Join Date: Oct 2007
Location: Ontario, Canada
You can use DIY CO2 24/7, usually even with 100% diffusion you can't get high enough CO2 to cause any harm for your fish. What people usually do with DIY CO2, since you can't turn it off, is to have an airstone running about an hour before lights turn off, and until the next morning when the lights turn back on. This gasses out the excess CO2 at night time when the plants aren't photosynthesizing, but producing CO2 themselves.
I have the same set-up as you, 2.5 gallon, 27w homedepot light, and I run 2x 2l DIY CO2, but that's only cause there's no livestock.
One 2 litre DIY CO2 bottle is fine for a 2.5 gallon.