Waste Away: Is it really bacterial? Or chemical? What does it do?

[taricha]

Really curious.......had a bad case of dinoflagellates. On Reefdudes youtube there was a protocol to follow that was very specific that involved Dr. Tim's and ATM, plus vodka dosing and some hydrogen peroxide. Worked perfectly....... so it really makes this so confusing. It works, it doesn't work and everyone is clueless. But the tests above was great to see and much appreciated.....

Yeah. Apologies for being unclear. I Absolutely think it does work - by which I mean the final results will be in line with most people's expectations: bacterial growth, consumption of nutrients, maybe local nitrogen stores depleted and nuisances growth less prominent. But it's the mechanism for HOW that is not what people expect.

I tested this same protocol you mentioned and made a thread to try to dissect it and analyze if and why it works (to figure out what tricks can be portable to other treatment protocols.)
https://www.reef2reef.com/threads/analyzing-a-bacterial-method-for-dinoflagellates-and-cyano.635165/
That's what Dan was referencing here...

I believe all this started with your investigation of a substrate cleaning protocol. Does this protocol which is based on WasteAway and carbon dosing need a reinterpretation in light of these findings?

And the answer is yes, there is a re-interpretation that makes sense in light of what's been said here. Put these two threads together and my conclusion is this:
The way the bacterial method works is to add huge carbon dose of multiple types (vodka+WA).
Bacteria (that are already in the aquarium system) multiply rapidly and consume N and P to go along with their C uptake.
Much of this Carbon consumption happens at the substrate, and therefore large quantities of nutrients (especially N) in the substrate get consumed by the bacterial bloom.
[In addition to the nutrients, there is likely a low O2 effect that also disrupts the nuisances at the substrate. You can aerate water, but massive consumption of organics at the sand surface layers will push moderate oxygen layers low, and push low O2 layers toward anoxic conditions. And yes, though dinos make a lot of oxygen in the day, low oxygen levels at night probably affect them negatively.]

I believe that the nitrogen stores in the substrate that drive nuisance growth are consumed by bacteria that are exported, and are denitrified out of the system, and changed in form away from the preferable types for nuisances, and simply redistributed elsewhere in the tank by bacterial uptake and activity.
That last sentence hasn't been demonstrated yet...

 

[Charley]

Yeah. Apologies for being unclear. I Absolutely think it does work - by which I mean the final results will be in line with most people's expectations: bacterial growth, consumption of nutrients, maybe local nitrogen stores depleted and nuisances growth less prominent. But it's the mechanism for HOW that is not what people expect.

I tested this same protocol you mentioned and made a thread to try to dissect it and analyze if and why it works (to figure out what tricks can be portable to other treatment protocols.)
https://www.reef2reef.com/threads/analyzing-a-bacterial-method-for-dinoflagellates-and-cyano.635165/
That's what Dan was referencing here...

And the answer is yes, there is a re-interpretation that makes sense in light of what's been said here. Put these two threads together and my conclusion is this:
The way the bacterial method works is to add huge carbon dose of multiple types (vodka+WA).
Bacteria (that are already in the aquarium system) multiply rapidly and consume N and P to go along with their C uptake.
Much of this Carbon consumption happens at the substrate, and therefore large quantities of nutrients (especially N) in the substrate get consumed by the bacterial bloom.
[In addition to the nutrients, there is likely a low O2 effect that also disrupts the nuisances at the substrate. You can aerate water, but massive consumption of organics at the sand surface layers will push moderate oxygen layers low, and push low O2 layers toward anoxic conditions. And yes, though dinos make a lot of oxygen in the day, low oxygen levels at night probably affect them negatively.]

I believe that the nitrogen stores in the substrate that drive nuisance growth are consumed by bacteria that are exported, and are denitrified out of the system, and changed in form away from the preferable types for nuisances, and simply redistributed elsewhere in the tank by bacterial uptake and activity.
That last sentence hasn't been demonstrated yet...

It seems that you do not believe that WA is bacteria. That's fine and your tests certainly suggests the same. But what is troubling is that on the label it is bacteria of some sort that should be in the bottle, not that is supercharging bacteria that already exists in the tank. Is it possible that your microscope simply cannot pick up the bacteria?

