DIY CO2 primer for planted tanks.

[Tug]

CO2 - au naturel !
There is nothing contrary to nature about CO2 (carbon) supplementation for plants. All living organisms need carbon as a food source and macrophytes typically exhibit an increase in both photosynthesis and growth in aquatic environments with elevated CO2 levels of 30 ppm and above.

Plants need carbon, but the levels provided in tank water are often limited due to nutrient demands under high light intensities (PAR) and plant uptake, surface agitation (gas exchange) and low levels of dissolved organic carbon (DOC). This most important nutrient carbon, is regularly lacking in a planted aquarium and CO2 supplementation provides vigorous, healthy plant growth. In fact, aquatic environments with ample CO2 and moderate light exhibit growth rates 4 - 5 times greater then those without CO2 enrichment. Under bright light (80 μmol/m2s), CO2 is essential or a planted tank quickly becomes CO2 limited, leading to growth deficiencies and unwanted algae.

Is CO2 enrichment necessary in low light environments?
CO2 provides a needed nutrient, even in low light. When CO2 is plentiful
plants invest less energy towards capturing CO2 and more energy for optimizing light capturing pigments.

"At low light and low CO2 there is not much energy to play around with for up or down-regulation of the pools of Chlorophyll or enzymes. If we then add a little more CO2 to the system the plant can afford to invest less energy and resources in CO2 uptake and that leaves more energy for optimizing the light - more Chlorophyll can be produced without fatal consequences from the energy budget. Hence, we have not raised the light and the plants can now utilize any available light - more efficiently." - Tropica

Tropica's study on Riccia and the affect of resource availability on growth.

Diagram courtesy of John LeVasseur

Before purchasing a pressurized CO2 setup, consider using a diy yeast powered generator. Wade through all the pertinent information on dual stage regulators at your leisure (before you spend all that hard earned cash). The experience will prepare you for many of the problems inherent to all types of CO2 supplementation. Fear not, CO2 generators provide plenty of CO2. How it is dispersed into solution is the main problem we have in common with pressurized systems.

☯​

The high light requirement myth
http://www.barrreport.com/showthread.php/7503-The-high-light-requirement-myth
CO2 and Light Stimulate Growth, - Tropica
http://www.tropica.com/advising/technical-articles/biology-of-aquatic-plants/co2-and-light.aspx
All You Ever Wanted To Know About CO2 But Were Afraid To Ask
http://aquaticconcepts.thekrib.com/Co2/co2_faq.htm

This treatise is not yet complete and I will be updating this thread.

If you need more information or have questions, I can probably still help. Contact me here or check out the forums.

Alternatively, if you think you can provide me with accurate information, please don't hesitate to contact me here.​

 

[Tug]

Design elements

DIY CO2 setups:

• 
  CO2 injection
  Your most important consideration will be how to get CO2 into solution and to your plants. Once this element works properly, you should see big changes in growth. There are several known options for water injection systems according to the needs of your tank. Hagen ladders, diffusers, modified powerhead/needle wheels and reactors all come to mind. For smaller tanks a Hagen ladder or diffuser and a small powerhead might be all you need. Choose wisely, injection systems are critically important. Because available CO2 in a yeast system is determined by biological processes, how it is dissolved into the water is crucial to your success. Add CO2 efficiently and you will be glad for your effort.
  
  
• Getting there
  Standard airline tubing quickly reacts with CO2 and breaks down. Tubing designed specifically for CO2 can be expensive. For this DIY project, silicon tubing moves to the front of the line of options. Silicon tubing is affordable, has good pressure retention characteristics and is slow to deteriorate from exposure to CO2. Making it an attractive option.
  
  Prevent air leaks around the bottle cap to the CO2 generator. Leaving the plastic backing inside the cap as a gasket will help improve the seal around the cap.
  
  Attaching the tubing to the bottle cap requires some type of mechanical seal. Glue or silicon sealants are often recommended but in truth I find they bond poorly and eventually give way to the pressure yeast can create. Using the diameter of the line to press against a smaller opening in the cap works sometimes, maybe. The best solution is a bulkhead fitting. They are worth every penny.
  
