Microbes: The Invisible Workforce

Everything visible in an aquatic ecosystem -- the algae, the tiny shrimp, the water fleas -- depends on organisms you cannot see with the naked eye. Bacteria and single-celled predators called ciliates do the essential behind-the-scenes work of recycling nutrients, breaking down dead material, and connecting the "waste stream" back to the living food web. Without them, nutrients would get locked up in dead matter and the whole system would grind to a halt.

This document covers five groups of microbes modeled in the simulator: nitrifying bacteria, heterotrophic bacteria, aquatic fungi, heterotrophic nanoflagellates (HNF), and ciliates. HNF and ciliates are protists -- single-celled organisms that are simultaneously microbes (by size and ecology) and consumers (by feeding strategy). They are introduced here because they are central to the microbial loop, but their feeding relationships also appear alongside other consumers in the food web and their per-species details live in the Species Catalog: Consumers.

Nitrifying Bacteria

What they do

Nitrifying bacteria perform a specific chemical conversion: they turn ammonium (NH4) into nitrate (NO3), a process called nitrification. In the real world, this is actually a two-step process carried out by two different types of bacteria (Nitrosomonas converts ammonium to nitrite, then Nitrobacter converts nitrite to nitrate), but the model combines them into a single group for simplicity.

How they get energy

Nitrifiers are chemoautotrophs -- a fancy word for organisms that get their energy from chemical reactions rather than from sunlight or from eating other organisms. Specifically, they harvest energy from the oxidation of ammonium (combining it with oxygen). They then use that energy to fix CO2 into organic carbon, building their own biomass. Think of them as similar to plants in that they build their bodies from CO2, except instead of using sunlight as their energy source, they use the chemical energy released when ammonium reacts with oxygen.

Their growth yield is very low: for every unit of ammonium they oxidize, only about 8% of the energy ends up as new bacterial biomass. The rest goes into powering the chemical reaction itself.

Why nitrification matters

Algae can use both ammonium and nitrate as nitrogen sources, so converting one to the other might seem like a trivial reshuffling. But nitrification has important side effects:

Oxygen consumption: Nitrification is a major oxygen consumer. For every unit of ammonium oxidized, two units of oxygen are consumed. In a small, sealed ecosystem, this can be a significant drain on dissolved oxygen.
pH effects: Nitrification releases acid (hydrogen ions), which lowers the pH of the water. The model tracks this through alkalinity changes -- nitrification decreases alkalinity by 2 equivalents for every unit of ammonium oxidized.
CO2 consumption: Because nitrifiers fix CO2 to build biomass, they are a (small) sink for dissolved inorganic carbon.

Growth characteristics

Nitrifiers are slow growers compared to most other bacteria. Their doubling time is around 30 hours under ideal conditions, which is sluggish by bacterial standards. Their growth is limited by:

Ammonium availability: They need ammonium as their energy source and substrate. In freshwater systems the nitrifying community is a mixture of ammonia-oxidising bacteria (AOB, e.g. Nitrosomonas) and ammonia-oxidising archaea (AOA, e.g. Candidatus Nitrosoarchaeum). The model uses a half-saturation constant of 0.35 mg N/L (2.5×10⁻⁵ mol/L), representing this mixed community. AOA, which often dominate at low ammonium (<1 mg N/L), have very high affinity (K ≈ 0.004–0.05 mg N/L), while AOB are less competitive (K ≈ 0.05–0.5 mg N/L). Algae typically have K values of 0.01–0.2 mg N/L, so algae still outcompete nitrifiers for ammonium at low concentrations, but not by as large a margin as previously modelled.
Oxygen availability: They are obligate aerobes -- they absolutely require oxygen and will die without it. The model includes specific hypoxia mortality: stress begins around 2 mg/L of dissolved oxygen, and conditions become lethal below about 0.5 mg/L.
Iron availability: The ammonia monooxygenase (AMO) enzyme that catalyses NH4 oxidation is an Fe-S / Cu metalloprotein, and nitrifier growth empirically half-saturates at ~20 nM Fe (Wagner et al. 2002). Iron limitation therefore throttles nitrification alongside O2 limitation — in very soft water or tanks built on RO water with no Fe supplementation, nitrifying bacteria can struggle to establish even when NH4 is plentiful. See Iron Cycle.

Mortality and death

Nitrifiers have a low base mortality rate (about 1% per day). They can also die from temperature extremes (they prefer 25-35 degrees C), pH stress (optimal around pH 7-8, stressed outside pH 6-9), and high salinity. When they die, their biomass becomes detritus -- 80% as fine suspended particles and 20% as settled material, reflecting their tiny cell size.

