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Probiotics Demystified
Above: detail of reef aquarium designed by Swiss Aquarist Iwan Lässer, who uses a hybrid probiotics approach. Below left: author Murray Camp, a Dallas aquarist.
By Murray Camp
Excerpt from the September/October 2010 issue of CORAL
THE BANE OF REEFKEEPERS has long been the control of dissolved nutrients that fuel nuisance algae growth and cause stony coral colors to shift to dull shades of brown. Large periodic water changes, using massive amounts of live rock in the display, building deep sand beds, running myriad types of filter systems, and running water through expensive amounts of chemical media have all been recommended as ways to win the battle against dissolved nitrates and phosphates. All of these tools have merit.
Over the last few years, however, many reef aquarists have embraced a more bacteria-centered approach utilizing what are termed “probiotic methodologies” to proactively manipulate the microbial communities involved in nutrient processing in their aquariums. Almost all of these methods involve providing a source of organic carbon to “feed” beneficial bacteria that thrive on nitrates and phosphates. Some of them call for the periodic inoculation of bacterial strains, and some of the more involved systems also utilize certain minerals, called zeolites, in an up-flow reactor.
In my own experiments with these systems, I have learned that there is much confusion about how these new methods work and a great deal of curiosity among the majority of aquarists who adhere to more traditional methods. Here I will try to present an overview of these systems, the basic biological science supporting their use, the added benefit of bacteria as a food source for some corals, and some of the major husbandry issues that arise.
PROBIOTICS:
WHAT ARE THEY AND HOW DO THEY WORK?
Probiotics can be broadly defined as the introduction or maintenance, or both, of live microorganisms that confer a benefit on the system in which they are applied. In human nutrition, the prime example is the consumption of yogurts with live Lactobacillus and Bifidobacterium cultures to foster healthier conditions in the digestive tract. In aquaria, the term generally applies to the application of a source of organic carbon, either with or without an inoculation of live microorganisms. The underlying concept of these systems is to enhance the populations of heterotrophic bacteria in the aquarium so that they can consume and metabolize dissolved nutrients.
Many aquarists have had noticeable success with these systems, often achieving what is commonly referred to as an “ULNS,” or Ultra-Low-Nutrient System. While ULNS is a hobbyist term, with no scientific definition, most hobbyists generally use it to describe an aquarium where dissolved nitrate and phosphate often reach levels so low they cannot be measured with commonly available hobby-grade test kits. These systems more closely approximate, but do not reach, the nutrient levels found in seawater around coral reefs.
Detail of a still-young reef designed by Sonny Harajly in Michigan using vodka dosing and Brightwell's MicroBacter7.
The benefits of a reduced nutrient system experienced by many aquarists include:
(1) Potential reduction or elimination of problem algae and other pest organisms in the display tank;
(2) Better growth in hermatypic corals where growth is otherwise inhibited by excess phosphate;
(3) More vibrant coral colors perceived as a result of increased reflection and refraction of light by underlying coral tissues that have decreased densities of zooxanthellae, especially in Acroporid and Pocilloporid corals. An additional benefit is the increased production of bacterioplankton, an important food source for many other corals and invertebrates.
Dosing of an organic carbon source is the core principle in almost all probiotic methodologies. The purpose of doing so is to raise the amount of dissolved organic carbon (DOC) in our systems, especially types of DOC that are readily utilized by heterotrophic bacteria.
Heterotrophic bacteria, in contrast to photosynthetic autotrophic bacteria, must assimilate organic carbon as a food source from the surrounding water, in addition to nitrogen and phosphorus sources, in order for cellular metabolism to occur. As a result of the growth of these organisms, complex microbial communities, called biofilms, are formed. Sources of nitrogen, in the form of nitrate ions, and phosphorous, in the form of phosphate, are readily bioavailable to these bacteria in most marine aquaria. Although subject to some dispute, it is recognized by many aquarists that reef aquaria may be carbon limited, at least in terms of types of organic carbon that are easily utilized by heterotrophic bacteria.