  
  
  A check valve (CV). I use glass or plastic CV for yeast setups. A glass CV can easily be cleaned with rubbing alcohol if it begins to stick and plastic check valves can easily be replaced. What ever you choose to use, prepare for the likely chance you will need to replace or swap it out on occasion.
  
  Above all else, streamline your design. The fewer things that cause blockage, leakage or seepage the better. My favorite setup has a CO2 generator, a single line running to a modified Duetto multi filter and one check valve in-between.
  
  
• Fermentation - the driving force behind CO2 generators.
  
  The reaction of importance in this process is the an-aerobic conversion of simple sugars to ethyl alcohol and carbon dioxide during alcoholic fermentation as shown below. Theoretically, 4 cups of sugar (about 2 lb) can provide 9.5 cu ft of CO2. By comparison a 10 lb. CO2 tank contains about 90 cu ft of CO2 or four 5lb. bags of cane sugar.
  
  
  C6H12O6 + Saccharomyces cerevisiae = 2C2H5OH + 2CO2
  (Sugar plus yeast yields alcohol and carbon dioxide)​
  
  Yeast Strains and what you need to know.
  Bakers and Ale yeast are referred to as top flocculants. That viscous, tan head on the top of the sugar water, clogging air lines and causing check valves to stick comes from flocculants suspended along the water surface.
  
  Wine or Champagne yeasts ferment at a wider range of temperatures. They tolerate higher alcohol and acid levels, toxic to most yeast. They produce nearly twice the CO2 and have a tendency to settle towards the bottom of a culture, making it easier to pour off the liquid and leave the flocculant behind to feed your new culture.

Design
Regulating and maintaining consistent levels of CO2 with yeast is it's greatest challenge. Reduce the number of things that go wrong and focus on improving the efficiency of your design. The less your DIY needs to be babied the better. You may want to add a bubble counter or gas separator to prevent the yeast from traveling into the diffuser. Features that incorporate pressure release valves, gang valves and anti-clogging devices are not covered here, but the same rules apply. You want a reliable and safe design. DIY generators provide between 8 ~ 25 psi of pressure, more then sufficient CO2 for planted aquariums. At these levels of CO2, a quick release valve or a way for the CO2 to gas off at night will insure no pressure builds up in the generator. For this reason the powerhead I use provides me with a bubble counter and keeps the pressure from building up in the generator when the powerhead isn't running. It runs on a timer and as long as the yeast remains active, I never need to touch it.



I use one gallon jugs with a bulkhead fitting. Although soda bottles are often used, these 4L cubes take up less room then you might think, don't have a tendency to tip over, and comply with the general principle that bigger is better (depending on how you use it). Near the end of one fermentation inoculate another CO2 generator with a yeast culture, let it sit open for two hours to build up the yeast colony and it's ready to put in place. To maintain consistent CO2 levels two or more generators can be replaced in series.


:gw My personal blend.

3.7L (~16 cups) of tank water
4 cups of sugar (sucrose)
4:1 ratios work with champagne yeast. Wine yeast stops growing at ruffly 25% ethanol,
allowing for more sugar in a recipe and longer fermentations.

1/4 tsp of 5.2 pH Stabilizer
This is not baking soda.
It is a pH stabilizer to maintain an optimal pH for yeast cultures.

1/2 tsp Champagne yeast
Using less yeast might provide a longer fermentation, but 1/2 tsp works for me.

1/16 tsp Fermaid K.
Adding Fermaid K will significantly increase the longevity of your culture.

Interesting tidbits & links;

Fermaid K, is a blended yeast nutrient containing;
Magnesium Sulfate, Inactive Yeast, Thiamine, Folic Acid, Niacin, Biotin,
Calcium, vitamin B5 (Pantothenate) and DAP (Diammonium phosphate).

Pantothenate, (vitamin B5) helps to keep open important metabolic pathways,
dramatically reducing the production of H2S.

Magnesium improves yeast alcohol tolerance.

