  F ¼ DðOMÃÞ anaerobic ( OMN ) for the same electron donor. With OMO > O2 0 and OMout 0), but the sinking of y OMN , the aerobic subsistence concentration is lower than particulate organic matter makes some of the organic matter à à the anaerobic subsistence concentration (OMO

Table 1 Equations for the model dBO ¼ B ðμ ÀDÞ with two metabolic functional dt |fflffl{zfflffl}O O (T1) types—aerobic heterotroph (BO) biomass synthesis and anaerobic heterotroph (BN) — dBN ¼ B ðμ ÀDÞ in a virtual chemostat dt |fflfflffl{zfflfflffl}N N (T2) biomass synthesis

dOM ¼ DOMð ÀOMÞ À 1 μ B À 1 μ B dt |fflfflfflffl{zfflfflfflffl}in y O O y N N (T3) |fflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflffl{zfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflffl}OMO OMN supply organic matter consumption d 1 O2 ¼ Dð À ÞÀ μ B dt |fflfflffl{zfflfflffl}O2in O2 y O O (T4) |fflfflfflfflffl{zfflfflfflfflffl}O2 supply oxygen consumption  dN ¼ DNð ÀNÞþ 1 À μ B þ 1 À μ B À 1 μ B dt |fflffl{zfflffl}in y 1 O O y 1 N N y N N (T5) |fflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflffl{zfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflfflffl}OMO OMN |fflfflfflffl{zfflfflfflffl}N supply remineralization

Growth rates of the two populations (μO and μN) are calculated with Eq. (1). To correctly estimate the consumption of both limiting and nonlimiting substrates, uptake is written in terms of the growth rate as À1 Vj ¼ yj μ for substrate j. For organic substrate, (1 − yj)Vj is partitioned towards respiration (remineraliza- tion) rather than biomass synthesis. Biomass B, organic matter OM, and dissolved inorganic nitrogen (N) are resolved in concentrations of nitrogen

OM OM + À→B + Here, represents the sum of both dissolved and sinking reduction to (DNRA: NO3 HetDNRA þ þ (particulate) organic matter supply, though we would expect NH4 ), chemoautotrophic aerobic ammonia oxidation (NH4 + →B + À the sinking portion to contribute to the majority of the O2 AOO NO2 ), chemoautotrophic aerobic nitrite oxi- fl r “ À + →B + À outgoing ux. Ratio is similar to the respiratory dation (NO2 O2 NOO NO3 ), and chemoautotrophic ” þ + À→B + À + quotient, or the amount of CO2 produced per mol O2 anammox (NH4 NO2 anx NO3 N2). consumed [46], although it additionally reflects the amount Following established methodology [47], we describe of organic matter assimilated into biomass. The value of r these metabolisms as metabolic functional types by com- appropriate for the ocean is a function of an average bining electron-normalized half-reactions to form the cata- heterotrophic growth efficiency given the complex soup of bolic and anabolic full reactions for each (Appendix 2). organic substrates (Eq. (A11), Fig. A1). It converges to the Electron fraction f then partitions the electron flow towards value of the respiratory quotient for the observed low biomass synthesis vs. respiration for energy. This results in efficiencies [46], and decreases if the average efficiency a whole- stoichiometry that quantifies the amount increases substantially (such as could happen with “fresh” of each substrate required to provide the electrons and organic matter input following a bloom). A decrease in r elements for synthesis of one unit of N-based biomass (ex: ϕ þ B increases , in which case less O2 supply is required to 112 mol NH4 per mol AOO for aerobic ammonia oxidation; maintain strictly aerobic activity. Using this balance, Fig. 1). We consider this amount of required substrate in À1 ϕ fi fi yNH ¼ locations where ocean < 1 can be identi ed as energetically terms of a substrate-speci c yield (ex: 4BAOO 112 ), favorable for anaerobic metabolisms. In other words, and so we can represent each metabolism in general form as anaerobic activity is sustainable once oxygen supply is  1 1 1 1 low enough that the ratio of available oxygen to organic Sred;i þ Xox;i ! Bi þ À 1 Sox;i þ Xred;i; yS yX yS yX matter is lower than the ratio of demand. i i i i ð5Þ Redox-based description of metabolic functional types where S (mol N L−1) is an electron donor substrate and X −1 (mol N L ) is the electron acceptor in reduced (Sred, Xred) −1 A minimum set of microbial metabolisms (in addition to or oxidized (Sox, Xox) form, and B (mol N L ) is the oxygenic photoautotrophy) mediates the climatically relevant biomass of type i. See Appendix 2 for a detailed description N-cycling in and around anoxic zones [1, 6]. For each of each metabolism and Fig. 1 and Table A1 for the metabolism, we relate the main N-based substrates and resulting stoichiometries and yields. excretion products to biomass B in units of N as: aerobic Redox chemistry links the yields to electron fraction f, OM + →B + þ heterotrophy ( O2 HetO NH4 ), heterotrophic nitrate which we constrain using a combination of theoretical and À þ À OM + →BHetNO + + reduction to nitrite ( NO3 3 NH4 NO2 ), empirical analysis (Appendix 2 and Table A1). For the fi OM + À→B heterotrophic denitri cation of nitrite ( NO2 HetNO2 chemoautotrophic metabolisms, after comparing ana- + þ + NH4 N2), heterotrophic dissimilatory nitrate (or nitrite) mmox stoichiometry with previous analysis of aerobic 292 E. J. Zakem et al.