It follows, then, that by increasing the amount of appropriate DOC, we can encourage heterotrophic bacteria to reproduce more quickly as the carbon limitation has been removed, thus allowing a net increase in their biomass.
Stated differently, we provide the missing carbon that will allow heterotrophic bacteria to thrive, reproduce, and build structures, and in doing so, uptake nitrate and phosphate from the water column and substrate (in addition to the dosed carbon source), thereby sequestering these dissolved nutrients from the system. These organisms increase in biomass, having “locked up” dissolved nitrate and phosphate as they live and grow. Some of this bacterial biomass is subsequently removed via foam fractionization (protein skimming), or consumed by invertebrates or other microbial organisms in the aquarium. Also, the added biomass allows a significant increase in the net metabolism associated with these bacteria, including the ultimate mineralization of some dissolved nutrients into inorganic—and less problematic—forms.
End view of a reef maintained by Iwan Lasser in Switzerland, using a hybrid mix of methods and products, primarily from ZEOvit and Prodibio.
BIOFILMS
AND NUTRIENT PROCESSING
Bacterial involvement in nutrient processing is a complex and not completely understood process. We know, however, that numerous species of bacteria are involved, each playing a role, or multiple roles, at different stages of the nutrient cycles. In areas where the right conditions exist, bacteria form biofilms—communities in which many different microbial organisms generate and embed themselves in an extracellular matrix comprised of proteins, polysaccharides and other organic molecules.
The complexity and metabolic capacity of these biofilms cannot be overstated. These are not just “germs and slimy stuff,” but extremely intricate communities in which different species and strains of bacteria, single-celled microorganisms, algae, and even small multicellular animals coexist, and in which most of the nutrient cycling in marine aquaria occurs. Live rock and live sand substrates, we should recall, first helped make reefkeeping possible by providing tremendous surface areas where beneficial bacteria could colonize and create metabolically active biofilms.
In order to create robust biofilms, heterotrophic bacteria need adequate nutrition in addition to adequate substrate, specifically sources of nitrogen, phosphorus and organic carbon. Substantial metabolic processing of dissolved nutrients occurs in the biofilms, both in aerobic and anaerobic areas of these communities. Biofilms exist everywhere in an aquarium, even on the glass, submersed equipment, and on living organisms.
BACTERIAL DENSITIES
Discussions about the role of bacteria in captive marine ecosystems often include the issue of bacterial densities, and whether we already have enough bacteria in our tanks. The bacterial biomass on and adjacent to coral reefs is enormous. Benthic bacterial densities (in biofilms, sediments, in and on algae and other organisms) have been estimated as high as 100 million bacteria per cubic centimeter. Bacterioplankton densities in reef waters, although lower, are still impressive—as high as 2 million bacteria per milliliter of seawater. Some studies have suggested that heterotrophic bacteria may comprise 90 percent of bacterioplankton biomass in waters adjacent to coral reefs, at least in .2–2 micron (picoplankton) size ranges.
Although definitive studies have not yet been conducted, the widespread reported success of probiotic systems leads to a reasonable conclusion that bacterial densities in marine aquaria are not as high as in natural reef waters, although our understanding of the diversity of microbial life in the waters adjacent to coral reefs is still in its infancy.
To boost the bacterial populations in their captive reefs, aquarists have utilized multiple sources of organic carbon, with varying degrees of success. These include ethanol (usually in the form of vodka), acetic acid (usually in the form of vinegar), sucrose (table sugar), glucose (in the form of dextrose or corn sugar), ascorbic acid, and acetate. Most commercial systems probably utilize one or more of these organic carbon sources, or related substances, although exact information is difficult to verify.
