Bacteria in a bottle, Myth or Fact

[BigJohnny]

Hello Everyone:

DrTim from DrTim's Aquatics and the developer of DrTim's One and Only. I thought I would try to answer some of the questions about how bacteria can stay alive in a bottle. I can only speak for my product and what we did at Marineland with BioSpira which I also developed. I have been studying and growing nitrifiers for over 20 years for reference so have a lot of experience with these bugs.

First and most obvious bacteria, including nitrifying bacteria, are not human. They do not have lungs or much of anything else humans or other multi-cellular organisms have in terms of needs to survive. So first, they do not need oxygen to survive. They need oxygen to convert ammonia to nitrite or nitrite to nitrate but that is to gain energy which to a bacterium is different than surviving. So the liquid in the bottle does not need a constant supply of oxygen unless one wants the bacteria to be dividing in the bottle. Thus if you don't add ammonia to the bottle there is not need to supplement with oxygen and the bacteria do quite well.

So if don't they feed (no ammonia or nitrite in the bottle) and don't require oxygen how to they survive. The answer is that all bacteria have cellular mechanisms to adapt and survive when conditions are not good for them. Many bacteria form spores. Nitrifiers do not form spores but produce a sugar inside that cell that helps them survive in poor conditions.

The other thing about bacteria is that they are hard to kill. Unless you break the cell open or disrupt the DNA/RNA or poison the cell (and some other things) the bacterium survive in a viable state. That is why they have been around for so long. Further, nitrifying bacteria live in a biofilm that acts as additional protection.

So how can they survive? Think of the nitrifier cell as a rechargeable battery. It's bottled at 100% energy level and since the organism does not have any ammonia or nitrite to feed on it does not have to spend any energy reproducing. Also since the conditions are not optimal for reproducing it goes into a resting stage and uses very little energy from its energy center waiting for conditions to improve. In this state they can last a long time in a bottle and work once poured into your tank. And the last key things is that the nitrifiers have to be the right species but that another story!

While many people think heat kills nitrifiers that is not true unless you get the liquid really hot what kills them is freezing. If the liquid is frozen solid for a period of time the ice crystals that form puncture the cell wall killing the bacteria.

So keep the nitrifying bacteria viable in a bottle you don't need secret sauce, you don't need to add amino acids or ammonia or nitrite. You need the have the right bacteria in the bottle to start (the key difference among products) and you need to keep the bottle from freezing.

I hope this explains a few things about nitrifiers - DrTim

Hey dr. Tim, can you explain why biospira originally required refrigeration but now does not?

Thanks

 

[Dr. Reef]

Also how many strands of bacteria are there in your product. Talking to people at microbelift they claim to have three strands while the bottle only mentions 2.
Thank you dr. Tim I really appreciate you coming up on this forum and explaining the science behind the bugs.

 

[DrTim]

Hey dr. Tim, can you explain why biospira originally required refrigeration but now does not?

Thanks

Hello - I have no idea why it is not refrigerated nowadays as I have had nothing to do with BioSpira for a long time now.

Originally, it was refrigerated at 50 F because at that temperature the bacteria are basically in suspended state and they will last a very long time in the bottle. But any colder is not good and most refrigerators are usually set to about 38-40 F.

 

[DrTim]

Also how many strands of bacteria are there in your product. Talking to people at microbelift they claim to have three strands while the bottle only mentions 2.
Thank you dr. Tim I really appreciate you coming up on this forum and explaining the science behind the bugs.

You're welcome - the marine (saltwater and reef) product has 5 nitrifiers while the freshwater has 3.

 

[brandon429]

Dr Tim if I could ask a couple clarifying questions about nitrifers regarding natural sourcing of marine groups from nature it would help round out the total picture for me.

Classic 80s freshwater cycling was fill, add plastic decor and epoxy rocks -wait- 30 days before buying guppies and swords and the tank won’t go cloudy, the base biofilm/nitrifers layer seeded and fed from nature, from our non asceptic fill technique and perhaps to some degree by air vectoring/attached to flotsam wafting about, aerosol xmission for a select few... however the input ~30 days submersion time and the tank would do fine

Many new aquarists often hedged a little bit...two weeks seemed good enough, but as soon as that load of fish went in / cloudy death by 48 hours cuz a little feeding never hurt as well

But something magical, universal it seems, about waiting thirty days for freshwater setups... = reliability anywhere we see people setting up tanks using old school submersion time only cycling.