−1 Fig. 1 Estimates of the stoichiometries of substrate demand (yield y ) of nitrate to ammonium (BHetDNRA), nitrate-reducing heterotrophy B B B and excreted products relative to synthesis of biomass for the ( HetNO3 ), denitrifying heterotrophy ( HetNO2 ), and anaerobic ammonia metabolic functional types (three aerobic in shades of blue, and four oxidation (anammox; Banx). Currency for generic organic matter sub- anaerobic in shades of red): aerobic heterotrophy (BHetO), ammonia strate OM and biomass B is moles of N oxidation (BAOO), nitrite oxidation (BNOO), dissimilatory reduction

Table 2 The interaction matrix of the metabolic functional types (blue: aerobic metabolisms, red: anaerobic metabolisms)

Lighter colored squares indicate required substrates, and are overlaid with the associated subsistence (R*) concentration calculated with loss rate L as the virtual chemostat dilution rate (D = 0.05 per day). Darker colored squares indicate waste metabolic products. For each column, all organisms requiring that substrate (all lighter colored squares in the column) can potentially compete with one another, while each pair of dark and light colors indicates the potential for a syntrophic (cross-feeding) interaction. DIC consumption and production is also noted. Yields and uptake kinetic parameters are listed in Tables A1 and A2 nitrification [37, 47, 48], we assigned the same value of stoichiometries of the electron-normalized redox reac- f = 0.03toanammoxaswellastothenitrifiers to most tions. We vary these values in an ensemble of model robustly test the competition between them (Appendix 2). solutions (Appendix 3). The resulting stoichiometry suggests that anammox is less Table 2 represents a matrix of all possible interactions in fi þ þ ef cient than aerobic NH4 oxidation with respect to NH4 , the model. Since the complex network prevents any simple þ requiring about 150 vs. 110 mol NH4 per mol biomass N, prediction of the outcome of the interactions, the diagnostic fi À R* but more ef cient than aerobic NO2 oxidation with framework provided by subsistence ( ) concentrations is a À À R* respect to NO2 , requiring about 220 vs. 330 mol NO2 per useful way to interpret model solutions. We list the mol biomass N. For all heterotrophs, we describe growth concentrations for each metabolism and substrate calculated on an average pool of marine organic matter, and so we with the yields, uptake parameters, and the chemostat assigned an average marine bacterial growth efficiency to dilution rate. For all functional types, we assume one uptake y ¼ : the aerobic heterotroph ( OMHetO 0 14) [46], which cor- parameterization for organic matter, another for DIN, and a responds to f ≈ 0.1 (Appendix 2). Informed by free ener- third for oxygen (Table A2). This essentially assumes a gies as above, we assigned a slightly lower organic matter similar cell size and proteome allocation for all populations, yield to all three anaerobic heterotrophs (10% lower). We and that differences in uptake kinetics do not determine the assigned them equal organic matter yields because we do outcome of any competition. This assumption is justified if not here consider the further characteristics that determine populations competing for the same substrate in the same the outcome of interactions among anaerobic hetero- environment have similarly optimized their enzyme allo- trophic metabolisms [49, 50]. With this simplification, cation, and that tradeoffs exist between traits, such as sub- differences in the amount of electron acceptor required by strate affinity, maximum growth rate, and defense strategies. each anaerobic heterotroph in Fig. 1 reflect only the However, the framework does allow for the possibility of Stable aerobic and anaerobic coexistence in anoxic marine zones 293

(and interpretation of) alternative outcomes if differences in a. Oxygen 1000 O these parameters do consistently distinguish populations 2 10 O *

2 2 carrying out competing metabolisms in ways that change O with facultative pop. 0.1 2 their relative subsistence concentrations. =1 µM O 0.001

Results b. Organic matter 0.08 OM OM * aerobe The transition from competitive exclusion to stable OM * anaerobe 0.06 OM with facultative pop.

coexistence µM N

0.04 We first examine the competition of aerobic and anaerobic Biomass metabolism in a simulated chemostat to make two points: 0.15 c. Aerobic (1) that the theoretical prediction of the metabolic shift is Anaerobic borne out and (2) that the same shift is predicted whether 0.1 Total Exclusion Coexistence Facultative associated with a reorganization of community metabolism µM N 0.05 by balancing the populations of obligate types, or by 0 facultative readjustment of individual metabolisms. We d. Respiration rates examine two parallel models, one with distinct (obligate) 400 O consumption 2 aerobic and anaerobic populations, and a second with a Denitrification

-1 Facultative O consumption facultative population. 200 2 The set of ordinary differential equations in Table 1 Facultative denitrification nM d B describes the biomass of the populations associated with 0 the distinct metabolisms, as well as organic matter, oxygen, 102 101 100 10-1 10-2 O : OM supply (mol/mol) and DIN. In the parallel simulation with the facultative 2 population, one bulk biomass Bfac carries out whichever Fig. 2 Steady state solutions for two metabolisms—aerobic and metabolism yields a higher growth rate at each time step anaerobic heterotrophy—in a virtual chemostat for a varying ratio of μ ¼ ðμ ; μ Þ ( fac max O N ). We assume abundant DIN avail- O2 to organic matter (OM; mol N) supply. Two parallel simulations are ability (Nin = 30 μM) as in the mesopelagic ocean, giving illustrated: one with discrete aerobic and anaerobic heterotrophic solutions independent of DIN. metabolic functional type populations, and one with a single faculta- tively anaerobic population. Respiration rates for the facultative We examine the equilibrium solutions to the equations population indicate the time-averaged use of O2 and DIN. The vertical for a varying ratio of oxygen to organic matter supply by dashed black line indicates ϕ = 1, the threshold at which anaerobic varying the incoming oxygen concentration (Fig. 2). In the metabolism becomes sustainable model with the two obligate types, the anaerobic type is competitively excluded at a high supply ratio (abundant oxygen) without a prescribed oxygen inhibition because its aerobic and anaerobic bacterial biomass competing for organic matter subsistence concentration is higher than that sulfide at varying oxygen to sulfide ratios [53]. à à of the aerobe (OMN >OMO). When the supply ratio The solution with the facultative population shows nearly decreases below threshold ϕ (black vertical line), the identical results (lighter colored lines in Fig. 2). The oxygen aerobic metabolism becomes limited by oxygen and is only concentration wavers slightly due to the synchronous able to oxidize a fraction of the available organic matter, switching from aerobic to anaerobic growth, and the organic à à and the anaerobic metabolism is sustained. matter concentration is between OMN and OMO for the Throughout this domain of coexistence, oxygen is period at which the facultative population utilizes both maintained at the subsistence concentration of the aerobe oxygen and DIN at similar fractions over time. à (O2; Fig. 2a). Organic matter is maintained at the higher The model shows that: (1) anaerobic sustainability is a à subsistence concentration of the anaerobe (OMN ; Fig. 2b), predictable function of the relative supply of oxygen and an which is qualitatively consistent with observations of electron donor such as organic matter, (2) the ratio of decreased attenuation of the particulate organic matter flux aerobic to anaerobic activity reflects this relative supply, (3) à in low oxygen environments (i.e., reduced particulate con- aerobic activity can be sustained at O2 where O2 is supplied, sumption) [51, 52]. The ratio of aerobic to anaerobic bio- such as through or physical transport, and mass and associated respiration rates decreases (4) a facultative population model gives a nearly equivalent proportionally with the relative decrease in oxygen supply biogeochemical outcome to a model with two obligate (Fig. 2c, d). This pattern is consistent with observations of populations at steady state. 294 E. J. Zakem et al.

