More than a decade ago the first evidence emerged that some foraminifera from O2-depleted environments are able to perform complete denitrification (Risgaard-Petersen et al., 2006). Heterotrophic denitrification describes the step-by-step reduction of nitrate (NO3-) to inert N2 gas (Reaction R1 according to Jorgensen, 2006, and Fig. 2). R15[CH2O]+4NO3-→2N2+4HCO3-+CO2+3H2O, where [CH2O] symbolizes organic matter of unspecified composition.

Heterotrophic denitrification provides energy to an organism for oxidative phosphorylation in a similar way to O2 respiration. The ΔG0 for heterotrophic denitrification per mole of carbon at a pH of 7 is -453 kJ mol-1, which is slightly less efficient O2 respiration (ΔG0=-479 kJ mol-1 according to Jorgensen, 2006).

The discovery of foraminiferal denitrification by Risgaard-Petersen et al. (2006) was also the first evidence for complete denitrification in eukaryotic cells in general, and it showed that they likely take up NO3- from the surrounding pore water and store it within intracellular seawater vacuoles. Nevertheless, no later study could actually prove a bona fide “complete” denitrification pathway in foraminifera, and the eukaryotic foraminiferal denitrification pathway is considered today to be incomplete (Woehle et al., 2018; Orsi et al., 2020; Gomaa et al., 2021; see discussion below). Other eukaryotes that are known to perform incomplete denitrification are the primitive eukaryote Loxodes (Finlay et al., 1983) and two species of fungi (Usuda et al., 1995).

Four years after the study by Risgaard-Petersen et al. (2006), Pina-Ochoa et al. (2010b) documented that intracellular NO3- storage and denitrification are not an exception, limited to a few specialized foraminiferal species, but actually a widespread phenomenon. Within a couple of years more studies either quantified denitrification rates or the intracellular NO3- storage of various foraminifera and gromiid species (Høgslund et al., 2008; Glud et al., 2009; Piña-Ochoa et al., 2010b, a; Koho et al., 2011; Bernhard et al., 2012b). The intracellular NO3- storage can reach concentrations up to 567 mM in gromiids (Piña-Ochoa et al., 2010b), and experiments with isotopically labeled NO3- showed that Globobulimina turgida takes up NO3- at a similar rate, independently of the presence or magnitude of the intracellular NO3- pool (Koho et al., 2011). It has been hypothesized that at least some denitrifying foraminifera seem to take up NO3- through the pores in their tests, and the pore density (number of pores per area) of some denitrifying species, such as Bolivina spissa, turned out to be a promising proxy for quantitative NO3- reconstructions (Glock et al., 2011, 2018). However, not all benthic foraminifera are able to denitrify, even if they live in environments that are periodically exposed to anoxia such as representatives of the intertidal species morphogroup Ammonia tepida (either Ammonia veneta, Ammonia aberdoveyensis or Ammonia confertitesta according to Hayward et al., 2021), which neither store NO3- nor show any denitrification activity (Piña-Ochoa et al., 2010b). Some foraminifera from the Bering Sea have been shown to store NO3- but did not denitrify in incubation experiments (Langlet et al., 2020). These species include Nonionella pulchella, Uvigerina peregrina and Bolivinellina pseudopunctata. Also, the NO3- storage in U. peregrina shows a high variability, depending on the environment. Individuals of U. peregrina from the Bay of Biscay lack significant NO3- storage, while U. peregrina from the North Sea and the Bering Sea both show intracellular NO3- enrichment (Piña-Ochoa et al., 2010b; Langlet et al., 2021). Other Uvigerina and Nonionella species have been shown to denitrify (Risgaard-Petersen et al., 2006; Høgslund et al., 2008; Piña-Ochoa et al., 2010b; Glock et al., 2019b; Gomaa et al., 2021). Many miliolids and allogromiids, several intertidal rotaliid species, and also some other rotaliids and textulariids completely lack intracellular NO3- storage (Piña-Ochoa et al., 2010b).

