# Hidden decomposers: Revisiting saprotrophy among soil protists and its potential impact on carbon cycling

François Maillard, Fredrik Klinghammer, Vincent E.J. Jassey, Bowen Zhang, Peter G Kennedy, Enrique Lara, Stefan Geisen, Lars Tranvik, Edith Hammer, Anders Tunlid

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► To cite this version:

François Maillard, Fredrik Klinghammer, Vincent E.J. Jassey, Bowen Zhang, Peter G Kennedy, et al. Hidden decomposers: Revisiting saprotrophy among soil protists and its potential impact on carbon cycling. Soil Biology and Biochemistry, 2025, 205, pp.109786. 10.1016/j.soilbio.2025.109786. hal-05238351

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Submitted on 25 Nov 2025

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Hidden decomposers: Revisiting saprotrophy among soil protists and its
potential impact on carbon cycling

François Maillard<sup>a,*</sup>, Fredrik Klinghammer<sup>a</sup>, Vincent E.J. Jassey<sup>d</sup>, Bowen Zhang<sup>b</sup>, Peter G. Kennedy<sup>e</sup>, Enrique Lara<sup>f</sup>, Stefan Geisen<sup>g</sup>, Lars Tranvik<sup>c</sup>, Edith Hammer<sup>a</sup>, Anders Tunlid<sup>a</sup>

ARTICLE INFO

Keywords:

Saprotrophic protists

Lysotrophic saprotrophy

Phagotrophic saprotrophy

Soil carbon cycling

Decomposition

Organic matter

A B S T R A C T

Soil protists are increasingly recognized as key players in organic matter turnover, yet their role as direct de-
composers (i.e., saprotrophs) remains underexplored compared to that of bacteria and fungi. Here, we synthesize
ecological, physiological, and genomic evidence to highlight the potential of protists to actively decompose
organic matter and influence soil carbon cycling. We distinguish two saprotrophic strategies within pro-
tists—lysotrophic (extracellular) and phagotrophic (intracellular)—with the latter being unique to protists
among microbial decomposers. By directly ingesting particulate or dissolved organic matter, phagotrophic
saprotrophic protists may bypass constraints associated with extracellular decomposition, potentially providing
an advantage in breaking down recalcitrant substrates. In contrast, lysotrophic saprotrophy in protists involves
the secretion of enzymes, similar to bacterial and fungal decomposers. We propose that integrating protist
saprotrophy into conceptual and quantitative models of soil organic matter decomposition could address critical
knowledge gaps. This integration involves employing functional genomics and functional ecology methodologies
to determine, *in vitro*, the capacity of protists to function as saprotrophs, elucidate the genetic pathways un-
derpinning saprotrophic activities, and assess, *in situ*, their direct contributions to organic matter decomposition
processes. Ultimately, a clearer view of the organic matter decomposition capacities of soil protists will refine our
understanding of microbially driven carbon fluxes.

1. Introduction

Saprotrophy is a specialized form of osmotrophy in which organisms
obtain carbon by actively decomposing organic matter (see Table 1 for
definitions) (Lewis, 1973; Worden et al., 2015; Baldrian, 2017). In
terrestrial ecosystems—particularly in temperate and boreal bio-
mes—soil-inhabiting microorganisms drive most decomposition pro-
cesses (Glassman et al., 2018; Kramer et al., 2016; López-Mondéjar et al.,
2018, 2020; Maillard et al., 2023). Carbon released from decaying
organic matter can be assimilated into microbial biomass, respired as

CO2, or stabilized as soil organic carbon (SOC) (Fig. 1) (Schimel and Schaeffer, 2012). Thus, soil saprotrophic microorganisms act as gatekeepers of organic carbon movement, regulating its flux into the atmosphere or its retention as SOC (Six et al., 2006; Domeignoz-Horta et al., 2020). Consequently, microbial saprotrophy has been extensively studied to enhance our understanding of the carbon cycle (Wieder et al., 2013).

Bacteria and fungi are recognized as key decomposers, yet the role of protists as direct saprotrophs remains poorly understood, in part due to their high phylogenetic and functional diversity (Geisen et al., 2020;

<footnote><sup>a</sup> Department of Biology, Lund University, Lund, Sweden</footnote>

<footnote>ᵇ Centre for Environmental and Climate Science, Faculty of Science, Lund University, Lund, Sweden</footnote>

<footnote>ᶜ *Linnology, Department of Ecology and Genetics, University of Uppsala, Uppsala, Sweden*</footnote>

<footnote n="4">⁴ Centre de Recherche sur la Biodiversité et l'Environnement (CRBE), Université de Toulouse, CNRS, IRD, Toulouse INP, Université Toulouse 3 - Paul Sabatier (UT3), Toulouse, France</footnote>

<footnote>Department of Plant and Microbial Biology, University of Minnesota, St. Paul, MN, USA</footnote>

<footnote>Department of Mycology, Real Jardín Botánico-CSIC, Madrid, Spain</footnote>

<footnote n="8">⁸ Laboratory of Nematology, Wageningen University and Research, Wageningen, the Netherlands</footnote>