Fig. 3 Steady state solutions for the diverse O2- and N-cycling metabolic functional types in a virtual chemostat for a varying ratio of O2 to organic matter (OM; mol N) supply. The shaded region indicates the 5th–95th percentile of the ensemble of solutions. Incoming OM concentration is 1 μMN

Diverse N-cycling metabolisms in a chemostat We illustrate the model solutions without DNRA in þ Fig. 3. When the DNRA functional type is included, NH4 À We next examine the equilibrium state of the interactions of but not NO2 accumulates in the anoxic state (Fig. A3), the diverse N-cycling metabolic functional types as a which is consistent with some observations, but not with the function of oxygen and organic matter supply. To consider characteristic state of AMZs [14]. We conclude that DNRA the uncertainty in the parameterizations, we computed an is likely less efficient at utilizing organic matter than the ensemble of solutions for which the parameter values dic- other anaerobic heterotrophic metabolisms, and that low or tating the yields (and thus also ratio r) were sampled ran- sporadic rates of DNRA must be sustained by a process not domly from plausible ranges of uncertainty (Equations and resolved in the current model, such as time-varying blooms detail in Appendix 3). of organic input (see Appendix 3 for further discussion). Stable aerobic and anaerobic coexistence in anoxic marine zones 295

Oxygenated state be excluded at higher oxygen supply than the nitrifiers. These results do suggest, however, that a subset of aerobic At high oxygen supply (O2:OM ≳ 10 mol/mol in Fig. 3), the heterotrophs can subsist in the anoxic core of AMZs, three aerobic metabolisms coexist syntrophically. All which is consistent with genetic and transcriptomic evi- anaerobic metabolisms are competitively excluded, except dence [17]. þ for some simulations in the ensemble in which the NH4 yield of anammox is similar to or higher than that of aerobic Intermediate state þ ammonia oxidation (Fig. 3d, e). Concentrations of NH4 and À NO2 are maintained at the subsistence concentrations of the In between the oxic and anoxic end-member states (O2: þ À OM ≈ – aerobic NH4 -oxidizing and NO2 -oxidizing populations, 1 10 mol/mol in Fig. 3), the simple model predicts a respectively [37] (Fig. 3b). This simulates microbial com- complex intermediate state. Oxygen supply is just low munity function in oxygenated environments below the enough to limit the growth of the nitrifiers, but not yet low euphotic zone. enough to limit the aerobic heterotroph, which continues to be limited by organic matter supply. Because of this, oxy- fi ’ à Anoxic state gen is maintained at the nitri ers higher O2 (Fig. 3a), and, analogous to the heterotrophic case study in Fig. 2, their At the lowest relative oxygen supply rates (O2:OM /≲ 1 oxygen-limited growth allows for residual reduced DIN that à mol/mol in Fig. 3), O2 is depleted to O2, and all anaerobic then sustains anammox. This represents a distinct threshold þ metabolisms are sustained. [NH4 ] is maintained at the for the onset of anaerobic anammox, matching experimental subsistence concentration of the anammox population, results [30]. In the model, anammox is a sustainable À fi while [NO2 ] accumulates higher than any subsistence metabolism at a higher oxygen supply than denitri cation concentration (Fig. 3b). The accumulation results from the because it competes against the chemoautotrophic nitrifiers À fl fi imbalance in NO2 supply and demand re ecting the stoi- for DIN. Chemoautotrophic nitri cation demands sig- chiometries of the redox reactions. Normalized by electron nificantly more O2 than heterotrophy to synthesize the same À À transfer, 1.5 times more NO3 is reduced to NO2 by the amount of biomass (Fig. 1), and so the oxygen supply that À À NO3 -reducing population than NO2 reduced by the deni- becomes limiting for them is higher than the oxygen supply À trifying population. Anammox also consumes NO2 , but not that becomes limiting for the average heterotroph. Nitrate enough to counter the imbalance since its growth is limited reduction and denitrification do not become energetically þ by NH4 . This perhaps explains the formation of the sec- favorable until aerobic heterotrophy is limited by oxygen, À À ondary NO2 maximum. and anammox is NO2 -limited until N2 has accumulated As oxygen supply decreases, the rates of denitrification (Fig. 3b). Thus, the redox-based model anticipates the and anammox converge to a constant ratio. The fraction of adaptation of anammox clades to tolerate higher con- anammox contribution to total fixed N loss is 29% (Fig. 3e), centrations of oxygen to exploit available chemical which is consistent with the theoretically and empirically potential. observed fraction of about 30% [6, 30, 32, 54]. This fraction remains constant across variations in yields and uptake A two-dimensional idealized AMZ parameters. A lower anammox yield translates to lower annamox-associated biomass, for example, but the water The virtual chemostat model provided an organized fra- column rate remains the same, demonstrating that bulk mework for interpreting the of the diverse O2- and water column rates are ultimately determined by organic N-cycling metabolisms in and around AMZs in isolation matter supply. from the impacts of ocean circulation. However, physical The aerobic heterotroph remains the only active aerobic supply and dispersal are important characteristics of the metabolism in the anoxic state. Once its growth is oxy- real system. As a final study, we included transport by gen-limited, it competes against the nitrifiers for oxygen. incorporating the ecological model into an idealized The nitrifiers are excluded because they demand more O2 circulation model. y à for growth (lower O2 ) and thus have a higher O2 (Fig. 3a, A 2D overturning circulation qualitatively simulates the blue dashed lines). This higher O2 demand reflects the 10°S transect across the S. Pacific basin (Fig. 4). A closed energetic cost of C fixation for chemoautotrophy com- flow field with a width of 10,000 km and a depth of 2000 m pared with the average heterotroph. However, in real is forced with wind stress mimicking the climatological environments, the heterogeneity of organic matter and mean (Fig. A4; see Appendix 4 for equations and detail). A diversity of the heterotrophic community should result in mixed layer is prescribed with an attenuating vertical dif- a diversity of populations each associated with a distinct fusivity coefficient, and eddy stirring is represented with a à à fl fi O2, and the portion of the community with higher O2 may horizontal diffusivity constant. The resulting ow eld 296 E. J. Zakem et al.