Heterotrophic Bacteria

What they do

Heterotrophic bacteria are the decomposers. They consume dissolved organic matter (DOM) -- essentially the dissolved remains of dead organisms -- and also break down suspended detritus particles floating in the water. If nitrifiers are chemical specialists, heterotrophic bacteria are the general-purpose recyclers.

How they feed

These bacteria consume two types of dead organic material:

Dissolved organic matter (DOM): This is organic material that has dissolved into the water, such as the contents of burst cells or leaked cellular products. Bacteria prefer DOM over detritus because it is easier to absorb (about 2x the preference).
Suspended detritus: These are tiny particles of dead organic matter floating in the water column. Bacteria can colonize and break down these particles, but it takes more effort than absorbing dissolved material.

Their maximum uptake rate is high -- they can consume about 6 times their own body carbon per day under optimal conditions. The half-saturation constant for DOM uptake is 0.096 mg C/L (8×10⁻⁶ mol/L) and for suspended detritus is 0.24 mg C/L (2×10⁻⁵ mol/L). These are specified as concentrations (mol/L) so that bacterial substrate kinetics are independent of the jar volume -- they scale correctly regardless of whether the simulation uses 0.1 L or 5 L.

Growth efficiency

Only about 28% of what heterotrophic bacteria consume becomes new bacterial biomass. The other 72% is respired as CO2. This efficiency (called bacterial growth efficiency) is a key parameter because it determines how much of the "dead" organic matter pool gets converted back into living biomass versus how much is simply burned off as CO2.

Nutrient recycling

When bacteria consume organic matter that is nitrogen-rich or phosphorus-rich relative to their own needs (bacteria have a C:N ratio of about 5:1 and an N:P ratio of about 10:1, meaning they are P-rich compared to Redfield), they excrete the excess nitrogen as ammonium (NH4) and excess phosphorus as phosphate (PO4). This mineralization is one of the most important processes in the ecosystem -- it returns nitrogen and phosphorus from the dead organic pool back into forms that algae and nitrifiers can use.

Additionally, when oxygen limits bacterial growth, the nitrogen and phosphorus that would have been assimilated into biomass are instead released back as NH4 and PO4. This ensures stoichiometric consistency -- bacteria cannot build biomass without the energy from aerobic respiration, so the unassimilated N and P are routed to the dissolved inorganic pools rather than vanishing.

Viral lysis

In the real world, bacteria face a unique form of mortality: they are killed by viruses (bacteriophages). The model includes this as a density-dependent process -- the higher the bacterial population density, the more frequently viruses encounter and infect bacteria. This creates a natural stabilizing feedback that prevents bacterial populations from growing without limit. The maximum lysis rate is 48% per day at saturating bacterial density (Michaelis-Menten kinetics), which keeps actual lysis at typical jar bacterial densities in the range of 15–25%/day -- consistent with literature estimates that viruses account for 10–50% of bacterial mortality (Fuhrman 1999, Weinbauer 2004).

Death products

When heterotrophic bacteria die (from any cause), their biomass is routed as follows:

80% becomes DOM (dissolved organic matter) -- bacteria are so small that when they die, their cell contents largely dissolve into the water
20% becomes detritus -- of this, 50% stays suspended and 50% settles

This routing is important because the DOM released from dead bacteria can immediately be consumed by other bacteria, creating a recycling loop.

Environmental tolerances

Heterotrophic bacteria are generally hardy organisms with wide tolerance ranges. They tolerate temperatures from about 5 to 35 degrees C (stress outside this range), pH from about 5.5 to 9.0, and prefer freshwater conditions. They can tolerate some hypoxia better than nitrifiers (some are facultative anaerobes), with stress beginning around 1 mg/L O2 and lethal conditions below about 0.15 mg/L.

Role in biofilm maturation

Heterotrophic bacteria are the primary architects of biofilm structural complexity. Their EPS secretion builds the scaffold that protects embedded organisms, creates refugia for prey, and provides habitat for benthic meiofauna — making them the single largest driver of biofilm maturation (Flemming & Wingender, 2010). As the biofilm matures, surface bacteria gain increasing shelter from planktonic bacterivores — at full maturity ~50% of heterotrophic bacteria (and up to ~80% of surface nitrifiers) are physically embedded in the EPS matrix and inaccessible to grazers (Flemming et al., 2016). The same per-species coefficient also shields the cell's own base + viral self-mortality, since deeply embedded cells experience less hostile chemistry and lower phage encounter rates. This creates a positive feedback loop: bacteria build biofilm, maturity increases, more bacteria are sheltered, bacteria accumulate faster. The logistic saturation of structural complexity prevents runaway. Fungi and nitrifiers also contribute to maturation — fungi through hyphal networks that add structural complexity, and nitrifiers through the EPS they secrete while colonizing surfaces.