<footnote>* Corresponding author.
E-mail address: francois.maillard2@gmail.com (F. Maillard).</footnote>
<table><caption>**Table 1**
Definitions of the key concepts introduced in this article.</caption><thead><tr><td>Term</td><td>Definition</td></tr></thead><tbody><tr><td>Organic matter</td><td>Dead organic material, including freshly dead and partially decomposed organic matter.</td></tr><tr><td>Osmotrophy</td><td>A nutritional strategy in which organisms absorb dissolved organic compounds directly through their cell membranes.</td></tr><tr><td>Saprotrophy</td><td>A specialized form of osmotrophy where organisms obtain carbon by actively decomposing organic matter, either through extracellular enzyme activity or intracellular digestion.</td></tr><tr><td>Non-saprotrophic osmotrophy</td><td>A specialized form of osmotrophy where organisms absorb dissolved organic compounds without contributing to the decomposition of organic matter (e. g., microbial “cheaters”).</td></tr><tr><td>Lysotrophic saprotrophy</td><td>A form of saprotrophy characterized by extracellular enzymatic breakdown of complex organic matter into smaller, readily absorbable compounds.</td></tr><tr><td>Phagotrophic saprotrophy</td><td>A form of saprotrophy involving the internalization of dissolved or particulate organic matter into food vacuoles, followed by enzymatic breakdown within the cell.</td></tr><tr><td>Lysophagotrophic saprotrophy</td><td>The capacity of a single organism to employ both extracellular (lysotrophic) and intracellular (phagotrophic) mechanisms of saprotrophy.</td></tr></tbody></table>

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Conceptual diagram of carbon movements in soils with saprotrophic protists, fungi, and bacteria obtaining carbon from decomposing organic matter, depending on its origin—either animal, plant, or microbial—and its fraction: dissolved organic matter (DOM), particulate organic matter (POM), or mineral-associated organic matter (MAOM). Predatory protists, by predating on saprotrophic microbial communities to obtain carbon, indirectly mediate organic matter decomposition processes. Carbon taken up by microorganisms, either from saprotrophy or predation, can be respired as CO₂, immobilized in the microbial biomass, or released as soil organic carbon (SOC), either as decomposition products, cellular waste products, or necromass after microbial death.</figcaption></figure>

Oliverio et al., 2020; Singer et al., 2020). Protists influence organic matter turnover both indirectly—by preying on decomposer bacteria and fungi—and directly, by acting as saprotrophs (Fig. 1) (Adl and Gupta, 2006). For example, studies have shown that top-down regulation by protist predators can alter microbial decomposer biomass, community composition, and diversity, thereby affecting organic matter decomposition rates (Kuikman et al., 1990; Jiang et al., 2023; Geisen et al., 2021; Liao et al., 2023a). Surprisingly, the direct role of saprotrophic protists in organic matter decomposition has received little attention.

Here, we synthesize the literature on soil protist saprotrophy by exploring and integrating its ecological, physiological, and genomic aspects. We also connect soil protist saprotrophy with that of aquatic protists and relate it to bacterial and fungal saprotrophy. By proposing a new conceptual framework that incorporates protist-driven organic matter decomposition, we aim to facilitate a more comprehensive understanding of microbial saprotrophy and promote the integration of protist functional diversity into soil biogeochemical models.

## 2. Saprotrophy vs. non-saprotrophic osmotrophy in soil protists

Osmotrophy—the absorption of dissolved organic compounds directly through cell membranes (Table 1)—is often confused with saprotrophy in protist research, even though the latter is a specialized form of osmotrophy that involves actively breaking down dead organic matter (Geisen et al., 2018). Unlike saprotrophy, which requires enzymatic decomposition, non-saprotrophic osmotrophy involves the direct absorption of dissolved compounds without prior breakdown (Worden et al., 2015). Nevertheless, saprotrophy and non-saprotrophic osmotrophy have historically been used interchangeably in protist research; for example, Woodcock (1916), observing non-phagotrophic flagellates, concluded that they were certainly saprotrophic, feeding by osmosis—without clarifying whether they actively participated in decomposition processes. Similarly, Geisen et al. (2018), in defining functional groups within protists, cited *Polytoma*, a flagellate genus within the Chlorophyta, and *Mycamoeba*, an Amoebozoa genus, as examples of saprotrophic protists. However, *Polytoma* has primarily been reported as engaging in non-saprotrophic osmotrophy (Rumpf et al., 1996; Pánek et al., 2022), while *Mycamoeba* are known to prey on bacteria (Blandenier et al., 2017), with no evidence of either genus decomposing organic matter. This confusion is further reinforced by the fact that many predatory protists can grow and multiply *in vitro* in nutrient-rich media without prey, relying exclusively on non-saprotrophic osmotrophy (Johnke et al., 2017).