The observations that some species store NO3- and denitrify in some environments and not in others might have two reasons. One reason could be that these species belong to an opportunistic group of foraminifera that can adapt well to both oxygenated environments where they respire O2 and do not denitrify and O2-depleted environments where they switch to denitrification. The other reason could be that some of these foraminifera belong to morphogroups that are identified as a single species but indeed are a mixture of cryptic and pseudocryptic species that include denitrifying and non-denitrifying species. An example for such a morphogroup that has recently had a revision is A. tepida. This morphogroup includes three species (A. veneta, A. aberdoveyensis and A. confertitesta) that can now be morphologically distinguished (Richirt et al., 2019; Hayward et al., 2021). A similar case concerns the morphogroup Nonionella stella, where representatives have been found to denitrify (Høgslund et al., 2008; Choquel et al., 2021). The morphogroup N. stella also consists of several cryptic to pseudocryptic species (Deldicq et al., 2019). The situation might be similar with other Nonionella species and the widespread species U. peregrina.

There is strong evidence for symbiosis between foraminifera and prokaryotes in many hosts from O2-depleted environments, which is most likely an adaptation to survive within the steep geochemical gradients close to the oxic–anoxic boundary (Bernhard et al., 2000, 2006, 2018; Bernhard, 2003; Nomaki et al., 2014). Most of the observed prokaryotic associates are endobionts within the foraminiferal cytoplasm, but some are ectobionts that are often observed close to the pores in the foraminiferal shell (Bernhard et al., 2001, 2010a, 2018). For about a decade after the first discovery of foraminiferal denitrification it remained unclear if foraminifera indeed denitrify themselves or if the bacterial symbionts are responsible for the denitrification. Evidence came up for both hypotheses. Bernhard et al. (2012b) showed that Bolivina argentea consumed its intracellular NO3- storage in O2-free incubations even after a very harsh treatment with antibiotics, which indicates that this species can denitrify even when the activity of potential bacterial symbionts would be inhibited. Other studies showed that bacterial endobionts likely perform denitrification in some allogromiid foraminifera and gromiid species (Bernhard et al., 2012a; Høgslund et al., 2017). Gromiida are a separate group of protists within the Rhizaria and are closely related to foraminifera. With the recent advances in molecular biology, however, it became possible to analyze the transcriptome of denitrifying foraminifera, and Woehle and Roy et al. (2018) showed that the enzymes responsible for denitrification in Globobulimina spp. from an O2-depleted Swedish fjord basin are indeed transcribed by eukaryotic RNA. These enzymes are homologues of enzymes that are also used by bacteria for denitrification, which indicates an ancient prokaryotic origin of denitrification in foraminifera. Nevertheless, the homologues of the enzymes that catalyze the first and the last step of foraminiferal denitrification (reduction of NO3- to nitrite (NO2-) and reduction of nitrous oxide (N2O) to N2 gas; Fig. 2) have not been identified yet. This indicates that foraminifera use other enzymes to catalyze these steps, that they rely on bacterial symbionts for these steps or that they use an alternative denitrification pathway in general.

One hypothesis, brought up by Woehle and Roy et al. (2018), is that the homologue of the nitric oxide reductase (Nor) is indeed a nitric oxide dismutase that has been proposed to catalyze the enzymatic reaction 2NO→N2+O2 (alternative pathway in Fig. 2) (Ettwig et al., 2012). The presence of the eukaryotic denitrification pathway found in foraminifera (Woehle and Roy et al., 2018) has been confirmed through other analyses of foraminiferal transcriptomes (Orsi et al., 2020; Gomaa et al., 2021). Gomaa et al. (2021) also identified an enzyme of yet unknown functionality that might be responsible for the first step in the foraminiferal denitrification pathway. Recent metagenomics and transcriptomic results of denitrifying foraminifera indicate that bacterial symbionts might perform the missing steps in the foraminiferal denitrification pathway or that they at least partly contribute to the amount of NO3- that is denitrified within foraminiferal cells (Woehle and Roy et al., 2022). It has already been hypothesized before that the ectobionts, found on Bolivina pacifica from the Santa Barbara Basin, are either sulfate-reducing or sulfur-oxidizing bacteria (Bernhard et al., 2010a). The possible complementation of the foraminiferal denitrification with bacterial symbionts appears to be contradictory to the results of Bernhard et al. (2012b), who showed that B. argentea consumed its intracellular NO3- storage (likely for denitrification) even after the antibiotic treatment. Gomaa et al. (2021) confirmed that B. argentea also lacks the first and last denitrification step in its transcriptome, although it lacks intracellular bacterial symbionts (Bernhard et al., 2012b). Future studies might decipher if indeed bacteria are responsible for the missing denitrification step and are immune to such antibiotic treatment; if an oxygenic nitric oxide dismutase skips the last denitrification step, as discussed by Woehle and Roy et al. (2018); and/or if foraminifera have unknown enzymes that catalyze the missing steps, as suggested by Gomaa et al. (2021). The study by Woehle and Roy et al. (2022) also showed that the last common ancestor of denitrifying foraminifera likely has its origin during the Cretaceous, possibly related to the occurrence of the Cretaceous anoxic events. Since the foraminiferal denitrification pathway is incomplete, and the first and last steps might be performed by Desulfobacteraceae in their microbiome, the authors suggested that the acquisition of denitrification ability in foraminifera occurred in multiple stages (starting during the Cretaceous) but is not yet complete (Woehle and Roy et al., 2022).

