# Doing DIY CO2 Differently



## ed seeley (Dec 1, 2006)

I'm hoping those of you with way more experience of DIY CO2 than me can answer a few questions for me.

First a little background:
I have a Nutrafin CO2 fermentation kit and am switching over to using my mix of yeast, sugar and Baking Soda rather than buying the ready-made kits!
I have set up a batch with about 1/2 teaspoon of yeast, a teaspoon of BS and then filled up to the line with sugar (I estimate this is about 4 big tablespoons of sugar).

Question 1.
As far as understand:
1. The amount of yeast will later the rate of CO2 production. More Yeast, more bps.
2. The amount of sugar governs the amount of CO2 produced altogether. More sugar, more CO2 (but the yeast will govern the rate it is produced.
3. The baking soda regulates pH and slows the acidity of the mixture. So more Baking soda the longer the mixture lasts????
Are these ideas in the right area? Obviously the relationships I have suggested only work in limits, I couldn't add a whole bag of sugar and get it all turned to CO2.

Question 2.
What I am wondering is does the Baking soda just do the above? Is it just to increase the pH of the mixture or does it have some other effect?
What I am thinking is that, if this is the cause of the DIY mix stopping working why not use aragonite or coral gravel? These would sit in the mixture and slowly dissolved to keep the pH up. Would this long term effect keep the mixture going longer if I upped the sugar?

Question 3.
As the yeast is respiring anaerobically to produce CO2 and this anaerobid aeration is inherently expensive biochemically for the Yeast and will produce alcohol, why don't people unplug their DIY bottles overnight to allow O2 into the mixture? Wouldn't this extend the life of the mixture?
I was wondering whether an air pump could be linked to the container via a one-way valve to pump air into the mixture during the night, adding O2 to the water and the mixture at night when the plants don't need the CO2.

I'm assuming there are good reasons why what I've written above is complete rubbish, but I'd love to know why!!!
Can anyone enlighten me?


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## snickle (Apr 8, 2007)

I am interested in the answers as well. I run the Hagen unit with a DIY Mix.

Sugar to the line
1/2 teaspoon baking soda
1/8 teaspoon champagne yeast (I have a homebrew shop near by, so better yeast is easy to find)

So far working well. First time I just dumped in some cahmpagne yeast without measuring burned through the sugar in 3 days.


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## Left C (Jun 14, 2005)

In addition to what Snickle mentioned.
-Use dechlored water heated up to ~95° F. Don't go much higher or you will kill the yeast. I believe that 105° F or higher will kill them.
-I put my yeast in a cup and added some of the warm water plus a pinch of sugar. I stirred it some and the I let it set out about 10 minutes. You'll see CO2 bubbles.
-After about 10 minutes, I added it to my mix container with the other ingredients.
-You can put the Hagen container of water with a heater in it to keep it warm. It helps it to be a little consistent at CO2 production.
-Optional. I added a tiny Rio 50 powerhead above the Hagen ladder to catch the bubbles that would normally escape to the surface. It would suck them up, chew them up and spit out a fine mist of bubbles.

The following info comes from Tarah Nyberg's Yeast CO2 powerpoint presentation from the AGA2K3 convention. http://www.aquatic-gardeners.org/Nyberg_yeast.ppt

It answers some of your questions, Ed. I hope it helps you.

*How to live on the cheap --CO2*
I have used yeast CO2 on tanks up to 180 gal. Also 75s, 65s etc. 
2 gallons of yeast changed once every 3wks to month depending on the temperature works well. I don't rotate the bottles either. 
The key to long lasting cultures is allowing them to grow and be happy. Also a little basic knowledge of yeast growth helps.

