The holobiont and the invisible balance: bacteria in the marine reef aquarium.

Index

1. Introduction: the aquarium as a microbiologically active ecosystem • Ecology of the home reef

• The microbiome in closed systems

• Balance, resilience and potential microbial instabilities

2. Marine Microbial Ecology: State of the Art

• Diversity and abundance of ocean bacteria

• Roles in biogeochemical cycles, symbiosis, decomposition

• Differences between natural and captive communities

3. Functional classification of the aquarium microbiome

• Chemo-lithotrophic autotrophs (nitrifiers)

• Heterotrophs (decomposers, denitrifiers, fermenters)

• Photoheterotrophs (non-sulphurous purple bacteria)

• PAO and PSB in the phosphorus cycle • Stabilizing mixotrophs and multispecies biofilms

4. Functional localization of biochemical sequences

• Nitrogen: ammonification, nitrification, denitrification, anammox

• Phosphorus: absorption, solubilization, bonds with carbon

• Dissolved organic carbon and microbial loop

• Sulfur, iron, manganese, integrated redox micro-cycles

5. Hypoxic zones and substrate microbiology

• Diffusive Boundary Layer and O₂/Redox gradients

• Stratification of sands and porous rocks

• Functional guilds in micro-oxia and anoxia

• Bioturbation, DSB, SSB, lithomorphic and sulphur reactors

6. Key bacterial species and preferred habitats

• Summary sheets of relevant genres

• Location: rocks, column, sand, mucus, technical installations

• Community dynamics dependent on light, nutrients and flow

7. The coral microbiome

• Coral holobiont and mucus microbiome

• Protective, nutritional, immunomodulatory functions

• Dysbiosis, RTN/STN and BMC probiotics

8. Manipulating the microbiome in the aquarium

• Commercial inocula: selection criteria and ecological limits

• Carbon dosing: logic, benefits, possible risks

• Integrated formulations (bacteria + enzymes + prebiotics)

• Conventional and molecular monitoring

9. Commercial bacterial formulations: production and quality

• Selection, cultivation, stabilization of strains •

Commercial forms: freeze-dried, liquid, encapsulated, gel

• Absence of species and CFU on the label: reasons and implications

• Towards “tailor-made” consortia led by eDNA

10. Open challenges and future prospects

• Knowledge and standardization gap

• Complex ecology of inoculums and data-driven management

• RTN/STN prevention and microbiome as a bio-indicator

• Precision Microbiology and Sustainability of Aquariums

Chapter 1

Introduction: The aquarium as a microbiologically active ecosystem.

In marine aquariums, it is now accepted that a reef aquarium is not simply a container of salt water and organisms, but a biologically complex ecosystem , characterized by an intricate network of chemical, physical and biological interactions. At the center of this network, often invisible and underestimated, are benthos, microfauna, various microorganisms , and in particular bacteria , which constitute one of the main functional bases of the entire system. A reef aquarium can be considered in all respects a microbiologically driven ecosystem , in which the global balance depends on dynamic, adaptable and functionally diversified bacterial communities.

The closed ecosystem and the role of bacteria

Unlike a natural marine environment, where self-regulating processes are supported by large volumes of water and a continuous exchange of nutrients, the home marine aquarium represents a closed or, at best, semi-open system . In this context, bacteria take on an even greater importance, as they are called upon to degrade waste substances, recycle nutrients, detoxify potentially lethal metabolites and contribute to the chemical homeostasis of the water column and substrates.

The main functions performed by bacteria in the aquarium include:

• nitrification (oxidation of ammonia to nitrite and then to nitrate)
  
• denitrification (reduction of nitrate to nitrogen gas)
  
• the degradation of organic matter (proteins, carbohydrates, lipids, organic debris)
  
• the phosphorus cycle (absorption, accumulation and release of phosphate)
  
• the production of bioactive compounds and chemical signals ( quorum sensing)
  
• competition with pathogens , both in space (exclusion from ecological niches) and for resources (nutrients)

These processes do not occur in isolation, but are the result of interactions between different bacterial populations , often organised in the form of biofilms and bacterial consortia.

The microbiome as a structural component of the system

The term microbiome , although very widespread today, finds here one of its most concrete and tangible expressions.

Microbiome refers to the set of microbial genomes present in a given environment, and which describe the immense variety of organisms that participate in these natural consortia. In everyday language it is often used to refer directly to the set of microorganisms that colonize our systems , mainly bacteria, archaea, microalgae, viruses and protozoa.

In a well-functioning reef aquarium, the microbiome:

• it gradually stabilizes over time, but is never static
  
• reacts to any environmental change (changes in lighting, chemical-physical parameters, nutrients, introduction of new organisms)
  
• plays a key role in preventing dysbiosis, imbalances that can lead to disease or systemic collapse (such as RTN/STN in hard corals).

Metagenomic studies (a technique that allows the genetic analysis of entire environmental DNA) conducted on reef aquaria have confirmed that bacterial diversity is a direct indicator of system health.

More diverse and biodiverse systems are more resistant to stress, infections and imbalances. In parallel, a reduction in specific bacterial taxa – such as Pelagibacteraceae, Rhodobacteraceae or Flavobacteriaceae – has been associated, in several metagenomic studies on home aquariums, with recurrent pathological conditions , including RTN episodes in hard corals, anomalous accumulation of nutrients or collapses of the redox balance (Kelly et al., 2014).

Why Study Bacteria in Marine Aquariums Today

Microbiology applied to aquariology is no longer an exclusively experimental and marginal field, but will increasingly become a central discipline , both in the management of professional protocols of ornamental aquaculture and environmental restoration, as well as in our aquariums.

The tools we have available today (from selective cultivation, to molecular identification, to the formulation of probiotic and prebiotic products) allow for careful and conscious management of the microbiome of our systems.

The introduction on the market of products containing specific strains, selective carbon sources, prebiotics, oligosaccharides, digestive enzymes and other biological adjuvants, paves the way for an active management of the microbiome , a concept already widely used in agriculture, animal husbandry and human medicine.

Chapter 2

Marine Microbial Ecology: What We Know Today

A Brief Introduction to Modern Marine Microbiology

Marine microbiology is a fascinating discipline that studies the diversity , functions and interactions of microorganisms in ocean environments.
Although invisible, marine bacteria are the most abundant life form on Earth in terms of numbers and biomass.
Each milliliter of seawater contains 10⁵ to 10⁶ bacterial cells, with even higher concentrations in the first few meters of the water column, where groups such as Pelagibacter ubique and other highly metabolically efficient oligotrophic bacteria dominate.

