Analyzing a Bacterial Method for Dinoflagellates (and cyano?)

[taricha]

You should do some figurin’ to see if your results are within the bounds of possibility. Here are some questions that might be useful?

What is the concentration of CO2 in the system when the pH drops from X to 7.55?

Would you expect the headspace above distilled water and tank water to be approximately the same? Show work

What concentration of CO2 in seawater would be in equilibrium with 1800 ppm CO2 in the headspace?

If all the carbon in the vodka and vinegar is converted to CO2 and did not leave the tank, what would be the concentration in the system?

How much oxygen is consumed in converting all the added ethanol and vinegar to CO2?

I'm still thinking about the headspace calculations. The papers on them get intense.

First, lets talk about how much Carbon I added and what that means for CO2 and O2 in the system.
12mL vodka - at 8x the carbon of vinegar - equivalent to about 100mL vinegar + 60mL actual vinegar, so about 160mL vinegar.
160mL (160g) * 5% acid = 8g of acetic acid.
[and I'm gonna ignore the direct acid effect because I didn't measure it, and focus on the CO2 effect which I do have data on.]

8g acetic acid / 60g molar mass = 0.133 moles Acetic acid (or acetate)
CH3COO- + 2O2 -> 2CO2 + H2O + OH-
2 CO2's for each acetate = 0.266 moles CO2 produced and 0.266 moles O2 consumed.
My system is ~265L so 0.266Mole/265L = 1millimole/L of CO2 produced and O2 consumed. We'll come back to CO2. on the Oxygen front, At 32g/mole = that's 32 mg/L of O2 consumed.
Oh Dear!
Maximum O2 level in seawater is about 7mg/L (see borneman), so that's enough Carbon to eat all the oxygen in my system 4.5 times. So that's the task for the aeration / photosynthesis.
Now, how does the CO2 production of 1millimole/L compare to the CO2 necessary to cause the pH shifts I observed?
This page from colorado state is amazing. Never seen it before. Calculate ocean pCO2 and pH from DIC, alkalinity, and temperature

Based on my (very low) alk, the observed shift of pH ~0.45 from mid 7.6ish to just over 8.0 in maximum aerated vs less aerated tank water corresponds to a total dissolved inorganic carbon (DIC = CO2 + HCO3 + etc.) change from 2178 to 1972 micromoles, a change of about 200 micromole/L of CO2 equivalents. And the vinegar I added could generate 1 millimole/L = 1000micromole/L of CO2. So I added enough Carbon to generate 5x the CO2 in the water that I actually observed via pH shift - again - saved by aeration & photosynthesis.

The bacteria consuming gunk ought to be producing some CO2.

My thought here is misguided. Any CO2 released from that process is overwhelmed by the CO2 from added carbon, so that's what would be observed.
That page with the awesome seawater calculator also allows you to see what ppm CO2 would be in equilibrium with those parameters.
It said that based on my Alk (2.27meq) and the pH shifts I put in, the 8.05 pH water would be in equill with 388ppm CO2 and the 7.6 pH water with 1290ppm CO2. My measured CO2 values for air in the bottles with those water samples were 680ppm and 1480ppm. Not the greatest agreement, but the calculator shows a 900ppm CO2 diff and I measured an 800ppm CO2 diff. So perhaps mostly reasonable.

 

[taricha]

A couple of general comments on the method as a follow-up. Addressing some of the questions I labeled as unknown previously.

The day 2 and 3 carbon dose is around 15% larger than the dose that I ran numbers on in the previous post. If we're adding enough carbon to an already cloudy-water bacterial bloom to consume all the O2 in the water ~5 times over, then things could move really fast. The bacteria are already in huge numbers and they likely have everything they need. O2 could drop very fast. For aeration, I don't think "airstone" is the right mental picture, more like skimmer with cup off and a lit up algae fuge connected to display with lots of turnover.

Another point, the suggestion in the method that strong aeration could starve the nuisance of CO2 doesn't look sensible. The system will be flooded in CO2 from added carbon.