Non-saprotrophic osmotrophic protists can also include phototrophs taking up dissolved compounds from their surrounding environment (Amblard, 1991; Selosse et al., 2017), and such non-saprotrophic osmotrophy can thus serve as an alternative or complementary feeding strategy for both predatory and phototrophic protists while remaining distinct from saprotrophy. However, to better understand soil carbon cycling, it is critical to identify which microorganisms actively decompose organic matter, as this is a rate-limiting step in terrestrial carbon cycling (Schimel and Weintraub, 2003; Schimel and Schaeffer, 2012; Wieder et al., 2013). Clarifying the distinction between these two carbon acquisition strategies—saprotrophy and non-saprotrophic osmotrophy—is therefore particularly important. To address this, we propose a revised framework in which saprotrophy entails acquiring carbon from organic matter while actively participating in decomposition processes (Fig. 2).

## 3. Lysotrophic saprotrophy in soil protists

Bacteria and fungi are recognized as primary decomposers due to their ability to degrade complex organic residues using extracellular enzymes—a process known as lysotrophic saprotrophy (Table 1) (Baldrian, 2014; Starke et al., 2021). This process comprises two main
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Conceptual diagram of the different saprotrophic types within soil microbial decomposers. Lysotrophic saprotrophs, or extracellular decomposers, including bacteria, fungi, and protists, secrete enzymes into the extracellular medium to degrade organic matter, then assimilate the resulting products. Phagotrophic saprotrophs (intracellular decomposers), which are exclusive to the protist domain, directly ingest particulate and dissolved organic compounds, subsequently degrading them intracellularly in phagosomes. At the intersection between lysotrophic and phagotrophic saprotrophy, a group of protists we term lysophagotrophs can utilize both saprotrophic strategies to degrade organic matter. Non-saprotrophic osmotrophs, including bacteria, fungi, and protists, uptake assimilable compounds originating from the decomposition activities of lysotrophic saprotrophs. Non-saprotrophic osmotrophs, as they do not directly contribute to the decomposition process, are not considered saprotrophs. Instead, they are regarded either as cheaters within the microbial community or as microorganisms employing osmotrophy as a facultative nutritional strategy.</figcaption></figure>

steps: the extracellular enzymatic depolymerization of organic matter followed by the uptake of the resulting dissolved compounds. This extracellular mode of decomposition underpins bacterial and fungal saprotrophic activity (Schimel and Schaeffer, 2012). In addition, certain protist taxa have been identified as capable of lysotrophic saprotrophy (Fig. 2) (Adl and Gupta, 2006; Herrmann et al., 2006).

Among soil protists, the most unequivocal lysotrophic saprotrophs are members of the Oomycetes within the Stramenopiles (Cavalier-Smith, 1997; Geisen et al., 2018). Although Oomycetes are primarily known for their pathogenicity in plants, ecological and physiological evidence suggests that some members also function effectively as lysotrophic saprotrophs (De Vries et al., 2020). For example, stable isotope probing of heavy carbon from plant residues into microbial DNA (DNA-SIP) has revealed the direct role of Oomycete taxa—particularly *Pythium*—in organic matter degradation (Murase et al., 2012; Kramer et al., 2016). Moreover, comparative genomic analyses have demonstrated that Oomycete genomes possess an extensive set of genes involved in degrading plant cell wall compounds, with a number of carbohydrate-active enzyme (CAZyme) genes comparable to those found in fungal genomes (Liu et al., 2023).

Other members of the Stramenopiles, particularly taxa within the Labyrinthulomycetes, have also been reported to secrete extracellular enzymes involved in the breakdown of polysaccharides, lipids, and proteins (Bongiorni et al., 2005; Nagano et al., 2011; Taoka et al., 2009;

Xiao et al., 2018). Although both Oomycetes and certain Labyrinthulomycetes produce extracellular enzymes, the latter primarily inhabit marine sediments and are less common in terrestrial ecosystems (Raghukumar, 2001; Olsen et al., 2003), whereas Oomycetes are more prevalent in soils (Murase et al., 2012; Kramer et al., 2016; Lin et al., 2022). Genomic analyses further reveal that Labyrinthulomycetes have a relatively limited repertoire of carbohydrate-active enzyme genes, possessing two to four times fewer glycosyl hydrolase-encoding genes compared to Oomycetes (Liu et al., 2023). Therefore, while both groups are categorized as saprotrophs by Adl et al. (2018), we extend this classification to lysotrophic saprotrophs and suggest that Oomycetes may play a more significant role in soil organic matter decomposition than Labyrinthulomycetes.