Aquatic Fungi

What they are

Aquatic fungi are a community of decomposers dominated by two groups: hyphomycetes (filamentous fungi that grow as branching hyphal networks on solid substrates) and chytrids (small, often unicellular fungi that produce motile zoospores). In the model, they are represented as a single functional group. Hyphomycetes contribute the bulk of the biomass; chytrids contribute the grazeable zoospore fraction that feeds into the food web.

What they do

Fungi are the specialists of refractory decomposition. Where heterotrophic bacteria excel at consuming fresh, labile dissolved molecules (amino acids, sugars), fungi break down complex, recalcitrant polymers -- humic compounds, cellulose, and lignin-like material -- that bacteria handle poorly. They do this via extracellular enzyme systems (cellulases, laccases, peroxidases) that depolymerize these tough substrates outside the cell before absorbing the products (Gessner et al. 2007; Krauss et al. 2011).

Their substrate preferences are essentially inverted compared to bacteria:

Substrate	Fungal preference	Bacterial preference
Refractory DOM	3.0x (strongest)	0.1x (weakest)
Soil OM refractory	2.5x	Not directly consumed
Settled detritus	2.0x	0.09x (effective)
Labile DOM	0.05x (weakest)	2.0x (strongest)

This niche separation means fungi and bacteria are largely complementary rather than competitive -- fungi process what bacteria cannot, and the two together decompose a broader range of organic matter than either could alone.

Fungal conditioning: the key ecological function

The most important thing fungi do is not growing -- it is what they produce while feeding. When fungi break down refractory organic matter, about 20% of the carbon that would otherwise be respired as CO2 is instead released as labile DOM: small, bioavailable molecules that bacteria can immediately consume (Suberkropp 1998; Gulis & Suberkropp 2003). This is the fungal conditioning effect -- the enzymatic pre-processing of recalcitrant carbon into forms accessible to bacteria.

In Walstad-style scenarios with organic soil substrate, this mechanism drives a characteristic decomposition succession:

Days 0-10: Bacteria bloom on labile substrates; fungi slowly establish on refractory material.
Days 10-30: Bacteria plateau as labile DOM is consumed; fungi grow on refractory substrates and produce labile DOM through conditioning.
Days 30+: Quasi-steady state. Fungi dominate refractory decomposition and sustain bacteria through a steady supply of conditioned labile DOM.

Without fungi, the refractory fraction of soil OM would turn over far more slowly, limiting long-term nutrient availability in planted tanks.

Growth characteristics

Fungi grow much more slowly than bacteria. Their doubling time is about 7 days under ideal conditions, compared to roughly 4 hours for heterotrophic bacteria (Suberkropp 1998). Their maximum uptake rate is about 0.84 times body carbon per day -- roughly 7 times slower than bacteria. Growth efficiency on refractory substrates is about 15% (compared to 28% BGE for bacteria on labile DOM), reflecting the energetic cost of extracellular enzyme production (Gulis & Suberkropp 2003).

Fungi have a C:N ratio of about 10:1 (compared to 5:1 for bacteria). Excess nitrogen and phosphorus from their substrate are released as ammonium and phosphate, providing a mineralization pathway operating on substrates that bacteria cannot efficiently exploit.

Bacterial competitive suppression

When bacterial density is high, bacteria suppress fungal uptake efficiency by up to 40%. This happens because bacteria consume labile intermediates produced by fungal extracellular enzymes before the fungi can assimilate them, and may competitively inhibit enzyme binding sites on particle surfaces (Mille-Lindblom, Fischer & Tranvik 2006). The suppression eases as bacterial density drops -- which is why fungi flourish after bacteria have depleted the labile substrate pool.

Environmental tolerances

Fungi are obligate aerobes -- they require dissolved oxygen for lignin degradation (Kirk & Farrell 1987). They are less hypoxia-tolerant than bacteria (stress at ~1.6 mg/L O2, lethal below ~0.5 mg/L) and have no facultative anaerobe capability. Their temperature optimum is cooler than bacteria (reference 20 C vs 25 C), giving them a competitive advantage in cool water (Suberkropp 1984; Chauvet & Suberkropp 1998). They tolerate mild acidity better than bacteria (pH stress below 4.5 vs 5.5), consistent with aquatic hyphomycetes that prefer pH 5-7 (Barlocher 1992).

Mortality and death products

Base mortality is low (~2%/day), reflecting the persistence of substrate-embedded hyphal networks (Barlocher 1992). When fungi die, their biomass splits three ways: 20% to labile DOM (cytoplasmic lysis), 15% to refractory DOM (chitin and melanin from cell wall fragments; Gooday 1990), and 65% to detritus (mostly settled hyphal fragments). The chitin-to-refractory-DOM routing is ecologically significant: unlike dead bacteria (which are 80% labile DOM), dead fungi contribute meaningfully to the refractory organic matter pool.