These microorganisms not only colonize every marine ecological niche — from rocky surfaces to anoxic sands — but are among the main players in global biogeochemical cycles.
Their activity regulates the fluxes of carbon, nitrogen, phosphorus , sulphur and other essential elements, directly influencing primary productivity (phytoplankton), water quality and the composition of marine food webs.

Metabolisms: Who Does What in Biogeochemical Cycles

Marine bacteria can be classified according to two main physiological characteristics:

• energy source: when they use light they use the prefix “photo-“, when they use chemical reactions “chemo-“
  
• carbon source: carbon dioxide (autotrophs) or organic compounds (heterotrophs).

Type	Energy source	Carbon source
Chemo-lithotrophic autotrophs	Inorganic compounds (e.g. NH₄⁺, NO₂⁻)	CO₂
Photoheterotrophs	Light + organic compounds	Dissolved organic compounds
Heterotrophs	Organic compounds	Organic compounds
Mixotrophs	Light and/or chemical compounds	Flexible

These metabolic categories are the basis of the functionality of aquatic ecosystems , both natural and recreated in aquariums.

The presence of adequate functional diversity ensures that the major chemical cycles can be effectively supported by the system.

Microbial Roles in Biogeochemical Cycles

1. Carbon cycle:

Heterotrophic bacteria continuously degrade dissolved organic matter (DOM) and particulate organic matter (POM) , releasing CO₂ , nutrients, and secondary compounds.
This process feeds the microbial loop , a trophic pathway that recycles organic carbon not directly accessible to higher consumers, returning it to the base of the food chain, particularly to phytoplankton.

Some bacteria such as Roseobacter and Flavobacterium also contribute to the production of volatile organic compounds (e.g. DMS, dimethyl sulfide), which are important for atmospheric chemistry and cloud formation, among other things.

2. Nitrogen cycle:

• Autotrophic nitrifiers convert ammonia to nitrite and then to nitrate, all in the presence of oxygen (e.g. Nitrosomonas marina, Nitrospira).
  
  
• Heterotrophic denitrifiers reduce nitrate to molecular nitrogen (N₂), especially in hypoxic or anoxic zones (e.g. Paracoccus denitrificans).
  
  
• Anammox bacteria perform the anaerobic oxidation of ammonium with nitrite, although they are rarely dominant in aquariums (e.g. Candidatus Brocadia)

The simultaneous presence of aerobic and anaerobic communities in biofilms and sediments allows for complete nitrogen cycling.

3. Phosphorus cycle:

Phosphorus enters the system mainly through the diet and is taken up by heterotrophic bacteria and PAOs (Polyphosphate Accumulating Organisms), which store it as intracellular polyphosphate granules.

This can be disposed of from the system thanks to the combined action of carbon sources and skimming, through absorption by macro and microalgae, stabilized in sediments or in specific adsorbent resins.

It should be noted that the reactions do not always go in the direction we expect: some bacteria called PSB (Phosphate Solubilizing Bacteria) are able to resolubilize phosphates bound to mineral or organic particles, making them bioavailable.

4. Sulfur cycle:

In oxygen-poor environments , sulfate-reducing bacteria such as Desulfovibrio reduce sulfate to hydrogen sulfide (H₂S), a toxic gas that can accumulate in deep sandy substrates.

Bacteria belonging to the PPB (Purple phototrophic bacteria) category such as Rhodobacter, Rhodospirillum, Rhodopseudomonas, (…) can contribute to detoxification thanks to their ability to metabolise sulphides in photic and anaerobic environments, such as the first millimetres of sandy substrates.

Microbial Consortia and Biofilms: The Power of Cooperation

In the marine environment, bacteria rarely live in isolation.
Most microbial communities form synergistic consortia, that is, groups of species that collaborate to maximize metabolic efficiency and resistance to environmental imbalances.

Microbial consortia operate through the interaction between different mechanisms and specifically:

• biofilm construction : three-dimensional structures composed of bacteria embedded in an extracellular matrix of mucilage, polysaccharides, proteins and extracellular DNA.
  
  
• cross-feeding : trophic exchanges between intermediate metabolites and by-products (a waste nutrient produced by one bacterium is used as food by another);
  
  
• quorum sensing : coordinates collective gene expression, regulating biofilms, enzyme production, virulence and competition (chemical signals that coordinate the overall behavior of consortia)
  
  
• ecological succession : some species prepare the substrate for subsequent ones.
  
  
• cooperative biofilms : different species integrate into stable structures, exploiting internal gradients to differentiate metabolic activities
  

The biofilm allows the coexistence of species with contrasting metabolisms:

• oxygenated surface → nitrifiers (e.g. Nitrosomonas);
  
  
• deeper layers → denitrifying, fermenting, sulphate reducing;
  
  
• transition zones → PAO, mixotrophs, PPB.

This stratification creates a redox micro distribution where each of the different species of the consortium finds its niche and which allows to treat different nutrients simultaneously and maintain the chemical stability of the entire system.

In the aquarium, biofilms form on glass, live rock, technical substrates, plastic surfaces, sand and in some special cases, even on coral mucus.

Implications for modern reef aquarium keeping

If we want to delve deeper into these new concepts in aquariums, it is not only out of passion for our work, but also to help enthusiasts understand that “bacterial balance” is not limited to the presence of a few strains of chemolithotrophic organisms that are now well known, but that it requires a very broad network of species, consortia and functions.

Just as it is very important to select bacterial inocula that reflect this functional diversity, it is essential to avoid the indiscriminate use of biocides (ozone, UV, drugs, chemicals) that can disturb and unbalance useful consortia.

Every technical change significantly influences microbial dynamics.

Microbiome management must therefore be dynamic, based on continuous observation, adaptive response and understanding of natural bacterial interactions.

Chapter 3

Functional classification of bacteria in aquarium

1. Autotrophic chemo-lithotrophic bacteria: nitrifiers

Autotrophic chemo-lithotrophic bacteria are organisms capable of obtaining energy from the oxidation of inorganic compounds (mainly nitrogenous) and of fixing atmospheric carbon in reduced form (CO₂ → organic compounds).

In the aquarium, their main role is nitrification, a two-stage process:

• Ammonia (NH₃/NH₄⁺) → Nitrite (NO₂⁻) : carried out by ammonium-oxidizing bacteria (AOB) such as Nitrosomonas marina and Nitrosococcus oceani (…).
  
  
• Nitrite (NO₂⁻) → Nitrate (NO₃⁻) : by nitrite-oxidizing bacteria (NOB) such as Nitrobacter winogradskyi and Nitrospira marina (…).

There are also “comammox” bacteria (complete ammonia oxidizers), such as Nitrospira inopinata, (…), capable of carrying out both reactions.