Other protist lineages have been identified as producers of extracellular enzymes, which suggests the potential for lysotrophic saprotrophy. For instance, ciliate species belonging to the genus *Tetrahymena* (Ciliophora), such as *Tetrahymena pyriformis* and *Tetrahymena thermophila*, have consistently been shown to secrete extracellular enzymes—including proteases, various glucosidases, and acid phosphatases (Blum, 1976; Vick and Blum, 1979; Banno et al., 1982, 1983; Tiedtke et al., 1988; Tiedtke and Rasmussen, 1989; Straus et al., 1992; De Coninck et al., 2000; Herrmann et al., 2006; Madinger et al., 2010). Furthermore, *Tetrahymena* are capable of degrading recombinant proteins in culture via secreted proteases, which confirms their efficient
extracellular protein breakdown (Herrmann et al., 2006). Although *Tetrahymena* spp. are primarily aquatic, the widespread diversity and abundance of Ciliophora taxa in soils indicate that some soil-dwelling ciliates may also engage in significant extracellular enzyme production and saprotrophic activities (Foissner, 1997). Typical soil protist groups, such as amoebae of the genus *Acanthamoeba* (Amoebozoa), have been reported to produce extracellular enzymes, particularly hydrolases (Hohman and Bowers, 1984; Siddiqui and Khan, 2012). Enzyme secretion by slime molds—a paraphyletic group of protists that have a unicellular living phase that transitions to a multicellular form known as plasmodia—has also been investigated for extracellular enzyme production, notably in *Physarum polycephalum* (Amoebozoa). Studies have documented the secretion of glucosidases, lipases, nucleases, proteases, and phosphatases (Braun and Behrens, 1969; Farr et al., 1974; Kilpatrick and Stirling, 1977; Kuehn et al., 1977; Saddler et al., 1979). Kobilansky and Schinner (1988) extended these findings to 17 slime mold strains, especially within the genus *Symphytocarpus* (Amoebozoa), which also exhibited extracellular enzyme secretion—including enzymes involved in degrading cellulose (cellulase) and hemicellulose (xylanase). Organic compounds deriving from mycelial residues (fungal necromass) can also likely be degraded by *Physarum polycephalum*, which possesses 19 genes encoding for endochitinases, central enzymes involved in the breakdown of chitin, a number of genes comparable to that found in fungal genomes (Renaud et al., 2023).

Phototrophic protists belonging to the Streptophyta and Chlorophyta have also been proposed to exhibit lysotrophic saprotrophy (Otto et al., 2015; Liao et al., 2023b). For instance, green algae can degrade recalcitrant organic matter by producing extracellular oxidases, such as lacases and tyrosinases (Semple et al., 1998; Otto et al., 2010; Ghasemi et al., 2011; Otto and Schlosser, 2014; Liao et al., 2023b). In addition to oxidases, green algae can degrade exogenous cellulose through the production of cellulases and β-glucosidases (Klassen et al., 2012; Velazquez et al., 2023; Dvoráková-Hladká, 1966; Burczyk et al., 1970). These observations suggest that phototrophic protists can also potentially contribute to saprotrophic activities via lysotrophic mechanisms as an alternative feeding strategy.

It is important to note, however, that detecting extracellular enzyme production in protists alone does not constitute proof for lysotrophic saprotrophy, as alternative hypotheses have often been favored (Siddiqui and Khan, 2012). For instance, in both *Tetrahymena* and *Acanthamoeba*, enzyme secretion has been suggested to play roles in defense against competitors or predators, chemotaxis for detecting other microorganisms, encystment and excystment processes, or even self-autolysis and recycling (Hohman and Bowers, 1984; Tiedtke et al., 1988). Moreover, Kobilansky and Schinner (1988), studying slime mold protists, argued that many studies into protist extracellular enzymes lacked strict axenic conditions, meaning that a fraction—or even most—of the extracellular enzyme signals could originate from co-cultivated bacteria serving as prey. Nevertheless, in *Tetrahymena*, genetic studies support a nutritional role for secreted enzymes: when protease genes were mutated, no significant effects on other cellular processes were observed, suggesting that these enzymes primarily function in degrading extracellular proteins for nutrition (Tiedtke et al., 1988). In summary, lysotrophic saprotrophy is well established in certain protist lineages—particularly within the Stramenopiles (e.g., Oomycetes and certain Labyrinthulomycetes)—while other functional groups, such as ciliates (Ciliophora), specific amoebae (Amoebozoa), and even some phototrophic taxa, are putative lysotrophic saprotrophs based on their extracellular enzyme production.

## 4. Phagotrophic saprotrophy in soil protists

Protists can also engage in phagotrophic saprotrophy, in which organic matter is transported intracellularly via the processes of endocytosis and phagocytosis within food vacuoles and subsequently degraded (Adl and Gupta, 2006). This mode of saprotrophy is unique to

protists compared to bacteria and fungi, which rely primarily on extracellular enzymes. Although endocytosis-like processes in bacteria and endocytosis in fungi have been documented, these mechanisms are regarded as not involved in soil organic matter decomposition (Lohhienne et al., 2010; Commer and Shaw, 2020). Studies on aquatic flagellates show that they can directly ingest dissolved organic matter (DOM) compounds such as proteins and polysaccharides, degrading them internally (Sherr, 1988; Tranvik et al., 1993). For instance, the marine clade Picozoa feeds mostly on colloids of sub-bacterial size suspended in the water column (Seenivasan et al., 2013). Additionally, larger organic compounds present in DOM have been found to be taken up by flagellates, suggesting that they could act as phagotrophic saprotrophs—either primarily or as a facultative nutritional mode alongside bacterivory (Tranvik, 1994). Given the significant abundance and diversity of flagellates in soils (Ekelund et al., 2001) and the likelihood that some share phylogenetic similarities with the aforementioned aquatic flagellates, it is plausible that certain soil flagellates function as primary or facultative phagotrophic saprotrophs.