It is noteworthy that denitrifying foraminifera from the Peruvian OMZ show a metabolic preference for NO3- over O2 as an electron acceptor (Glock et al., 2019b). These foraminifera show an increasing cell volume with increasing ambient NO3- and decreasing O2 concentrations. Similar observations have been made at the California Borderland, where some benthic foraminifera also increase their cell volume with decreasing ambient O2 concentrations (Keating-Bitonti and Payne, 2017). Additional evidence for the metabolic NO3- preference came from comparing denitrification and O2 respiration rates and scaling them to their cell volume (Glock et al., 2019b). The scaling is lower for O2 respiration than for denitrification, indicating that the NO3- metabolism during denitrification is more efficient than the O2 metabolism during aerobic respiration in foraminifera from the Peruvian OMZ. This might explain why some infaunal denitrifying foraminifera follow the oxycline within sediments (Linke and Lutze, 1993; Duijnstee et al., 2003). We have to keep in mind that O2 can be quite harmful for organisms that are not adapted to higher O2 concentrations due to its strong reactivity. Even trace amounts of O2 can inhibit denitrification, and O2 can repress the denitrifying enzyme synthesis (Smith and Tiedje, 1979; Knowles, 1981; Tiedje, 1988; Mckenney et al., 1994). Thus, if denitrifying foraminifera are exposed to small amounts of O2 they cannot denitrify but also do not have enough O2 to supply their demands for electron acceptors. Larger amounts of O2 might supply this demand but also harm the cell. For example, O2 can inhibit the growth of some obligate anaerobes' poison enzymes that are important for their metabolism (Lu and Imlay, 2021). Also, for aerobes, O2 can be harmful. “Hyperoxia”, an excess supply of O2, leads to damaging effects by highly reactive metabolic products of O2 (free O2 radicals) that inactivate enzymes in the cell, damage DNA and destroy lipid membranes (Frank and Massaro, 1980). Furthermore, foraminifera are able to store NO3- within vacuoles due to its lower reactivity and still have an electron acceptor reservoir if NO3- is depleted in their microhabitat. This is not possible for O2 due to its high reactivity (Auten and Davis, 2009). Finally, a review by Zimorski et al. (2019) addresses the common misconception that the presence of O2 improves the overall energetic state of the cell. It is a fact that the energy yield from remineralizing glucose or amino acids is higher in the presence of O2 (“O2 respiration”), but it is also a fact that the synthesis of biomass consumes 13 times more energy per cell if O2 is present compared to anoxic conditions. This is related to the chemical equilibrium between organic matter and CO2, which strongly shifts to the side of CO2 in the presence of O2 (Zimorski et al., 2019). All this might explain why the metabolism of at least some foraminifera is better adapted to denitrification than to O2 respiration.

The circumstance that some foraminifera have a metabolic preference for NO3- over O2 as an electron acceptor (Glock et al., 2019b) and that other species like U. peregrina denitrify in some environments but completely lack intracellular NO3- storage in others (Piña-Ochoa et al., 2010b) might partly explain the microhabitat selectivity of benthic foraminifera in the sediment. According to the conceptual TROX model, benthic foraminifera can be divided into groups due to their microhabitat preference: epifauna, shallow infauna, intermediate infauna and deep infauna (Jorissen et al., 1995). The presence of this species-specific microhabitat structure was first documented by Corliss (1985). These microhabitats are mainly controlled by bottom-water O2 concentrations and the supply of non-refractory organic matter (i.e., food; Jorissen et al., 1995). Due to our increasing understanding about the anaerobic metabolism of foraminifera we can now assume that NO3- availability is another controlling factor (Fig. 3). This is also indicated by a study that coupled early diagenetic modeling with foraminiferal ecology to model the microhabitats of benthic foraminifera (Jorissen et al., 2022). According to their metabolic preference for NO3- or O2 as electron acceptors, many benthic foraminifera species that typically occupy a certain microhabitat (epifauna, shallow infauna and deep infauna) might partly be assigned to three different attributes (aerobe, facultative anaerobe and facultative aerobe). There are most likely exceptions to these classifications, which are discussed below. Another controlling factor of the microhabitat is the specific trophic strategy of the foraminiferal species, which is further discussed in Sect. 3.