These organisms grow slowly, require well-oxygenated environments and stable surfaces (live rocks, specific filter biomedia) to settle, but their presence is crucial to avoid toxic accumulations of ammonia and nitrite.

2. Heterotrophic bacteria: decomposers, denitrifiers and fermenters

As previously mentioned, heterotrophic bacteria obtain energy and carbon from dissolved or particulate organic matter (DOM and POM).

They are divided into three main subgroups:

• Aerobic decomposers: such as Bacillus subtilis, Alteromonas, Flavobacterium, (…), capable of degrading proteins, sugars, lipids and cellular debris. They form the basis of the transformation of organic matter: they are the ones who take care of the first phases of degradation of nutrients (the famous “meat” I always talk to you about)
  
  
• Facultative denitrifying agents: such as Paracoccus denitrificans, Pseudomonas stutzeri, (…), which under hypoxic or anoxic conditions reduce nitrate (NO₃⁻) to nitrogen gas (N₂), completing the nitrogen cycle.
  
  
• Anaerobic fermenters: such as Clostridium spp., (…), which degrade organic compounds in the absence of oxygen producing volatile fatty acids, H₂ and CO₂.

Recall that the activity of heterotrophs is strongly influenced by the availability of organic carbon and the C:N:P relationship in the system.
An excess of Carbon can quickly lead to bacterial blooms and redox imbalances.

3. Photoheterotrophic bacteria: non-sulfur purple bacteria (PPB)

Non-sulfur purple bacteria are a group of microorganisms capable of using light as an energy source (phototrophy) in combination with organic carbon (heterotrophy). Representative examples are Rhodobacter sphaeroides and Rhodopseudomonas palustris, whose metabolisms are well studied and documented in the scientific literature, although in reality a considerable number of species may be involved.

These bacteria colonize well-lit but simultaneously oxygen-poor environments, such as the first millimeters of the sandy bed or shade/light transition zones. They are versatile, capable of using various organic substrates and producing bioactive compounds (vitamins, antioxidants).

They contribute to the reduction of nutrients, detoxification of the substrate and microbiological stability.

PS: They are great in zooplankton cultures, refugiums and deep sand layers.

4. Phosphorus cycling bacteria: PAO and PSB

In nature, phosphorus is a nutrient that is not very available and often acts as a limiting factor. In aquariums, however, the situation is often reversed: if the system is not managed correctly, it is easy to observe a gradual and continuous accumulation.

As mentioned in chapter 2, bacteria involved in its regulation are divided into:

• PAO (Polyphosphate Accumulating Organisms) : such as Candidatus Accumulibacter phosphatis, (…), which absorb orthophosphate (PO₄³⁻) and store it as intracellular polyphosphate, acting as a buffer against variations in availability.
  
  
• PSB (Phosphate Solubilizing Bacteria): such as Pseudomonas fluorescens, (…), capable of solubilizing phosphorus bound to organic particles or complexes, making it assimilable by other organisms.

Their activity is essential for regulating phosphate levels, especially in systems rich in DOM.

Chapter 4

Functional localization of biochemical sequences

In a marine aquarium , each essential element – ​​nitrogen, phosphorus, carbon, sulfur, iron and micro-trace elements – goes through biochemical sequences that depend largely on bacterial activity.

Knowing these cycles, the microbial communities that govern them but also the micro-environments in which they take place allows us to correctly interpret the variations in water parameters and the health status of the animals, intervening in a targeted way on the management of the system.

1. Nitrogen cycle

Stadium	Reaction (simplified)	Key bacteria (simplified)	Typical micro-habitat
Ammonification	Proteins → NH₄⁺	Bacillus , Alteromonas	Water column, surface biofilms
Nitrification	NH₄⁺ → NO₂⁻ ( Nitrosomonas , Nitrosococcus ) NO₂⁻ → NO₃⁻ ( Nitrobacter , Nitrospira , comammox)	Oxygenated biofilms, filter materials	Oxygenated substrates, specific biomedia, filter materials
Denitrification	NO₃⁻ → N₂ (intermediate steps NO₂⁻, NO, N₂O)	Paracoccus , Pseudomonas , Ruegeria	Hypoxic zones of sand and rocks, biofilm depth, biomedia
Anammox	NH₄⁺ + NO₂⁻ → N₂ + H₂O	Candidatus Brocadia , Kuenenia	Anoxic pocket in mature DSB, dedicated reactors

2. Phosphorus cycle

Process	Main bacteria (simplified)	Operational notes
Absorption	PAO ( Candidatus Accumulibacter , Tetrasphaera )	They store PO₄³⁻ as polyphosphate (up to 20% of dry mass).
Solubilization	PSB ( Pseudomonas fluorescens , Bacillus megaterium )	They release organic acids and phosphatases which release PO₄³⁻ from minerals or complex organic P.
Controlled release	Same PAO (shortage phase)	In the absence of external phosphate they split the granules and release it into the environment, acting as “buffers”.

• C–P Interaction A C:P ratio around 100:1 (molar) maximizes PAO uptake without overfeeding opportunistic heterotrophs.

3. Dissolved organic carbon (DOC) cycle

• Mineralization: Alteromonas, Vibrio, Shewanella, (…) convert DOM into CO₂, ammonium and phosphate.
  
  
• Biomass sequestration: During carbon dosing, heterotrophs incorporate NO₃⁻/PO₄³⁻ into new biomass; the skimmer removes bacterial particulates while reducing inorganic nutrients.
  
  
• Harmful residues: Part of the DOM is transformed into recalcitrant compounds (RDOC) that accumulate slowly – practical management consists of changing a fraction of the water, using scavenger resins or activated carbon.

4. Sulfur cycle

Phase	Bacteria	Conditions
Reduction	Desulfovibrio , Desulfobacter	Anoxic sediments, DSB > 6 cm
Phototrophic oxidation	PPB ( Rhodobacter , Rhodopseudomonas )	Surface layers of illuminated sands, low O₂
Chemo-lithotrophic oxidation	Beggiatoa , Thiobacillus	Surface of living rocks and sandy layers, oxygen-sulfide interface

• Managing the sandy bed with a gentle but constant interstitial flow prevents H₂S accumulation.

5. Iron, manganese and trace elements

• Siderophores produced by Vibrio, Marinobacter chelate Fe³⁺, making it assimilable to phytoplankton and corals.
  
  
• Fe(II)/Mn(II) oxidizing bacteria (Gallionella, Leptothrix) form oxides that adsorb phosphates and heavy metals: useful in porous substrates or in ferro-manganese filters.
  