Fig. 4 Observations and model simulations of the 10° S transect across transport. Steady state solutions of the metabolic fi a À d fl fi b fi the Paci c Ocean. Observations of O2 ( ) and NO3 ( ) are from the incorporated into the ow eld include: O2 ( ), xed N loss from fi c À e World Ocean Atlas 2013 climatology [65], with distance from 175° heterotrophic denitri cation and anammox ( ), NO3 ( ), and che- longitude in the W. Pacific. Arrows in e indicate the idealized 2D flow moautotrophic C fixation calculated from biomass production rates field, with intensified upwelling driven by wind-stress-induced Ekman with a biomass C:N of 5 (f) simulates Ekman transport, “eastern” coastal upwelling, and through the anoxic zone where all metabolisms are sustained dispersed downwelling in the “west” (Fig. 4e). at various depths (Fig. 5e–h). The six microbial metabolic functional types in Fig. 3 are The characteristic profiles through the oxygenated zone advected and diffused by the circulation, along with two resulting from the metabolic model have been previously phytoplankton types and three zooplankton grazing types discussed [37]. Here we highlight aspects of the modeled that produce and consume oxygen, respectively. (Equations, anoxic zone, their parallels with observations, their detail, and parameter values in Appendix 4 and Table A3). mechanisms in the model, and implications for AMZ Zooplankton grazers consuming phytoplankton are inhib- biogeochemistry: ited at low oxygen concentrations. Vertical migration of zooplankton preying on the non-photosynthetic microbes is AMZ formation is controlled by relative rates of oxygen and simulated by allowing them to consume microbial biomass organic matter supply throughout the anoxic zone and accounting for their oxygen consumption above and below [55–57]. (See Appendix 4 In the simulated anoxic zone, oxygen is depleted to nano- for detail.) Thus total zooplankton activity is reduced, but molar concentrations or lower, with the lowest values due to does not cease, in the AMZ. Two pools of organic matter— dispersal of aerobic cells into the anoxic zone. Immigrant one sinking and one non-sinking—are sourced from the cells can drive down the limiting resource below the sub- mortality of all populations. Oxygen fluxes across the sistence concentration of a purely local population because air–sea interface. Fixed N lost to denitrification or anammox immigration makes up for the reduced local growth implied À is summed and distributed as a source of NO3 equally over by the lower resource concentration [27]. The modeled the domain at all depths, simulating distant N fixation. vertical profile simulates the sharp oxycline characteristic of À The solutions demonstrate how the combination of circu- pelagic anoxic zones (Fig. 5e) [4]. Modeled NO3 has the lation and surface —due to intrinsic links characteristic concave profile that indicates N loss (Fig. 5f). between intensified coastal upwelling and a lack of ventilation The fraction of anammox contribution to total fixed N loss below in eastern boundaries [58]—results in anoxic zone increases at the periphery of the AMZ (Fig. A7), although formation (Figs. 4 and 5; complete solutions in Figs. A5 and this intermediate state is less pronounced than in the che- A6). Intensified upwelling enhances primary productivity, mostat model due to the mixing. Some organic matter sinks which produces more sinking organic matter input to the below the anoxic zone, fueling a deep aerobic community unventilated zone below where aerobic respiration depletes for an additional few hundred meters (Fig. 5g, h). The oxygen. Figure 5 illustrates profiles through two water col- solution state can be anticipated by calculating ϕocean using umns: one in the oligotrophic, oxygenated zone, where only the model flow field. The contour ϕocean = 1 (the dashed the aerobic metabolisms are sustained (Fig. 5a–d), and one lines in Figs. 4 and 5) delineates—to first order—the Stable aerobic and anaerobic coexistence in anoxic marine zones 297

Fig. 5 Steady state solutions through two water columns in the 2D model: one in the “western” oxygenated zone and one in the anoxic portion of the “eastern” upwelling zone. The vertical dashed and dotted lines indicate subsistence concentrations (R*s). The horizontal black dashed lines (ϕocean = 1) indicate the zone of stable anaerobic activity, which does not account for transient coexistence due to dispersal from vertical mixing