Deep-infaunal species can most likely be considered to be facultative aerobes that have a metabolic preference for NO3- over O2 (Glock et al., 2019b) and try to avoid trace amounts of O2. They cannot be counted as obligate anaerobes though since they can withstand periods of oxygenation. Many experiments show that denitrifying foraminifera can switch to O2 respiration if they are exposed to O2 (i.e., Piña-Ochoa et al., 2010b). Still, they follow the oxycline in the sediments to avoid the inhibition of denitrification by trace amounts of O2. The δ13C signature of shells of deep-infaunal globobuliminids indicates that they calcify in sediment depth where the pore water O2 level reaches zero or even deeper in the sediments. The offset between δ13C of Globobulimina spp. tests and δ13C of epifaunal foraminifera or of bottom-water dissolved inorganic carbon (DIC) is nearly equal to the offset between DIC at the zero-O2 layer and the bottom water (Schmiedl and Mackensen, 2006) and often can be even higher (Costa et al., 2023), indicating that many globobuliminids live even below the oxycline. Even though they can switch to O2 respiration (Piña-Ochoa et al., 2010b), these species would most likely try to avoid crossing the oxycline since denitrification would already be inhibited by O2 concentrations within the nanomolar range (Dalsgaard et al., 2014), and the O2 concentration slightly above the oxycline is not high enough to fulfill their metabolic demands. Indeed, the model by Jorissen et al. (2022) describes the distribution of deep infauna very well by using the presence of O2 as an inhibiting factor, which also suggests that they can be considered facultative aerobes instead of facultative anaerobes. Taxa belonging to the deep-infaunal group that might be considered to be facultative aerobes that prefer NO3- over O2 include, for example, Valvulineria inflata and bradyana, Bolivina seminuda, Globobulimina pyrula, Globobulimina affinis, and Cancris carmenensis (Jorissen et al., 1995; Schmiedl and Mackensen, 2006; Mojtahid et al., 2010; Glock et al., 2019b).

Shallow infauna can in many cases be considered to be facultative anaerobes that are well adapted to the presence of low O2 concentrations but can switch to denitrification if they are exposed to anoxic conditions or need to enter the deeper sediment parts to find food or avoid competitive stress. These species have the advantage that they can utilize both fresh phytodetritus from the top of the sediments and organic matter of lower quality from the deeper parts of the sediments. A good example for a shallow-infaunal–facultative anaerobe species is U. peregrina, which is well known for its shallow-infaunal lifestyle (Schmiedl and Mackensen, 2006) and has been found with and without intracellular NO3- storage in different environments (Piña-Ochoa et al., 2010b; Langlet et al., 2020). Of course, it cannot be generalized that all foraminifera from a shallow infaunal habitat are indeed facultative anaerobes. At least some species that can be considered shallow-infaunal have been shown neither to be able to store NO3- nor to denitrify. As mentioned above, all specimens from the Ammonia tepida morphogroup that have been analyzed so far lack intracellular NO3- storage and cannot denitrify (Piña-Ochoa et al., 2010b). Nevertheless, these taxa are often exposed to anoxia and can sometimes even be found alive in 4 to 26 cm sediment depth (Alve and Murray, 2001; Thibault de Chanvalon et al., 2015). It is possible that these foraminifera indeed only have an aerobe metabolism and just become dormant under exposure to anoxia (dormancy is discussed in another section). However, another possibility is that intertidal species such as A. veneta, A. aberdoveyensis and A. confertitesta have other adaptations to anoxia than denitrification. Recent studies revealed other possible anaerobic metabolic pathways in foraminifera such as fermentation and dephosphorylation of creatine phosphate, which are discussed in Sect. 2.1.3 (Orsi et al., 2020; Gomaa et al., 2021). Also, an Ammonia sp. has been shown to take up nitrogen from 15N-labeled NO3- under O2 depletion and to assimilate it within cell organelles known as electron dense bodies (Nomaki et al., 2016). Eventually, studies on the transcriptome of non-denitrifying species from infaunal environments might be able to show if some of these species can switch to an alternative anaerobe metabolism under exposure to anoxia.