Related Links:
Tower Hobbies bulkhead fitting
http://www3.towerhobbies.com/cgi-bin/wti0001p?&I=LXG849&P=ML
Exploding diy Co2 containers
http://www.thekrib.com/Plants/CO2/yeast-disaster.html
DIY CO2 Systems for Freshwater-Planted Aquaria, by John LeVasseur
http://www.qsl.net/w2wdx/aquaria/diyco2.html
CO2 Injection Methods - pros and cons
http://www.barrreport.com/showthread.php/5851-CO2-Injection-Methods-pros-and-cons
Tom's DIY internal Reactor, great for Yeast CO2 users!
http://www.barrreport.com/showthread.php/41-DIY-internal-Reactor-great-for-Yeast-CO2-users
My DIY internal Reactor, also great for Yeast CO2 users!
http://www.barrreport.com/showthread.php/6231-DIY-CO2-reactor-surface-skimmer?p=40663#post40663

 

[Wet]

Especially awesome here is that you've placed so much priority and article space to a) the relationship between light and CO2 levels, b) the importance in planning what it is the user wants to acheive, and c) efficiency in the mixture including links to specific products in stock. I hope you are taking suggestions!

1) After the wonderful setup with a) and b) above, I was disappointed that "Getting There" listed more minutia in what can go wrong (a bad seal, tubing, etc) than a plan to move forward. I imagine a new section in the article where we go step-by-step in the execution of deploying a CO2 system (preferably with pictures) for given bugets of, say, $20 and $50.

For example:
Let's imagine a gardener who's willing to DIY, is curious about CO2, and has a budget of $50, and they are reading this primer. How would you suggest that user invest in their system? Would you recommend spending, say, ~$10 on the bulkheads, ~$10 on two of those nice 1 gallon square containers? Would you spring for the Fermaid K or simply change the mix more frequently?

I think examples and thinking of user questions like these will reach an audience that other articles cannot. And I think you're particularly suited to write it because your decisions here scale very well: for example, if an aquarist did allocate $20 on the containers, s/he makes a great investment that they can carry onto their next (presumably larger) tank.

2) If your recipie maximizes CO2 production, won't the number of bottles required scale linearly with tank size? I mean that if 2x1 gallon jugs supports a 29 gallons, won't 4x1 gallon jugs support a 55 gallon?

But I think the trick is you don't use 4 bottles to one diffusor: instead, you pair bottles to separate diffusors, then place those diffusors on opposite sides of the tank. In other words, the Tug CO2 system scales not just to gallonage, but the challenges of larger tanks.

3) I have this idea that there's at least two types of DIY CO2 folks out there: folks who spend time making extremely efficient mixtures and folks who use the brute force approach of weekly (two bottles, each changed every two weeks, alternating each week) mixture changes. Because of the nature of yeast, this often is the difference between folks who seek a gradual (inevitable) decline in CO2 and folks who think of CO2 in terms of thresholds*. And I think instead of a middle ground we should look at designing the most awesome DIY CO2 system ever.

*What I mean by thresholds is sort of EI as I love it, and was my personal epiphany back when I used DIY CO2. Let's pretend C is no different than N. EI tells me that I don't care about C levels: I just can't have C hit too low. Let's say "too low" is 15ppm CO2. We know DIY CO2 -- aka, yeast -- fluctuates. So, we should stop targeting 15ppm CO2; instead, let's target 25ppm CO2. Our fluctuation (the inevitable deline in yeast output in a closed system) of, let's say, 7ppm CO2, is then unimportant since it remains above our threshold. So, I may as well just keep changing bottles...

In application, I think this thought experiment is supported by my personal experience and belief that the fluctuation between 20-30ppm CO2 can be a headache but the fluctuation between 35-45ppm CO2 is just dandy. Perhaps other folks do not agree.

4) I think of paired/chained CO2 bottles as in parallel (to one diffusor) than in series, for what it's worth. I only mention this because if someone uses a check valve between each bottle and the union to those bottles, either bottle can be disconnected without affecting the other. This may only be my misunderstanding and poor mental visualization for the term "in series," but maybe I'm not the only one?

5) More pictures and details of your set-up would be valuable. Again, those containers and the ideas are awesome and affordable!

