What carbon sources are more selectively digestible?

[Dan_P]

It is also possible, and even likely, that any growing bacteria could be getting their N from nh4 and no2 and the no3 drop is from anoxic bacteria (or other things more willing to use no3) where less no3 is being produced.. Do not assume that anything new has to use no3 for it to drop - even the same no3 use in the tank will drop no3 if nh4 and no2 is getting consumed at a higher rate. I probably rambled, so my apologies if this is jibberish.

I will have to relook at energy and mass balances to support the notion that it takes so long to consume nitrate because the amount of acetate used for energy is much larger than biomass production and thus nitrogen uptake. Because acetate dose increase is performed so slowly, the bacteria population grows very slowly. Nitrate consumption is not detectable for many weeks.

What I am very unsure about is whether the bacteria growth is primarily doing its job consuming ammonia and denitrification is mopping up the accumulated nitrate in the water at its normal rate, or maybe a bit faster because of the presence of acetate.

The experimental design is still in the works to tease this out.

 

[Kenneth Wingerter]

Good argument, the flip side is that if I compare equal carbon of acetate to ethanol, I get less visible bacterial growth with acetate (Skimmate, film on glass). I'm reasoning that because the acetate is so widely consumed by everything, there's less uptake by bacteria.
So whether other organisms need the carbon or not, they seem to grab it when it's there in an available form.

Whoa, excellent point that hadn't yet ever occurred to me. I'd add though that not all microbes can efficiently utilize ethanol. That might be good or bad I guess, depending upon which microbes are benefiting (or not) from the addition of ethanol or any other less labile carbon source.

 

[Kenneth Wingerter]

Thinking about how to refine the question a bit...
Here's some things that are technically solutions to my question but are actually not helpful at all.
Cellulose: clearly not a usable energy source for algae, but bacteria can break it down - slowly, partially, under the right conditions.
Here's a hobby product that's not too far removed from that idea:
"Composed of an all-natural chitin substrate that mimics copepod exoskeleton fragments and coated with PNSB, Hydrospace™ PNS YelloSno™ effectively simulates a marine snowfall."

also Formate is of low digestibility to most things, but bacteria can be grown to scale up to the challenge - (one would conclude, given the long time that it takes to ramp up All For Reef.)

but I think neither of those could really be used to grow bacterial populations the way we think of for "Carbon dosing."

Maybe the best way to address this is to select a carbon source that favors the type of microbial metabolism that favors coral health. A major source of carbon on a typical coral reef is coral mucus. So, maybe heavily stocked tanks should already have what they need for this purpose (in the absence of excessive skimming, UV, ozone, etc.)? Would it be possible to replicate coral mucus as a food for microbes in new/sparsely stocked tanks (I have no idea what coral mucus is mainly composed of, or how hard it would be to 'make')? I know, this is a way out there idea, but I just had to put it out there.

If this produced lots of bacterioplankton, we might be on to something big (the bulk of a coral's planktonic diet is not phytoplankton or zooplankton, but rather bacterioplankton). https://reefs.com/magazine/the-carbon-continuum-heterotrophic-bacterioplankton-and-reef-food-webs/

 

[Hans-Werner]

Maybe @Hans-Werner can explain if he thinks this product fits the criteria you are wondering about.

It is a very intersting question indeed. The carbon source for our liquid organic carbon dosing products was found simply by experimenting. Which carbon source has the best effect to the overall tank condition without causing excessive slimy bacteria coatings?

In scientific papers the ability of bacteria to make use of and degrade certain substrates is tested and described.

A challenge for bacterial degradation usually are biopolymers like alginate and cellulose. After such biopolymers are formed they are not degraded by the same organism that formed them. When leaves of trees are shed they still contain the cellulose and lignin. It are fungi and bacteria that degrade most biopolymers. In the intestines of fish, fermenters, mainly bacteria, degrade biopolymers like alginate and cellulose to "volatile fatty acids" like acetic acid which are further degraded by other bacteria or taken up by the fish. It is similar as in ruminants. In this way these biopolymers have a prebiotic effect, supporting the growth of "good bacteria".