domain in which anaerobic metabolism is sustainable. In A secondary chlorophyll maximum formation from O2- Fig. A2, we plot resulting O2 consumption, N loss, and O2 limited grazing concentrations against the incoming supply of O2 and organic matter to each grid box, which further demonstrates Phytoplankton sustainability is determined by the balance the resource ratio control. of light supply, supply, and loss rates to grazing and other mortality. Starting from the surface, phyto- Vertical mixing sustains anaerobic metabolisms in biomass increases with depth to the deep (primary) oxygenated chlorophyll maximum, where both nutrient and light supply is optimized, then decreases as light begins to limit growth, In the model, significant rates of all three anaerobic meta- then increases again as O2 is depleted and grazing becomes bolisms are sustained at very high (tens of μM) oxygen O2-limited, and then decreases again because of light lim- concentrations above the anoxic zone (Fig. 5h). Since ϕocean itation (indicated by the chlorophyll concentration in does not account for transport, the N loss outside the dashed Fig. 5e). This constitutes one hypothesis for the formation line ϕocean = 1 (and some of the oxygen consumption of the secondary chlorophyll maximum (SCM), and further within, as mentioned above) is a consequence of the dis- hypothesizes that phytoplankton should experience a local persal of biomass. This may be a mechanism supporting the minima in their loss rate there. Changes in viral lysis rates high diversity of metabolisms in the oxycline in real AMZs with depth may be a particularly likely hypothesis given the [59, 60]. observation that distinct cyanophage communities exist at Dispersal-driven anaerobic respiration remains when the SCM [63]. The model predicts simultaneous O2 pro- including the facultative type in the 2D model: as in duction, O2 consumption, and sustained anaerobic activity Fig. 2, with respiration rates averaged over time, the at the SCM, exhibiting a “cryptic oxygen cycle” [19]. steady state solutions with facultative aerobic hetero- À trophs are indistinguishable. When obligate aerobic, Formation of a secondary NO2 maximum obligate anaerobic, and facultative heterotrophs are À allowed to compete in the 2D model, the facultative As above, NO2 accumulates in the modeled AMZ to con- population competitively excludes both obligate types centrations higher than any subsistence concentration throughout the entire domain unless it is penalized for (Fig. 5f). This broad tendency arising from the stoichio- having more metabolic capabilities. This may anticipate metry of the redox reactions may constitute a clue for why À why many heterotrophs, including the ubiquitous SAR11, the secondary NO2 maximum forms [35]. Actual mechan- À are facultatively anaerobic [61, 62]. isms may include time-varying concentrations of NO2 298 E. J. Zakem et al.