The specific metabolic pathways involving these sources of organic carbon have not been identified to a significant degree, and are not completely understood. For our purposes, however, it is sufficient to understand that these sources are metabolized via multiple reactions to form precursor metabolites, which are then used by heterotrophic bacteria to build cellular and extra-cellular structures such as DNA, cell membranes, cytoplasm and biofilms, all needed in the process of bacterial reproduction and biofilm generation. Of importance to the aquarist, these processes also utilize dissolved nitrate and phosphate, the end result being that these substances are removed from the water column by bacteria. Dissolved organic carbon also may play a role in satisfying the energy budget of some marine invertebrates.
Flourishing stony corals in an aquarium kept by Greg Timms of Calgary, Alberta, Canada, using Polyp Lab probiotic products.
BACTERIOPLANKTON AS A FOOD SOURCE FOR CORALS
Bacteria constitute a major source of nutrition for some corals, including certain species in the genera Acropora, Montipora, Pocillopora, Seriatopora and Stylophora. Many other marine invertebrates, such as sponges, fan worms and tunicates, consume bacteria. Bacteria in the form of free-floating bacteria (bacterioplankton), dislodged or sheared biofilms and other bacterial aggregates, and bacteria connected with other materials, both organic and inorganic, are consumed by a wide range of corals. It some studies, it has been suggested that bacteria in these forms can satisfy over 70 percent of the nitrogen and carbon requirements of the subject corals, although our understanding of the range of species that actually digest bacterioplankton is, at best, incomplete.
The use of probiotic methods, in all likelihood, increases the availability of planktonic bacteria sources for consumption by those marine invertebrates that use bacteria as a source of nutrition. The zeolite-based systems discussed below are perhaps the most efficient at bacterioplankton production, as they require the regular “shaking” of the zeolite stones in the reactor, which shears the biofilms growing on the zeolites for subsequent transport to the display tank. These systems may also be useful in the keeping of azooxanthellate (non-photosynthetic) corals, and potentially open up a wide range of difficult-to-keep species to the dedicated aquarist.
So-called Red Spider, Candy Cane or White-lined Sponge with encrusting Parazoanthus sp. polyps in a ZEOvit system kept by Danny Myers in Quebec.
COMMERCIAL SYSTEMS
In general, commercial probiotic systems involve dosing both a bacteria source and an organic carbon source. Some systems, such as ZEOvit and Ultralith, also use zeolites in a flow-through reactor. The zeolite stones are periodically shaken in order to shear the biofilms, which are then released in the display. Zeolites are a broad class of very porous aluminosilicate minerals widely used as commercial adsorbants, composed largely of aluminum, silica and oxygen. In a reactor, they act as a media upon which biofilms develop.
Some manufacturers claim that zeolites adsorb significant quantities of ammonia and ammonium, thereby reducing the initial load on the nitrogen cycle in the aquarium. There is significant debate as to whether this claim is valid from a geochemical perspective, as certain ions adsorbed by zeolites in seawater are subject to rapid displacement by other ions. One possible, although remote, explanation is that enough ammonia compounds are adsorbed for a long enough period of time, albeit minimal, to create a higher microgradient of nitrogenous ions that enhance development of biofilms.
Regardless of these divergent schools of thought, certain zeolites have a significant capacity to function as robust bacterial media. Importantly, the actual mineral content of zeolites varies, and those sold for use in freshwater applications may not be appropriate for marine applications. Accordingly, the reader is cautioned to only use those clearly designated for marine aquarium use.
As noted above, another benefit of zeolite-based systems is their capacity to function as bacterioplankton generators via operation of the periodic “shaking” of the zeolites in the reactor, the consequent shearing of the biofilms, and the release of the resulting bacterial aggregates into the system.