When I used Dr. Tim's protocol to get rid of dinoflagellates using his products only, it did not work for at all. Only Cruz's protocol worked. First week almost all gone, I was worried but second week it completely knocked it out. Thank goodness because everything else failed...blackouts, etc

 

[Charley]

Further thought......if you scoped another product that uses bacteria would it be the same issue. No bacteria detected just particles floating around. Might be interesting to compare to Dr. Tims

 

[taricha]

Good Q's @Charley
I'm going to pass on whether I think there's actual bacteria in the bottle. I can't prove the absence of bacteria. I did try to do tests that recreate expectations for what bacteria in the product would be capable of doing, and found no evidence of bacteria from the bottle doing those things, but plenty of bacteria from tank water did behave as expected.

Not seeing bacterial wiggles under the microscope is extremely weak evidence, and my scope isn't great. Totally easy to fool myself with that. The stronger evidence is that the boiled product does not have measurably less bacterial activity than the raw product under any test I can find.
I don't at all think that's exclusive to waste away, and I would love to see how many "bacterial" products work exactly the same if you boil them first to sterilize.

Doesn't mean they don't work. It just means they work differently than assumed.

[BTW. Read the bottles of Waste Away and One and Only side by side. They discuss live bacteria very differently.]

 

[K7BMG]

Following

 

[Victoria M]

Wow, @taricha. I am just amazed at the depth and effort of this experiment and your write up. As a nurse I have enough chemistry and laboratory testing knowledge to know I am just gonna focus on the taking care of the people side of things. ;Hilarious But I love the science behind what I do for a living and in this hobby. As you may remember I LOOSELY followed the elegant corals treatment and was very happy to report it worked on the amphidinium I otherwise could not beat.
BTW they are showing up a little bit again though so as soon as the weather warms I am gonna do that protocol again.

 

[taricha]

So What DOES Waste Away Do?
Most of my posts in this thread have been showing that bacteria in Waste Away bottle seemingly don't do anything measurable, but just as importantly - the media in the WA bottle does a lot.

So here's a demo of what WA (a carbon-rich media) does in terms of nutrient reduction and comparing that with a straight carbon source, vodka.

I started with a gallon of tank water, added Fritz F/2 media at half strength and half a salifert scoop of crushed fish flake then bubbled the result for a day or two to get the water nutrients semi-stable.

Starting nutrient level: 51ppm NO3, 2.61ppm PO4
I then split it into 3 bottles that I bubbled continuously in the dark. Control got nothing, 2nd got double recommended doses of Waste Away, and the 3rd bottle got vodka that was 1/6th of the WA (approximately carbon equivalent based on earlier O2 consumption measurements)

So here's what it looks like

Stars indicate which days WA and Vodka were added.
The control became more Carbon Limited around day 5 and nutrient reduction slowed a lot.
The Waste Away and Vodka treatments behaved really similarly to each other. In Phosphate, after day 6, they stayed within about one test error of each other, though the WA was almost always the lower one.
In nitrate, the difference was a little more clear - WA was consistently lower and became (and stayed) zero/undetectable on days 11&12.

Maybe the data simply says that a little more vodka was needed to have same carbon content as the WA. But it also might suggest that Waste Away is better at lowering NO3 vs PO4 than an equivalent carbon dose. Why do I say that?
Well, a recommended dose of WA adds about 0.03ppm PO4, so my 4 double doses added ~0.24ppm PO4 total. Yet, the final WA PO4 level was the same or lower than the vodka. This indicates that more PO4 uptake is happening in the WA treatment, and therefore the NO3 uptake ought to also be higher, as the data appears to indicate it is.

Where did the nutrients go?
Biomass, mostly.
Although some N and C gasses off, most went into cells.
Every day I "skimmed" the bottles by submerging an airstone for 10 minutes and putting a paper towel at the bottle opening to absorb the foam that rose up the bottle neck. This removed a few tenths up to a mL every day. So some nutrients left by skimming.
But the biggest place the nutrients went was into bacterial growth.

This colorless filamentous fuzzy bacterial stuff was at the bottom of each bottle, and more at the bottom of the WA and Vodka bottles than the control. Also the airline for each bottle grew a biofilm thick enough to make it feel really slimy. The sides of the bottles probably have similar growth.

As far as how applicable this is, the nutrient drops here might seem extreme and the doses might seem heavy and perhaps they are. But not unheard of.
The bottles got a Carbon equivalent of 0.33mL vodka per L of water spread over 10 days.
For comparison, this Bacterial Method for Dinos (and cyano) using WA and vodka that I played with before results in a carbon equivalent of 0.35mL vodka per L of tank water spread over 6 days (and half of that is added in just 2 days)

Summary: Ongoing heavy Waste Away use can be expected to reliably create low PO4 and even lower (N-limiting) NO3 levels. This is most likely the mechanism for why Waste Away can sometimes blunt nuisance growth.