Who eats fungi

Fungi are grazed by ciliates and HNF, but only partially. The bulk of fungal biomass is in substrate-embedded hyphal networks (50-500 um) that are far too large for microzooplankton to ingest. Only the chytrid zoospore fraction (2-10 um) is accessible -- about 20% for ciliates and 12% for HNF (Kagami et al. 2007). This partial grazing pathway (sometimes called the mycoloop) channels energy from refractory organic matter through fungi and zoospores into the broader food web, complementing the microbial loop that runs through bacteria.

Heterotrophic Nanoflagellates (HNF)

What they are

Heterotrophic nanoflagellates (HNF) are small protists, typically 2-20 micrometers in size, that use flagella for both swimming and feeding. They are the dominant consumers of bacteria in freshwater ecosystems, consuming 30-50% of bacterial production. Common examples include Bodo, Spumella, and Paraphysomonas. In the model, they are represented as a generic freshwater HNF community.

What they eat

HNF are primarily bacterivores -- their main food source is heterotrophic bacteria. They are specialized for capturing bacterial-sized prey (0.5-5 micrometers). Their feeding preferences, as modeled, are:

Food source	Preference	Assimilation efficiency
Bacteria	1.0 (primary)	55%
Suspended detritus	0.2	10%
Planktonic algae	0.15	35%
Periphyton	0.15	30%

HNF have even higher access to bacteria than ciliates (~75% vs ~70%) because their smaller body size lets them penetrate finer biofilm structures. Most periphyton cells (5-50 micrometers) are too large for HNF to ingest -- they only consume the smallest cells or fragments (5% access).

Why HNF matter: the microbial loop

HNF are the critical first link in the microbial loop. They sit between bacteria (which are too small for most larger grazers) and ciliates (which are large enough for copepods to eat). By consuming bacteria and concentrating that biomass into a larger package, HNF make bacterial production accessible to higher trophic levels. Ciliates eat HNF with a preference of 0.7 and 60% assimilation efficiency. HNF are also eaten by rotifers (preference 0.5) and to a lesser extent by Copepods (preference 0.4).

The microbial loop pathway now runs: dead matter -> bacteria -> HNF -> ciliates -> copepods, adding an extra trophic step that more accurately reflects real freshwater food webs.

Growth and reproduction

HNF reproduce by binary fission and can do so rapidly. Their maximum ingestion rate is about 2.4 times their body carbon per day -- higher than ciliates on a mass-specific basis, consistent with allometric scaling from their smaller body size. They are nearly continuously active, feeding at 95% of daytime rates at night. The half-saturation constant for bacterivory is 0.048 mg C/L (~4.8×10⁶ bacterial cells/mL), calibrated to the elevated bacterial densities expected in small productive jars while remaining consistent with the upper end of Fenchel's (1982) published Ks for natural HNF assemblages (10⁵–10⁶ cells/mL).

Mortality

HNF face several sources of mortality:

Base mortality: About 5% per day (higher turnover than ciliates)
Starvation: HNF are naked flagellates with no resting stage; experimental studies show starving populations die at 20–50%/day (Zubkov & Sleigh 1995, Weisse 2002). The model uses 30%/day at complete starvation.
Predation by ciliates, rotifers, and copepods and Daphnia: Handled in those species' code
Hypoxia: Stress below about 1.3 mg/L O2, lethal below 0.3 mg/L
Temperature extremes: Stress outside 8-30 degrees C, lethal outside 2-38 degrees C
Ammonia toxicity: Moderately sensitive to unionized ammonia (NH3)
pH stress: Stress outside pH 5.8-9.0

When HNF die, their biomass is split: 30% dissolves directly to DOM (cytoplasmic contents released by cell lysis) and 70% becomes particulate detritus (85% suspended, 15% settled). The DOM fraction re-enters the bacterial uptake cycle, partially recreating the viral-shunt-like rapid recycling that occurs when small naked protists lyse. Their fecal material is also very fine -- 90% stays suspended.

Nutrient recycling by HNF

Like other consumers, HNF excrete excess nitrogen as ammonium and excess phosphorus as phosphate. Because they consume large quantities of bacteria (which are nitrogen-rich), HNF are major nutrient regenerators, rapidly recycling NH4 and PO4 back into the water for algae to use.

Ciliates

What they are

Ciliates are single-celled organisms (protozoans) ranging from about 10 to 300 micrometers in size. They are covered in tiny hair-like structures called cilia that they use for both swimming and feeding. Common freshwater examples include Paramecium, Tetrahymena, and Vorticella. In the model, they are represented as a generic bacterivorous ciliate community.