The suggestion that it's the nitrifier (One and Only) that fills the water and gets skimmed out seems unsupported. Dr Tim's warns over and over about Waste Away causing blooms and to use much less than you think to start. Also they warn at first sign of cloudiness to turn on skimmer/UV. Contrast that with the instructions for One and Only where it says an overdose is impossible. And that it colonizes surfaces, not the water. So what is the role of the nitrifier? I guess nitrification.

Waste Away creates "small amount" of ammonia according to Dr Tim. How much it'll create in an intentional runaway bloom? I don't know. I apparently don't have an Ammonia test kit. But maybe there's a possibility that some of the livestock loss is ammonia production and not just O2 drop. The nitrifier does process ammonia and nitrite, but the timing of the bloom over just 2-3 days is faster than what the makers of nitrifier bacteria say is needed to scale up to the ammonia demands of a tank.

I'd add it anyway. If you are trying to purge nuisance algae, then ammonia production is an enemy. A bottle of nitrifier is at least fighting on the right side even if may not be up to the task.

 

[Dan_P]

I'm still thinking about the headspace calculations. The papers on them get intense.

First, lets talk about how much Carbon I added and what that means for CO2 and O2 in the system.
12mL vodka - at 8x the carbon of vinegar - equivalent to about 100mL vinegar + 60mL actual vinegar, so about 160mL vinegar.
160mL (160g) * 5% acid = 8g of acetic acid.
[and I'm gonna ignore the direct acid effect because I didn't measure it, and focus on the CO2 effect which I do have data on.]

8g acetic acid / 60g molar mass = 0.133 moles Acetic acid (or acetate)
CH3COO- + 2O2 -> 2CO2 + H2O + OH-
2 CO2's for each acetate = 0.266 moles CO2 produced and 0.266 moles O2 consumed.
My system is ~265L so 0.266Mole/265L = 1millimole/L of CO2 produced and O2 consumed. We'll come back to CO2. on the Oxygen front, At 32g/mole = that's 32 mg/L of O2 consumed.
Oh Dear!
Maximum O2 level in seawater is about 7mg/L (see borneman), so that's enough Carbon to eat all the oxygen in my system 4.5 times. So that's the task for the aeration / photosynthesis.
Now, how does the CO2 production of 1millimole/L compare to the CO2 necessary to cause the pH shifts I observed?
This page from colorado state is amazing. Never seen it before. Calculate ocean pCO2 and pH from DIC, alkalinity, and temperature

Based on my (very low) alk, the observed shift of pH ~0.45 from mid 7.6ish to just over 8.0 in maximum aerated vs less aerated tank water corresponds to a total dissolved inorganic carbon (DIC = CO2 + HCO3 + etc.) change from 2178 to 1972 micromoles, a change of about 200 micromole/L of CO2 equivalents. And the vinegar I added could generate 1 millimole/L = 1000micromole/L of CO2. So I added enough Carbon to generate 5x the CO2 in the water that I actually observed via pH shift - again - saved by aeration & photosynthesis.

My thought here is misguided. Any CO2 released from that process is overwhelmed by the CO2 from added carbon, so that's what would be observed.
That page with the awesome seawater calculator also allows you to see what ppm CO2 would be in equilibrium with those parameters.
It said that based on my Alk (2.27meq) and the pH shifts I put in, the 8.05 pH water would be in equill with 388ppm CO2 and the 7.6 pH water with 1290ppm CO2. My measured CO2 values for air in the bottles with those water samples were 680ppm and 1480ppm. Not the greatest agreement, but the calculator shows a 900ppm CO2 diff and I measured an 800ppm CO2 diff. So perhaps mostly reasonable.

Hey, nice calculatin’!

When I looked into acetic acid dosing, I observed that the daily dose was consumed in 24 hrs. My maximum dose was 2 mL/gal or ~0.5 mL/L.

You report an estimated 32 mg/L is consumed from your dose. Assuming this happened over 24 hours and assuming your system’s replenishment rate could not keep up, at an average consumption per rate of1.3 mg/L (an average O2 concentration decrease to 5.7 mg/L) that gives an O2 level of 80% of the maximum. If I read your post correctly, just before stirring up the sand and shutting off the lights, the O2 level was at 60%. Interesting. What happened after stirring up the substrate indicates the danger of disturbing the substrate too much.