*Yeast Growth/metabolism* 
Yeast can either produce energy by fermentation or oxidative phosphorylation (ox/phos)
Yeast greatly prefer fermentation over ox/phos and will not start ox/phos until all the sugars are converted to ethanol.
Ethanol is a good source of energy and in the presence of oxygen yeast use it up as well. 
Fermentation also produces 2 CO2 molecules per molecule of sugar-- as a by product. =)

*Why is just sugar and water not ideal?*
When you just add sugar and water to yeast, they are essentially starving to death.
However the enzymes for converting sugar to ethanol are still in the cell and will work for a limited amount of time. 
Since the cells do not have what they need to make new enzymes as cells starve/run out of enzymes the culture produces less and less CO2.

*Also, we add too much sugar.*
Ethanol and sugar are increasingly toxic to yeast at greater than 10% concentrations. 
Therefore 2cups (~500ml) of sugar in 2L of water (~25% sugar) is unhealthy for the yeast. 
It is also a waste of sugar because 10% sugar will yield roughly 10% Ethanol at which point the yeast stop growing anyway. 
Special strains of yeast, like champaine and wine yeast, have stronger cell walls that protect them from the Ethanol -- so they grow longer.

*Happy yeast give you long, productive cultures.*
The solution is to give yeast less sugar and also supply them with the nutrients they need to grow. 
This will give you a long lived culture that produces a consistent amount of CO2, (they are not challenged by toxic conditions at the beginning and end)
Also your yeast mass at the end will be alive and well and able to quickly start growing again when you add more sugar.

*The Recipe!*
Improvise at will, but here is a good start:
Use 1 cup sugar per 2L H20 (tank water is great or dechlorinated tap -- chlorine, but not ammonia, kill yeast. ) (they like ammonia)
Add 1-2 tsp of a protein drink mix
(optional) Add 1 tsp of ammonium sulfate, otherwise use 1 T mollasses. (or both)
1 tsp baking soda is also nice to keep the pH from crashing (they like it >pH3-4)
Leave yeast from previous mix in the bottom.

*Benefits/conclusons*
Growing yeast this way saves you time (less re-starting) and money on sugar and yeast.
The only real extra cost is the protein mix, but you can use really old/cheap stuff. 
The key thing is that it has protein and vitamin/minerals, Anything that has "yeast extract" in it is perfect.
(Even non-fat powdered milk will do the trick, but it will smell funny)


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## ed seeley (Dec 1, 2006)

Thanks guys.

That's a lot of info LeftC, cheers.

I reckon that means that coral gravel would work well to boost the pH. Might not be any better than Baking Soda though obviously!

Still not sure of potential benefits of aerating the mixture at night. From my degree I thought that yeast was a facultative anaerobe, i.e. it would use O2 to respire the sugar if it was present, but would switch to anaerobic respiration when the O2 ran out. As this is a more efficient process surely it would benefit the culture to add some air to the mix?


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## Left C (Jun 14, 2005)

Hi Ed

I hope the info helped you.

There was someone a few years ago that was working on a process/procedure to allow yeast to produce CO2 aerobically. I never read anything about it afterward. I don't know if this person succeeded or not.

IMO, I would try to help the yeast to maximize the production of CO2 anaerobically and be done with it.

A few years ago, I did some tests on the various 6 carbon sugars thinking that glucose would be the best at CO2 production. It worked out that sucrose (table sugar), a 12 carbon sugar, worked better. I quit then. That was enough for me to just use table sugar.

I've never tried adding any O2 to the mix; so I don't know what the benefits/results/negatives would be.

There's a site that sells a 48 hour "turbo charged" yeast that comes with it's own nutrients. It's supposed to be awesome. It's Alcotec 48-Hour Turbo Yeast. http://www.beer-wine.com/category_page.asp?categoryID=90&sectionID=2

Good luck!


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## snickle (Apr 8, 2007)

I don't see the need to add O2. I was a home brewer for years and as long as you kept the nutrients up, the yeast was happy and productive. Alcohol content is the biggest problem as long as the yeast basic needs are met.

Now I need to use that knowledge to maximize the CO2 output over the long run.


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## Left C (Jun 14, 2005)

ed seeley said:


> ...Still not sure of potential benefits of aerating the mixture at night. From my degree I thought that yeast was a facultative anaerobe, i.e. it would use O2 to respire the sugar if it was present, but would switch to anaerobic respiration when the O2 ran out. As this is a more efficient process surely it would benefit the culture to add some air to the mix?