What they eat

Ciliates are primarily bacterivores -- their main food source is heterotrophic bacteria. They are specialized for capturing bacterial-sized prey (0.5 to 5 micrometers). Beyond bacteria, they also eat:

Heterotrophic nanoflagellates (HNF) (a major secondary food source -- HNF are optimal prey size for ciliates)
Small planktonic algae (a secondary food source)
Periphyton microalgae (minor -- they are swimmers, not scrapers, so they have limited access to surface-attached cells)
Suspended detritus (they can ingest fine particles but digest them poorly)

Their feeding preferences, as modeled, are:

Food source	Preference	Assimilation efficiency
Bacteria	1.0 (primary)	55%
Nanoflagellates (HNF)	0.7	60%
Planktonic algae	0.3	45%
Suspended detritus	0.3	10%
Periphyton	0.25	40%
Settled detritus	0.1	25%

The low assimilation efficiency for detritus (only 10% for suspended particles) reflects the fact that bacterivorous ciliates lack the enzymes to efficiently break down dead organic matter -- they are built for eating bacteria, not decomposing debris.

Why ciliates matter: the food web link

Ciliates play a critical role as the bridge between the microbial world and the larger food web. Bacteria are too small for most larger grazers to eat efficiently. Copepods copepods, for example, have a feeding apparatus optimized for prey in the 5-50 micrometer range, which makes individual bacteria (0.2-2 micrometers) largely inaccessible. But ciliates, at 10-300 micrometers, are the perfect size for copepods to eat.

This means ciliates act as a "packaging service" -- they eat bacteria (directly) and HNF (which have already consumed bacteria) and concentrate that biomass into a larger, copepod-edible package. Copepods eat ciliates with a preference of 0.9 (nearly as high as their top food source) and an assimilation efficiency of 65%.

Growth and reproduction

Ciliates reproduce by binary fission (they simply split in two) and can do so rapidly -- potentially doubling their population in as little as 3-24 hours. Their maximum ingestion rate is about 1.9 times their body carbon per day. They are active feeders both day and night (95% activity at night versus daytime).

Mortality

Ciliates face several sources of mortality:

Base mortality: About 4% per day
Starvation: Active (non-encysted) ciliates starve to extinction within 3–7 days without prey (Fenchel 1987). The model uses 20%/day at complete starvation, which represents a mixed community where some individuals encyst while others die rapidly.
Predation by Copepods, Daphnia, and rotifers: Handled in those species' code
Hypoxia: Stress below about 1.3 mg/L O2, lethal below 0.3 mg/L
Temperature extremes: Stress outside 8-30 degrees C, lethal outside 2-38 degrees C
Ammonia toxicity: Ciliates are moderately sensitive to unionized ammonia (NH3)
pH stress: Stress outside pH 5.8-9.0

When ciliates die, their biomass is split: 25% dissolves directly to DOM (cytoplasmic contents from cell lysis) and 75% becomes particulate detritus (70% of the detritus stays suspended, 30% settles). Their fecal pellets are also tiny -- 80% stay suspended in the water column rather than settling.

Nutrient recycling by ciliates

Like other consumers, ciliates excrete excess nitrogen as ammonium and excess phosphorus as phosphate. Because their prey (bacteria) is nitrogen-rich (C:N ratio of about 5:1) and their own C:N ratio is about 4.5:1, the amounts of N excreted per unit of feeding are relatively small. But their rapid feeding and growth rates mean they contribute meaningfully to the overall nutrient recycling in the system.

Anaerobic Sediment Microbes: The Diagenetic Ladder

Everything described above lives in the oxic part of the tank — the water column, the surface biofilm, and the topmost millimetres of substrate that bulk-water O₂ can reach. Below that, the substrate is anoxic: bacterial respiration consumes O₂ faster than it can diffuse down, and pore-water O₂ collapses to near zero within days of any organic matter accumulating in the soil.

Anoxic does not mean dead. It means a different community runs the show — a sequence of obligate anaerobes that respire whatever electron acceptor is left after O₂ has been used up. Each guild specialises in one acceptor, and they line up in a strict energy-yield order called the diagenetic ladder (Reddy & DeLaune 2008; Berner 1980):

   surface  ┌─────────────────────────────────────────────┐
            │  oxic zone — heterotrophs and nitrifiers    │  breathe oxygen   (top)
            ├─────────────────────────────────────────────┤
            │  denitrifiers      nitrate → nitrogen gas   │
            ├─────────────────────────────────────────────┤
            │  iron reducers     rust → dissolved iron    │
            ├─────────────────────────────────────────────┤
            │  sulfate reducers  sulfate → sulfide        │
            ├─────────────────────────────────────────────┤
            │  methanogens       carbon → methane         │  (bottom)
   bottom   └─────────────────────────────────────────────┘

The ladder is an energy ranking. Nitrate sits just below oxygen and yields nearly as much energy per electron; iron oxide and sulfate yield substantially less; and methanogenesis, at the very bottom, yields the least of all. So given a choice, every guild prefers the acceptor above it on the ladder. The result is emergent niche partitioning: in any soil layer, whichever acceptor is most plentiful decides which guild dominates, and the guilds below it idle until their own acceptor's turn comes up.