That same nature and wait technique for reef tanks in the middle of cotton fields, highly dry climates, no where near the ocean, when we get dry Marco rock systems and dried pool filter sand bedded systems and fill them up with sw we mixed, all of this originating as dry start materials, we have to wait somewhere in the order of 90-120 days or so but the marine system still base-cycles off nature alone...how in the world are marine nitrifers circling the globe and staying alive enough to be delivered among the cotton fields and Amarillo and Lubbock I’d certainly like to know? If the mix materials we are using are vectoring in the filtration bacteria, which source is it? Surely dried pool sand and Marco rocks won’t bring in bac...is it our mix water originating as freshwater but still delivering marine able nitrifers?


I saw one your published studies /dna assays on nitrifiers from fw filters and in the freshwater ones there were still marine outliers present but in smaller numbers. Tiny numbers if I recall

On a bottle of tetra fresh start it says add 2x as much for marine tanks...one bottle inoculates both sources, both salt extremes, somehow these details tie into unassisted cycling even for marine tanks across the globe and that’s fantastic microbiology.

I’m thinking as you’ve hinted about additional nitrifying strains being available beyond the common ones we harness, perhaps some yet to be discovered, we’ve gained benefit from clades that are euryhaline tolerant or are some way equipped to function in both sw and freshwater? The fact unassisted saltwater cycling takes four time longer ish, that freshwater inoculates still work for reefs, and that your sampling still showed some marine bac present in fw filters (if I read correctly) all seems to imply that reef bacteria travel everywhere beyond reefs at least to some measurable degree.
Brandon

 

[DrTim]

Hello Brandon

Your post was quite incisive. Let me fill in the missing parts and answer your questions.

The primary source of nitrifying bacteria for most people is their tap water. Especially since the start of using chloramines to disinfect tap water the unintended consequence has been the nitrification of drinking water. This happens because the chloramine added to dis-infect the water dissociates along the journey to your house producing ammonia. The ammonia is then nitrified by nitrifying bacteria living in biofilms on the walls of the pipes. This is why water reaching your house can have high nitrate levels. There are several published studies showing how resistant nitrifiers (in their biofilm) are to chloramines -- much more so than free swimming bacteria.

Further, as you noted, research has shown that even in freshwater nitrifying cultures there are some cells that will dominate when the salinity is increased. There are always some 'rogue' bugs in every culture that adapt to changing conditions. As I have written before these bugs are very patient and work on a time scale that we have a hard time imagining.

So why does unassisted saltwater take 3-4 times longer. Two factors are involved in this. 1) Numbers - the bacteria might be circling in the air but the prime source is the water but the number of saltwater tolerant cells in the water is very low so it takes a long time for the numbers to increase to the point where they can take care of the ammonia in the tank, why?; 2) Physiology - there is a nice study predicting the upper salt tolerance of ammonia- and nitrite-oxidizing near 40-50 ppt with nitrite-oxidizing bacteria (NOB) being less tolerant then the ammonia-oxidizing bacteria (AOB). This is exactly what has been found when culturing these bacteria. The saltier the water the slower the AOB and NOB grow. This is due to the cell having to spend more and more energy just maintaining the cell shape (meaning keeping it intact a process called cell turgor). NOB are more affected then AOB which is why nitrite rather than ammonia is the one that takes so long to cycle.

So the practical advice of this - when first setting up a marine tank use a salinity of 20-24 ppt. The bacteria will work faster and once established you can increase the salinity to 30-34 ppt.

Best

 

[RichtheReefer21]

13 weeks ago I was a first time salt water reef rookie. I used a combination of 2 turbo snails, 4 hermits and an emerald crab to get my cycle started.

I have a 55gallon. 30lbs of dry rock, and one 10 pounx chunk of cultured live rock from the LFS. Used an old aqueon 75g hotb filter packed with matrix and dosed Aquavitro stability and API bacteria in a bottle twice a day, and i had a fully cycled in 3 weeks.

I now have 34 species of coral, and 7 fish. 13 weeks in.

It works, period.

 

[clark griswold]

Hello Everyone:

DrTim from DrTim's Aquatics and the developer of DrTim's One and Only. I thought I would try to answer some of the questions about how bacteria can stay alive in a bottle. I can only speak for my product and what we did at Marineland with BioSpira which I also developed. I have been studying and growing nitrifiers for over 20 years for reference so have a lot of experience with these bugs.

