Using PNS Substrate Sauce

We all agree that it is critically important to “cycle” an aquarium before stocking sensitive animals such as fish, clams, etc. But we don’t always agree about what that actually means. Arguably, those who talk of the nitrogen cycle simply as the conversion of “toxic” ammonia into “harmless” nitrate (i.e. nitrification) are missing part of the picture. To the point, low concentrations of ammonia are essential and high concentrations of nitrate are deleterious. Even more to the point, nitrogen conversions in Nature truly constitute a cyclical process; there is no beginning or end. Integral to the full nitrogen cycle are the processes of denitrification which converts nitrate to biologically inert nitrogen gas (N2) and nitrogen fixation which converts N2 into biologically usable ammonia.

Recirculating aquarium systems certainly do not function like natural ecosystems. Most often, it is relatively easy to maintain safe levels of ammonia and nitrite through the establishment of nitrifying microbes. However, the byproduct of nitrification--nitrate--often climbs to extremely high levels. At even just a few parts per million, nitrate can spur nuisance algae blooms. By the time it reaches 50 ppm, it begins to interfere with the normal cellular function of most animals, thereby restricting their growth and negatively impacting their health. The surest way to control runaway nitrate accumulation is to perform regular, large water changes. For sure, this has other benefits--replenishing essential trace elements, for example. But if you’re replacing massive volumes of water just to dilute nitrate (that is, if all other parameters consistently remain within acceptable ranges), then maybe some more targeted means of nitrate removal would be far less costly in terms of both money and time.

And what of the introduction of ammonia? Don’t most of us “dose” more than enough ammonia through the feeding of our oversized populations of fish? Maybe… But nitrogen fixation might be a preferable means of supplying ammonia, especially in a reef aquarium environment (more about that in a bit).

All of these nitrogen transformations, including assimilation, are carried out by an astonishing diversity of microbes. Few microbes, on the other hand, are capable of carrying out all the transformations. One such group of exceptionally versatile microorganisms is the purple non-sulfur bacteria (PNSB).

Grab a beverage and nestle into your comfy chair because you’re going to learn a hell of a lot about PNSB. You’ll be glad you did.

Purple planet

Purple non-sulfur bacteria are ubiquitous on Earth and for sure are quite abundant in natural coral reef environments; specifically, they are abundant in healthy coral reef environments. One survey of a typical healthy reef including (1) the sandy substrate, (2) coral surfaces in the lagoon, (3) coral surfaces in the reef crest and (4) the open water revealed that PNSB account for a large part of its microbial community. Indeed, two PNSB (namely Rhodopseudomonas and Rhodospirillum) were found to be among the 16 most abundant bacterial genera there (out of hundreds or perhaps even thousands).

Great. So, these microbes probably just find their way into most aquaria via live rock and live sand, right? Unfortunately, that is not often the case. As revealed by analyses conducted by AquaBiomics, these genera are not included in the “core microbiome” of the typical saltwater aquarium system. In fact, they do not even appear to be present in any substantial density much less in their natural densities. This is--again--because aquaria are inherently unnatural environments, skewed to favor other, competing, types of bacteria such as Vibrio, Flavobacterium, Alteromonas, Mycobacterium, etc. These frequently are aerobic copiotrophic bacteria, which flourish in the organic carbon-rich environment of the typical home aquarium. Unfortunately, numerous species from this group are known to be opportunistically pathogenic in corals.

Can one then infer that it is possible to make the aquarium environment function more naturally by restoring select PNSB genera to their natural densities? Yes, we think so--at least to some extent. There are models through which many of the potential benefits of PNSB can be illustrated. And yes, there are countless anecdotal reports of them improving aquarium animal health. Still, in staying within the the scope of our discussion on nitrogen transformation, we shall focus specifically on these microbes’ usefulness in cycling and conditioning the aquarium.

Consider the biochemical environment of a newly set up reef tank. If it is undergoing a fishless cycle, and particularly if it has been scaped with dry rock and dry sand, it is a more or less sterile environment. Consider also that if the saltwater in this system is comprised of RO/DI water and a high-quality salt mix, then it should be very close to phosphate-free to start with. Most often, we inoculate the system with bottled nitrifying bacteria (e.g. ammonia oxidizers such as Nitrosomonas and nitrite oxidizers such as Nitrobacter) along with a source of “fuel” ammonia. Then we sit back and await the development of the so-called biofilter (which can be painfully slow due to phosphate deficiency).