The model represents this with four anaerobic guilds, plus an inhibition cascade that holds each guild off until the rungs above it are spent.

Denitrifier — the top of the anaerobic ladder

What they do. Denitrifying bacteria (typical genera: Pseudomonas, Paracoccus, Bacillus) run the reaction nitrate + organic carbon → nitrogen gas + carbon dioxide in anoxic sediment microzones. It is the dominant biological pathway by which fixed nitrogen leaves an aquatic ecosystem — the nitrogen gas bubbles harmlessly into the atmosphere or vents as bubbles. In a planted tank with a soil substrate this is one of the major reasons nitrate stays manageable without water changes; in a sealed jar it's the only way nitrate ever decreases.

Why they're anaerobic. Denitrifiers are facultative anaerobes — they prefer oxygen when it's available (because oxygen yields more energy per electron) but switch to breathing nitrate when oxygen runs out. Because they can ride out oxygenated spells without dying, they keep a small but persistent presence in the substrate even in a well-aerated tank. The model captures this by letting denitrifiers down in the anoxic pore zone respire hard, while any sitting up in the oxygenated boundary layer just tick over on maintenance.

What they eat. They burn dissolved organic carbon — the breakdown products of the substrate's own slow decomposition — together with settled detritus. In a bare-bottom tank they take it straight from the open water; in a soil tank they draw on the organic matter dissolved in the sediment pore water, the same supply the other anaerobic guilds share. Because that pore-water supply is largely out of reach of the aerobic heterotrophic bacteria up in the water column, the denitrifiers don't have to compete with them for it.

Iron-reducer (Geobacter analog)

What they do. Iron-reducing bacteria (typical genera: Geobacter, Shewanella) run the reaction rust (iron oxide) + organic carbon → dissolved iron + carbon dioxide once nitrate is drained, using the dissolved organic carbon in the pore water and the soil's buried ferrihydrite reservoir. Each unit of organic carbon reduces four units of iron — a high ratio that lets a small population turn over a large rust reservoir over weeks.

Why they matter. Iron-reducers drive two coupled fluxes that the aquarium hobbyist sees:

Internal P-loading. As Fe(III)-oxide dissolves, the phosphate (PO₄) it had been scavenging from pore water is released back into solution. This is the dominant mechanism by which planted tanks access P reserves locked in the substrate — ~60% of soil-adsorbed P in walstad-style soils is bound to Fe-oxide surfaces, and Fe reduction unlocks it.
Soil iron supply. Fe²⁺ released into pore water diffuses upward and either re-oxidises into a fresh ferrihydrite coating (the cryptic iron cycle), gets taken up by macrophyte roots as a micronutrient, or escapes into the bulk water column where it oxidises in oxic conditions and fertilises algae and producers there.

Inhibition. Iron reducers idle while nitrate is plentiful — the denitrifiers above them hold the substrate. Only once nitrate is drawn down to a fraction of a milligram per litre does iron reduction switch on and the population start to climb.

Geochemical fingerprint. Geobacter cells carry an unusually heavy load of the iron-binding proteins (c-type cytochromes) that shuttle electrons out to rust surfaces, so their iron requirement runs about three times that of a typical microbe — one of the largest such deviations in the model.

Sulfate-reducer (Desulfovibrio analog)

What they do. Sulfate-reducing bacteria (typical genus: Desulfovibrio) run the reaction sulfate + organic carbon → sulfide + bicarbonate once both nitrate and the buried rust reservoir are drawn down. The sulfide they make is what the substrate's iron-sulfide chemistry is built around. As sulfide accumulates in the pore zone it meets dissolved iron and precipitates almost instantly as iron monosulfide — the black colour that develops in old anoxic substrates. That black layer acts as a buffer: it captures sulfide before it can diffuse up into the water column, where it would oxidise back to sulfate and, at higher concentrations, become toxic hydrogen sulfide gas.