When you get around to it, I hope you can take the time to take a picture and write down every step you take the next you change a mixture. I think this portion of your time will save many others many hours of time.

6) I know we're online friends and everything, and I don't think it needs to be said, but let me say all of the above are only unbaked ideas for your consideration! It's a great article and this is only a response from a reader who found your article generated lots of ideas!!

 

[Tug]

DIY CO2 deserves the attention to detail you are suggesting.

How would you suggest that user invest in their system? Would you recommend spending, say, ~$10 on the bulkheads, ~$10 on two of those nice 1 gallon square containers? Would you spring for the Fermaid K or simply change the mix more frequently?

• $10 plus shipping would purchase 3 of the containers. Any high-grade and food-grade plastic (polyethylene, polypropylene) will also work.
  
  
• Yeast nutrient like Fermaid K or Tronozymol (if you're in the UK) will ensure a healthy, active fermentation and a good steady supply of CO2. As an alternative, a small amount (½ tsp) of Malt Extract such as Marmite is an effective yeast nutrient.
  
  
• The bulkhead fittings are awesome. I would recommend them to anyone over any other method I have tried. They are relatively cheap and very reliable.

 

[quatermass]

People in the UK may wish to know that the equivalent nutrient replacement for Fermaid K is Tronozymol and you can get this from any decent Wine brewer shop.

For example: http://www.thebrewmart.com/tronozymol-nutrient-i601428.html

Of course some of us just use a Malt extract like Marmite! Hey if it works for Wine Makers...?

I've tried a Wine Yeast instead of the normal bread yeast and I must say the CO2 fair bursts out in comparison.

There are links to species of yeasts at the bottom of : http://theaquariumwiki.com/Yeast

 

[Tug]

Close.

Tronozymol does not contain any amino nitrogen.
What it does have:
Diammonium Phosphate (DAP)
Monopotassium Phosphate
Magnesium Sulphate
Nicotinamide (vitamin B3 / niacin)
Aneurine Hydrochloric (thiamine)
Trace minerals
Trace vitamins

Inactive yeast or Marmite
Adding a little inactive yeast or Marmite (another Liebig discovery ) should improve yeast development. This is because as fermentation progresses yeast have diminished ability to utilize DAP. After about the half-way point in the fermentation, it is better to provide nitrogen in the form of FAN (free amino acids) as a nutrient.

Find a yeast "energizer" that contains more amino nitrogen (inactive yeast) and less DAP. Fermaid K by Lallemand/Lalvin is just one example. They also make Fermaid 2133 which does not contain any DAP. Yeast extracts like Marmite and inactive yeast have some of the same characteristics (amino nitrogen and B vitamins). Marmite might taste better on toast.

 

[quatermass]

I've often thought adding some plant trace element powder to my mixtures might help as its full of trace minerals.
I've also wondered if adding Epsom salts and maybe Potassium nitrate for the nitrogen as I've got these on hand?

 

[Tug]

EI For Yeast

The trick seams to be using products that are readily assimilated by the yeast and avoiding those that are not.

Not everyone has access to Fermaid K or Tronozymol.
In this case, inactive dry yeast products (complete B complex supplements) along with some Epsom salts might be all that is needed to maintain a longer fermentation. If ammonia salts, Fermaid K or Tronozymol are difficult to purchase, give it a try.

More about Epsom salts:
Magnesium has been shown to improve yeast alcohol tolerance.
As it is seams to be one of the main ingredients in Tronozymol and Fermaid K, I do not see the advantage to adding more if you use those products. However, it is an essential ingredient for any diy nutrient mix.

Calcium (Ca):
50 - 200 ppm is typical considered beneficial.

Sulphate (SO4):
Normally best below 150 ppm but can go as high as 700 ppm.

Carbonates (CO3):
Best kept below 50 ppm, although my water is closer to 5.6-dKH (100ppm).

KNO3:
S. cerevisiae is incapable of using nitrate as a nitrogen source. More importantly, half-way through the fermentation nitrogen in the form of amino acids is more beneficial due to yeast's diminished ability to utilize ammonia salts. In most cases inactive yeast and ammonia salts can be added early in the fermentation through formulas like Fermaid K.