 

[VR28man]

FYI, my tank crash was preceeded by a (I believe) full dose of waste away about 60 hours from the time I type this, into a tank I believe was loaded with dead/dying algae. Within 12 hours, the fish were showing signs of oxygen depletion (which I didn't correctly note :mad, 24 hours the fish were all dead, and 36 hours I had this:



Ref: this thread:

Tank Crash :(

So before my tank (29g/110L aqueon, no sump but with a was running OK, but loaded with algae and DOC, which I think I sort of had under control. Several SPS and the like were growing and had PE. Wednesday night I decided to do a massive clean out of algae on the tank walls and on rocks, to...

www.reef2reef.com

I don't "blame" waste away, per se. Mostly that I probably used too much given the circumstances; this was a case where I had not applied it in a long time (though I never had the slighest problem before), there was way more stuff than usual, and I should have used it cautiously. (or used vodka carefully, according to your experiments. )

 

[Dan_P]

So What DOES Waste Away Do?
Most of my posts in this thread have been showing that bacteria in Waste Away bottle seemingly don't do anything measurable, but just as importantly - the media in the WA bottle does a lot.

So here's a demo of what WA (a carbon-rich media) does in terms of nutrient reduction and comparing that with a straight carbon source, vodka.

I started with a gallon of tank water, added Fritz F/2 media at half strength and half a salifert scoop of crushed fish flake then bubbled the result for a day or two to get the water nutrients semi-stable.

Starting nutrient level: 51ppm NO3, 2.61ppm PO4
I then split it into 3 bottles that I bubbled continuously in the dark. Control got nothing, 2nd got double recommended doses of Waste Away, and the 3rd bottle got vodka that was 1/6th of the WA (approximately carbon equivalent based on earlier O2 consumption measurements)

So here's what it looks like

Stars indicate which days WA and Vodka were added.
The control became more Carbon Limited around day 5 and nutrient reduction slowed a lot.
The Waste Away and Vodka treatments behaved really similarly to each other. In Phosphate, after day 6, they stayed within about one test error of each other, though the WA was almost always the lower one.
In nitrate, the difference was a little more clear - WA was consistently lower and became (and stayed) zero/undetectable on days 11&12.

Maybe the data simply says that a little more vodka was needed to have same carbon content as the WA. But it also might suggest that Waste Away is better at lowering NO3 vs PO4 than an equivalent carbon dose. Why do I say that?
Well, a recommended dose of WA adds about 0.03ppm PO4, so my 4 double doses added ~0.24ppm PO4 total. Yet, the final WA PO4 level was the same or lower than the vodka. This indicates that more PO4 uptake is happening in the WA treatment, and therefore the NO3 uptake ought to also be higher, as the data appears to indicate it is.

Where did the nutrients go?
Biomass, mostly.
Although some N and C gasses off, most went into cells.
Every day I "skimmed" the bottles by submerging an airstone for 10 minutes and putting a paper towel at the bottle opening to absorb the foam that rose up the bottle neck. This removed a few tenths up to a mL every day. So some nutrients left by skimming.
But the biggest place the nutrients went was into bacterial growth.

This colorless filamentous fuzzy bacterial stuff was at the bottom of each bottle, and more at the bottom of the WA and Vodka bottles than the control. Also the airline for each bottle grew a biofilm thick enough to make it feel really slimy. The sides of the bottles probably have similar growth.

As far as how applicable this is, the nutrient drops here might seem extreme and the doses might seem heavy and perhaps they are. But not unheard of.
The bottles got a Carbon equivalent of 0.33mL vodka per L of water spread over 10 days.
For comparison, this Bacterial Method for Dinos (and cyano) using WA and vodka that I played with before results in a carbon equivalent of 0.35mL vodka per L of tank water spread over 6 days (and half of that is added in just 2 days)

Summary: Ongoing heavy Waste Away use can be expected to reliably create low PO4 and even lower (N-limiting) NO3 levels. This is most likely the mechanism for why Waste Away can sometimes blunt nuisance growth.

Nice work as always.

I have been growing aquarium water bacteria lately, comparing oxygen consumption and ammonia production. The ratio of O2:NH3 changes over the coarse of the experiment. Just wondering how the ratio of phosphate to nitrate decline behaved throughout your experiment? How did the ratio compare across treatments? I was too lazy to extract the numbers from your plots, so I ask

 

[taricha]

FYI, my tank crash was preceeded by a (I believe) full dose of waste away about 60 hours from the time I type this, into a tank I believe was loaded with dead/dying algae. Within 12 hours, the fish were showing signs of oxygen depletion (which I didn't correctly note :mad...