But it works. It has always worked. But, could we do better? As in, not change anything about this proven approach, but rather add to it? (Yeah, you see it coming…) Yes, we do believe that nitrification and indeed the entire nitrogen cycle of any habitat (whether wild or captive) is greatly enhanced by the presence of PNSB. Here’s why.

Happy together

The traditional nitrifiers we use are all obligately aerobic lithoautotrophs. In other words, they (1) live only in oxygenated environments (2) using an inorganic chemical reaction, ammonia/nitrite oxidation, to obtain energy while (3) utilizing an inorganic source of carbon (i.e. CO2) for growth. Light inhibits the productivity of both ammonia oxidizers and nitrite oxidizers (albeit to different degrees between species). They characteristically live attached to some substrate such as sand grains. While they are quite adept at performing nitrification, nitrification is itself a low-energy yielding life strategy. Therefore, nitrifiers grow at a very slow pace (with doubling times that may take as long as several weeks in the natural environment)--so, not much assimilation.

In comparison, PNSB typically are facultatively anaerobic heterophototrophs. In other words, they (1) prefer anoxic environments but can survive (and even proliferate) in the presence of oxygen (2) relying primarily on photosynthesis for energy while (3) utilizing either inorganic or (preferably) organic sources of carbon for growth. Though PNSB can survive in total darkness, they prefer illuminated environments which enable them to carry out photosynthesis. While mature cells may attach to a solid medium and form a biofilm, young cells are motile and swim around in the water column. PNSB can perform nitrification, denitrification and nitrogen fixation. In large part owing to their versatility, they are incredibly productive with doubling times as short as one hour(!)--so, lots of assimilation.

The usefulness of the commonly bottled nitrifiers is well demonstrated. Not only are these microorganisms really good at oxidizing ammonia and nitrite, but also, just as importantly, they are highly competitive against other microorganisms. They thus form populations that persist (without need of ongoing inoculation) over the long term. That all being said, there are several distinct advantages to using PNSB alongside the nitrifiers when cycling new systems.

PNSB enhance the nitrogen cycling capacity of a captive system by complimenting the activity of nitrifiers. For example, because they prefer illuminated anaerobic environments, they colonize areas that nitrifiers cannot. Unlike nitrifiers, they aggressively consume organic waste (and in so doing compete directly with many potentially pathogenic copiotrophs). Also, PNSB remove far more nitrogen through assimilation due to the fact that they grow much faster and have a planktonic life stage; some free-swimming individuals are exported via protein skimming and some are recycled by predation (the assimilated nitrogen is passed up the food chain as the PNSB are consumed by bacterioplanktivores such as corals, copepods, sponges, tunicates, etc.). Better yet, unlike the nitrifiers, PNSB curb the rising nitrate levels via denitrification.

Perhaps best of all, PNSB perform nitrogen fixation, most of which occurs within the corals which take them up as endosymbionts. Here, the bacterium takes up organic wastes from the coral and zooxanthellae and in return supplies the zooxanthellae with a bit of extra ammonia (their preferred form of nitrogen). In the wild, corals thrive in nitrogen-poor environments; here they are safe from overgrowth by benthic algae yet do not starve for nitrogen thanks to the tiny but limitless ammonia factories living inside of them. Can PNSB produce too much ammonia? Good question, but the answer is nope. They can sense ammonia in their environment; if it is present, they shut down their nitrogen-fixing machinery and simply start snatching ammonia from the water. What this means is that they actually help to regulate ammonia availability from within the coral, maintaining low but steady internal concentrations. Pretty cool, right? The idea here is to replicate Nature, where reef waters are extremely nitrogen-starved whilst corals maintain their own, higher, optimal levels of biologically available nitrogen internally thanks to these symbiotic microbes.

The perfect start

PNS Substrate Sauce, which contains both Rhodopseudomonas and Rhodospirillum, was developed to best meet the needs of aquarists and aquaculturists when cycling and conditioning new systems. This product allows the keeper to inundate the system with these bacteria as to (1) promote faster cycling and also to (2) give the PNSB a competitive edge that helps ensure that they establish lasting populations.