In addition to DOM, protists can also ingest particulate organic matter (POM) (Schönborn, 1965, 1978). This phenomenon has been consistently documented across many protist lineages, although it is rarely classified as saprotrophy. It is often explained as the incidental incorporation of particles into food vacuoles—similar to how protists sometimes incorporate plastic particles or latex beads (Rillig and Bonkowski, 2018) or particles thought to carry living bacteria on their surfaces, with the bacteria being the primary target of digestion (Pratt and Cairns, 1985). However, research on testate amoebae has proposed that the ingestion of plant residues and humus particles into phagosomes could be associated with phagotrophic saprotrophy (Schönborn, 1965, 1978; Chardez, 1985; Wilkinson and Mitchell, 2010).

Testate amoebae are widely distributed protists, with some described as inhabiting organic matter—rich environments such as the organic soil layers in forests and peatlands (Jassey et al., 2012, 2016; Lamentowicz et al., 2020). Schönborn et al. (1987) demonstrated that the testate amoeba species *Schoenbornia humicola* creates “food-bundles” of humus particles, which are gradually internalized into food vacuoles. Although organic residue incorporation in protist food vacuoles is frequently proposed, its microscopic documentation is scarce. To illustrate this process, we used microfluidic chips that mimic soil microstructures and were inoculated with forest topsoil and plant residues, as described in Mafla-Endara et al. (2021). As shown in Schönborn et al. (1987), we observed the same process of food-bundle creation and slow uptake of particulate residues within food vacuoles by testate amoebae, with no apparent bacteria being ingested (Fig. 3A and B).

Furthermore, phagotrophic saprotrophy in testate amoebae—based on food vacuole content—has been documented across different Arcellinida taxa, including the genera *Bullinularia*, *Centropyxis*, *Cyclopyxis*, *Diplochlamys*, *Hyalosphenia*, *Nebela*, *Pseudawerintzewia*, *Schwabia*, and *Trigonopyxis* (Bonnet, 1964; Schönborn, 1965; Laminger, 1977; Gilbert et al., 2000). For most of these genera, it remains unclear whether testate amoebae inhabiting plant-derived organic matter—rich habitats, such as soil humus layers, primarily feed on these plant residues (Schönborn et al., 1987). However, for some taxa—such as *Nebela tincta* and *Hyalosphenia papilio*—observations of feeding habits and ¹³C and ¹⁵N isotopic signatures suggest that they use plant-derived organic matter as an occasional food source (Gilbert et al., 2000; Jassey et al., 2012; see Fig. 3C for *Hyalosphenia papilio* ingesting plant residues). Interestingly, Laminger (1977) noted that during periods of low soil moisture, the phagosomes of the rhizarian testate amoeba *Trinema enchelys* were enriched in plant debris. Collectively, these findings provide strong support for phagotrophic saprotrophy within testate amoebae for plant-derived POM. Curiously, this functional role appears widely underappreciated, with no testate amoebae currently recognized as potential saprotrophs within existing frameworks for protist functional groups (e.g., Dumack et al., 2020).

Given that fungal necromass can constitute up to 50 % of soil organic
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Bright field microscopy of testate amoebae (orange arrows) (A) and (B) in soil chips, and *Hyalosphenia papilio* (C) from peatland soil taking up plant or humus particles (yellow arrows) in their phagosomes. Scale bars indicate 20 µm for (A) and (B) and 80 µm for (C). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

carbon in some ecosystems (Liang et al., 2019), understanding whether protists also participate in its decomposition is important. It is well documented that specific soil protist taxa feed on living fungi, targeting either hyphae or spores (Adl and Gupta, 2006; Gilbert et al., 2000; Jassey et al., 2012; Estermann et al., 2023). It is conceivable that all protist taxa performing fungal phagotrophic predation also engage in phagotrophic saprotrophy, particularly on freshly dead hyphae, which might retain a biochemical composition similar to that of living hyphae at the moment of death (Estermann et al., 2023). In cases of fungal starvation or partial senescence, however, fungal residues may be highly depleted in high-energy, easily degradable non-structural compounds such as polysaccharides and proteins, and enriched in compounds such as glucans, chitin, and melanin that are more challenging to degrade (Sokol et al., 2022; Camenzind et al., 2023). Thus, it seems likely that phagotrophic saprotrophy of fungal necromass may be restricted to protists with a high intracellular enzymatic capacity for degrading fungal cell wall compounds. Using microfluidic chips that mimic soils inoculated with a natural soil community and fungal necromass for illustration purposes, we observed protist taxa ingesting pieces of fungal necromass into their food vacuoles, including amoebae (Fig. 4A) and *Nuclearia* sp. (Fig. 4B and C). The same reasoning regarding fungal necromass also applies to bacterial residues; it is likely that certain soil protists might also act as decomposers of senesced bacterial cells.