Many epifaunal species can most likely be considered to be aerobes that typically occur at the sediment–water interface or on elevated surfaces. Typical epifaunal–aerobe taxa include Cibicides spp. and Planulina spp. (Corliss and Chen, 1988; Lutze and Thiel, 1989). These species have the advantage that they are well adapted to collect fresh food supply from above (Wollenburg et al., 2021) but usually cannot withstand longer O2-depleted periods (Mackensen et al., 1995). Nevertheless, recent genetic data indicate that Cibicidoides wuellerstorfi clusters very close to known denitrifying species in the phylogenetic tree, so it cannot be excluded that some Cibicides spp. may denitrify under certain circumstances (Woehle and Roy et al., 2022). In the same way as for the other microhabitats, not all species with an epifaunal lifestyle should be automatically considered to be aerobes. There are examples of epifaunal benthic foraminifera that have not been found in well-oxygenated environments but reach high abundances in O2-depleted environments. One example is Epistominella smithi, which has been described in low-O2 environments, such as the Santa Barbara Basin (Harman, 1964) or the Peruvian OMZ (Erdem and Schönfeld, 2017). Nevertheless, the morphology of E. smithi strongly suggests an epifaunal lifestyle. Another example is the epifaunal species Planulina limbata. This species is abundant only in O2-depleted environments on continental margins within the eastern Pacific (Natland, 1938; Erdem and Schönfeld, 2017; Glock et al., 2022). Recent P. limbata specimens are present in severely O2-depleted water masses within the Peruvian OMZ ([O2] = 3–12 µmol kg-1; Glock et al., 2022). Nevertheless, P. limbata also adapts its pore density to the availability of O2 (Glock et al., 2022), which might indicate that it has an aerobic metabolism, despite the fact that its presence appears to be limited to low-O2 environments. Another possibility is that species such as E. smithi or P. limbata may denitrify under certain circumstances and therefore can also be considered to be facultative anaerobes. Hopefully, measurements of metabolic rates, intracellular nutrient content and enzymatic activity might bring further evidence in the future if at least some epifaunal species can switch to an anaerobe metabolism when O2 is too depleted.

The intermediate infauna is somehow an exceptional case. Common representatives of intermediate-infaunal taxa are Melonis barleeanus and Pullenia spp. (Corliss, 1991). The typical example for intermediate-infaunal species, M. barleeanus, is interesting since it stores either no or only very small amounts of NO3- (see Tables 2 and 3). Still, several studies indicate that M. barleeanus lives deeper in the sediments than some Uvigerina spp. (Corliss, 1991; Ní Fhlaithearta et al., 2018), although many Uvigerina species have been shown to store NO3- and denitrify (Tables 1 and 2). This might give room to speculate if M. barleeanus has other metabolic adaptations to O2 depletion than denitrification or if it simply does not store large amounts of NO3- but denitrifies NO3- directly after the uptake from the seawater. Indeed, a recent study predicted the microhabitats of infaunal benthic foraminifera using an early diagenetic model and showed that the intermediate-infaunal clusters around the NO3- maximum in the pore water (Jorissen et al., 2022). Future perspectives on understanding the biology of intermediate infauna might include transcriptome analyses to decipher other anaerobe metabolic pathways and testing the denitrification capacity after incubation in NO3--free and NO3--containing seawater.

Note that the deep infauna can even migrate deeper into the sediments below the depth of NO3- penetration if they must due to their ability to intracellularly store NO3- as a reservoir (Fig. 3). The deeper boundary of the deep infauna might be controlled by the zone of sulfate reduction, where free sulfide is produced, which could be toxic for the foraminifers. Research to measure denitrification rates in different benthic foraminiferal species continues (Langlet et al., 2020; Choquel et al., 2021). This will add to the scarce available data and contribute to estimates of the role of foraminifera in oceanic N cycling. This topic is discussed a bit further in Sect. 4.