Trace minerals like CSM+B:
Adding CSM+B made my fermentation sluggish and I would not recommend adding it at anytime. There are rehydration nutrient mixes like "Go-Ferm" that contain a balance of micronutrients. They are added when rehydrating yeast cultures and allowed to rehydrate for 20-25 minutes.

Week four and the generator is burping out 2~3bps.
[video=youtube;RH4Po_gSNCQ]http://www.youtube.com/watch?v=RH4Po_gSNCQ[/video]

 

[quatermass]

"If your using EI dosing methods, the tank water should provide plenty of nitrogen and trace for the yeast to stay healthy. "

Are you using tank water right from the start to fill your fermenting bottle?

If I open my yeast bottle mid-term the reaction stops as it's no longer in anaerobic conditions. So I tend not to open the bottle once it's started.

 

[Tug]

I use tank water right from the get go.

The main advantages to using water from the aquarium would be elevated levels of PO4, SO4 and some trace minerals. One other possible advantage might be higher levels of O2 at the onset of fermentation then what is found in tap water.

If I open my yeast bottle mid-term the reaction stops as it's no longer in anaerobic conditions. So I tend not to open the bottle once it's started.

Well, when Oxygen is present, a colony of yeast doubles every 4 hours.

This may be useful information. My fermentation was sluggish and the BC was low, so I took off the cap and left it off overnight. The next morning I replaced the cap and had more output of CO2 then the day before. Anecdotal, I know. But, worth a closer look.

 

[quatermass]

Thanks for that Tug,
I've never had problems adding Malt Extract right from the start. Never tried these special additives as when one of the yeast bottles I use starts to slow up I simply replace it and the other one continues.

I use ordinary tap water as it's very soft here, GH 1d, KH 1 and 0.3ppm of chloride which is ridiculously low and has never hindered a Yeast fermentation to my knowledge.

 

[Tug]

Adding CSM+B when rehydrating a yeast culture.

So, I tried adding CSM+B when rehydrating my yeast culture. Otherwise, the recipe was unchanged and the yeast was from a new packet, but the fermentation was sluggish to the point of despair. I would not recommend adding CSM+B and have gone back to edit a earlier post when I considered trying it as a yeast supplement. The good news is that adding just a little Fermaid K has brought it back from the dead.

There are rehydration nutrient mixes like "Go-Ferm" that contain a balance of micronutrients. I am not sure what is in them, but CSM+B they're not.

 

[Tug]

Organic compounds

The major source for energy production in Saccharomyces cerevisiae is glucose. Glycolysis is the general pathway for conversion of glucose to pyruvate, whereby production of energy in form of ATP is coupled to intermediates in form of NADH for biosynthetic pathways.

Simple sugars: Glucose and fructose are directly assimilated by yeast through penetration of the cell membrane. Simple sugars are transformed into alcohol and carbon dioxide by zymase, an enzyme naturally present in yeast cells. These sugars are the first ones used in the fermentation process.

Complex sugars: Because of the 
complex composition of saccharose (sucrose) 
and maltose, these sugars are used later in the fermentation 
process. Approximately 30
minutes at the beginning of the 
fermentation period is necessary to 
achieve their enzymatic transformation
 into simple sugars. The enzymes involved are saccharase, which
 transforms saccharose into glucose and 
fructose, and maltase, which transforms maltose into glucose.

:gw Ultimately, the type of yeast we choose for the purpose of CO2 production and yeast longevity is more relevant.

A detailed list of different sugars and their properties can be found at http://www.geocities.com/lesjudith/HomeBrewingTips/AlternativeBrewingSugars.html,

 

[quatermass]

Hexose sugars such as glucose (favored food of Saccharomyces cerevisiae) and fructose, or disaccharides such as sucrose (table sugar) and maltose are an energy resource and carbon source for yeast.

A detailed list of different sugars and their properties can be found at http://www.geocities.com/lesjudith/HomeBrewingTips/AlternativeBrewingSugars.html,

That's an old and undead link.