Thanks for that painful illustration.
The WA recommended dose is 10mL/10Gal and based on my oxygen demand tests, it seems to have about the O2 consumption or Carbon equivalent of 1/6 the volume of vodka.
So it may be similar to starting carbon dosing from zero by throwing 1.5 to 2 mL of vodka per 10 Gallons. It's certainly capable of causing problems for those who aren't ready to jump in with UV and heavy aeration when things start going sideways. Also it looks like you may not have a skimmer?
And you say there was more dead algal material than usual, And the sand bed was stirred up in the same time frame.
Lots of factors decreasing the oxygen. yikes!

 

[taricha]

The ratio of O2:NH3 changes over the coarse of the experiment. Just wondering how the ratio of phosphate to nitrate decline behaved throughout your experiment? How did the ratio compare across treatments?

You were mentioning how mole ratios can be helpful, and here you're quite right. You'll like this, I think...

from this graph I posted earlier it looks like a limitation (Carbon?) happens around day 5.

So here's what it looks like

Stars indicate which days WA and Vodka were added.

So I split the mole ratios of N, P, and C reductions into before and after day 5.

	N/P drop				C/P drop			C/N drop
	Control	W.A.	Vodka		W.A.	Vodka		W.A.	Vodka
Day 0-5	25.0	26.4	24.9		52	53		2.0	2.1
Day 5-12	31.0	162	135		1870	1430		11.5	10.6

Caveat on the C ratios, I don't know how much available C was in the samples to start with. I calculated based on the C that I added. So for days 0-5 there's way more C consumed within the sample than just what I added. But after day 5, probably the Carbon I added to the bottles was most of the carbon available.

 

[Dan_P]

You were mentioning how mole ratios can be helpful, and here you're quite right. You'll like this, I think...

from this graph I posted earlier it looks like a limitation (Carbon?) happens around day 5.


So I split the mole ratios of N, P, and C reductions into before and after day 5.

	N/P drop				C/P drop			C/N drop
	Control	W.A.	Vodka		W.A.	Vodka		W.A.	Vodka
Day 0-5	25.0	26.4	24.9		52	53		2.0	2.1
Day 5-12	31.0	162	135		1870	1430		11.5	10.6

Caveat on the C ratios, I don't know how much available C was in the samples to start with. I calculated based on the C that I added. So for days 0-5 there's way more C consumed within the sample than just what I added. But after day 5, probably the Carbon I added to the bottles was most of the carbon available.

You are right, I do like these numbers. I need to dream about these ratios tonight.

This clarifies what I need to do in the next glutamine digestion experiment. I will try to find a low budget way to monitor glutamine, and also monitor ammonia, phosphate, nitrate, nitrite and oxygen consumption. This should get me pretty close to closing the loop on the mass balance. I know that I can monitor acetate, so, I might work that in later when I carbon dose. I might in the near future run the experiment in a giant syringe and push out samples every so often instead of running multiple samples so that I can sacrifice one per time period.

 

[taricha]

...So I split the mole ratios of N, P, and C reductions into before and after day 5.

	N/P drop				C/P drop			C/N drop
	Control	W.A.	Vodka		W.A.	Vodka		W.A.	Vodka
Day 0-5	25.0	26.4	24.9		52	53		2.0	2.1
Day 5-12	31.0	162	135		1870	1430		11.5	10.6

Been thinking about this some more, and putting the graphs and the data table together, here's my take aways from it.
1. Similarity of N/P reduction ratios in the first 5 days suggest the same kind of nutrient reduction was happening in each - likely biomass creation. This means nutrients in the control, the Waste Away, and Vodka samples were mostly growing biomass in this time.
2. But from the graph, the amount grown in days 1-5 was not similar. WA and Vodka grew much more biomass.
3. After day 5, the N/P reduction ratio in the control stayed basically the same as before day 5, despite the nutrient reduction dramatically slowing down even though plenty of N & P remaining. Probably still mostly growing biomass, but the carbon to do so became more scarce.
4. After day 5 the ratios with Phosphorus blew up in the WA and Vodka samples. PO4 had gone very low and didn't budge much once it got down to ~0.10 ppm.
5. The N/P, C/N and C/P ratios of the WA and Vodka samples after day 5 show that P decrease had basically stopped (PO4 quite low) while N decrease in response to Carbon dose still continued - but not at same rates or ratios as before.
6. N/P decrease ratios in WA and Vodka went from looking like biomass ("redfield-ish") before day 5 to looking nothing like cell biomass after day 5.
7. I think (5. and 6.) means that P was being re-cycled within the samples, but N was leaving the sample. So N decrease after day 5 was mostly not biomass.
8. The WA was slightly better than pure carbon dose (Vodka) at reducing N, and the fact that WA comes with a PO4 dose probably helped that after day 5 when P became scarce.