Ed

These are my thoughts about trying this:
-When you remove the top to let O2 in, much of the CO2 escapes.
-Then, when you put the top back on; the CO2 level needs to increase to a certain pressure again so that bubbles are produced at your ladder/diffuser.
-Yeast is facultative. It can live aerobically or anaerobically. Anaerobic is easier for our use.


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## Catherine (Feb 22, 2006)

Left C, when your mix has completely stopped producing CO2, do you dump it all except for the yeast sludge and put in new aged water/sugar/additives, or just readd sugar?

I have tried making a container out of the 2L juice bottles but I can't seem to get the yeast going well enough to make it to the ladder. Is it possible that the silicone tubing is allowing it to leak/diffuse out? I have siliconed the heck out of the cap. It is meant for a 135g tank.


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## snickle (Apr 8, 2007)

I would suspect a leak. My Hagen cylinder produces enough CO2 in about an hour after I add the mix.

As for dumping, you need to dump the water as it is loaded with alcohol that will kill the yeast.


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## ed seeley (Dec 1, 2006)

Left C said:


> Ed
> 
> These are my thoughts about trying this:
> -When you remove the top to let O2 in, much of the CO2 escapes.
> -Then, when you put the top back on; the CO2 level needs to increase to a certain pressure again so that bubbles are produced at your ladder/diffuser.


Completely agree here, but I'm thinking of having an extra non-return valve glued in the lid to pump air into the mixture and keep the container pressurised. There would be no CO2 escaping, other than going into the tank, and no need to re-pressurise.
In fact doing this you could constantly slowly bubble air into the mix even during the day.

So there would be a non-return air inlet connected to an air pump and the usual outlet to the CO2 ladder.



Left C said:


> -Yeast is facultative. It can live aerobically or anaerobically. Anaerobic is easier for our use.


It may be easier I agree, but if the main killer of yeast is alcohol levels, aerobic respiration has the great advantage of not producing alcohol! Adding air should prevent the build up of alcohol in the first place.

As it's more efficient then you should also get more CO2, if yeast behaves like most facultative anaerobes?

In normal aerobic respiration this simplified reaction occurs;
C6H12O6 + 6 02 → 6 CO2 + 6 H2O + 36ATP (2880KJ)

In Ethanol fermentation this reaction occurs;
C6H12O6 → 2 C2H5OH + 2 CO2 + 2ATP

So each molecule of sugar (Glucose in the above equations) will give more CO2 when O2 is present!


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## Left C (Jun 14, 2005)

Catherine said:


> Left C, when your mix has completely stopped producing CO2, do you dump it all except for the yeast sludge and put in new aged water/sugar/additives, or just readd sugar?


I used to dump it all. That's what I did after I learned better. Then I added sugar, water, baking soda, etc. to the yeast sludge.



Catherine said:


> I have tried making a container out of the 2L juice bottles but I can't seem to get the yeast going well enough to make it to the ladder. Is it possible that the silicone tubing is allowing it to leak/diffuse out? I have siliconed the heck out of the cap. It is meant for a 135g tank.


How deep is your diffuser placed? Yeast/CO2 produces such little pressure that it can't produce CO2 bubbles through the diffuser when placed at higher depths. Try ~6 to 8 inches or so.


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## Left C (Jun 14, 2005)

ed seeley said:


> ...It may be easier I agree, but if the main killer of yeast is alcohol levels, aerobic respiration has the great advantage of not producing alcohol! Adding air should prevent the build up of alcohol in the first place.
> 
> As it's more efficient then you should also get more CO2, if yeast behaves like most facultative anaerobes?
> 
> ...


The "Pasteur effect" was discovered Louis Pasteur, who showed, that aerating the yeasted broth causes the yeast cell growth to increase, while the fermentation rate to decrease.