I have used the ZEOvit system on multiple tanks with successful results. This system and other zeolite-based systems are targeted primarily to Acroporid enthusiasts, although the basic nutrient reduction principles have much wider application. Of all the many probiotic systems sold commercially, zeolite-based methodologies require the most diligence in application and observation, as they tend to be the most aggressive in terms of nutrient reduction. As a result, they can affect rapid and substantial changes in the bacterial dynamics of a closed aquatic system, not only with respect to the bacterial communities on the substrate, but also those residing on and in the tissues of the corals themselves. The latter populations of bacteria are involved in multiple biological processes involving the coral, including nutrient transport across tissue boundaries and zooxanthellae metabolism. Accordingly, rapid changes in those localized populations can negatively impact coral health, although the exact mechanisms at play are not fully understood. The sudden over-application of organic carbon or other system components has been repeatedly reported to cause shock to stony corals, in the form of bleaching or colony death. Fine-tuning the amount of zeolite used is part of the art of using this methodology.
POLYMER CARBON SOURCES
More recently, multiple solid products have been introduced into the market, and many users report success. Instead of dosing a liquid organic carbon source into the aquarium, these products use small beads of polymers. Some of the polymers used for these “bio-pellets” include polyhydroxyalkanoates (PHAs) and polycaprolactones (PCLs). Generally, these materials are polyesters manufactured from the process of bacterial fermentation of sugars or other organic substances, and are, ostensibly, biodegradable. These beads are typically placed in an up-flow reactor, and not only serve as a surface area for biofilm development, but also as the source of organic carbon. Heterotrophic bacteria consume the pellets, requiring periodic replenishment, typically every three to six months. With this method, the bacteria are consuming, in effect, the very material on which they build biofilms. (Image below: Julian Sprung's office lookdown aquarium with mangroves, LPS corals, and sponges, running with a reactor filled with NPX BioPlastic biodegradable polymer pellets.)
The perceived benefit of solid polymers is that these products do not require dosing of an organic carbon source to the whole system, as the carbon source is localized in the reactor. Additionally, the flow through the reactor dislodges some of the bacteria in a process similar to a zeolite reactor, although the actual amount dislodged is uncertain. Finally, use of these pellets does not require daily dosing of a carbon source, and is a viable solution to the aquarist who is not able to conduct daily tank maintenance. I have transitioned one of my mixed coral systems from a liquid organic carbon dosing system (using a mixture of ethanol, glucose, and acetic acid) to a pelletized polymer (Warner Marine’s EcoBak), and the results look promising. I have also installed a reactor using this same product, in addition to a ZEOvit reactor, on a system exclusively containing azooxanthellate invertebrates that incorporates a continuous application of various feeds.
The ultimate end-stage metabolites of these polymer products when used in seawater have not been conclusively identified, as they were initially developed for terrestrial biodegradability. Additionally, many, or perhaps most, of these products use some type of fillers.
Nevertheless, given their ease of use, more products using a solid source of organic carbon will undoubtedly come to market, as more sources of biodegradable polymers—and other solid sources of organic carbon—are investigated. It is clearly part of the future of probiotic methodologies in marine aquaria.
DO-IT-YOURSELF CARBON SOURCES
HUSBANDRY ISSUES
Given the capacity of these methodologies to drive dissolved nutrients to very low levels, unique issues arise of which the marine aquarist should be aware:
1. Maintain natural seawater parameters. It is important that abiotic water parameters are as close to natural levels as possible. The suggested levels are:
• Calcium 410–430 ppm
• Magnesium 1250–1300 ppm
• Alkalinity 6.5–7.5 dKH
• Potassium 380–400 ppm
• Salinity of 34–36 ppt
(Specific Gravity 1.024—1.026)
Perhaps the most important of these parameters is alkalinity. Many aquarists dosing organic carbon who have maintained consistent alkalinity levels above 8 dKH have reported varying degrees of tissue necrosis in scleractinian corals, especially corals in the genus Acropora, usually described as “burnt tips.” I have personally seen this effect, but the exact cause of this phenomenon is not known. Some suspect a phosphate deficiency. The prudent aquarist employing a probiotic methodology will pay close attention to alkalinity levels, especially as nutrients fall to nearly undetectable limits. Additionally, be aware that probiotic systems may cause a more rapid depletion of alkalinity levels than you may have seen in normal calcification processes.