  
• Cu, Zn, Mo cycles are less studied but influence enzyme cofactors and bacterial growth.

6. Biofilm as a “cascade reactor”

In mature biofilms, vertical gradients are established that allow opposite reactions within a few millimetres:

• From 0 to 100 microns: O₂ ~ 5–6 mg L⁻¹ (full aerobic regime) → nitrification, Fe²⁺ oxidation
  
• From 100 to 300 microns: O₂ ~ 0.2–1 mg L⁻¹ (micro-oxic) → maximum activity of PPB and PAO
  
• Greater than 300 microns: O₂ < 0.1 mg L⁻¹ (anoxic) → denitrification, SO₄²⁻ reduction, fermentation

A slow renewal of water between the pores and the exopolysaccharide matrix (EPS) ensures adequate retention times for complete transformations of the elements.

Management advice in pills

Objective	Microbial lever
Reduce NO₃⁻	Promote hypoxic zones → thick biofilms, sand > 4 cm, sulphur or controlled alcohol reactors
Stabilize PO₄³⁻	Cultivate PAO (encourage O2/C fluctuations in the reactor), macroalgae, insert iron-manganese supports, avoid excess carbon sources that inhibit PSB
Prevent H₂S	Moderate DSB thickness or manage with SSB, introduce photic PPB, ensure slow but constant recirculation within the substrates
Avoid bacterial bloom	Gradual carbon dosing, efficient skimmer, UV light only as a spot containment measure

Chapter 5

Hypoxic zones and substrate microbiology

1. The importance of hypoxic and microaerophilic zones

In a mature reef aquarium, over 90% of nutrient transformation occurs within the substrates – sand, live rock, porous materials, technical biofilms – where oxygen drops rapidly and redox < +50 mV is established.

These hypoxic micro-niches are not a management defect: they are the only place where the biochemical cycles started in the aerated portions can be closed (see Chap. 4).
The challenge is to control its thickness, porosity and turnover in order to promote useful processes (denitrification, anammox, phosphorus immobilization) and avoid accumulations of toxic metabolites (NO₂⁻, H₂S).

2. Fine stratigraphy of the substrate

Small spec: The Diffusive Boundary Layer (DBL)
Between free water and sand there is a layer of 200–500 µm in which diffusion dominates over the advective flow.

Here the oxygen drops from ~5.8 mg L⁻¹ (saturated water column, 25 °C, 35 ‰) to < 0.2 mg L⁻¹ already at 1 mm depth.

Yes, as I mentioned a few years ago, I know that this is a difficult statement to digest for a long-time enthusiast.

Vertical redox sequence

Typical substrate depth	O₂ (mg L⁻¹) / Redox (mV)	Dominant processes	Ideal retention time*
0–2 mm	5.8 → 1.6 / +350 → +150	Nitrification, oxidation Fe²⁺	seconds–minutes
2–10 mm	1.6 → 0.2 / +150 → + 50	Partial denitrification, PAO	minutes–hours
10–50 mm	< 0.2 / + 50 → – 50	Complete denitrification, anammox	6–12 h
> 50 mm	~0 / < – 50	Sulphate reduction → H₂S, fermentation	> 12 pm

*Time required for interstitial water to complete a complete cycle in the respective zones.

3. Characteristic microbial communities

Functional guild	Key Genre(s)	Optimal O₂ (mg L⁻¹ ≈ ppm)	Other key conditions	Ecosystem products/services
Deep NOBs	Nitrospira  class IV	0.16 – 0.48 mg L⁻¹	pH ≈ 8	NO₂⁻ consumption in DBL
Denitrifying	Paracoccus , Ruegeria	< 0.32 mg L⁻¹	C-org/N ratio ≥ 3	NO₃⁻ → N₂, simultaneous uptake of PO₄³⁻
Anammox	Ca. Scalindua , Brocadia	~ 0 mg L⁻¹ anoxia	Redox ≈ 0 mV, NO₂⁻ > 0.5 mg L⁻¹	NH₄⁺ + NO₂⁻ → N₂, no C required
Deep PAOs	Ca. Accumulibacter	Oscillation 0.3 → 0 mg L⁻¹	O2/anoxia alternation, low C-labile	Polyphosphate storage
Marginal PDB	Rhodopseudomonas , Rhodobacter	< 0.64 mg L⁻¹	Light 5–20 µmol phot m⁻² s⁻¹	Detox H₂S, vitamin synthesis. B₁₂
Sulphate reducers	Desulfovibrio , Desulfobulbus	~ 0 mg L⁻¹	Redox < –50 mV, SO₄²⁻ ≈ 28 g L⁻¹	They produce H₂S + CO₂
Fe/Mn oxidants	Leptothrix , Gallionella	0.2 – 1.0 mg L⁻¹  (micro-oxic)	Fe²⁺/Mn²⁺ > 0.1 mg L⁻¹	Adsorbent oxides precipitate PO₄³⁻

 

4. Bioturbation: macrofauna at work

• Benthic and/or burrowing organisms (e.g. Amblygobius, Archaster, …) create galleries and depressions, oxygenating the intermediate layers and moving up to 25% of the sediment/day.
  
  
• Detritivorous worms stir up fine particulate matter, allowing oxygen and nutrient flow into substrates and reducing the creation of sulfidic pockets.
  
  
• Molluscs and fossorial animals scrape surface biofilms and mix the first surface layers, maintaining the permeability of the sand.

Minimalist SPS tanks without burrowing macrofauna show on average, NO₂⁻-NO₃⁻ +30% and H₂S +60% in the first 6 months compared to tanks with mixed benthic populations (UNIMIB internal study 2023, n = 12).

For those who would like to learn more, we invite you to view our articles on benthos and microfauna.

Chapter One - The secret life of the reef: the roles of benthos and zooplankton.

Chapter Two - The Secret Life of the Reef: The Roles of Benthos and Zooplankton

 
5. Substrate engineering tools and management

System	Principle	Advantages	Critical issues
DSB (Deep Sand Bed)	10–15 cm sand 0.8 mm	Strong denitrification, PO₄³⁻ buffers	Risk of compaction, toxic accumulations, H₂S release if disturbed
SSB (Shallow sand bed)	3-5 cm sand 0.4-1.2 mm	Moderate denitrification, increased stability, ease of management	Reduced depth for burrowing organisms, reduced denitrification compared to DSB
Lithomorphic reactor	Slow flow fed macro-porous rocks/specific biomedia	Anammox + controlled denitrification	Biofilm clogging, flow monitoring, mandatory pre-filtration
Sulfur reactor	Elemental sulfur as an electron donor	NO₃⁻ → N₂ without carbon-dosing	Acidifies effluent, requires post CaCO₃ stage, technology not suitable for domestic aquariums

Chapter 6

Common bacterial species and their preferred habitats

After examining the ecological functions and element cycles mediated by bacteria, it is essential to identify the most relevant species in marine aquariums and natural reefs, not only to recognize them, but to understand where they live, how they act and what favors their growth.