 Explanation
The effect can be easily explained, as the yeast being facultative anaerobes can produce energy using two different methabolycal tracts. While the oxygen concentration is low, the product of glycolysis pyruvate is turned into ethanol and carbon dioxide and the energy production efficiency is low (2 moles of ATP per one moles of glucose). If the oxygen concentration grows, pyruvate is converted to acetyl CoA that can be used in Krebs Cycle, which increases the efficiency to 38 moles of ATP per 1 moles of glucose.

Under anaerobic conditions, the rate of glucose metabolism is faster, but the amount of ATP produced (as already mentioned) is smaller. When exposed to aerobic conditions, the rate of glycolysis slows, because the increase in ATP production acts as an allosteric inhibitor for the pathway (See Allosteric regulation and more specifically Allosteric inhibition). So, it is advantageous for yeast to undergo glycolysis in the presence of oxygen, as more ATP is produced with less glucose.
http://en.wikipedia.org/wiki/Pasteur_effect


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## Rex Grigg (Jan 22, 2004)

ed seeley said:


> I'm hoping those of you with way more experience of DIY CO2 than me can answer a few questions for me.
> 
> First a little background:
> I have a Nutrafin CO2 fermentation kit and am switching over to using my mix of yeast, sugar and Baking Soda rather than buying the ready-made kits!
> ...


1. The more yeast at start the more CO2 at start. In the long run it makes little difference since the yeast will reproduce geometrically till either the alcohol kills them or the food runs out.
2. Not necessarily true. Bread yeast will die of alcohol poisoning before they use all the sugar. A good wine yeast that ferments to dryness will use all the sugar.
3. Never bothered with it.

It's not necessary to use dechlorinated water. I've never seen a baker use anything but tap water. Nor have I ever used anything but tap water in baking or DIY CO2 systems. I have a bit of experience here since my father was a professional baker for over 40 years and my ex-brother-in-law is a large scale commercial baker.

You will have much better luck with a wine yeast like Premier Cuvee. And you can reuse the yeast over and over.


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## ed seeley (Dec 1, 2006)

Left C said:


> The "Pasteur effect" was discovered Louis Pasteur, who showed, that aerating the yeasted broth causes the yeast cell growth to increase, while the fermentation rate to decrease.
> 
> Explanation
> The effect can be easily explained, as the yeast being facultative anaerobes can produce energy using two different methabolycal tracts. While the oxygen concentration is low, the product of glycolysis pyruvate is turned into ethanol and carbon dioxide and the energy production efficiency is low (2 moles of ATP per one moles of glucose). If the oxygen concentration grows, pyruvate is converted to acetyl CoA that can be used in Krebs Cycle, which increases the efficiency to 38 moles of ATP per 1 moles of glucose.
> ...


Thanks Left C! But isn't the Pasteur effect concerned with the production of alcohol and suagr glycolosis (unless I read and remembered that bit of Biochemistry wrongly!), when discussing the fermentation rate rather than CO2? Less sugar will be metabolised via glycolosis, but the continued metabolisation of the products of glycolosis, via Kreb's cycle and other pathways, will also produce CO2, so wouldn't this mean the rate of CO2 production may well stay at a similar rate, if not even increasing? After all we don't care about the alcohol produced (in fact we'd like to minimise it really) and just want more CO2 per unit!


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## longhornxtreme (Feb 20, 2007)

Hi all.

Beer, wine, and distillery fermentors don't aerate their mixtures because the pyruvates from glycolysis would then feed into the TCA cycle instead of becoming ethanol. Even though we don't care about ethanol production, I think a system to aerate said yeast would be an exasperating experience considering the relatively cheap cost of going pressurized CO2. You'd have to inject o2 at a rate that was slower than co2 production or the net effect would be just be pumping o2 into your tank. Way more difficult than just buying a cheap co2 bottle and regulator and then ordering the rest of the parts from Rex. Not to mention pressurized o2 would cost even more than pressurized co2 as you'd also have the concern that o2 now adds combustion to the equation of safeguards.