[49, 50], and further differences in rate would result from input, the oxygen supply required to stay above ϕ also significant differences in efficiencies among the anaerobic increases for constant r (Fig. A2). Thus, the oxygen con- heterotrophic metabolisms. centration in the ocean alone is, at best, a good approx- imation for locations of anaerobic activity. Deep nitrification and carbon fixation at the AMZ periphery Results suggest that the ratio of aerobic to anaerobic respiration will generally decrease from the periphery to the Just above and below the simulated anoxic zone, significant core of an anoxic zone. This is consistent with the obser- rates of aerobic chemoautotrophic nitrification are sustained vations of foraminifera in the Peruvian AMZ [64]. The (Fig. 5g, h). This is consistent with observations [12, 14], perspective here suggests that the inferred “preference” for fi À and, along with anammox, drives deep CO2 xation NO3 utilization by the cells in conditions where oxygen is (Fig. 4f). In the illustrated realization, this nitrification is rarely supplied constitutes the consequence, not the dicta- supported by local heterotrophic remineralization of organic tion, of the outcome of the metabolic competition among a matter because the one pool of utilizable (labile) organic facultative population. matter remains available throughout the anoxic zone (Fig. Though the present 2D simulation does not capture the À A6d, e). enhanced NO2 oxidation observed in AMZs [12], we can use the framework to hypothesize the conditions that would À allow it. NO2 oxidation may be sustained at higher rates Discussion þ fi than NH4 oxidation in the AMZ periphery where suf cient À À concentrations of both NO2 (accumulated from NO3 Theory and modeling suggest that the onset of anaerobic reduction) and O2 co-occur due to mixing. This is perhaps metabolism in aquatic environments is best characterized as more likely deeper in the anoxic zone or at its bottom a transition from competitive exclusion to stable coex- boundary where labile organic matter is depleted, inhibiting þ istence. Even trace amounts of aerobic activity are theore- heterotrophy and NH4 oxidation. Instead, the 2D model tically stable if trace amounts of O2 are supplied, suggesting here resolves only one type of sinking organic matter that the potential for low rates of aerobic activity concurrent remains available beneath the simulated AMZ. with anaerobic activity in anoxic environments. Adding Intriguingly, the intermediate state of the chemostat stable coexistence to the list of explanations for co- model resembles some aspects of recent observations in the occurring aerobic and anaerobic metabolisms (which Bay of Bengal [33], where anammox but not denitrification includes particle segregation [22, 25], time-varying circu- was measured at intermediately low oxygen concentrations lation [20, 21], and dispersal (Fig. 5h)) expands the degree (hundreds of nM). In both model and observations, ana- À to which we expect that aerobic metabolisms should be mmox is NO2 -limited in the intermediate state. However, “ ” À sustained cryptically in anoxic zones [19]. Results sup- unlike the observations, the model predicts that NO3 port the speculation that a significant amount of organic or reduction and denitrification are strictly coupled, which may reduced inorganic substrate may be metabolized aerobically indicate that in reality that their associated organic matter within anoxic zones [17]. This impacts predictions of fixed yields are not similar, that anoxic niches inside particles nitrogen loss based on the amount of organic matter oxi- contributes to their spatial segregation, or that the obser- dized in anoxic conditions, such as in biogeochemical vations reflect a non-steady state environment and so other models that resolve organic matter fluxes. characteristics are key [49, 50]. By parameterizing diverse aerobic and anaerobic meta- While the subsistence concentration R* governs fitness in bolisms with their underlying redox chemical reactions, the steady state environments, the maximum growth rate of a model allows for the thresholds determining the absence or population is a better measure of fitness in dynamic, time- presence of each metabolism to emerge dynamically as the varying environments [65]. The steady state assumption is consequence of ecological interactions. Avoiding prescribed valid when the rates of activity of microbial populations are thresholds is necessary to simulate the stable coexistence of large relative to the changes in the biomass of the com- competing metabolisms. While natural assemblages do munity, and this should characterize much of the stagnant exhibit oxygen inhibitions [18, 30], the predictions here aim waters where anoxic zones form. However, a pulse of fresh to understand these inhibitions more fundamentally, i.e., to sinking organic matter into an anoxic zone may spur a anticipate why enzymes evolved particular oxygen sensi- “bloom” of microbial activity that is better characterized as tivities over time. a time-varying state, and the dynamics and the distinctions The model suggests that to quantify the relative degree of among the communities relevant to this state are not aerobic and anaerobic activity, the relative supply rates of articulated here. electron donor and oxygen must be known (or resolved in a Here, we have provided a theoretical framework to physical circulation model.) For increasing organic matter quantify the distinctions between yields among microbial Stable aerobic and anaerobic coexistence in anoxic marine zones 299 metabolisms. Though we focus on yields, we explained that stably coexisting aerobic and anaerobic metabolism as a the subsistence concentration R* is the appropriate metric of function of substrate supply, and the resulting model fitness. Therefore, the other variables comprising R* also simulates key aspects of AMZs. The approach progresses matter for the competition. We expect large variation in microbial ecological modeling towards resolving bulk uptake affinity and in loss rates due to maintenance costs, community metabolism dynamically and systematically grazing, and viral lysis in the microbial community. In using underlying chemical and physical constraints, ulti- particular, we expect the traits of the functional types to mately improving predictions of microbial activity in vary consistently in different conditions, as they do for unobserved and future environments. phytoplankton [65], with variation in cell size and allocation of enzyme. While we expect huge variation to characterize Acknowledgements We are grateful for the thorough and thoughtful differences in realized phenotypes among any one meta- comments of two anonymous reviewers. We also thank Andrew Babbin for helpful comments. EJZ was supported by the Simons bolic strategy, we do not however anticipate systematic Foundation (Postdoctoral Fellowship in Marine ). patterns of variation between metabolic strategies for these AM was supported by the Office of Naval Research (ONR #N000- traits in ways that impact the ordering of their R*s. Rather, 14-15-1-2555). JML was supported by U.S. National Science we speculate that for any given environment, an aerobe and Foundation (NSF #OCE-1259388). MJF was supported by the Gordon and Betty Moore Foundation (GBMF #3778) and the an anaerobic analog competing for the same electron donor Simons Foundation: the Simons Collaboration on Ocean Processes are dealt the same supply of elements and electrons, and and Ecology (SCOPE #329108) and the Simons Collaboration on have both adapted to optimize their fitness so that the only Computational Biogeochemical Modeling of Marine remaining distinction is the difference in the energy (CBIOMES #549931). acquired by the respiration pathways (i.e., the free energy released by the redox reactions underlying their metabo- Compliance with ethical standards lisms). However, organisms “hard-wired” with phenotypes Conflict of interest The authors declare that they have no conflict of adapted to different environments may demonstrate a dif- interest. ferent outcome: an anaerobe adapted to environment A, for example, may outcompete an aerobe adapted to environ- Publisher’s note Springer remains neutral with regard to ment B if they are both subjected to environment A, and this jurisdictional claims in published maps and institutional affiliations. type of interaction may characterize communities with Open Access This article is licensed under a Creative Commons populations supplied by dispersal from strong mixing or Attribution 4.0 International License, which permits use, sharing, other transport. Of course, these differences should not adaptation, distribution and reproduction in any medium or format, as matter for facultatively anaerobic populations. long as you give appropriate credit to the original author(s) and the A useful aspect of this modeling approach is the pre- source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this diction of concentrations of active biomass associated with ’ −1 article are included in the article s Creative Commons license, unless each metabolism. This biomass (mol N L ) can be con- indicated otherwise in a credit line to the material. If material is not − verted to cellular (cells L 1) with an estimate of included in the article’s Creative Commons license and your intended a cell quota Q (mol N cell−1) and compared quantitively use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright with genes, transcripts, and other biomolecular data (e.g., holder. To view a copy of this license, visit http://creativecommons. [37]). The resulting insight into the relationship between org/licenses/by/4.0/. community composition and function is one benefitof resolving populations explicitly in ecosystem models. For References example, though substrate supply dictates that the water column anammox rate is about 30% of the denitrification 1. Devol AH. Denitrification including anammox. In: Capone DG, rate, modeled anammox biomass is about 20x lower than Bronk DA, Mulholland MR, Carpenter EJ, editors. Nitrogen in the denitrifying biomass in Fig. 3 because of their different Marine Environment. 2nd ed. Academic Press, Amsterdam; 2008. p. 263–301. substrate yields (Fig. 1). Thus, the model articulates how 2. Paulmier A, Ruiz-Pino D. Oxygen minimum zones (OMZs) in the populations with relatively low abundances are responsible modern ocean. Prog Oceanogr. 2009;80:113–28. for high ambient metabolic rates and thus can be as bio- 3. Brandt P, Bange HW, Banyte D, Dengler M, Didwischus SH, geochemically significant as populations with higher Fischer T, et al. On the role of circulation and mixing in the ventilation of oxygen minimum zones with a focus on the eastern abundances. tropical North Atlantic. Biogeosciences. 2015;12:489–512. To conclude, we used redox chemistry and physiological 4. Ulloa O, Canfield DE, DeLong EF, Letelier RM, Stewart FJ. parameterizations of resource uptake to construct a self- Microbial oceanography of anoxic oxygen minimum zones. Proc – consistent ecosystem model that aims to deepen our Natl Acad Sci USA. 2012;109:15996 6003. 5. Wright JJ, Konwar KM, Hallam SJ. Microbial ecology of understanding of microbial ecology and biogeochemistry. expanding oxygen minimum zones. Nat Rev Microbiol. The theory predicts a dynamic transition from aerobic to 2012;10:381–94. 300 E. J. Zakem et al.