Old-tank syndrome. Once sulfate reduction is firing, every bit of dissolved iron the iron reducers liberate gets captured by sulfide and locked into iron sulfide. Over months in a sealed organic-rich substrate, that black iron-sulfide layer can build to surprisingly high stocks. If it is suddenly exposed to the oxygen in the open water — by disturbing the substrate during a deep clean, or by aggressive burrowing — it re-oxidises explosively, releases a pulse of sulfide, and crashes pH and dissolved oxygen together. This is the textbook "old-tank syndrome" Walstad and Diana describe.

Inhibition. Sulfate reduction switches on only after both nitrate and the buried rust reservoir are largely spent. In a young soil tank full of fresh iron oxide it stays off for weeks to months; in an aged tank, where the buried oxide is mostly consumed, it becomes the dominant anaerobic process.

Geochemical fingerprint. Desulfovibrio carries a special nickel-iron enzyme that sets it apart from other anaerobes, so its nickel requirement runs about five times that of a typical microbe.

Methanogen (Methanosaeta analog)

What they do. Methanogenic archaea (typical genera: Methanosaeta, Methanosarcina) are the bottom of the ladder. With no external acceptor left to breathe, they split the leftover organic carbon directly — organic carbon → methane + carbon dioxide, in roughly equal parts. The methane then takes one of three routes:

Cryptic re-oxidation — the thin oxic-anoxic interface above the methanogenic zone hosts methane-eating bacteria that capture much of the rising methane and burn it back to carbon dioxide. This is what keeps water-column methane low in most healthy tanks.
Diffusive escape — methane that survives the trap diffuses up into the open water, where it is slowly oxidised to carbon dioxide.
Ebullition — when pore-water methane exceeds its solubility limit (around 1.4 mmol/L at room temperature), bubbles nucleate and rise straight up, bypassing both the trap and the slow diffusion path. In a sealed jar the bubbles collect in the headspace; in an open-top tank they vent to the air. The visible "Walstad bubbles" hobbyists report from maturing planted tanks come mostly from this path.

Inhibition. Methanogens get their turn only when nitrate, buried rust, and pore-water sulfate are all exhausted at once. In a planted Walstad-style tank with steady nitrate recharge from nitrification, that combination rarely arises and methanogens stay scarce — which is the correct ecology, since planted tanks seldom smell of swamp gas. In an organic-rich sealed peat substrate, with no light and no fresh nitrate, the rungs above empty out in sequence over weeks and methanogens become the dominant anaerobic process.

Geochemical fingerprint. Methanogens carry two metal-bearing cofactors found in almost nothing else — a nickel cofactor at the heart of the methane-making enzyme and cobalt-bearing coenzymes — so they need about five times the nickel and ten times the cobalt of typical biomass, making them the most cobalt-rich cells in the model.

Why the diagenetic ladder runs as a sequence, not in parallel

A fresh soil substrate doesn't host all four guilds at full activity from day 1. The ladder runs as a temporal succession, driven by which acceptor is currently abundant:

Substrate age	Dominant anaerobic flux	Ecological signature
Days 0–30	Denitrifier	NO₃ trends downward; some N₂ ebullition
Months 1–6	Iron-reducer	Pore Fe²⁺ accumulates; soil-adsorbed PO₄ unlocks; algae find more P
Months 6–24	Sulfate-reducer	FeS sediment buffer fills; pore turns black; trace H₂S smell at substrate edge
Years 1+	Methanogen	Visible bubble nucleation; Walstad's "tank breathes"

Because each guild's substrate is also competed for by the guild above it, and because the inhibition gates encode the energy-ladder ordering thermodynamically rather than as hardcoded ordering rules, the succession emerges from the chemistry rather than being scripted. A planted tank with strong nitrification gets stuck on the top rung; a sealed dark peat jar walks down the entire ladder over weeks; a tank that accidentally goes anoxic at the surface (from overfeeding) collapses fast and shows symptoms of the bottom three rungs simultaneously.

Two structural reminders for how this fits with the rest of the model:

The re-oxidation steps are chemistry, not biology. The cryptic traps at the oxic-anoxic interface (dissolved iron back to rust, sulfide back to sulfate, methane back to carbon dioxide), the precipitation of iron sulfide, and the bubbling of methane are all chemistry — they belong to the iron, sulfur, and methane stories, not to the microbes. Only the reduction step is the work of these guilds.
The four ladder guilds don't compete with the aerobic microbial loop. They feed on the dissolved organic carbon held deep in the sediment pore water, which the aerobic bacteria up in the open water can't reach, so the two communities divide the substrate by where they live. The microbial loop runs above; the ladder runs below; the only crossover is the slow upward seep of the pore products — ammonium, phosphate, dissolved iron, methane — into the water column, where the rest of the food web picks them up.