 

[taricha]

I will try to find a low budget way to monitor glutamine, and also monitor ammonia, phosphate, nitrate, nitrite and oxygen consumption. This should get me pretty close to closing the loop on the mass balance.

Ha! Just a complete tracking of all Nitrogen species from addition of organics to the end states would be daunting enough. So of course you want to do O and P too.

 

[Dan_P]

Been thinking about this some more, and putting the graphs and the data table together, here's my take aways from it.
1. Similarity of N/P reduction ratios in the first 5 days suggest the same kind of nutrient reduction was happening in each - likely biomass creation. This means nutrients in the control, the Waste Away, and Vodka samples were mostly growing biomass in this time.
2. But from the graph, the amount grown in days 1-5 was not similar. WA and Vodka grew much more biomass.
3. After day 5, the N/P reduction ratio in the control stayed basically the same as before day 5, despite the nutrient reduction dramatically slowing down even though plenty of N & P remaining. Probably still mostly growing biomass, but the carbon to do so became more scarce.
4. After day 5 the ratios with Phosphorus blew up in the WA and Vodka samples. PO4 had gone very low and didn't budge much once it got down to ~0.10 ppm.
5. The N/P, C/N and C/P ratios of the WA and Vodka samples after day 5 show that P decrease had basically stopped (PO4 quite low) while N decrease in response to Carbon dose still continued - but not at same rates or ratios as before.
6. N/P decrease ratios in WA and Vodka went from looking like biomass ("redfield-ish") before day 5 to looking nothing like cell biomass after day 5.
7. I think (5. and 6.) means that P was being re-cycled within the samples, but N was leaving the sample. So N decrease after day 5 was mostly not biomass.
8. The WA was slightly better than pure carbon dose (Vodka) at reducing N, and the fact that WA comes with a PO4 dose probably helped that after day 5 when P became scarce.

Good. This will be my starting point in designing my next study.

 

[Miller535]

I always wondered why you have to keep dosing bacteria. If there is food in the aquarium and you introduce bacteria that like the food, end of story. They reproduce and do their job. The gel product makes no sense in this light.

I have always wondered the same thing, of all the bacteria additives.

 

[Miller535]

The other day I was on a thread that was discussing a company that sells a test kit and service of testing the bacteria and microbial content in a reef tank. The owner of that company even was answering questions on the thread. I can not remember the name of that company. Would be interesting to add some of these bacteria products that we use to the sample water and have it sent away. Along with a control sample that does not have the bacteria added.

 

[Lasse]

The other day I was on a thread that was discussing a company that sells a test kit and service of testing the bacteria and microbial content in a reef tank. The owner of that company even was answering questions on the thread. I can not remember the name of that company. Would be interesting to add some of these bacteria products that we use to the sample water and have it sent away. Along with a control sample that does not have the bacteria added.

@AquaBiomics ?

@taricha Even if it have been bacteria in that bottle - i have never understand why they should outcompete the already adapted bacteria biomass in the aquarium. A biomass that is rather constant because DOC and other organic carbon source is limited. It is a total other situation when you start a new aquarium from scratch - now you have the chance to introduce new bacteria because the bus is empty

Sincerely Lasse

 

[Miller535]

@Lasse , YES, that it is. Thank you

 

[taricha]

Even if it have been bacteria in that bottle - i have never understand why they should outcompete the already adapted bacteria biomass in the aquarium.

That line of argument was always very persuasive, and in hindsight it seems blindingly obvious.

Let me turn it around another way. We add all kinds of goodies into our systems, and we keep adding it daily. We also have hundreds of strains of bacteria that come into our systems through livestock, rock, sand, algae (400 strains on average in the core aquarium microbiome see aquabiomics).
And yet, I'm going to buy a bottle of a handful of kinds of bacteria. And I hope that bottle is going to have the bacteria in it that will eat goodies that none of the other hundreds of strains in my tank every day are capable of eating.
That's a really big ask for a bottle!
oh, and also despite the fact that those bacteria in the bottle can presumably eat goodies that nobody else eats and that I'll keep adding those goodies every day, the bacteria will all die, and I'll have to get more of them.
What an unlikely proposition!