6. Ward BB. Oceans. How nitrogen is lost. Science. 2013; 26. Clayton S, Dutkiewicz S, Jahn O, Follows MJ. Dispersal, eddies, 341:352–3. and the diversity of marine phytoplankton. Limnol Oceanogr 7. Keeling RE, Körtzinger A, Gruber N. Ocean deoxygenation in a Fluids Environ. 2013;3:182–97. warming world. Ann Rev Mar Sci. 2010;2:199–229. 27. Lévy M, Jahn O, Dutkiewicz S, Follows MJ. Phytoplankton 8. IPCC. Climate change 2014: Synthesis report. Contribution of diversity and community structure affected by oceanic dispersal working groups I, II and III to the Fifth Assessment Report of the and mesoscale turbulence. Limnol Oceanogr Fluids Environ. Intergovernmental Panel on Climate Change. IPCC, Geneva; 2014;4:67–84. 2014. 28. Mestre M, Ruiz-gonzález C, Logares R, Duarte CM, Gasol JM. 9. Oschlies A, Brandt P, Stramma L, Schmidtko S. Drivers and Sinking particles promote vertical connectivity in the ocean mechanisms of ocean deoxygenation. Nat Geosci. 2018;11: microbiome. Proc Natl Acad Sci USA. 2018;115:6799–807. 467–73. 29. Bristow LA, Dalsgaard T, Tiano L, Mills DB, Bertagnolli AD, 10. Lipschultz F, Wofsy SC, Ward BB, Codispoti LA, Friedrich G, Wright JJ, et al. Ammonium and nitrite oxidation at nanomolar Elkins JW. Bacterial transformations of inorganic nitrogen in the oxygen concentrations in oxygen minimum zone waters. Proc Natl oxygen-deficient waters of the Eastern Tropical South Pacific Acad Sci USA. 2016;113:10601–6. Ocean. Deep Sea Res Part A. 1990;37:1513–41. 30. Dalsgaard T, Stewart FJ, Thamdrup B, Dalsgaard T, Stewart FJ, 11. Lam P, Jensen MM, Lavik G, McGinnis DF, Muller B, Schubert Thamdrup B, et al. Oxygen at nanomolar levels reversibly sup- CJ, et al. Linking crenarchaeal and bacterial nitrification to ana- presses process rates and gene expression in anammox and mmox in the . Proc Natl Acad Sci USA. 2007; denitrification in the oxygen minimum zone off Northern Chile. 104:7104–9. MBio. 2014;5:e01966–14. 12. Füssel J, Lam P, Lavik G, Jensen MM, Holtappels M, Günter M, 31. Ward BB, Tuit CB, Jayakumar A, Rich JJ, Moffett J, Naqvi et al. Nitrite oxidation in the Namibian oxygen minimum zone. SWA. Organic carbon, and not copper, controls denitrification ISME J. 2012;6:1200–9. in oxygen minimum zones of the ocean. Deep Res Part I. 13. Stewart FJ, Ulloa O, DeLong EF. Microbial metatranscriptomics 2008;55:1672–83. in a permanent marine oxygen minimum zone. Environ Microbiol. 32. Babbin AR, Keil RG, Devol AH, Ward BB. Organic matter 2012;14:23–40. stoichiometry, flux, and oxygen control nitrogen loss in the ocean. 14. Kalvelage T, Lavik G, Lam P, Contreras S, Arteaga L, Löscher Science. 2014;344:406–8. CR, et al. Nitrogen cycling driven by organic matter export in the 33. Bristow LA, Callbeck CM, Larsen M, Altabet MA, Dekaeze- South Pacific oxygen minimum zone. Nat Geosci. 2013;6:228–34. macker J, Forth M, et al. N2 production rates limited by nitrite 15. Peng X, Fuchsman CA, Jayakumar A, Oleynik S, Martens- availability in the Bay of Bengal oxygen minimum zone. Nat Habbena W, Devol AH, et al. Ammonia and nitrite oxidation in Geosci. 2016;10:24–9. the Eastern Tropical North Pacific. Glob Biogeochem Cycles. 34. Buchwald C, Santoro AE, Stanley RHR, Casciotti KL. Nitrogen 2015;29:2034–49. cycling in the secondary nitrite maximum of the eastern tropical 16. Babbin AR, Peters BD, Mordy CW, Widner B, Casciotti KL, North Pacific off Costa Rica. Glob Biogeochem Cycles. Ward BB. Novel metabolisms support the anaerobic nitrite budget 2015;29:2061–81. in the Eastern Tropical South Pacific. Glob Biogeochem Cycles. 35. Beckmann A, Hense I. The impact of primary and export pro- 2017;31:1–14. duction on the formation of the secondary nitrite maximum: a 17. Kalvelage T, Lavik G, Jensen MM, Revsbech NP, Löscher C, model study. Ecol Modell. 2017;359:25–33. Schunck H, et al. Aerobic microbial respiration in oceanic oxygen 36. Martin TS, Primeau F, Casciotti KL. Modeling oceanic nitrate and minimum zones. PLoS One. 2015;10:e0133526. nitrite concentrations and isotopes using a 3-D inverse N cycle 18. Tiano L, Garcia-Robledo E, Dalsgaard T, Devol AH, Ward BB, model. Biogeosciences. 2019;16:347–67. Ulloa O, et al. Oxygen distribution and aerobic respiration in the 37. Zakem EJ, Al-Haj A, Church MJ, Van Dijken GL, Dutkiewicz S, north and south eastern tropical Pacific oxygen minimum zones. Foster SQ, et al. Ecological control of nitrite in the upper ocean. Deep Sea Res Part I. 2014;94:173–83. Nat Commun. 2018;9:1206. 19. Garcia-Robledo E, Padilla CC, Aldunate M, Stewart FJ, Ulloa O, 38. Penn J, Weber T, Deutsch C. Microbial functional diversity alters Paulmier A, et al. Cryptic oxygen cycling in anoxic marine zones. the structure and sensitivity of oxygen deficient zones. Geophys Proc Natl Acad Sci USA. 2017;114:8319–24. Res Lett. 2016;43:9773–80. 20. Brandt P, Greatbatch RJ, Claus M, Didwischus SH, Hormann V, 39. Louca S, Hawley AK, Katsev S, Torres-Beltran M, Bhatia MP, Funk A, et al. Ventilation of the equatorial Atlantic by the Kheirandish S, et al. Integrating biogeochemistry with multiomic equatorial deep jets. J Geophys Res. 2012;117:C12015. sequence information in a model oxygen minimum zone. Proc 21. Pena-Izquierdo J, Van Sebille E, Pelegri JL, Sprintall J, Mason E, Natl Acad Sci USA. 2016;113:201602897. Llanillo PJ, et al. Water mass pathways to the North Atlantic 40. Tilman D. Resource competition and community structure. Prin- oxygen minimum zone. J Geophys Res Ocean. 2015; ceton, N.J: Princeton University Press; 1982. 120:3350–72. 41. Roden EE, Jin Q. Thermodynamics of microbial growth coupled 22. Karl DM, Knauer GA, Martin JH, Ward BB. Bacterial chemo- to metabolism of , ethanol, short-chain organic acids, and lithotrophy in the ocean is associated with sinking particles. . Appl Environ Microbiol. 2011;77:1907–9. Nature. 1984;309:54–56. 42. LaRowe DE, Van Cappellen P. Degradation of natural organic 23. Woebken D, Fuchs BM, Kuypers MMM, Amann R. Potential matter: a thermodynamic analysis. Geochim Cosmochim Acta. interactions of particle-associated anammox with bacterial 2011;75:2030–42. and archaeal partners in the Namibian upwelling system. Appl 43. Zakem EJ, Follows MJ. A theoretical basis for a nanomolar cri- Environ Microbiol. 2007;73:4648–57. tical oxygen concentration. Limnol Oceanogr. 2016;62:795–805. 24. Klawonn I, Bonaglia S, Brüchert V, Ploug H. Aerobic and 44. Ward BA, Dutkiewicz S, Moore CM, Follows MJ. Iron, phos- anaerobic nitrogen transformation processes in N2-fixing cyano- phorus, and nitrogen supply ratios define the biogeography of bacterial aggregates. ISME J. 2015;9:1456–66. nitrogen fixation. Limnol Oceanogr. 2013;58:2059–75. 25. Bianchi D, Weber TS, Kiko R, Deutsch C. Global niche of marine 45. Dutkiewicz S, Ward BA, Scott JR, Follows MJ. Understanding anaerobic metabolisms expanded by particle microenvironments. predicted shifts in diazotroph biogeography using resource com- Nat Geosci. 2018;11:263–8. petition theory. Biogeosciences. 2014;11:5445–61. Stable aerobic and anaerobic coexistence in anoxic marine zones 301