1. Descriptive sheets of the most common bacterial species

Below is a small (but not exhaustive!) overview of the main bacterial species and genera commonly found in marine reef aquariums, selected for their ecological function and relevance. The actual variety of species present in natural ecosystems is immensely greater than that simplified here.

Species / Genus	Main function	Metabolic type	Notes
Marine nitrosomonas	Nitrification (NH₃ → NO₂⁻)	Autotroph	Slow growing; highly specialized
Nitrobacter winogradskyi	Nitrification (NO₂⁻ → NO₃⁻)	Autotroph	Common in biofilters
Marine nitrospira	Comammox (NH₃ → NO₃⁻)	Autotroph	It carries out both stages of nitrification
Paracoccus denitrificans	Denitrification	Heterotrophic	Facultative anaerobic, present in sandy bottoms
Pseudomonas stutzeri	Denitrification, PSB	Heterotrophic	Highly adaptable; degrades complex compounds
Bacillus subtilis	Decomposition, probiotic	Heterotrophic	Spore-forming, active in biofilms
Rhodobacter sphaeroides	Photoheterotrophic, probiotic, B12	Mixotroph	Useful in hypoxic photic areas
Rhodopseudomonas palustris	Photoheterotroph	Mixotroph	High capacity to degrade DOM
Candidatus Accumulibacter	PAO, PO₄³⁻ accumulation	Heterotrophic	Involved in the phosphorus cycle
Pelagibacter ubiquitous	DOC Recycling, Probiotic	Oligotrophic	Dominant in open ocean; almost absent in aquarium

The vast majority of these species never act in isolation, but within structured microbial consortia , often organized in multispecies biofilms.

2. Preferential habitats and ecological niches

Each bacterial species or group has specific environmental needs that determine its location in the aquarium:

Microhabitats	Dominant species	Environmental features
Living rocks and porous surfaces	Nitrosomonas , Nitrospira , Bacillus , Roseobacter	Moderate flow, good oxygenation, biofilm support
Water column	Pelagibacteraceae , Vibrio , Alteromonas , Flavobacterium	High exposure, DOC available, constant flow
Sandy bed (aerobic zone)	Rhodobacter , Rhodopseudomonas , Bacillus , Shewanella	Presence of light, moderate O₂, accumulated DOM
Sandy bed (hypoxic zone)	Paracoccus , Desulfovibrio , PAO , Various denitrifiers	Low O₂, reducing environment, NO₃⁻ and SO₄²⁻ available
Coral mucus	Endozoicomonas , Ruegeria, Alteromonas, Bacillus, Rhodobacteraceae	Host-specific symbiosis, highly selective conditions
Technical biofilms (pipes, filters)	Pseudomonas , Comamonas , Acinetobacter	Nutrient-rich water, continuous physical-chemical gradients

Remember that these distributions are extremely dynamic and always respond to changes in nutrition, water chemistry, light, hydrodynamics and technical management.

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Chapter 7

The coral microbiome

In reef aquaria, generally in a mature tank, much of the biomass consists of sessile invertebrates and especially corals.

These organisms are not isolated entities, but true metaorganisms : complex associations between the host animal, its photosynthetic symbionts (Symbiodiniaceae), and a network of bacteria, archaea, viruses and protists that form the coral microbiome.
This complex of intrinsically related species is called the Holobiont.

The role of bacteria associated with sessile invertebrates is as fundamental as it is still partially unexplored , but in recent years metagenomics and selective culture techniques are shedding new light on these interactions.

1. The coral slime microbiome

• Coral tissue is constantly covered with a layer of mucus consisting of glycoproteins, polysaccharides and lipids. This:
  
  
• acts as a physical and chemical barrier against pathogens and particulate matter;
  
  
• it is colonized by specific bacterial communities , which vary between coral species and between individuals;
  
  
• It serves as a niche for interaction between coral, algal symbionts and microbes.

Mucus bacterial communities are relatively stable under healthy conditions , but sensitive to thermal stress, nutrient variation and water quality. When dysbiosis (alteration of microbial balance) occurs, it opens the way to pathologies such as RTN (Rapid Tissue Necrosis) or STN (Slow Tissue Necrosis).

Among the genera commonly found in coral slime:

• Endozoicomonas – symbiont highly represented in healthy corals
  
  
• Ruegeria, Alteromonas – involved in microbial competition
  
  
• Pseudovibrio, Vibrio – can be opportunistic or pathogenic in altered contexts
  

Genus specificity is remarkable, just to give a quick example: Acropora hosts on average 50–60% Endozoicomonas, while Pocillopora presents more varied communities with an abundance of Ruegeria.

2. Microbial functions in corals

Bacteria associated with coral mucus and tissue perform protective, metabolic and regulatory functions:

• Production of natural antibiotics (e.g. tropodithietic acid, produced by Ruegeria, …), useful for example against Vibrio spp.
  
  
• Synthesis of essential vitamins (B₁₂, biotin), required by both coral and Symbiodiniaceae
  
  
• Mucus degradation and carbon recycling: bacteria such as Flavobacterium (…) recycle the DOM produced by the coral itself
  
  
• Modulation of the innate immune system by MAMPs: MAMPs are a “molecular language” between microbes and host, which allows the coral to activate the immune system, tolerate symbiotic bacteria and establish a balanced microbiome-host relationship

Function	Bacteria protagonists	Mechanism
Antimicrobial	Ruegeria , Pseudoalteromonas	Natural antibiotics (TDA, bromopyrolides, …) inhibit Vibrio  spp.
Vitamins & Co-Factors	Endozoicomonas , Flavobacterium	Synthesis of B₁₂, Biotin, Thiamine essential for octopus & algae
DOM Recycling	Alteromonas , Marinobacter	They degrade mucopolysaccharides → simple sugars re-absorbed by the coral
Reduced nitrogen	Diazotrophs ( Azospirillum , Vibrio diazotrophicus )	They fix N₂ → NH₄⁺ for the benefit of Symbiodiniaceae
Detox ROS	Rhodobacter , Shewanella	Catalase/SOD Enzymes Reduce High Radiation Oxidative Stress

3. Dysbiosis, stress and pathologies

Under stressful conditions (increased temperature, accumulation of nutrients, exposure to heavy metals or UV), the coral microbiome:

• loses functional diversity;
  
  
• sees a growth of opportunistic bacteria, often latent pathogens;
  
  
• can trigger tissue necrosis (RTN/STN), sometimes in synergy with bacterial virulence and cellular apoptosis.