 

[Hans-Werner]

A major source of carbon on a typical coral reef is coral mucus. So, maybe heavily stocked tanks should already have what they need for this purpose (in the absence of excessive skimming, UV, ozone, etc.)? Would it be possible to replicate coral mucus as a food for microbes in new/sparsely stocked tanks (I have no idea what coral mucus is mainly composed of, or how hard it would be to 'make')? I know, this is a way out there idea, but I just had to put it out there.

To my knowledge coral mucus is quite rich in nitrogen and proteins. A substantial amount of the nitrogen corals take up are excreted with the slime, highlighting that phosphorus is the nutrient limiting coral growth and corals strive most for.

In his excellent book on Coral Reef Ecology, Yuri Sorokin describes coral reefs as a kind of filter for enriching phosphates, driven by the corals. Coral mucus is a kind of phosphate trap, where bacteria with superior abilities to enrich phosphate sit and feed on the mucus. Since the phosphate is enriched in the reef in this way, corals benefit from it in one way or another (bacteria, plankton, fish feces etc.).

 

[taricha]

Going the other direction from biopolymers....

attached paper: Effect of Organic Solvents on Microalgae Growth, Metabolism and Industrial Bioproduct Extraction: A Review

Such a wide review lays out a mixed picture. Most solvents mentioned can be used by microalgae as a carbon source to grow under certain circumstances, but could also inhibit some algae under some conditions. Microalgae seem ridiculously versatile in organics they can break down and use.

Compared to acetate and glucose though, these solvents are generally found less favorable for algae growth.

Some fascinating (to me) snippets from the discussion at the end...
"Contrary to methanol, which can stimulate microalgae growth only in the presence of
light, ethanol was reported to serve as a carbon source also in dark, during heterotrophic cultivation."

"Acetone, acetonitrile, hexane, DMSO and DMF did not improve microalgae growth, with one
exception for DMF [46], and were neutral and/or inhibitory at various concentrations. Hence, the presence of these solvents in industrial wastewaters would not be beneficial for microalgae growth.

Higher alcohols (Cn, n ≥ 3), also caused inhibition of microalgae growth, and inhibitory effect increased drastically, with the carbon number in the alcohol molecule [75], although Polytomella strain was reported to assimilate alcohols (C4, C5, C6) as carbon sources in dark [82]."

 

[Dan_P]

Good argument, the flip side is that if I compare equal carbon of acetate to ethanol, I get less visible bacterial growth with acetate (Skimmate, film on glass). I'm reasoning that because the acetate is so widely consumed by everything, there's less uptake by bacteria.
So whether other organisms need the carbon or not, they seem to grab it when it's there in an available form.

Another angle for us to consider. When an organism is grabbing organic carbon, let’s assume it is for growth. If it is growing, it is also grabbing nitrogen for biomass production, whether it is a heterotroph or mixotroph.

We typically do not detect nitrate reduction until feeding organic carbon in increasing amounts over many weeks, sometimes never for some. We seemingly have an odd situation of organic carbon consumption without nitrogen consumption. The problem is resolved by removing the assumption that the lack of nitrate consumption is the lack of nitrogen consumption. Also, the ratio of organic carbon for energy to organic carbon for biomass is large. There could very well be nitrate consumption we are not detecting until the organic carbon dose is of the amount needed for exponential growth. There is a way to look at biomass accumulation that might also be useful in understanding carbon grabbing with minimal nitrogen depletion.

When we feed our aquarium bacteria with a fixed dose of acetate, the bacteria population can increase. When we add the same amount of acetate again and again, the bacteria population increase slows down to a crawl. This is because there is a fixed amount of energy per dose that supports only so many bacteria. Until a higher dose is administered, a kind of stationary state develops. During this state, growth might stop, the number of deaths might equal the number of births, but essentially little new biomass accumulates. That means the consumption of nitrogen slows, maybe just enough to maintain the bacteria cells. When the larger dose is delivered, there is a growth spurt and nitrogen grab, but then the nitrogen consumption drops off again. During these punctuated growth spurts with their small nitrogen grabs, the small nitrate depletions are quickly replaced. This scenario explains why dosing may appear to be ineffective. What I still don’t understand is the apparent tipping point, when a certain large dose initiates easily detectable nitrogen depletion and the goal of reducing nitrate is achieved.