Kleptoplasty describes a symbiosis between algal chloroplasts and a host organism that sequesters the chloroplasts from algae (Clark et al., 1990). The word originates from the Greek word Kleptes, which means “thief”. Kleptoplasty in foraminifera is most extensively studied for shallow Elphidium and Haynesina species that often thrive within the photic zone, and this research originated in the 1970s (Lopez, 1979; Lee et al., 1988; Correia and Lee, 2000, 2002b, a; Goldstein et al., 2004; Pillet et al., 2011, 2013; Cevasco et al., 2015; Jauffrais et al., 2016, 2017, 2018; Cesbron et al., 2017; Goldstein and Richardson, 2018; Jesus et al., 2021). Several studies showed that the sequestered chloroplasts in the intertidal species Haynesina germanica are still capable of photosynthesis under light exposure (Lopez, 1979; Cesbron et al., 2017). H. germanica often shares a habitat with species from the Ammonia tepida morphogroup (Ammonia aberdoveyensis or Ammonia confertitesta according to Hayward et al., 2021), which also tend to ingest chloroplasts, but these chloroplasts do not show any photosynthetic activity anymore (Jauffrais et al., 2016).

The kleptoplasts in foraminifera originate from diatoms, which has been confirmed on the basis of the chloroplast shape in transmission electron microscopy (TEM) observations and by sequencing the chloroplasts using molecular-biological methods (Lopez, 1979; Lee et al., 1988; Cedhagen, 1991; Lee and Anderson, 1991; Bernhard and Bowser, 1999; Grzymski et al., 2002; Goldstein et al., 2004). Austin et al. (2005) hypothesized that the tooth plates in H. germanica are morphological adaptations to crack diatom frustules for access to their chloroplasts. Recently, LeKieffre et al. (2018) showed in (aerated) incubation experiments with H13CO3- and 15NH4+ during a light–dark cycle that Haynesina germanica is indeed able to fix inorganic carbon and nitrogen under light exposure. Intertidal foraminifera are often exposed to O2-depleted or even anoxic conditions when water stagnates during low tide or if they are transported to deeper anoxic sediment layers by bioturbation (Rybarczyk et al., 1996; Cesbron et al., 2017). Oxygen penetration depths in tidal flats can vary from a few millimeters during low tide to several centimeters during high tide (Jansen et al., 2009). Thus, intertidal foraminifera are often exposed to anoxia, even within the first centimeter of the sediment column. H. germanica is also supposed to occur in black sediments of the British salt marsh tide pools (Bernhard and Bowser, 1999), which likely become anoxic during tidal cycles (Rybarczyk et al., 1996), and it was among the first recolonizers of a fjord suffering from organic pollution (Cato et al., 1980; Bernhard and Bowser, 1999). Kleptoplasty might thus be an additional adaptation of foraminifera from photic environments to stay active during periods of O2 depletion, which has already been hypothesized by Cesbron et al. (2017).

Less well understood is the phenomenon of kleptoplasty, observed in the benthic foraminifers Nonionella stella, Virgulina fragilis and Nonionellina labradorica, which can thrive below the photic zone and often inhabit O2-depleted sediments (Cedhagen, 1991; Bernhard and Bowser, 1999; Grzymski et al., 2002; Bernhard, 2003; Tsuchiya et al., 2015; Jauffrais et al., 2019; Gomaa et al., 2021; Powers et al., 2022). Experiments to test if N. labradorica is able to photosynthesize with its sequestered chloroplasts have been inconclusive. While Cedhagen (1991) found active photosynthesis in N. labradorica specimens incubated with 14C, Jauffrais et al. (2019) showed an increased O2 respiration rate instead of O2 production and chloroplast degradation in specimens exposed to light. Recently, Gomaa et al. (2021) found chloroplast encoded in transcripts of N. stella, indicating that the kleptoplasts in this species are still active. Genetic analyses revealed that the kleptoplasts in N. stella and N. labradorica are also mainly sequestered from diatoms, most likely after ingestion and selective digestion of phytodetritus (Grzymski et al., 2002; Jauffrais et al., 2019; Gomaa et al., 2021). Grymzki et al. (2002) calculated that the required amount of light for N. stella specimens collected from aphotic depths at the Santa Barbara Basin is too low to sustain active photosynthesis. Instead, they suggested that the kleptoplasts in foraminifera from aphotic environments provide the ability to fix inorganic nitrogen via the glutamine synthetase and glutamate 2-oxoglutarate amidotransferase (GOGAT) pathway. Indeed, Jauffrais et al. (2019) showed that kleptoplastic N. labradorica are able to fix inorganic nitrogen, but coupled TEM–nanoscale secondary ion mass spectrometry (nanoSIMS) revealed that the assimilated nitrogen is associated with electron opaque bodies instead of sequestered chloroplasts. Analyses of the transcriptome of N. stella by Gomaa et al. (2021) support the observations by Grimzki et al. (2002), since N. stella appears to be able to fix ammonia by itself. They also found that the fucoxanthin–chlorophyll binding protein (FCP) was expressed in the transcriptome of N. stella and speculated that the ability to synthesize FCP was derived from the kleptoplasts by horizontal gene transfer. FCP is a pigment, commonly found in chloroplasts of brown algae, and allows a more efficient photosynthesis with a light absorption bandwidth, which is especially useful in aquatic environments (Papagiannakis et al., 2005; Premvardhan et al., 2008). The true function of the kleptoplasts in deep-sea benthic foraminifera from aphotic, often O2-depleted environments still remains enigmatic, though.