2. Discontinue UV sterilization and ozone applications when using these systems. Almost all commercial probiotic systems recommend the discontinuance of UV sterilization and ozone on the basis that these applications will adversely impact the bacterial populations that the probiotic methodology is attempting to increase. With respect to UV sterilization, this reasoning may seem suspect, as the UV applications would not directly impact the biofilms and other benthic bacterial communities on the substrate. Nevertheless, ozone and UV applications are not recommended if employing a probiotic methodology, commercial or DIY.
3. Phosphate absorbers not recommended in some systems. The use of phosphate-binding agents, such as granular ferric oxide (GFO) is not recommended in some commercial systems. As an initial matter, many find it is unnecessary as some systems have the capability to reduce phosphate to very low levels. Additionally, there is concern that rapid depletion of phosphate caused by using GFO in conjunction with some probiotic systems may result in coral tissue necrosis, presumably caused by rapidly shifting the bacterial dynamics in the areas on or adjacent to the coral tissues. Many aquarists, however, do use GFO in conjunction with DIY carbon dosing applications and do not experience these problems. If continuing to use GFO, I suggest reducing the amounts and reactor flow rates, and observing the system inhabitants closely.
4. Use and maintain a good protein skimmer. A productive protein skimmer should be used in all probiotic methodologies. Foam fractionization removes some dead bacteria prior to decomposition, and may be a significant export pathway for the nutrients that have been “locked up” by the enhanced bacterial populations resulting from organic carbon dosing, although other important nutrient export pathways are involved, as discussed. Additionally, a protein skimmer adds an additional safeguard in the event of a bacterial bloom caused by an overdose of organic carbon. If your protein skimmer performance is marginal, consider upgrading before beginning a probiotic regimen.
5. Don’t overdose organic carbon. Although less common in commercial probiotic systems than in DIY applications, overdosing of organic carbon is certainly possible. Remember, these methodologies have the capability of causing significant shifts in the bacterial dynamics in the aquarium—in the water column, on the substrate, and on and in the tissues of coral. Excessively rapid nutrient depletion can cause stress in corals. In case of a substantial overdose, a bacterial “bloom,” or “whiteout,” can occur, usually presenting as a semi-opaque or milky white change to the water. The primary concern in this instance is oxygen depletion in the water column, caused by respiration by the bacteria cells. In most instances, the tank inhabitants will survive. However, a quality protein skimmer is the best defense against catastrophic oxygen depletion. The main caveat here is to go slow. Incremental increases in the dose and careful observation are the best safeguards.
6. Do your water changes. Many aquarists have cut down on the volume of water changed during periodic water changes, as water changes are no longer used as a means of significant nutrient export. Water changes are still recommended, however, in order to address certain mineral depletions, and for other reasons, such as reduction of allelopathic metabolites released by corals and other organisms. Once dissolved nutrients are reduced to target levels, at least a 5-10% weekly water volume change is recommended. It is particularly important to match the abiotic parameters of the change water to the aquarium water, given the potential problems associated with higher alkalinity levels in some instances.
7. Beware potassium depletion. In zeolite-based systems, significant potassium depletion may become an issue. Several explanations for this phenomenon may come into play, including those involving the role that potassium ions play in regulating pH gradients along cell membranes. Regular testing of potassium is therefore indicated when using these systems. Another reported indicator of potassium depletion is faded coloration in certain Acroporids, particularly Montipora capricornis. In the event of depressed potassium ion levels (below 370 ppm), a high quality potassium supplement is recommended.
8. Watch for nitrogen limitation. Almost all of the probiotic systems described are effective at reducing nitrate in the water column. Based on my observations, some are more effective than others at phosphate reduction. To a large extent, this effect is contingent upon the extant bacterial populations in the system and other chemical balances in the system. Nevertheless, the aquarist may encounter a nitrogen limitation issue, which may subsequently inhibit the rate of phosphate reduction. If nitrogen limitation is suspected, I recommend gradually increasing feedings, or institution of an amino acid supplementation program. This process requires a careful observation and a delicate balance, however, as rapid increases in food wastes and other sources of dissolved nutrients can overwhelm the system, especially initially.