@taricha had an idea that could describe a tipping point mechanism: oxygen depletion. At a certain point of heterotrophic bacteria growth depletes the oxygen to a level where denitrification occurs very efficiently. Until that depletion occurs, very little nitrate is removed. The oxygen level depletion rate could differ across aquaria, requiring different levels of heterotrophic bacteria growth, rates and therefore, dose rates to observe nitrate reduction. And I presume that the population of denitrification bacteria is not the same across all systems.

That’s today’s ripple in the pond hitting the shore (Explanation: @taricha ‘s post was like a stone dropped in a quiet pond of thought. It set up many concentric rings of thought. Some made it to shore to create a little splash)

 

[biom]

Very interesting discussion. I think it is not that important how much energy given carbon source can give in theory but it is more important if there is established way to metabolize it. Glucose and acetate are part of citric acid cycle and every organism that respire can use them as energy source. Ethanol can ensure more energy than acetate but not every organism (and not every human ) has the enzymes first to metabolize it to acetate which then can be used in the Krebs (citric acid) cycle.

I can confirm what @taricha said about the usage of acetate and ethanol. During my experiments with different carbon sources I found out that when dosing acetate there was nitrate reduction but my acros were losing colors and became brown, it was the same with glucose dosing. But when ethanol dosing there were similar reducing of nitrate but no noticeable browning of acros, the same with methanol dosing. I think the reason of this browning was the zoox which were also using acetate and glucose to multiply faster, but they were not able to use ethanol/methanol, or at least not as much as acetate.

 

[Randy Holmes-Farley Verified Community Expert]

But when ethanol dosing there were similar reducing of nitrate but no noticeable browning of acros, the same with methanol dosing. I think the reason of this browning was the zoox which were also using acetate and glucose to multiply faster, but they were not able to use ethanol/methanol, or at least not as much as acetate.

That's a very interesting possibility. I had very little acro when dosing vinegar, but if that is generally true it's a good reason to not use vinegar with them, except perhaps in a coral farm where faster growth may be more important than color during the growth period.

 

[biom]

To my knowledge coral mucus is quite rich in nitrogen and proteins. A substantial amount of the nitrogen corals take up are excreted with the slime, highlighting that phosphorus is the nutrient limiting coral growth and corals strive most for.

In his excellent book on Coral Reef Ecology, Yuri Sorokin describes coral reefs as a kind of filter for enriching phosphates, driven by the corals. Coral mucus is a kind of phosphate trap, where bacteria with superior abilities to enrich phosphate sit and feed on the mucus. Since the phosphate is enriched in the reef in this way, corals benefit from it in one way or another (bacteria, plankton, fish feces etc.).

Coral mucus consists of a glycoprotein chain with oligosaccharide side chains. But it was found that phosphate in coral mucus exceeding seawater concentrations by a factor of 133 (!), which is remarkably high and probably is coming from the mucus itself - maybe phospholipids for modifying the viscosity of the mucus.

 

[Randy Holmes-Farley Verified Community Expert]

. But it was found that phosphate in coral mucus exceeding seawater concentrations by a factor of 133 (!), which is remarkably high and probably is coming from the mucus itself - maybe phospholipids for modifying the viscosity of the mucus.

That's sounds like a lot, but compared to other tissues, it is not. Whole corals (xenia, sarcophyton) and macroalgae (caulerpa) contain phosphorus on the order of hundreds of ppm, according to Ron's tests:

https://reefkeeping.com/issues/2002-12/rs/feature/index.php

 

[biom]

That's sounds like a lot, but compared to other tissues, it is not. Whole corals (xenia, sarcophyton) and macroalgae (caulerpa) contain phosphorus on the order of hundreds of ppm, according to Ron's tests:

https://reefkeeping.com/issues/2002-12/rs/feature/index.php

That is true, but I was referring Hans-Werner's post about coral mucus serving as phosphate trap. In contrary it looks coral mucus already is very rich on phosphate, which is very interesting knowing phosphorus is often main limiting factor in the reef ecosystem. That said mucus secretion of corals seems to be extremely important for their survival if they are happy to release that big amount of their precious phosphorous, glucose and proteins in the surrounding waters.