Several recent publications based on advances in molecular-biological methods (e.g., next generation sequencing) have revealed some other metabolic adaptations of foraminifera that thrive under O2 depletion (see examples in Fig. 4) (Woehle and Roy et al., 2018, 2022; Orsi et al., 2020; Gomaa et al., 2021). In N. stella and Bolivina argentea, Gomaa et al. (2021) found evidence for the expression of proteins, including pyruvate:ferredoxin oxidoreductase (PFOR) and [FeFe]-hydrogenase, which are characteristic of anaerobic metabolism. These PFOR sequences were indeed eukaryotic and closely related to those of the facultative anaerobe polychaete Capitella teleta and the anaerobic protistan parasite Blastocystis. The [FeFe]-hydrogenase is very similar to those in the amoeba/flagellate Naegleria gruberi, which has experimentally been shown to be active and to produce molecular hydrogen even under aerobic conditions (Tsaousis et al., 2014). Due to these observations, Gomaa et al. (2021) suggested that N. stella and B. argentea might be able to produce H2 gas and have the capacity for an anaerobic energy metabolism.

Another important observation was made by Orsi et al. (2020). They used metatranscriptomics on sediments from the Namibian shelf, where the foraminiferal community is dominated by Bolivina and Stainforthia species. Presumably living foraminifera were present in the sediment column up to 28 cm depth in an anoxic habitat with high sulfide concentrations. The gene expression of those foraminifers increased under sulfidic conditions, which indicates that they not only survive but thrive under anoxic conditions. The anaerobic energy metabolism of these foraminifers seems to be sufficient enough to support calcification and phagocytosis even under anoxic conditions. Evidence for foraminiferal calcification under anoxia already came up in a study by Nardelli et al. (2014). Orsi et al. (2020) suggested that the Namibian foraminifera use phagocytosis (vacuolic ingestion of food particles) to ingest prey cells even under anoxic conditions. These processes (calcification and the ingestion of prey cells by phagocytosis) require bursts of high energy, which the authors suggest is generated by dephosphorylation of intracellular creatine phosphate storage to regenerate ATP from ADP. Evidence for the capacity for the dephosphorylation of creatine phosphate under anoxia was indicated by the metatranscriptomes. In addition, high intracellular dissolved inorganic phosphate storage has been found in benthic foraminifera from the Peruvian OMZ, which might serve as a reservoir to synthesize creatine phosphate and/or to synthesize polyphosphates that might be broken down to harvest energy (Glock et al., 2020). Orsi et al. (2020) and Gomaa et al. (2021) also found evidence for another anaerobic metabolism. Their data indicate that the foraminifers metabolize hydrolyzed organics to produce ATP using fermentation and fumarate reduction.

Most foraminifera species from O2-depleted habitats possess numerous peroxisomes that are usually associated with mitochondria and the endoplasmatic reticulum (Bernhard and Bowser, 2008). Bernhard and Bowser (2008) hypothesized that these peroxisome proliferations might be used to either metabolize H2O2 and other highly reactive oxygen species that are produced within the chemocline close to the oxic–anoxic boundary or to reduce the oxidative stress by these compounds. Indeed, they showed in an experiment that ATP concentrations in foraminifera increased proportionally to ambient H2O2 concentrations. A recent study on the transcriptome and metatranscriptome of N. stella and B. argentea from the Santa Barbara Basin revealed that these species utilize an adaptable mitochondrial and peroxisomal metabolism, depending on the chemical treatment in the experiment (Powers et al., 2022). The high plasticity of their peroxisomal and mitochondrial metabolism might be substantial for survival under the highly variable conditions at the chemocline in the sediments. The results of Powers et al. (2022) indicate that at least some processes that are involved in foraminiferal denitrification are associated with mitochondria. Interestingly, the expression of denitrification-related genes in both species was upregulated after incubation with elevated H2O2 but without NO3- and downregulated if they were incubated without H2O2 but with NO3-, compared to a control treatment with both H2O2 and NO3-. In the same way several peroxisomal processes were upregulated in the H2O2-only treatment. In addition, despite the fact that both species are able to denitrify, Powers et al. (2022) found distinct metabolic adaptations to anoxia in both species. For example, a quinol:fumarate oxidoreductase, which is considered to be an adaptive mechanism for anaerobic respiration in eukaryotic organisms, was present in N. stella but not in B. argentea. On the other hand, B. argentea has the capacity to digest food vacuole contents under O2 depletion, while N. stella was lacking food vacuoles (Powers et al., 2022).