Common triggers:

· Trigger	· Microbiological effect
·  NO₃⁻/PO₄³⁻ peaks	·  Vibrio dominance , Endozoicomonas decline
·  Heat stress (+2 °C/48 h)	·  Alteromonas overgrowth  opportunistic
·  Poorly filtered UV radiation, drugs and biocides	· loss of Pelagibacteraceae, increase in ROS

 

Many episodes of RTN in aquariums are associated with conditions of microbial imbalance , rather than specific pathogens. The absence or strong reduction of “key” bacteria such as Pelagibacteraceae or Endozoicomonas has been documented in affected corals.

4. Perspectives: probiotics and microbiome manipulation

From this new knowledge comes a new approach to coral health management: microbiome modulation through specific probiotics.
This approach is known as BMC – Beneficial Microorganisms for Corals , a concept borrowed from human medicine and animal husbandry.

Potential applications include:

• preventive or therapeutic bacterial baths (e.g. after RTN or invasive manipulations)
  
  
• direct inoculation into the system by selected strains (e.g. Ruegeria, Bacillus, Pseudoalteromonas)
  
  
• combined use of prebiotics , such as oligosaccharides and other similar compounds, to promote beneficial bacteria already present
  Ongoing studies (e.g. Peixoto et al., 2022) are verifying the efficacy of probiotic consortia in:
  
  
• accelerate tissue regeneration,
  
  
• prevent pathogenic colonization,
  
  
• improve response to heat stress.

Although still in the experimental stage, some manufacturers are already developing liquid or encapsulated formulations containing specific strains for coral immune support.

Chapter 8

Manipulating the Microbiome in the Aquarium

Consciously managing the microbiome in a marine aquarium is therefore not limited to letting “bacteria do their job”: today it is possible, and in many cases advisable, to actively intervene to direct the composition, functionality and stability of microbial communities.

We will now try to explain in a simplified way, the methods and logics that can be used to specifically manipulate the microbiome of a reef aquarium.

1. Inoculation of specific bacterial mixtures: capabilities and limitations

The use of products containing live bacterial consortia (in liquid suspension, freeze-dried or encapsulated) is a common practice in aquariums.
However, the actual effectiveness of these inocula depends on several factors:

• Survival and Viability: Many strains will not survive the packaging or storage phase unless properly conditioned and refrigerated.
  
  
• Ecological competition: Introduced strains must be able to compete or integrate with the existing microbiome. In mature environments, this is often difficult without targeted nutritional support.
  
  
• Environmental compatibility: temperature, salinity, substrate and nutrient availability must be suitable for the growth of the inoculated species.
  A bacterial inoculation is more likely to be successful when:
  
  
• it is repeated over time (cumulative effect)
  
  
• It is accompanied by prebiotics (oligosaccharides, carbon sources, etc.)
  
  
• It is directed towards a clear functional purpose (e.g. denitrification, anti-RTN, organic digestion).

2. Use of carbon sources (carbon dosing)

One of the most common forms of microbial manipulation is the addition of labile carbon sources to stimulate the growth of heterotrophic bacteria. The main goal here is almost exclusively the rapid reduction of NO₃⁻ and PO₄³⁻.

A small amount of easily biodegradable carbon (ethanol, acetate, or mixtures such as NOPOX or the classic VSV recipe) is introduced into the water.

The process is simple: by providing a rapid “fuel” to heterotrophic bacteria characterized by an assimilative metabolism, their growth is encouraged and consequently the assimilation of nitrates and phosphates into new biomass.

This biomass is then removed by the skimmer and the nutrients leave the system in the form of skimmed bacterial flocs.

The method is inexpensive, relatively easy to administer, and, if dosed appropriately, very effective in bringing NO₃⁻ and PO₄³⁻ back to acceptable levels.
Its very simplicity, however, hides some pitfalls.

Overdosing or dosing too quickly can trigger a bacterial bloom (with all the consequences that this entails), and the sudden abundance of non-specific organic substrates can favor undesirable opportunistic strains and upset the delicate balance of the microbiome living in coral slime.

Administration should always be started at minimal and controlled doses , with regular monitoring of nutrients and animals.

3. Combined approaches: bacteria + enzymes + prebiotics

More advanced products today include multifactorial formulations , which combine:

• selected bacterial strains
  
  
• digestive enzymes (e.g. protease, amylase, lipases) to accelerate the degradation of organic particulate matter
  
  
• prebiotic oligosaccharides (e.g. inulin, FOS, GOS, etc.) to selectively and specifically feed the desired strains

These multifactorial formulations work synergistically: enzymes make simple substrates available, prebiotics promote the growth of beneficial bacteria, and the inoculated strains colonize more effectively.

It is a more advanced approach inspired by functional microbiology , with logics similar to those used in animal husbandry, agriculture and human medicine.

4. Monitoring and managing the microbiome

Manipulating the microbiome does not mean acting blindly: an experienced aquarist can learn to interpret the system's indirect signals by observing:

• transparency and smell of water
  
  
• visible organic residues
  
  
• animal behavior
  
  
• color and growth of algae and coral
  
  
• response to carbon or probiotic administration

In the professional field, there are also molecular monitoring techniques (eDNA, qPCR, metagenomics) that allow us to identify variations in the microbiome even before visible problems appear. Fortunately, some companies in the sector are just starting to offer this type of analysis, which unfortunately is still quite expensive at the moment.

Chapter 9

Commercial formulations: production, quality, content

The growing interest in microbiome management in marine aquariums has driven the development of commercial bacterial formulations , now available in multiple forms and oriented to different purposes: nutrient reduction, disease prevention, digestion support, system maturation.

But how are these bacteria produced? What is their real effectiveness? And above all: how can the enthusiast evaluate their quality, content and safety?

1. Cultivation, selection and stabilization

Bacteria intended for commercial formulations come from two main sources:

• strains isolated from natural marine environments (e.g. reefs, sands, coastal muds)
  
  
• certified microbial collections (e.g. ATCC, DSMZ)
  
  Once selected, the strains are:
  
  
• grown under controlled conditions (nutrient media, pH, temperature)
  
  
• analyzed for purity, vitality and biochemical characteristics
  
  
• stabilized for storage (lyophilization, encapsulation, suspension in liquid supports with osmotic salts or gel)

Some producers use co-cultured strains , that is, grown together to stimulate synergistic interactions or mimic natural communities.