46. Robinson C. Heterotrophic bacterial respiration. In: Kirchman DL, 56. Wishner KF, Outram DM, Seibel BA, Daly KL, Williams RL. editor. Microbial Ecology of the Oceans. 2nd ed. Wiley-Black- Zooplankton in the eastern tropical north Pacific: boundary effects well, Hoboken; 2008. p. 299–334. of oxygen minimum zone expansion. Deep Sea Res Part I 47. Rittman BE, McCarty PL. Environmental biotechnology: princi- 2013;79:122–40. ples and applications. McGraw-Hill, New York City; 2001. 57. Bianchi D, Babbin AR, Galbraith ED. Enhancement of anammox 48. Strous M, Heijnen JJ, Kuenen JG, Jetten MSM. The sequencing by the excretion of diel vertical migrators. Proc Natl Acad Sci batch reactor as a powerful tool for the study of slowly growing USA. 2014;111:15653–8. anaerobic ammonium-oxidizing microorganisms. Appl Microbiol 58. Karstensen J, Stramma L, Visbeck M. Oxygen minimum zones in Biotechnol. 1998;50:589–96. the eastern tropical Atlantic and Pacific oceans. Prog Oceanogr. 49. Kraft B, Tegetmeyer HE, Sharma R, Klotz MG, Ferdelman TG, 2008;77:331–50. Hettich RL, et al. The environmental controls that govern the end 59. Zaikova E, Walsh DA, Stilwell CP, Mohn WW, Tortell PD, product of bacterial nitrate respiration. Science. 2014;345:676–9. Hallam SJ. Microbial community dynamics in a seasonally anoxic 50. Lilja EE, Johnson DR. Segregating metabolic processes into dif- fjord: Saanich Inlet, British Columbia. Environ Microbiol. ferent microbial cells accelerates the consumption of inhibitory 2010;12:172–91. substrates. ISME J. 2016;10:1568–78. 60. Bryant JA, Stewart FJ, Eppley JM, DeLong EF. Microbial com- 51. Van Mooy BAS, Keil RG, Devol AH. Impact of suboxia on munity phylogenetic and trait diversity declines with depth in a sinking particulate organic carbon: enhanced carbon flux and marine oxygen minimum zone. Ecology. 2012;93:1659–73. preferential degradation of amino acids via denitrification. Geo- 61. Zumft WG. Cell and molecular basis of denitrification. chim Cosmochim Acta. 2002;66:457–65. Microbiol Mol Biol Rev. 1997;61:533–616. 52. Cavan EL, Trimmer M, Shelley F, Sanders R. Remineralization of 62. Tsementzi D, Wu J, Deutsch S, Nath S, Rodriguez-R LM, Burns particulate organic carbon in an ocean oxygen minimum zone. Nat AS, et al. SAR11 bacteria linked to ocean anoxia and nitrogen Commun. 2017;8:14847. loss. Nature. 2016;536:179–83. 53. van den Ende FP, Laverman AM, van Gemerden H. Coexistence 63. Fuchsman CA, Palevsky HI, Widner B, Duffy M, Carlson MCG, of aerobic chemotrophic and anaerobic phototrophic bac- Neibauer JA, et al. and cyanophage contributions teria under oxygen limitation. FEMS Microbiol Ecol. to carbon and nitrogen cycling in an oligotrophic oxygen-deficient 1996;19:141–51. zone. ISME J. 2019. https://doi.org/10.1038/s41396-019-0452-6 54. Koeve W, Kähler P. Heterotrophic denitrification vs. autotrophic 64. Glock N, Roy AS, Romero D, Wein T, Weissenbach J, Revsbech anammox—quantifying collateral effects on the oceanic carbon NP, et al. Metabolic preference of nitrate over oxygen as an cycle. Biogeosciences. 2010;7:2327–37. electron acceptor in foraminifera from the Peruvian oxygen 55. Escribano R, Hidalgo P, Krautz C. Zooplankton associated with minimum zone. Proc Natl Acad Sci USA. 2019;116:2860–5. the oxygen minimum zone system in the northern upwelling 65. Dutkiewicz S, Follows MJ, Bragg JG. Modeling the coupling of region of Chile during March 2000. Deep Sea Res Part II ocean ecology and biogeochemistry. Glob Biogeochem Cycles. 2009;56:1049–60. 2009;23:1–15.