It is plausible that certain protist taxa can perform both lysotrophic and phagotrophic saprotrophy (i.e., occupying the intersection of lysotrophic and phagotrophic saprotrophy as illustrated in Fig. 2). Specifically, this may be the case for slime molds—potential lysotrophic saprotrophs—which are also described as phagotrophic predators, incorporating bacteria and fungi into their food vacuoles during their single-celled stage (Renaud et al., 2023). Altogether, we introduce the term “phagotrophic saprotrophy” to categorize protists that intracellularly degrade organic matter, whether dissolved or particulate. This mode of nutrition has been explored for aquatic flagellates and soil testate amoebae, serving either as a primary or alternative feeding strategy depending on environmental conditions. Moreover, the general observation of organic matter ingestion across diverse polyphyletic protist functional groups—such as flagellates, testate and naked amoebae—suggests that phagotrophic saprotrophy might be relatively widespread among soil protists.

**5. Potential contribution of protist saprotrophy to soil organic matter decomposition**

The role of protist saprotrophy in soil organic matter decomposition relative to bacteria and fungi, along with its contribution to the

transformation of different organic matter fractions (DOM, POM, and mineral-associated organic matter, MAOM) and sources (plant-, animal-, or microbially derived), remains poorly explored. DNA or RNA-SIP studies, in which heavy carbon from organic matter is traced into microbial nucleic acids, are particularly effective at identifying microorganisms directly participating in decomposition processes (López-Mondéjar et al., 2020; Maillard et al., 2023). However, because protists also engage in predatory activities on bacteria and fungi, predatory phagotrophic protists may be confused with decomposers, thereby limiting conclusions on phagotrophic saprotrophy. In contrast, for protists lacking predatory phagotrophic activity—where absorbed carbon derives from lysotrophic saprotrophy—DNA-SIP and RNA-SIP studies have consistently found participation of Oomycetes protists, notably *Pythium* spp., in plant residue decomposition processes, with enrichment patterns similar to those observed in fungal taxa (Murase et al., 2012; Kramer et al., 2016). Interestingly, Kramer et al. (2016) observed that when isotopically heavy glucose was added as a substrate in soil, it was very poorly incorporated into protist RNA relative to bacteria and fungi, suggesting that non-saprotrophic osmotrophy, although often observed *in vitro* for predator protists, might not be ecologically relevant when bacteria and fungal competitors are present. However, in the same study, Oomycetes protists were found to take up carbon from glucose relatively well, suggesting a functional parallel with bacteria and fungi, whereby soil lysotrophic protists can both efficiently degrade plant-derived compounds and effectively take up the decomposition products within complex decomposer communities. These results support a substantial role of Oomycetes as lysotrophic saprotrophs in soil organic matter decomposition processes, sharing functional similarities with bacterial and fungal decomposers.

While protist lysotrophic saprotrophy can be studied using similar techniques as those employed for bacterial and fungal decomposers, phagotrophic saprotrophy is more challenging to determine *in situ* and is conceptually drastically different from extracellular organic matter decomposition. Indeed, as noted above, what distinguishes protist saprotrophy from that of bacteria and fungi is the ability of certain protist taxa to ingest dissolved, colloidal, and particulate organic compounds into food vacuoles (Schönborn et al., 1987; Tranvik et al., 1993). This is significant because, in microbial ecology, extracellular enzymes involved in decomposition are theorized as a public good that benefits the microbial community at the expense of the individual investing energy in their production, which has major implications for decomposition limitations, carbon movements, and microbial community functioning (Smith and Schuster, 2019).

Firstly, assimilable compounds released from extracellular decomposition might be taken up by non-saprotrophic osmotrophic
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Bright field microscopy of fungal necromass residues (yellow arrows show examples) inoculated in soil chips being taken up by (A) amoeba (orange arrow) and (B) *Nuclearia* sp. (orange arrows). Note that some fungal necromass residues are present within phagosomes, while others are located intracellularly but outside of phagosomes. Scale bars indicate 40 µm for (A) and 80 µm for (B) and (C). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

microorganisms that did not contribute to enzyme secretion, effectively acting as “cheaters” or passive consumers (Fig. 2) (Allison, 2005). Secondly, extracellular organic degradation requires the secretion of enzymes, a process that is costly in terms of both carbon and nitrogen. These enzymes are not necessarily recycled by the secreting individuals, as they can be degraded by other microorganisms as a carbon and nitrogen source or become immobilized through interactions with organic compounds such as tannins in organic matter-rich soils (which complex with proteins) (Adamczyk et al., 2017) or adsorb onto secondary minerals like iron and aluminum oxides found in mineral soils (Tian et al.,

2020). Thirdly, extracellular enzymes have specific optimal pH ranges and often require cofactors for catalytic activity, conditions that are difficult to control extracellularly for microbial decomposers (Op De Beeck et al., 2021). For all these reasons, degradable organic matter can accumulate in soils.

For phagotrophic saprotrophs, these limitations are alleviated because their enzymes function as a private good. All assimilable compounds liberated from the intracellular degradation of organic matter are retained by the individual producing the enzymes, potentially leading to a higher energy gain compared to extracellular degradation.
Moreover, organic compounds ingested into food vacuoles can be effi-
ciently degraded under optimal conditions for enzyme catalysis—such
as stable pH, appropriate cofactor concentrations, and coordinated
multi-enzyme pathways—and the enzymes within food vacuoles can be
efficiently recycled, thereby limiting energy losses. Thus, phagotrophic
protists may have inherent advantages in degrading recalcitrant organic
matter relative to microorganisms employing lysotrophic saprotrophy.