As for the CO2 level in the head space analysis, one interpretation of the high numbers is that your system is not in equilibrium and is supersaturated in CO2. I guess if you aerated the pH 8.05 sample and the pH increased, that would suggest the system is not equilibrating quickly, possibly the case for oxygen too.

 

[Dan_P]

The suggestion that it's the nitrifier (One and Only) that fills the water and gets skimmed out seems unsupported. Dr Tim's warns over and over about Waste Away causing blooms and to use much less than you think to start. Also they warn at first sign of cloudiness to turn on skimmer/UV. Contrast that with the instructions for One and Only where it says an overdose is impossible. And that it colonizes surfaces, not the water. So what is the role of the nitrifier? I guess nitrification.

A couple thoughts.

About the notion that bacteria being produced by carbon dosing are removed by skimming. Then why did my nitrate level jump back to 50 ppm when I instantly stopped dosing if the bacterial biomass was being skimmed off during the dosing period? I am having doubts about the received wisdom that the skimmer removes bacteria to any significant degree.

The addition of a nitrifier might be a fail safe mechanism for situations when heterotrophic bacteria consume nitrogen rich waste and excrete the excess nitrogen as ammonia.

 

[taricha]

As for the CO2 level in the head space analysis, one interpretation of the high numbers is that your system is not in equilibrium and is supersaturated in CO2. I guess if you aerated the pH 8.05 sample and the pH increased, that would suggest the system is not equilibrating quickly, possibly the case for oxygen too.

I think for any of my measurements to make sense requires that the water is not equilibrated with the air above it. I also put my CO2 meter in the fuge a few inches above where the skimmer foams. I wanted to see if it looked more like room/outside air (skimmer pulls from outside), or more like the headspace in the shaken bottles of tank water with at least 2x as much CO2. It was basically the same as room air. So exchange may be kinda slow.

About the notion that bacteria being produced by carbon dosing are removed by skimming. Then why did my nitrate level jump back to 50 ppm when I instantly stopped dosing if the bacterial biomass was being skimmed off during the dosing period? I am having doubts about the received wisdom that the skimmer removes bacteria to any significant degree.

The cloudy water going into my skimmer foamed big frothy foam that stuck around near the skimmer (cup was off). That certainly would have been skimmed out. But all this....

...is not getting into the skimmer. (this is where the bacteria found the goodies that a tuft of green hair algae had been hoarding).

I generally scratch my head at the mass conservation problem of turning undesirable aquarium material - be it grunge or nuisance growth - into nothing, as everyone would like to do. But I'll leave that for another time.

 

[taricha]

By the way, the intentional 2.5 day bacterial bloom of Waste Away seems to have been somewhat effective. Cyano seems to have been knocked back all over the tank. This is a representative after/before shot of a patch of mixed red and green cyano.

Not obvious to me why it would have worked.
I mean Ammonia was probably generated. CO2 flooded the system, nothing really was skimmed out. I dunno what stressed the cyano here.

 

[Dan_P]

By the way, the intentional 2.5 day bacterial bloom of Waste Away seems to have been somewhat effective. Cyano seems to have been knocked back all over the tank. This is a representative after/before shot of a patch of mixed red and green cyano.

Not obvious to me why it would have worked.
I mean Ammonia was probably generated. CO2 flooded the system, nothing really was skimmed out. I dunno what stressed the cyano here.

Nice pictures on which to speculate

We know cyanobacteria are highly mobile. What we see here might just be the result of cyanobacteria migrating from the mat. We have seen this rapid movement on small scale. What if we assume that what is in the water is not very important to mat formation (heresy, I know)?

Our question might be how is a clean culture dish surface like a natural surface just ravaged by a hoard of bacteria?

 

[Dan_P]

I think for any of my measurements to make sense requires that the water is not equilibrated with the air above it. I also put my CO2 meter in the fuge a few inches above where the skimmer foams. I wanted to see if it looked more like room/outside air (skimmer pulls from outside), or more like the headspace in the shaken bottles of tank water with at least 2x as much CO2. It was basically the same as room air. So exchange may be kinda slow.