 

[Randy Holmes-Farley Verified Community Expert]

That is true, but I was referring Hans-Werner's post about coral mucus serving as phosphate trap. In contrary it looks coral mucus already is very rich on phosphate, which is very interesting knowing phosphorus is often main limiting factor in the reef ecosystem. That said mucus secretion of corals seems to be extremely important for their survival if they are happy to release that big amount of their precious phosphorous, glucose and proteins in the surrounding waters.

Ah, yes, I see the point.

 

[Hans-Werner]

Coral mucus consists of a glycoprotein chain with oligosaccharide side chains. But it was found that phosphate in coral mucus exceeding seawater concentrations by a factor of 133 (!), which is remarkably high and probably is coming from the mucus itself - maybe phospholipids for modifying the viscosity of the mucus.

There is another possibility: The bacteria in the mucus, on the coral and after suspending of the slime. Bacteria have superior abilities to enrich phosphate.

I can confirm what @taricha said about the usage of acetate and ethanol. During my experiments with different carbon sources I found out that when dosing acetate there was nitrate reduction but my acros were losing colors and became brown, it was the same with glucose dosing. But when ethanol dosing there were similar reducing of nitrate but no noticeable browning of acros, the same with methanol dosing. I think the reason of this browning was the zoox which were also using acetate and glucose to multiply faster, but they were not able to use ethanol/methanol, or at least not as much as acetate.

There is another difference between ethanol and acetate: Ethanol is an uncharged molecule while acetate is an ion. Acetate could help dissolve precipitated trace metals like iron or keep them dissolved.

 

[Koty]

A few remarks: Ethanol, unlike acetate, spontaneously crosses membranes and thus is distributed in and out of cells freely.
Mocus viscosity, as far as I know, results from proteoglycans, not phospholipids.
I would say that the goal of carbon dosing is way beyond reducing N and P but a means to enrich the microbiome to the benefit of our corals. So it would be great if someone would ever pick up the challenge and test experimentally with proper controls the effect of various types of carbon sources on coral growth and correlate it to the type of bacteria enriched in the water column and coral mucus. This probably needs an NIH grant size of funding.

For me, although I used them consecutively, none of the carbon types I dosed had an effect similar to the TM products, in my case, Elimi-NP and ReefActif. Actually, I did not find them way more efficient in reducing nitrate and phosphate. To keep my phosphate below 0.2 ppm, I actually need to use a GFO reactor. However, the positive effect on my corals was very clear. It can still be that I am wrong in my conclusions and that it is my tank that reached maturity and not the type of carbon dosed. Still, mid-day, after I drop a spoonful of Reef Actif corals, especially LPS, greet me by looking fully inflated.

 

[biom]

There were many ways of carbon dosing that were used/ tested during the years - sugar, glucose, vodka, vinegar, different combinations, but I think 1:1 vodka + vinegar dosing proved itself and is still used. It was commercialized by Red Sea as NOPOX, just more concentrated.

 

[Randy Holmes-Farley Verified Community Expert]

FWIW, I started with vodka/ethanol, but in my tank it led to a cyano increase that did not remain after switching to vinegar. That result may depend on the cyano strains present in the aquarium. Neither seemed to lead to coral browning.

A single dose of sucrose led to coral browning in my tank.

 

[biom]

FWIW, I started with vodka/ethanol, but in my tank it led to a cyano increase that did not remain after switching to vinegar. That result may depend on the cyano strains present in the aquarium. Neither seemed to lead to coral browning.

A single dose of sucrose led to coral browning in my tank.