Dormancy is another strategy to survive anoxia or extreme O2 depletion for some benthic foraminifera that cannot denitrify. Dormancy is defined as the reduced or suspended metabolic activity in response to exogenous factors (Ross and Hallock, 2016). Observations that indicate the potential of dormancy in foraminifera have been documented since the 1950s and are extensively reviewed by Ross and Hallock (2016). Nevertheless, many aspects of foraminiferal dormancy, such as its role in the foraminiferal life cycle or its role in structuring foraminiferal assemblages, remain unexplored (Ross and Hallock, 2016).

In the 1990s some studies suggested that some foraminifera may become dormant when exposed to anoxia. Bernhard and Alve (1996) observed that the ATP concentration ([ATP]) of the benthic foraminiferal species Bulimina marginata, Stainforthia fusiformis and Adercotryma glomerata flushed with N2 gas to drive out O2 was significantly lower than in specimens from well-aerated conditions. They interpreted this observation as an indication that dormancy is a survival strategy for some foraminiferal species when they are exposed to periods of anoxia. Linke and Lutze (1993) observed cysts of Elphidium incertum from putative anoxic habitats that might be interpreted as a sign for dormancy, and Hannah and Rogerson (1997) hypothesized that foraminifera transported to an anoxic sediment layer might become dormant until they return to aerated conditions by transport through bioturbation.

Recently, dormancy of foraminifera exposed to anoxia has gained more attention again. LeKieffre et al. (2017) did a feeding experiment with specimens from the Ammonia tepida morphogroup (A. confertitesta according to Koho et al., 2018, and Hayward et al., 2021) using a 13C-labeled diatom film as a food source. They compared the metabolic differences in Ammonia sp. between oxic and anoxic conditions by mapping the distribution of 13C within the cells using coupled TEM–nanoSIMS and by analyzing the carbon concentration and stable-carbon-isotope composition of the total organic matter and individual fatty acids in the foraminifer. Nearly the complete diatom biofilm was consumed, and the foraminiferal cytoplasm was strongly enriched in 13C under oxic conditions. Specimens from the anoxic incubation ingested only few of the diatoms, and those were neither assimilated nor metabolized further. In addition, the specimens from the oxic incubation produced a significant quantity of specific polyunsaturated fatty acids, which was not the case under anoxic conditions. A. confertitesta reacted to the induced anoxia with a severely reduced metabolic rate within less than 24 h. All these observations provide solid evidence that dormancy is a survival strategy of A. confertitesta under anoxia.

Koho et al. (2018) further analyzed cell structural changes in Ammonia spp. under exposure to anoxia collected from the field as well as from incubations. The specimens from anoxia showed an increase in lipid droplets and electron dense bodies within their cytoplasm. The cytoplasm itself was thinned out, which was interpreted as metabolization of the cytosol. In addition, while absent within the specimens from oxic environments, various bacteria were present within the cytoplasm of the specimens from anoxia. These were interpreted as endobionts but might also be parasites that could not be fended off due to the drastically reduced metabolism during dormancy under anoxia. A continuum of intracellular bacteria including prey in food vacuoles, endobionts, parasites and necrophages has been documented before in benthic foraminifera from cold seeps (Bernhard et al., 2010b). It has already been hypothesized by the authors that bacteria can switch their function from endobionts to predators, depending on the vitality of the host cell. Considering all the studies about dormancy, it is likely that dormancy is a common survival strategy for foraminiferal species whose supply of suitable electron acceptors is exhausted (i.e., O2 or NO3-) or who are exposed to periods of extreme environmental conditions. Since there is evidence for dormancy in both S. fusiformis and B. marginata (Bernhard and Alve, 1996), it is likely that even denitrifying species can become dormant under unfavorable conditions. Another Stainforthia sp. has been shown to denitrify, and B. marginata stores NO3- in some environments (Piña-Ochoa et al., 2010b).