The Microbial Loop

The microbial loop is one of the most important concepts in modern aquatic ecology. It describes how nutrients that would otherwise be lost to the "dead matter" pool get recycled back into the living food web through a chain of microscopic organisms.

Here is how it works, step by step:

Something dies. An alga cell bursts, a copepod produces waste, or a bacterium gets killed by a virus. The organic matter from these events enters the pool of dissolved organic matter (DOM) and detritus.
Bacteria consume the dead matter. Heterotrophic bacteria absorb the DOM and break down suspended detritus. About 28% of what they consume becomes new bacterial biomass; the rest is respired as CO2. They also release excess nitrogen as ammonium, which algae can use.
HNF eat the bacteria. Nanoflagellates, being specialized bacterivores, consume bacteria and convert bacterial biomass into HNF biomass. They excrete excess nitrogen as ammonium, rapidly recycling nutrients.
Ciliates eat HNF and bacteria. Ciliates consume both HNF (preference 0.7) and bacteria directly (preference 1.0), converting this biomass into ciliate biomass. Rotifers also feed on HNF and bacteria at this level.
Copepods eat the ciliates and rotifers. Copepods prey on ciliates (preference 0.9) and rotifers (preference 0.5), converting their biomass into copepod biomass. The copepods are now part of the "main" food web.
The cycle repeats. When copepods die, produce waste, or excrete, their organic matter re-enters the DOM and detritus pools, and the loop starts again.

The pathway looks like this:

Dead matter / DOM ---> Bacteria ---> HNF ---> Ciliates ---> Copepods
       ^                  |           |          |    ^          |
       |                  v           v          v    |          v
       +------------- death, waste, excretion --------+--- Rotifers

Why the microbial loop matters

Without the microbial loop, a large fraction of the organic matter in the system would simply accumulate as dead material that nothing could eat. The loop recovers this energy and these nutrients and feeds them back into the living system. In the model, this pathway is essential for ecosystem stability because:

It prevents detritus from piling up endlessly
It recycles nitrogen that would otherwise be locked in dead organic matter
It provides a food source for copepods even when algae are scarce
It creates multiple pathways for nutrients to flow, making the whole system more resilient

The addition of HNF as an intermediate trophic step between bacteria and ciliates more accurately reflects real freshwater food webs, where bacteria are rarely consumed directly by ciliates without an intermediate nanoflagellate step.

The viral lysis of bacteria adds an interesting twist: when viruses kill bacteria, the bacterial contents are released as DOM, which other bacteria then consume. This creates a tight recycling loop within the bacterial population itself -- the viral shunt -- where nutrients cycle rapidly between living bacteria and dissolved organic matter without ever reaching the ciliates or copepods.

The same shunt operates on planktonic phytoplankton. When cyanobacteria, green algae, or diatoms are lysed by cyanophages, chloroviruses, or diatom viruses, their cytoplasm enters the labile DOM pool rather than sinking as detritus. Bacteria immediately consume that DOM and remineralise the released N and P, which is why a phytoplankton crash often produces a transient bacterial bloom in simulator output. Phytoplankton viral lysis is one of the largest single inputs to the microbial loop in real lakes, and frequently the proximate trigger for bloom collapse — see mortality mechanisms for the per-group rates.

A smaller but real DOM source comes from sloppy feeding: whenever any consumer eats, approximately 10% of what it ejects as "feces" actually dissolves immediately into the water as DOM rather than forming compact fecal pellets. Cell disruption during the feeding act (flagellates disrupting bacterial cells, copepods shredding algal cells) releases cytoplasmic contents that are highly labile and instantly available to bacteria. This is a real pathway in aquatic ecosystems (Møller 2005) and is modelled for all consumer species.

Additionally, when HNF and ciliates die, a fraction of their biomass dissolves to DOM rather than becoming particulate detritus (30% for HNF, 25% for ciliates). These naked cells lyse easily, and the released DOM feeds back into the bacterial uptake cycle, completing the loop.

The mycoloop: a parallel pathway through fungi

A separate pathway channels energy from refractory organic matter through fungi into the microbial food web. Aquatic fungi (primarily chytrids) produce motile zoospores (2-10 um) that are consumed by ciliates and HNF. This "mycoloop" (Kagami et al. 2007) runs in parallel with the classical microbial loop:

Refractory OM ---> Fungi ---> zoospores ---> Ciliates/HNF ---> Copepods
                     |
                     v
               labile DOM ---> Bacteria ---> (microbial loop)

The mycoloop is smaller in magnitude than the microbial loop because most fungal biomass is in substrate-embedded hyphae that protists cannot ingest. But it provides a pathway for nutrients locked in refractory organic matter to reach higher trophic levels -- material that would otherwise only enter the food web after slow abiotic decomposition.