9. Maintaining sand beds and algal filters. Most zeolite-based commercial systems suggest a shallow sand bed to increase the available substrate for biofilm development. Some users with deep sand beds have reported difficulty in obtaining consistent dissolved nutrient reduction when transitioning into a probiotic system, for reasons yet to be conclusively determined. Likewise, the aquarist may experience a long-term inability to maintain macroalgae-based filtration methods due to the lack of nutrients available to the algae. If the dosages are carefully balanced, however, it is possible to keep macroalgae alive, and sustaining some growth, although the balancing will have to be relatively precise.
10. Managing smaller systems. I have used various probiotic systems, including ZEOvit, in aquaria as small as 20 gallons. In my experience, a “balanced” microbial biology is more difficult to achieve with a probiotic methodology in smaller (less than 50-gallon) systems, presumably due to a more limited amount of substrate available for biofilm development. Nonetheless, the benefits of probiotic methodology can be achieved in smaller systems with patience and careful observation. On a related note, I have found that a mixed-source DIY application (utilizing vodka, glucose and vinegar), along with periodic bacterial inoculations, gave the best, and most stable results in smaller reef aquaria, although the pelletized polymers certainly appear to be amenable to this type of application as well.
CRITICISMS OF PROBIOTICS
There is considerable scientific support for the conclusion that excess dissolved organic carbon may be associated with coral mortality on natural reefs. The amount of dissolved organic carbon that constitutes a dangerous level in a reef aquarium has not been determined.
Additionally, a common criticism of probiotic methodologies is that some pathogenic bacteria are heterotrophic. In order words, organic carbon dosing feeds the “bad” bacteria along with the “good” bacteria. There appear to be few, if any studies that suggest that pathogenic bacteria are more efficient than desirable species in the ability to uptake dissolved organic carbon. If the aquarist proceeds slowly and incrementally, the risk of coral mortality appears low.
These methodologies are also criticized as being “unnatural.” The fact that we pull marine organisms off a reef and stick them in a glass box with artificial seawater makes that entire philosophical discussion pointless in my view. The real issue is what we can do to create a captive environment that maximizes the health of the inhabitants.
Certainly, excess nutrient levels and the problems associated with them—algal overgrowth of corals, inhibition of calcification due to elevated phosphate levels, and proliferation of pest organisms, among other things—are not “natural.” Conventional wisdom often supposes that other husbandry practices, such as algal filters and related filtration techniques, are more “natural,” and hence more effective, than the active management of bacterial populations that probiotic systems foster.
That supposition is, unfortunately, misplaced.
Communities of bacteria, archaea, protists, and unicellular fungi account for most of the oceanic biomass. These organisms may be responsible for as much as 98 percent of primary production, and mediate all biogeochemical cycles in the oceans to a significant extent. On a “natural” coral reef, to use the term broadly, bacterial communities are responsible for most of the nutrient cycling, more than algal communities, not only as a result of having more biomass, but also because heterotrophic bacteria are potentially more efficient nutrient scavengers than algae due to their small size and large surface-to-volume ratio, among other factors.
The conventional “more natural is better” paradigm is therefore turned on its head.
Finally, the average hobbyist spends significant time and money in creating the best obtainable environment for his or her macroorganisms. The same effort and investment should also be directed to the most important organisms in the aquaria—the bacteria and the biofilms they build and populate.
CONCLUSION
Probiotic methodologies are not for everyone, and are not without risks. They generally require careful monitoring of abiotic parameters, and, in the case of some commercial systems at least, can be considered somewhat expensive. However, with a slow and incremental approach, and careful husbandry, these systems are an extremely effective means of controlling dissolved nutrient levels in the marine aquarium.
Murray Camp is a trial lawyer and marine aquarist with more than 15 years’ experience in reefkeeping. He speaks frequently at aquarium society events and lives in Dallas, Texas.
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