While internalizing the decomposition process may confer physio-
logical advantages, it remains unclear whether phagotrophic protists
possess an efficient enzyme repertoire to extract energy from soil
organic matter. *Orciraptor agilis* (Cercozoa), an aquatic flagellate, uses
contact digestion—closely resembling phagotrophic saprotrophy—to
efficiently degrade algal cell walls using a combination of hydrolases
(cellulase and pectinase) and likely Lytic Polysaccharide Mono-
oxygenases (LPMOs) (Gerbracht et al., 2022). Anaerobic protists
inhabiting the gut of lower termites have also been characterized as
highly efficient degraders of wood particles through phagotrophic sap-
rotrophy and the secretion of enzymatic cocktails, including cellulases
and hemicellulases (Brune, 2014). More recently, such protists have
been noted for degrading fungal cell wall compounds like chitin via the
production of chitinases (Nishimura et al., 2020). These examples un-
derscore that certain protist taxa inherently possess intracellular enzy-
matic systems for degrading recalcitrant organic matter.

Given these potential advantages, one can question whether phag-
otrophic saprotrophy among soil protists presents limitations, particu-
larly in terms of physiological or ecological trade-offs. Regarding the
ingestion and intracellular decomposition of dissolved compounds, one
significant limitation is that many compounds constituting DOM—such
as soluble short-chain polymers (e.g., cellobiose and chitobiose) or sol-
uble proteins and peptides (Guggenberger and Zech, 1993; Kaiser et al.,
2004)—are readily broken down by saprotrophic bacteria and fungi,
even those that are relatively poor lysotrophic degraders, as they require
minimal enzymatic attack. Therefore, soil phagotrophic protists might
be outcompeted by bacteria and fungi for the degradation of labile DOM.
However, recent findings indicate that a significant fraction of soil DOM
comprises carbohydrate-containing colloids resistant to bacterial
decomposition (Andersson et al., 2023; Andersson, 2023); these colloids
could represent a potential target for phagotrophic saprotrophic protists.

For POM of both plant and fungal origin, the most apparent limita-
tion is size. Protist food vacuoles are restricted by the cell cytoskeleton,
preventing the uptake of large particles. Consequently, phagotrophic
saprotrophy is limited to small particles, often necessitating prior frag-
mentation of organic matter by abiotic and biotic processes. Moreover,
observations in soil chips have revealed significant cytoskeletal

deformation during fungal necromass ingestion by amoeboflagellate protists (Fig. 5). These protists change their shape to a more spherical form while ingesting fungal necromass (Fig. 5A and B), which may limit their mobility and render them more susceptible to predation by larger protists such as amoebae (Fig. 5C), illustrating a second potential limitation of phagotrophic saprotrophy. Overall, we show that both lysotrophic and phagotrophic saprotrophic protists might substantially contribute to soil organic matter decomposition alongside bacterial and fungal decomposers. In particular, phagotrophic saprotrophy within protists—although still under-investigated—may play a crucial yet underexplored role in decomposing highly recalcitrant organic matter associated with the potential advantages of intracellular decomposition.

**6. Future research directions**

While direct evidence for saprotrophy in soil protists remains limited to a few lineages or functional groups (e.g., Oomycetes, testate amoebae), indirect evidence—including extracellular enzyme production and the ingestion of organic matter particles in food vacuoles—suggests significant potential and warrants further exploration. We propose that the study of soil protist saprotrophy should align with methodologies used for bacterial and fungal decomposers, focusing on three complementary areas: (1) determining carbon transfer from organic matter to potential decomposer protists and how it translates in terms of biomass or cell abundance; (2) identifying the mechanisms used, such as enzyme production both intracellularly and extracellularly; and (3) analyzing the chemical modifications of degraded substrates as indicators of decomposition activity.

In terms of methodologies, DNA-SIP studies using organic com-
pounds of plant, animal, or microbial origin—with relatively short in-
cubation periods to limit cross-feeding—are likely to provide key
insights into lysotrophic saprotrophy in soil protists (Murase et al., 2012;
Kramer et al., 2016). Studies on protist physiology can combine tran-
scriptomics, proteomics, and genomics on putative saprotrophic isolates
grown in pure cultures with different organic compounds as sole carbon
sources, building on methods used for protist predation activities
(Gerbracht et al., 2022). Together with spectroscopic analyses, such
studies can reveal how decomposing activities affect the structure and
potential long-term persistence of decomposed organic matter in soil
(Tunlid et al., 2022). Specifically for phagotrophic saprotrophy, micro-
fluidic soil chips appear particularly suitable for documenting the
ingestion and degradation processes of POM by soil protists
(Mafla-Endara et al., 2021), while fluorescent reporters combined with
organic substrates (e.g., FITC-casein, FITC-dextran) can be used to