2. Available commercial forms

The main forms of bacteria on the market are:

Form	Advantages	Limits
Freeze-dried	Long shelf life, well-known titles, quick activation	Sensitive to humidity, less vitality if poorly stored
Liquid	Live strains, ready to use	Short shelf life, cold chain or stabilizing additives required
Encapsulated	Mechanical protection, controlled release	Higher cost, difficulty in dosage homogeneity
Colonized gels or substrates	Ready biofilms, effective in substrate maturation	Not very standardizable, variable effectiveness

Labels almost never report the number of CFU (colony forming units), and very rarely indicate the exact species.

This lack of information, however, makes it difficult to compare products and evaluate their compatibility with your system.

3. Risks of uncontrolled formulations

• Strains not adapted to salt water can upset the existing balance or simply fail to take root.
  
• In the absence of species information, there is a risk of introducing opportunistic or interfering strains into the coral slime microbiome.
  
• Many strains currently on the market have been developed for the management of industrial wastewater and are not adapted for use in reef aquariums.
  
  In short: the quality of a bacterial blend is not measured by the number of declared species and claims, but by the research behind its formulation, the balance between the different species and the adaptability to the target environment.
  
  

4. Why labels generally don't report species and CFU (and why it may be understandable!)

Many enthusiasts wonder why, unlike probiotics intended for human or livestock use, bottles of aquarium bacteria do not indicate the complete list of species or the vital load in CFU per milliliter.

There are essentially four reasons, all linked to the still “immature” and poorly regulated stage of the sector: there is not yet a specific regulatory or certification framework for bacterial products in ornamental aquaculture.

1. Intellectual Property Protection
   Companies invest time and resources to isolate marine strains that tolerate 35 ‰ salinity, pH 8 – 8.3, high osmotic pressure and, perhaps, produce useful metabolites. Publishing the detailed composition would facilitate “reverse engineering”: a competitor could easily replicate the mixture in the laboratory in a few weeks, bypassing years of R&D.
   
   
2. Dynamic mixtures and co-cultures
   Some products do not contain single purified strains, but stable co-culture communities (multilayer biofilms, bacterial bioflocs) that change slightly during fermentation. In these cases, the classic plate count underestimates the adherent bacteria or those that cannot be cultivated on standard media; declaring a “fixed” number of CFU would be unrealistic and scientifically uninformative.
   
   
3. Lack of a specific regulatory framework
   For zootechnical supplements there are FAO/WHO guidelines and legislation (e.g. EU Reg. 2015/327). In the marine ornamental sector there is not yet an entity that imposes minimum labeling standards; companies operate in a gray area where transparency is desirable, but not mandatory. Until the sector is framed and mature, variability will remain high.
   
   
4. Young market, different needs
   “Modern” reef aquariums are barely twenty years old; many formulations are under constant revision. Declaring a CFU title today and changing it with each batch would confuse the user. Some manufacturers prefer to communicate functional efficacy (“reduces nitrates in X days”, “inhibits Vibrio”, etc.) instead of taxonomic details, which would be difficult for an enthusiast to interpret.
   
   
   Looking ahead – With the spread of low-cost 16S sequencing and the growth of the market, it is likely that guidelines similar to those for veterinary probiotics will emerge within the next few years.
   
   When this happens, indicating at least the main dominant genres and a minimum viability range will become the norm;
   
   Today, however, the aquarist must rely on other evidence (reviews, brand reputation, third-party results) to make an informed choice.
   
   

5. Towards a “precision” microbiology in the aquarium

The future of bacterial formulations is in functional customization:

• bacterial consortia tailored to aquarium types (SPS, LPS, FO)
  
  
• engineered supports (zeolites, ceramics, gels) to modulate release and localization
  
  
• Microbiome analysis via eDNA and metagenomic analysis to choose the most suitable strains for each ecosystem

The most advanced lines of research are already moving in this direction, some of which also involve biotech startups and collaborative projects with oceanographic universities.

Chapter 10

Open challenges and prospects

Although, as we have seen, in recent years microbiology applied to marine aquariums has made enormous progress, numerous technical, theoretical and operational challenges remain open.

In recent years, microbiology applied to marine aquariums has moved from a descriptive phase to a functional and engineering dimension.

The growing interest in manipulating the microbiome for preventive, therapeutic and functional purposes requires an increasingly precise, integrated and documented approach.

This final chapter collects the main unresolved issues, the current limits of knowledge and future directions of development.

1. Fragmented knowledge and lack of standardization

Much of the information available on the effectiveness of bacteria in aquariums comes from empirical experience or data provided by manufacturers, often without peer-reviewed scientific publication.

Molecular testing in home systems (metagenomics, eDNA) is still inaccessible to most users , due to cost, logistics and interpretation reasons.

There is no shared standard protocol for assessing the colonization , efficacy or functionality of bacterial formulations.

The challenge lies in developing accessible and reliable methodologies to map and monitor the aquarium microbiome , even in the absence of laboratory instruments.

2. Complex and unpredictable ecological dynamics

Introducing exogenous strains into an established microbiome can produce counterintuitive effects: some species disappear, others proliferate, or nothing measurable happens.

Interactions between beneficial, symbiont and opportunistic bacteria are complex, highly influenced by environmental parameters and trophic flows.

The effect of technical tools such as ozone, UV, skimmers, chemical molecules and biocides on the microbiome is real, but difficult to quantify.

In this case, the research is aimed at understanding the conditions that favor the establishment and persistence of the introduced bacteria , and developing criteria for their selection on a case-by-case basis.

3. Prevention and management of RTN/STN

We have seen that coral mucus dysbiosis is one of the main triggers of tissue necrosis in stony corals (SPS) , but there are no standardized microbiological treatments.

Loss of microbial biodiversity or key taxa (e.g. Endozoicomonas, Ruegeria) may precede the clinical event, but the signals are still difficult to interpret operationally.

Bacterial baths or microbiome transplants (“microbiota transfer”) are promising, but require broader experimental validation.

Today, the focus is on developing effective preventive and emergency probiotic protocols, calibrated on coral physiology and tested on a multi-species basis.

4. Towards adaptive and data-driven management

Microbiological management of barrier systems must move from a “one size fits all” model to an adaptive model , based on:

• system characteristics (management, bioload, flows, coral typology);
  
  
• functional objectives (nutrients, prevention, recovery, disease control);
  
  
• empirical data collected over time (NO₃⁻, PO₄³⁻ trends, coral response, transparency, odors, etc.).

Emerging technologies (e.g. biochemical sensors, testers, controllers, continuous monitoring) may one day transform the aquarium into a partially “self-diagnostic” system.