Agree on cyano worsening with only vodka dosing but FWIW not that strong increase of cyano if 1:1 vodka/vinegar is used. Never tried vinegar only dosing if cyano is present - good idea to experiment next time

 

[taricha]

Another angle for us to consider. When an organism is grabbing organic carbon, let’s assume it is for growth. If it is growing, it is also grabbing nitrogen for biomass production, whether it is a heterotroph or mixotroph.

We typically do not detect nitrate reduction until feeding organic carbon in increasing amounts over many weeks, sometimes never for some. We seemingly have an odd situation of organic carbon consumption without nitrogen consumption. The problem is resolved by removing the assumption that the lack of nitrate consumption is the lack of nitrogen consumption. Also, the ratio of organic carbon for energy to organic carbon for biomass is large. There could very well be nitrate consumption we are not detecting until the organic carbon dose is of the amount needed for exponential growth. There is a way to look at biomass accumulation that might also be useful in understanding carbon grabbing with minimal nitrogen depletion.

When we feed our aquarium bacteria with a fixed dose of acetate, the bacteria population can increase. When we add the same amount of acetate again and again, the bacteria population increase slows down to a crawl. This is because there is a fixed amount of energy per dose that supports only so many bacteria. Until a higher dose is administered, a kind of stationary state develops. During this state, growth might stop, the number of deaths might equal the number of births, but essentially little new biomass accumulates. That means the consumption of nitrogen slows, maybe just enough to maintain the bacteria cells. When the larger dose is delivered, there is a growth spurt and nitrogen grab, but then the nitrogen consumption drops off again. During these punctuated growth spurts with their small nitrogen grabs, the small nitrate depletions are quickly replaced. This scenario explains why dosing may appear to be ineffective. What I still don’t understand is the apparent tipping point, when a certain large dose initiates easily detectable nitrogen depletion and the goal of reducing nitrate is achieved.

@taricha had an idea that could describe a tipping point mechanism: oxygen depletion. At a certain point of heterotrophic bacteria growth depletes the oxygen to a level where denitrification occurs very efficiently. Until that depletion occurs, very little nitrate is removed. The oxygen level depletion rate could differ across aquaria, requiring different levels of heterotrophic bacteria growth, rates and therefore, dose rates to observe nitrate reduction. And I presume that the population of denitrification bacteria is not the same across all systems.

That’s today’s ripple in the pond hitting the shore (Explanation: @taricha ‘s post was like a stone dropped in a quiet pond of thought. It set up many concentric rings of thought. Some made it to shore to create a little splash)

I've been running a mix of vinegar ethanol and methanol simultaneously, and it wouldn't be too hard for me to switch back and forth between 80% one, and 10% the others (by carbon), and rotate between them. I'd make some observations about skimmate volume, maybe water turbidity, and perhaps nitrate drawdown rate.

This wouldn't help untangle any of the mechanisms that you have laid out, but it might at least be interesting if the size of the differences is large enough. And maybe it'll improve the questions that we ask.

 

[Dan_P]

I've been running a mix of vinegar ethanol and methanol simultaneously, and it wouldn't be too hard for me to switch back and forth between 80% one, and 10% the others (by carbon), and rotate between them. I'd make some observations about skimmate volume, maybe water turbidity, and perhaps nitrate drawdown rate.

This wouldn't help untangle any of the mechanisms that you have laid out, but it might at least be interesting if the size of the differences is large enough. And maybe it'll improve the questions that we ask.

Yeah, it may take experiments to sharpen up our question,

i don’t recommend the volume of skimmate as a precise measure only because as analytical method there are too many uncontrolled variables. I did buy a small hang on the side skimmer with the notion that I could better control the variables. The method would involve dunking it into the aquarium for a fixed time to make an observation. The volume of skimmate would be one measure and measuring the COD of the skimmate might be one of the ways to characterize what was collected.

Also to your point, dosing a sample of aquarium water or aquarium water plus sand might be a away to quickly probe the dynamics of nitrate reduction while controlling of variables more tightly without worrying about harming any exotic life. Measuring heterotrophic bacteria activity with methylene blue medium might be a simple way to get an estimate of the relative size of the bacteria count per dose size.