Exopolymers in Aquaculture Hatcheries: An Overlooked Factor in Hygiene and Feed Quality03 August 2015
Transparent exopolymers (TEPs) or Extracellular Polymeric Substances (EPS) are exudates of aquatic organisms that generally elude detection by traditional microscopical techniques. This review by A Joyce and S Utting describes the overlooked roles of these materials in aquaculture hatcheries.
In mesocosm and natural ecosystem research, microalgae, macroalgae and most mature marine invertebrates (e.g. molluscs, sea cucumbers, corals) contribute some quantity of EPS to the water column.
In shellfish hatcheries, bacteria in conjunction with dinoflagellate and diatom cultures are the primary contributors to EPS, while in all hatcheries (e.g. fish, crustacean and shellfish), broodstock and larvae also play a role in mucin contributions through pseudofaeces, faeces or the epidermal mucus. All such mucins can qualify as EPS on account of their macromolecular gel-forming properties.
EPS have been well-researched in marine, fresh water and estuarine environments as they are significant contributors to the conversion of dissolved organic matter (DOM) to particulate organic matter (POM), with implications for sedimentation, carbon/nitrogen cycling, and nutrient availability.
EPS have been acknowledged for their importance in aquaculture, but primarily studied for their contribution to waste buildup and suspended solids removal in recirculating culture systems.
In hatcheries, EPS are implicated in biofilm formation, but it would be a mistake to conflate them with hatchery hygiene issues, as their applicability in aquaculture is much broader and more complex.
For instance, much of the research on optimising physical and biochemical characteristics of larval feeds arguably provides only a partial description of feed quality unless exopolymers are taken into account.
The adhesive and semiochemical properties of EPS may affect:
- food preferences;
- particle sizes ingested;
- food digestibility;
- predator grazing.
EPS are important determinants of stability and quality of microalgal cultures in shellfish hatcheries, and by implication, are also important to the quality of live feeds such as copepods and rotifers used in fish hatcheries.
EPS play an important role in particle selection and transport in many grazers and filter feeders, but further research is warranted to also examine their role in development of bacterial flora in the larval gut, with implications for nutrient assimilation and pathogen resistance in both fish and invertebrates.
This review examines recent literature for ways in which EPS affect hatchery processes, with suggestions as to how further research and development of biosensor technologies for EPS have the potential to improve production processes.
Definitions of exopolymers
In marine, fresh water, and estuarine environments, both single-cell and multicellular organisms produce sticky, extracellular polymers that have been variously referred to in the literature as exudates, ectocrines, transparent exopolymer particles (TEPs), extracellular polymeric substances (EPS), and now more commonly, as exopolymers (EPS).
In this paper the authors use the most current convention of referring to them as EPS or exopolymers, although the other terms remain interchangeable.
The transparency of EPS with regard to many traditional microscopic techniques has made them elusive, even though they were detected and characterized biochemically more than a half century ago.
In this review, we examine a wide literature on EPS and their roles in aquatic systems, suggesting ways in which these understudied components of feed quality and culture systems deserve further investigation and monitoring in an aquaculture context.
Structures and biofilms formed by exopolymers
From a nutritional perspective for larvae, EPS are high in carbohydrate content but also include proteins, nucleic acids, and lipids. They generally constitute 100 times more solids/solvent (~ 10 g/L) than seawater dissolved organic carbon (DOC) (~ 10- 1 g/L), thus are buoyant forms of reduced organic carbon and other potential nutrients available to aquatic organisms.
The saccharides comprising exopolymers are typically anionic and therefore sites for adsorption of cationic nutrients, such as calcium, all-important iron , and other metal ions for extracellular enzymatic processes.
Cationic functional groups provide spatial architecture in which microbes proliferate and transfers of organic molecules take place.
Chemically, EPS contain variable amounts and kinds of monosaccharides in polymeric chains that typically constitute 20-50 per cent of the extracellular carbohydrates.
As polymers elongate, they tend to form dissolved entanglements of ~ 5-50 nm that become interpenetrating and assemble to form nanogels (~ 100-200 nm).
As concentrations increase and environmental and physical parameters come into play (e.g. ratios of hydrophilic/hydrophobic domains, topology, charge densities, conformations, solvent interactions), these nanogels anneal to form microgels (~ 3-6 μm).
The processes of assembly and annealing are in reversible equilibrium so removal, for instance by bacteria, may result in additional formation by microalgae, whereas shear or excess production may result in fragmentation and dispersion into DOM.
Nano- and microgel formation by this relatively low-energy physical process is distinct from that of chemical gels which form higher-energy covalent bonds and yield particles that are more refractory and less likely to interpenetrate as entanglements.