The live PNSB in Substrate Sauce are cultured under conditions that specially adapt them for cycling right out of the bottle. For one, they are cultured in a saltwater medium; this shortens the lag period undergone before they enter exponential growth phase when used in marine aquaria (PNS ProBio is also suitable for marine use, but takes slightly longer to “wake up” in marine aquaria because it is cultured in a freshwater medium). Also (in contrast to ProBio with is grown on organic sources of nitrogen), Substrate Sauce is grown on ammonia salts; not only does this adapt the bacteria to utilize inorganic nitrogen sources, but also shuts down their nitrogen-fixing machinery (no need to create ammonia in an environment where the aquarist has purposefully dosed a bunch of ammonia to initiate the cycling process, right?).

One unique feature of Sauce is that it contains a culture medium that purposefully includes an excess of phosphate. In other words, these bacteria are cultured in an environment where they stop growing after consuming all the ammonia but before they’ve consumed all of the phosphate. Thus, at full dose, this product raises PO4 concentrations by approximately 0.05 ppm. This nutrient boost serves to accelerate microbial growth, not just of the PNSB, but also of the nitrifiers and indeed of all beneficial microbes that you have seeded (every organism requires phosphorus for survival and growth). Provided that there there are no other major inputs of phosphorus into the system, you will observe PO4 decrease to undetectable levels as the bacteria assimilate phosphorus into their biomass. Some PO4 remains locked up in the microbial loop, while some get exported along with bacterial biomass via water changes and protein skimming. In this respect, Sauce ramps up your phosphorus cycle as well as your nitrogen cycle!

Planting the seed

Due to the apparent inability of PNSB to compete effectively against copiotrophs in the captive environment, it may be necessary to add booster doses (ProBio is typically used for this purpose post-cycle). Since every aquarium system has its own unique physical, chemical and biological environment, it’s impossible to say exactly what long-term dosing plan, if any, will be need to maintain a dense population of PNSB. Still, there is one thing you can do to help these bacteria seed the substrate successfully. The first thing is to add (if possible) the first dose directly to the rock, sand and/or biomedia when first building the scape. This is as simple as, say, soaking a dose into your MarinePure block just before you drop it in your sump. This extra step assists the cells in locating a suitable (e.g. anaerobic) place to live and hopefully reproduce. The higher concentration of cells also helps them to fight off competitors!

In case you’re wondering… One 16-oz. bottle of PNS Substrate Sauce treats 400 gallons. That could be one dose for a 400-gallon tank, two doses for a 200-gallon tank, and so on. How many doses are best? Frankly, the best answer to that (just like with nitrifying bacteria) is as many as possible. Of course, the first dose (as with the nitrifiers) is by far the most important. But adding more bacteria, as well as giving the system more time to “settle in” and stabilize, will reward you handsomely when you begin stocking animals. Sauce may be added at the same time that you add your nitrifiers. After the first dose, you may add a second when PO4 has dropped to near undetectable levels. You can do the same for a third dose, and then a fourth, etc. as desired until excess nitrate has also dropped to very low levels. So, in other words, the ideal method is to re-dose as PO4 drops to low levels, and to keep re-dosing until nitrate reaches low levels.

What about lighting during all of this? The early part of the cycle at least should be carried out under dark conditions (remember that nitrifying bacteria are generally inhibited by light, and that PNSB can easily live without light). We would suggest leaving the lights off (and not even turning them on briefly) for at least the first couple of days after adding the nitrifiers; this will give them ample opportunity to colonize suitable microhabitats without undue stress. To avoid causing a major algal plague, it may be better to keep the lights off at least until nitrate levels have dropped considerably. When you do start running the lights, run them at their lowest levels and only gradually increase levels over the course of a few days to a week. Even once you are running the lights at the intensity and photoperiod you’ll use when you introduce corals, we suggest letting the inevitable “uglies” stage run its course before you stock your first coral.

Conclusion

Sure, there are many different ways that one could build a successful reef tank. This certainly applies to choice of cycling method. We’d be the first to admit that there is no magic potion that reliably cycles a system in an instant. Really, rather than any particular product, your best asset when cycling a new aquarium is patience. This applies to the denitrifying stage even more than the nitrifying stage. That all being said, the purple non-sulfur bacteria in PNS Substrate Sauce are an incredibly useful tool when it comes to preparing a safer and healthier environment for all of your livestock. And, if you can establish a productive permanent population of PNSB, they will continue to protect your investment and reduce the need for intensive maintenance (such as aggressive water exchange) for years and years down the road!

PNS Substrate Sauce is available exclusively through AlgaeBarn and participating LFSs.