The cloudy water going into my skimmer foamed big frothy foam that stuck around near the skimmer (cup was off). That certainly would have been skimmed out. But all this....

...is not getting into the skimmer. (this is where the bacteria found the goodies that a tuft of green hair algae had been hoarding).

I generally scratch my head at the mass conservation problem of turning undesirable aquarium material - be it grunge or nuisance growth - into nothing, as everyone would like to do. But I'll leave that for another time.

Have you tried either mixing the white bacteria strings with cyanobacteria and inoculating a medium that you know grows cyanobacteria, or taken a piece bacteria mat and placing it on or next to a cyanobacteria mat?

Clues to what might happen to aquarium grunge can be found in the rich literature of waste water treatment. It would fun to study aquarium grunge digestion. This is how I isolated sludge/grunge from aquarium substrate. Vigorously shake the substrate in tank water and centrifuge. The pellet may contain both organic material and substrate dust which was there or created during the shaking process. You could try a round of mild shaking followed by vigorous shaking to obtain pellets of different composition. You could do a grunge digestion and see what is left. I suspect bacteria mass will have formed from the grunge, the rest CO2, ammonia, phosphate, etc. I seem to recall that you have a “neglected” aquarium. Maybe it could be your source of grunge. You can also see if cyanobacteria can grow on it. I just remembered that I have a several mL collection of substrate grunge in the freezer Maybe I will feed it to my Spirulina cultures.

 

[AngryOwl]

You guys started speaking another language here

 

[taricha]

You guys started speaking another language here

Maybe a little.
The main projects are always spinning off side projects, and the side projects are always crossing over to connect to other side projects.
It can be hard to keep up with

 

[taricha]

Have you tried either mixing the white bacteria strings with cyanobacteria and inoculating a medium that you know grows cyanobacteria, or taken a piece bacteria mat and placing it on or next to a cyanobacteria mat?

I seem to recall that you have a “neglected” aquarium. Maybe it could be your source of grunge.

I'm all out of beakers. Gotta let some of my tests end first.
But yes I have lots of sources of aquarium sludge, and after I finish testing this I may have some good ideas for how to test sludge digestion.

 

[AngryOwl]

Once you guys figure this out please let all of us have the English translations ;Hilarious

 

[taricha]

Here's some pictures! (well, graphs)
The hazy water from the bloom cleared up, So I restarted it with the recommended (Day 3) dose of Carbon and Waste Away.

I figured out how to tighten the calibrations a little bit and here's data for O2.

The Blue data represents the tank with lights off, and no exchange with the fuge or skimmer. No aeration, only circulation from pumps. O2 got down to 11% of the max measured level. Maximum measured level was (7.2mg/L).
The Red data is where I turned on the flow to/from fuge with a little foaming/aeration in the fuge. Still no lights.
Where I stirred and blew around the sandbed and added an additional 20mL of vinegar is noted on the graph. So the rate is not carbon proportional, and disturbing the sandbed isn't a dramatic effect when you've been running the bloom for a few days and you just blew around the sandbed a couple of days earlier.

For CO2 production, I compared the theory from the site calculator I mentioned in this post, calculating the equilibrium CO2 level based on my measured pH and Alk (I re-did more careful calibrations there as well).
Before the bloom was restarted, the theoretical calculated CO2 was 447ppm and my measured was an average of 508ppm CO2 - within 50 ppm of the room.
I sampled again at the height of the bloom (4hr) when the O2 hit 11% of Max. Theory based on Alk and pH predicts 1316ppm
I did a series of bottle measurements to estimate what the value would be if I could make the air in the 300mL bottle nearly zero. Worked pretty well!
Theory 1316, measured extrapolates to 1404ppm CO2.

Overall, the late stages of the bloom - after 4 days or so - aren't as intense or dangerous and very modest approaches to flow and aeration are enough to stabilize the system. But when the bloom first gets rolling, much more caution is required.

Thinking about some recommendations for how to safely navigate the start of the bloom, and make some large uncertainties smaller.