<figure><img src="image_7.png" /><figcaption>**Fig. 5.** Bright field microscopy of (A) amoeboflagellate (orange arrows) without fungal necromass (yellow arrows) present in phagosomes; (B) the same amoeboflagellate taxa with fungal necromass present in phagosomes; and (C) amoeboflagellate with fungal necromass particles in their food vacuoles being consumed by a predator amoeba (green arrow). Scale bars indicate 20 µm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>
document intracellular decomposition of DOM (Sherr, 1988; Tranvik et al., 1993; Arellano-Caicedo et al., 2023). In addition, by utilizing ¹³C-labeled plant or microbial residues, nanoscale secondary ion mass spectrometry (NanoSIMS) may be used to track the transfer of carbon between organic matter and protists at the single-cell level, thereby characterizing both lysotrophic and phagotrophic saprotrophy (Sheik et al., 2013; Carpenter et al., 2018). For *in situ* methods, metatranscriptomics and metaproteomics show promise by focusing on genes involved in carbohydrate, lipid, and protein degradation, which allows differentiation between bacterial, fungal, and protist contributions to soil organic matter decomposition (Starke et al., 2021; Auer et al., 2024).

To better integrate the role of protists in biogeochemical processes, it will also be crucial to determine whether saprotrophy can serve as an alternative feeding strategy for certain phototrophic and heterotrophic protists, as some studies suggest, and under what conditions (Schönborn et al., 1987; Tranvik, 1994). Furthermore, exploring potential competitive or synergistic interactions between protists and other saprotrophic microorganisms—such as bacteria and fungi—during organic matter decomposition is essential, particularly given growing evidence for interdomain commensalism and mutualism within soil microbial communities (Vohník et al., 2009; Deveau et al., 2018; Ge et al., 2023; Pérez-Pazos et al., 2024). Finally, beyond the ecology and physiology of protist saprotrophy, it will be interesting to include an evolutionary component, as evidence of partial or full saprotrophy exists across many lineages (Amoebozoa, Stramenopiles, Picozoa), which may help us better understand the evolutionary history of this nutritional mode within protists.

Utilizing the aforementioned methodologies and the newly introduced conceptual framework for protist saprotrophy, the following key research questions arise, moving from fundamental understanding to broader ecological and modeling implications.

* Which soil protist taxa function as saprotrophs, and to what extent is saprotrophy their primary or facultative feeding strategy?

* What fraction of organic matter decomposition processes is mediated by decomposer protists compared to bacteria and fungi?

* How do the contrasted protist saprotrophic strategies (lysotrophic versus phagotrophic saprotrophy) affect decomposition processes and the formation of soil organic carbon stocks?

* Which fractions of soil organic matter (DOM, POM, MAOM) and what types of residues (plant-, animal-, or microbially-derived) are protist saprotrophs most effective at decomposing relative to bacterial and fungal decomposers?

* Can metrics such as carbon-use efficiency, enzyme production rates, or trait-based classifications of protist decomposers be integrated into soil biogeochemical models to improve predictions of carbon fluxes?

## 7. Conclusions

Here we propose a new conceptual framework for understanding saprotrophy among soil protists, aligning it with established concepts of bacterial and fungal saprotrophy. We advocate for a clear definition of protist saprotrophy that distinguishes it from non-saprotrophic osmophy, and for differentiating two types of protist saprotrophy based on the physical location of organic matter decomposition: extracellular (lysotrophic saprotrophy) and intracellular (phagotrophic saprotrophy). This distinction is ecologically important, as it likely has significant implications for carbon cycling in soils. We also synthesize the growing evidence that soil saprotrophic protists are functionally diverse and target multiple organic matter sources and fractions; while direct evidence remains limited, this is a promising frontier for soil microbial ecology. Integrating protist saprotrophy into established research on microbial decomposition will ultimately help improve our ability to predict and manage soil carbon storage under changing environmental conditions.

### CRediT authorship contribution statement

**François Maillard:** Writing – review & editing, Writing – original draft, Visualization, Supervision, Project administration, Investigation, Funding acquisition, Conceptualization. **Fredrik Klinghammer:** Writing – review & editing, Visualization, Validation, Investigation, Formal analysis. **Vincent E.J. Jassey:** Writing – review & editing, Validation, Investigation, Formal analysis. **Bowen Zhang:** Writing – review & editing, Investigation. **Peter G. Kennedy:** Writing – review & editing, Validation, Investigation. **Enrique Lara:** Writing – review & editing, Validation, Investigation. **Stefan Geisen:** Writing – review & editing, Validation, Investigation. **Lars Tranvik:** Writing – review & editing, Validation, Investigation. **Edith Hammer:** Writing – review & editing, Validation, Investigation. **Anders Tunlid:** Writing – review & editing, Supervision, Investigation.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgments

The authors acknowledge financial support from the Swedish Research Council (Starting Grant in Natural and Engineering Sciences, VR 2023-04643, to François Maillard, and Research Grant in Natural and Engineering Sciences, VR 2021-05188, to Anders Tunlid) and the Crafoord Foundation (Research Grant, Crafoord 20241084, to François Maillard). We thank the two anonymous reviewers for their valuable comments and suggestions, which have greatly improved this article.

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