@LC, I'm curious about the particulars of your experiment where you found that sucrose was better than glucose considering sucrose is really just a glucose molecule joined to a fructose molecule.



> Under anaerobic conditions, the rate of glucose metabolism is faster, but the amount of ATP produced (as already mentioned) is smaller. When exposed to aerobic conditions, the rate of glycolysis slows, because the increase in ATP production acts as an allosteric inhibitor for the pathway (See Allosteric regulation and more specifically Allosteric inhibition). So, it is advantageous for yeast to undergo glycolysis in the presence of oxygen, as more ATP is produced with less glucose.


Also, this passage is sort of flawed concerning single celled organisms such as Saccharomyces cerevisiae. Glycolysis is indeed slowed when ATP concentration is high. But it is not 'advantageous' for yeast to undergo glycolysis as they MUST undergo glycolysis in order to move on to utilize their aerobic respiration capability. Yes in the presence of oxygen more ATP per molecule of glucose is produced. But, even with oxygen present, glycolysis is required to produce pyruvate to feed into the TCA.


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## Squawkbert (Jan 3, 2007)

For bread yeast, use (by vol.) 1 part sugar to 3 parts water. Bread yeast will pretty well consume the last of the sugar as they start to die off from ethanol poisoning. You can run modestly higher amounts of sugar with wine/beer yeasts, but they're a PITA to get started (they're sold in liquid form, have a short shelf life, and are relatively expensive). Also - if you miss changing your solution and adding fresh sugar by very long, you may well kill it and have to start over. That and the best of them die off once the etOH gets up to (I think) about 15%. That may get you another week vs. a good strain of bread yeast, and it uses the same amount of sugar per unit CO2.

So, the question is is it worth the extra expense & hassle of using a wine/brewers yeast instead of a bakers yeast, just to make your changes a little less frequent?


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## longhornxtreme (Feb 20, 2007)

After fiddling around with DIY co2, I found that my wasted time was much more expensive than just ordering a 5# co2 bottle, and built my own regulator. Pressurized is just wonderful. Fermentation will be used to brew BEER for me, not for feeding my plants


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## snickle (Apr 8, 2007)

Beer/Wine Yeast come in both liquid and packet form. I use Lalvin EC-118, champagne yeast. It is 85 cents a packet. It will support up to about 15% alcohol. Easy to start. Just add the dry ingredients, fill with warm water and dump an 1/8 a teaspoon of yeast on top. Done.

Starts quick.


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## ed seeley (Dec 1, 2006)

Great stuff here guys, I'm really loving all this.

Long horn, I agree that pressurised is easier, and I have that on my larger tank. I'm kinda trying to think laterally about the whole DIY CO2 things and see if I can't do a little better!

For the set-up I currently have (CO2 into a ladder with HOB filter nearby) I don't think having more air in the bubbles will be a major disadvantage, but I may be wrong! The CO2 should still go into solution.

I'm not going to bother with wine yeast as I don't have an easy source for it and the main criteria behind my ideas are laziness! I.e. can i get better results with less effort!!!

I think, from what I know and what everyone has said here, that I will try and give this a go and see if it will work! Got to be worth a try! If anyone has more to add, please do!


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## Left C (Jun 14, 2005)

longhornxtreme said:


> [email protected], I'm curious about the particulars of your experiment where you found that sucrose was better than glucose considering sucrose is really just a glucose molecule joined to a fructose molecule...
> 
> ...After fiddling around with DIY CO2, I found that my wasted time was much more expensive than just ordering a 5# CO2 bottle, and built my own regulator. Pressurized is just wonderful. Fermentation will be used to brew BEER for me, not for feeding my plants


Hi LHX

I can't remember now why sucrose was better. I don't remember if it was an enzyme or the shape of the molecule, etc. I've been scratching my head, but I can't remember now. Before the test, I would of sworn that D-glucose (dextrose) would be the best.

I put pressurized systems on my tanks near the end of this test and I forgot about DIY CO2 completely.

Like you said LHX, yeast has much better uses!


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