Unlike chemical gels, EPS cross-linkages in self-assembling gels are weak enough that changes in environmental conditions like hydration, pH, salinity, and pressure/mixing can affect their colloidal properties, hence resulting in flux between the sol and gel states. Such structural properties also affect how ‘sticky’ they are in relation to each other and substrates.
EPS originate as an encapsulating matrix around the organisms that produce them and consist of hydrophobic and hydrophilic microdomains leading to their classification as macroporous polymers.
Their phase transitions between solvated and condensed forms can alter their volume by as much as 20-fold. As porous structures, they allow for bacterial colonisation, contain pockets of enzymatic activity and can trap toxins and detritus.
When EPS become visible macroscopically (> 1mm), they have been variously described as aggregates, floc, and marine snow.
There have been numerous mesocosm and microalgal culture studies demonstrating the need for careful attention to parameters like bacterial contamination, pH, salinity, gas exchange, and densities to explain biofilms that visibly manifest as ‘slime’.
The formation of biofilms can be explained by the equilibrium between DOM and POM that is dependent upon the chemical components interacting with biophysical properties of the EPS microenvironment.
When conditions allow for proliferation of EPS in quantities, visible slime forms on tanks and piping, but several important stages of biofilm formation will occur even before this slime becomes visible.
In algal cultures, foaming and scum are often used as indicators of a need for better hygiene protocols to reduce bacterial contamination, or may be indicators that cultures have aged and should be discarded.
In spite of this awareness, there is comparatively little research on the nutritive value or requirement for invisible EPS micro- and nanogels as dietary components for larvae that are fed microalgae (e.g. shellfish), for the copepods and rotifers used as feed, or even for larval fish.
For filter-feeders, the size, stickiness and chemical composition of EPS are important factors in adhesion by lectins in the secretory mucus on gill surfaces. This, in turn, plays a role in whether particles are rejected or selected for ingestion, digestion and ultimately assimilation.
The same is likely true for zooplankton given their dietary preferences. However, there is currently very little research on EPS phase transitions and how they affect feeding behaviours or gut microflora at various life stages of hatchery organisms.
Role of EPS in hatcheries: from hygiene to feed quality
EPS are significant contributors to carbon, nitrogen, and micronutrient cycling in aquatic systems. Both microalgae and bacteria are capable of producing exudates which exist in both dissolved and particulate phases, and contain significant amounts of carbohydrate as well as proteins, nucleic acids, and lipids.
EPS are typically sticky, anionically charged aggregates of nano- and micro-gel extracellular exudates, which are not generally observable by light microscopy.
Mutualistic assemblages of bacteria may exist in the gel matrix of microalgal EPS and exhibit diffusive and adhesive interactions that vary with environmental conditions and age.
The complex microenvironment of EPS is a site of enzymatic activity, often involving sequestration of micronutrients essential for microalgal growth and/or production of substances allelopathic to competing organisms.
The full range of mutualistic, commensal and allelopathic relationships among different species of bacteria and microalgae is poorly understood, but visualization of these relationships is crucial to understanding hatchery dynamics, particularly within microalgal cultures.
Imbalances in the microbial assemblages associated with EPS aggregates may result in over- or under-production of exudates resulting in suboptimal culture conditions and even population crashes.
Toxins produced by bacteria and/or furanones produced by microalgae in response to grazers or competitors can be embedded in the porous structure of EPS aggregates.
Such toxins may elude normal hatchery filtration mechanisms associated with intake water sources but be lethal to fish and shellfish larvae. In suboptimal microalgal production systems, these toxins can be unknowingly incorporated into feeds.
Some zooplankton and filter feeders ingest and assimilate components of EPS. As a result, EPS may play a more important role in nutrition of molluscs, copepods and rotifers than previously assumed; further investigations of EPS in hatchery settings are warranted to determine when, how, and why they either contribute to, or interfere with, optimal production of quality live feeds.
EPS likely have an important role in determining the microbial flora of the larval gut, with aggregates not only implicated in feed selection and digestibility, but also transport of bacteria to regions of the gut where they can proliferate and provide immunostimulatory protection from pathogens and aid in micronutrient assimilation.
Surface biofilms contain EPS that can alter nutrient bioavailability and culture viability. In large quantities, such biofilms become a nuisance when they form macroscopic mats that foul culture apparatus.
It would be possible in future to develop smart sensors in hatcheries that are capable of continuously monitoring growth stages, nutrient availability, EPS and biofilm production, as well as more standard environmental parameters (e.g. pH, salinity, density, turbulence, metal ion bioavailability) with the possibility for biofeedback to deliver precise control of culture conditions.
Further research into the role of EPS in hatcheries is clearly needed in order to benefit from the potential utility of such biosensors.
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