See their latest site at: http://www.aussiehomebrewing.com/HomeBrewingTips/AlternativeBrewingSugars.html


BTW, little mention is made of Fructose. Is this because it effectively gives the same result as Sucrose do you think?

 

[Tug]

An Experiment

Made from corn syrup, pure, dry fructose.

For the purpose of an experiment, a generator could be fitted with an adjustable check valve and a pressure gage. This should show if different sugars provide more CO2 over extended periods of time.

 

[quatermass]

Or do a log of the daily count of the bubbles in two minutes at the same time of day?

 

[Tug]

Tinny bubbles, are in my mind...

Different sugars add different characteristics to the alcohol - flavor mostly. Unless you drink the brew from your DIY generator, developing a flavor component from one of the sugars mentioned does not effect CO2 production significantly.

Recording bubble counts and/or the pressures created could effectively show the efficacy of common yeasts used for DIY reactors; i.e., active dry yeast, instant yeast like "RapidRise", brewer's yeast and yeast used in winemaking.

Optimal temperatures: Yeasts vary in what temperature range they grow best. For example, Leucosporidium frigidum grows at -2 to 20 °C (28-68 °F), Saccharomyces telluris at 5 to 35 °C (41-95 °F) and Candida slooffi at 28 to 45 °C (82-113 °F).

The reason I use Champagne yeast is as much about temperature as anything else. Not only do they form less foam, less sulfur and ferment more sugars (tolerate higher alcohol levels) - they grow at a range of temperatures (50-86 °F) that suit my home environment.

 

[pat w]

Tug, Stuart,

Not a DIY yeast user, but I like a good DIY project. Excelent thread BTW.

I was thinking ... If you could save the unused/waste CO2 generated at night in some sort of pressure vessel and effectivly use it during the day couldn't you slow the pace of the yeast reaction and extend the usefull life of each batch? If you made the yeast reactor from a more robust material couldn't you keep at least some of the night's reserve and increase the economy.

If so, I've got an idea for a DIY pressure step down delivery system if it's worthwhile. It'd be a few $$'s over a std DIY budget (2 solenoid valves and a float switch). It might be a nice step for someone who's thinking about migrating to pressurized CO2. They could get some of the pieces and be using them while they're saving up for the other stuff.

Pat

 

[quatermass]

Hi Pat,
Really not practical to capture the CO2 without spending thousands of pounds.
I did try placing 3 CO2 bottles together onto a single CO2 ceramic diffuser and unfortunately the pressure wasn't enough and the CO2 reaction in all 3 bottles simply stopped.

One thing I did think of was to place an inverted cup over the top of the CO2 ladder I use so the wasted CO2 collects in there and underwater currents could try to dissolve it.
Shame the manufacturers of these ladders didn't think about that aspect in their designs. Still, fairly easy to glue on a piece of plastic to the top I guess.

 

[Tug]

Hi Pat,
How about a five-gallon, stainless steel soda keg?
The problem is that pressures over 35 psi can be toxic to yeast and if kept under pressure carbon dioxide can cross the cell walls and kill yeast cells. Plus, I would not expect it to extend the usefulness of the yeast. A pressure step down delivery system might be useful if CO2 was saved and used a week or two down the road. It's an expensive idea but it has a huge wow factor.

Extending the useful life of a fermentation can be done in a number of ways;

1. a large generator or dilution solution
2. yeast that ferment higher alcohol levels like Champagne yeast
3. providing optimal conditions for the yeast.

Another DIY CO2 strategy is to stagger more then one generator, overlapping their running and switching out one generator at a time, in an alternating pattern.

DIY project (experiment).
Using an adjustable check valve a generator to test different yeast strains. It should help slow down the number of bubbles I have to count when I check the bubble rate of the generator.

quatermass @

I did try placing 3 CO2 bottles together onto a single CO2 ceramic diffuser and unfortunately the pressure wasn't enough and the CO2 reaction in all 3 bottles simply stopped.

There must have been some back pressure created from somewhere. Did you add a check valve for each bottle, clean the ceramic disk and look for blockages?