So, to do a little recap:

The whole article aims to explain how the marine aquarium cannot be understood – nor managed – ignoring the role of the microbiome, which:

• regulates the cycles of nitrogen, phosphorus, carbon and sulfur;
  
  
• competes with pathogens for space and nutrients;
  
  
• produces vitamins, enzymes and bioactive metabolites;
  
  
• modulates the well-being of coral and other invertebrates.

Consequently, the microbiome is not a simple “biological background”, but a management lever that can be used to get the best results from your system.

The use of bacterial inocula, carbon sources, enzymes, oligosaccharides, and engineered substrates has paved the way for active manipulation of the microbiome.

However, as in any complex system:

• uncalibrated interventions can cause unwanted effects (e.g. bacterial bloom, dysbiosis);
  
  
• bacterial formulations “recycled” from other sectors can alter delicate balances;
  
  
• management must be targeted, gradual, and guided by clear parameters (nutrients, visual response, animal behavior, etc.).
  
  Future developments in the discipline are aimed at precision microbiology, with products specific to each type of tank, and molecular diagnostic supports accessible even to advanced hobbyists.
  
  In the near future, the microbiome will be increasingly used as a bioindicator of the health of a system based on precepts such as:
  
  
• low diversity = low resilience
  
  
• loss of key taxa = increased risk of disease
  
  
• excess DOM + carbon sources = risk of dominance of opportunistic heterotrophs

Bona, it was long and quite “technical” but we did it.

I hope I have clarified things a little and not bored you too much.

Stay tuned, stay salty and happy reefing to all.

PS: if you don't read the ENTIRE bibliography don't even talk to me or I won't answer you ❤️

I love you! Bye

Author(s)	Year	Title	Editorial office	DOI / URL
Allison SD, Martiny JBH	2008	Resistance, resilience, and redundancy in microbial communities	PNAS 105: 11512-11519	https://doi.org/10.1073/pnas.0801925105
Azam F., Malfatti F.	2007	Microbial structuring of marine ecosystems	Nat. Rev. Microbiol. 5: 782-791	https://doi.org/10.1038/nrmicro1747
Bourne DG, Morrow KM, Webster NS	2016	Insights into the Coral Microbiome: Underpinning the Health and Resilience of Reef Ecosystems	Annual Review of Microbiology, Volume 70, Pages 317–340	https:// doi.org/10.1146/annurev-micro-102215-095440
LaRowe DE et al.	2020	The potential for microbial life in marine sediments and the deep subseafloor biosphere.	NASA Technical Report, NTRS ID 20230002462	https://ntrs.nasa.gov/api/citations/20230002462/downloads/LaRowe%20etal%202020.pdf
Amenyogbe, Eric.	2023	Application of probiotics for sustainable and environment-friendly aquaculture management - A review.	Cogent Food & Agriculture.	9. 10.1080/23311932.2023.2226425.
Daims H. et al.	2015	Complete nitrification by Nitrospira bacteria	Nature 528:504-509
DeLong EF	2009	The microbial ocean from genomes to biomes	Nature 459: 200-206	https://doi.org/10.1038/nature08059
Fenchel T., Finlay B.J.	2008	Oxygen and the spatial structure of microbial communities	Biol. Fertile. Soils 44: 589-596	https://doi.org/10.1111/j.1469-185X.2008.00054.x
Flemming H.-C., Wingender J.	2010	The biofilm matrix	Nat. Rev. Microbiol. 8:623-633	https://doi.org/10.1038/nrmicro2415
Glasl B. et al.	2017	Microbial indicators of coral health	ISME J. 11: 1506-1519	https://microbiomejournal.biomedcentral.com/articles/10.1186/s40168-019-0705-7
Hall-Stoodley L. et al.	2004	Bacterial biofilms: from the natural environment to infectious diseases	Nat. Rev. Microbiol. 2:95-108	https://doi.org/10.1038/nrmicro821
Hernández-Agreda A., Gates RD, Ainsworth TD	2017	Defining the core microbiome in corals	Trends Microbiol. 25: 125-140	https://doi.org/10.1016/j.tim.2016.11.003
Jørgensen BB, Revsbech NP	1985	Diffusive boundary layers and the oxygen uptake of sediments and debris	Limnol. Oceanogr. 30: 111-122	https://doi.org/10.4319/lo.1985.30.1.0111
Kelly LW et al.	2014	Local genomic adaptation of reef microbiomes	PNAS 111: 10227-10232	https://doi.org/10.1073/pnas.1403319111
Levin SA	1998	Ecosystems and the biosphere as complex adaptive systems	Ecosystems 1: 431-436	https://doi.org/10.1007/s100219900037
Madigan MT et al.	2018	Brock Biology of Microorganisms (15th ed.)	Pearson	Book, DOI not assigned
Mino T., van Loosdrecht MCM, Heijnen JJ	1998	Microbiology and biochemistry of EBPR	Water Res. 32: 3193-3207	https://doi.org/10.1016/S0043-1354(98)00129-8
Peixoto RS et al.	2017	Beneficial microorganisms for corals (BMC): a new approach	Nat. Microbiol. 2: 16091	https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2017.00341/full
Prosser JI, Nicol GW	2012	Archaeal and bacterial ammonia oxidisers in soil	Nat. Rev. Microbiol. 10:701-712	https://doi.org/10.1038/nrmicro2832
McDevitt-Irwin. et al.	2017	Responses of Coral-Associated Bacterial Communities to Local and Global Stressors.	Frontiers in Marine Science.	https://doi.org/10.3389/fmars.2017.00262
Robertson LA, Kuenen JG	2006	The ecology of nitrifying bacteria  (chapter in The Nitrogen Cycle in the Biosphere )	Springer	https://doi.org/10.1007/978-1-4020-4524-2_7
Rosado PM et al.	2019	Marine probiotics: increasing coral resistance to bleaching through microbiome manipulation	Sci. Adv. 5: eaaw1022	https://doi.org/10.1038/s41396-018-0323-6
Shade A. et al.	2012	Fundamentals of microbial community resistance and resilience	Front. Microbiol. 3: 417	https://doi.org/10.3389/fmicb.2012.00417
Wegley L. et al.	2007	Metagenomic analysis of the microbial community associated with the coral Porites astreoides	https://doi.org/10.1111/j.1462-2920.2007.01383.x
Ziegler M. et al.	2019	Coral holobiont plasticity under environmental change	Nat. Commun. 10: 2537	https://doi.org/10.1038/s41467-019-10969-5
FAO/WHO	2006	Probiotics in food: Health and nutritional properties and guidelines for evaluation	FAO/WHO Report	DOI not available