 

[Dan_P]

Here's some pictures! (well, graphs)
The hazy water from the bloom cleared up, So I restarted it with the recommended (Day 3) dose of Carbon and Waste Away.

I figured out how to tighten the calibrations a little bit and here's data for O2.

The Blue data represents the tank with lights off, and no exchange with the fuge or skimmer. No aeration, only circulation from pumps. O2 got down to 11% of the max measured level. Maximum measured level was (7.2mg/L).
The Red data is where I turned on the flow to/from fuge with a little foaming/aeration in the fuge. Still no lights.
Where I stirred and blew around the sandbed and added an additional 20mL of vinegar is noted on the graph. So the rate is not carbon proportional, and disturbing the sandbed isn't a dramatic effect when you've been running the bloom for a few days and you just blew around the sandbed a couple of days earlier.

For CO2 production, I compared the theory from the site calculator I mentioned in this post, calculating the equilibrium CO2 level based on my measured pH and Alk (I re-did more careful calibrations there as well).
Before the bloom was restarted, the theoretical calculated CO2 was 447ppm and my measured was an average of 508ppm CO2 - within 50 ppm of the room.
I sampled again at the height of the bloom (4hr) when the O2 hit 11% of Max. Theory based on Alk and pH predicts 1316ppm
I did a series of bottle measurements to estimate what the value would be if I could make the air in the 300mL bottle nearly zero. Worked pretty well!
Theory 1316, measured extrapolates to 1404ppm CO2.

Overall, the late stages of the bloom - after 4 days or so - aren't as intense or dangerous and very modest approaches to flow and aeration are enough to stabilize the system. But when the bloom first gets rolling, much more caution is required.

Thinking about some recommendations for how to safely navigate the start of the bloom, and make some large uncertainties smaller.

A well thought out and executed demonstration. Love the quantitation. I wish you had access to 10 more aquaria to increase the data set to >N=1.

There are chemical tests for measuring oxygen. Would you recommend their use to reduce suffocating the system?

 

[taricha]

A well thought out and executed demonstration. Love the quantitation. I wish you had access to 10 more aquaria to increase the data set to >N=1.

There are chemical tests for measuring oxygen. Would you recommend their use to reduce suffocating the system?

Yes! Knowing the general shape of the curve now, and that a well aerated tank will start at ~95% O2, then getting just one or two O2 values at a set time after the trigger of the bacterial bloom like 1 or 2 hours later can decently predict when/if there will be problems.
The issue with using pH to monitor as the guide suggested is that if the calibration is off by just 0.1 pH unit then what looks like a low but not dangerous level of say 30% could really be in the single digits % of O2.

 

[taricha]

Here's another After/Before shot of a cyano patch - bottom is from day 2 of bacterial addition. Top is 5 days later - including 3 days where I maintained continuous cloudy water conditions.

Wildly speculating about what's happening here, I'd say the original cyano blanket is holding tons of fluffy debris underneath which slowly decomposes and fuels the cyano growth. After continuous bacterial bloom, it looks like the white bacterial mass has colonized the debris pile, maybe consuming nutrients out of it, and the cyano might have retreated from film to clumps.
If the cyano were being starved out, I'd expect it to grow thin and wide in search of nutrients. This looks more like being displaced by an unfriendly organism.
I really need to harvest some of the white bacterial film material and apply it to a healthy culture of cyano, like @Dan_P suggested. Koch's postulates and all that.

 

[Ling_Thing]

This sounds exactly like dr. Tims cyano recipe, give that a read

 

[taricha]

One more pic. Amazing how much structure bacteria can create. Looks like a large organism, almost sponge-like

.

 

[taricha]

This sounds exactly like dr. Tims cyano recipe, give that a read

I've been thinking about that too, and how to bring it in to this thread. It has many similarities and some important differences. (Black out, and Re-fresh instead of One and Only.)

 

[Dan_P]

One more pic. Amazing how much structure bacteria can create. Looks like a large organism, almost sponge-like

.

This is amazing. This is not the usual “bacterial slime” photo. The nodes and nets might be interesting under the microscope. There seems to be a line nodules as well on the lower right. Is this some sort of complex mat?