First and most obvious bacteria, including nitrifying bacteria, are not human. They do not have lungs or much of anything else humans or other multi-cellular organisms have in terms of needs to survive. So first, they do not need oxygen to survive. They need oxygen to convert ammonia to nitrite or nitrite to nitrate but that is to gain energy which to a bacterium is different than surviving. So the liquid in the bottle does not need a constant supply of oxygen unless one wants the bacteria to be dividing in the bottle. Thus if you don't add ammonia to the bottle there is not need to supplement with oxygen and the bacteria do quite well.

So if don't they feed (no ammonia or nitrite in the bottle) and don't require oxygen how to they survive. The answer is that all bacteria have cellular mechanisms to adapt and survive when conditions are not good for them. Many bacteria form spores. Nitrifiers do not form spores but produce a sugar inside that cell that helps them survive in poor conditions.

The other thing about bacteria is that they are hard to kill. Unless you break the cell open or disrupt the DNA/RNA or poison the cell (and some other things) the bacterium survive in a viable state. That is why they have been around for so long. Further, nitrifying bacteria live in a biofilm that acts as additional protection.

So how can they survive? Think of the nitrifier cell as a rechargeable battery. It's bottled at 100% energy level and since the organism does not have any ammonia or nitrite to feed on it does not have to spend any energy reproducing. Also since the conditions are not optimal for reproducing it goes into a resting stage and uses very little energy from its energy center waiting for conditions to improve. In this state they can last a long time in a bottle and work once poured into your tank. And the last key things is that the nitrifiers have to be the right species but that another story!

While many people think heat kills nitrifiers that is not true unless you get the liquid really hot what kills them is freezing. If the liquid is frozen solid for a period of time the ice crystals that form puncture the cell wall killing the bacteria.

So keep the nitrifying bacteria viable in a bottle you don't need secret sauce, you don't need to add amino acids or ammonia or nitrite. You need the have the right bacteria in the bottle to start (the key difference among products) and you need to keep the bottle from freezing.

I hope this explains a few things about nitrifiers - DrTim

Follow up questions about heat and expiration dates. All of my LFS have Dr. Tim’s bottles past their expiration date. What effect does this have on its efficacy? Would ordering direct from your site ensure getting a fresh batch?

I’ve also been waiting to order as I live in Texas and worry about a shipment sitting out in the 100 plus degree weather. Any worries there? Thank you. Great to see you on reef2reef.

 

[brandon429]

That's just fantastic information and in the first paragraph alone you covered items we've pondered and chatted about as a great paradox, tap water nitrifiers, in detail I'd never read before. You have given me lots more to read on now much appreciated

The salinity adjustment, wonderful strategy how fun is that for tank tuning!

Thank you much for the reply Dr T it means a lot
B

 

[DrTim]

13 weeks ago I was a first time salt water reef rookie. I used a combination of 2 turbo snails, 4 hermits and an emerald crab to get my cycle started.

I have a 55gallon. 30lbs of dry rock, and one 10 pounx chunk of cultured live rock from the LFS. Used an old aqueon 75g hotb filter packed with matrix and dosed Aquavitro stability and API bacteria in a bottle twice a day, and i had a fully cycled in 3 weeks.

I now have 34 species of coral, and 7 fish. 13 weeks in.

It works, period.

Unfortunately, we can tell what 'it' is. Is 'it' the cultured rock, is 'it' the old aqueon filter with matrix, is 'it' the Stability, is 'it' the API bacteria (dosed twice days for how many days?) or is 'it' a combination of one or more.

In the end doesn't matter since you are happy but no way which 'it' it was!

 

[cracker]

Dr Reef, I think Your original post & test is the best. Best fits the the average salt keeper. There are a lot of posts here suggesting this & criticizing that. I personally look forward to your test results !
Also Dr Tim's insight about bacteria living in our water source is something I would never had considered.
Last round on cycling a Q/t, I used Sea chem stability. I have a bottle of Dr Tim's on hand for the next QT setup.

 

[DrTim]

Follow up questions about heat and expiration dates. All of my LFS have Dr. Tim’s bottles past their expiration date. What effect does this have on its efficacy? Would ordering direct from your site ensure getting a fresh batch?

I’ve also been waiting to order as I live in Texas and worry about a shipment sitting out in the 100 plus degree weather. Any worries there? Thank you. Great to see you on reef2reef.

Thanks, glad to be here.

Product past the expiration date will work better than nothing. If you buy directly from us you get fresh material bottled that week. As for high heat there is an optional extreme weather kit - gel ice in a styrofoam box to keep the bacteria from overheating.

 

[NY_Caveman]

Hello Brandon

Your post was quite incisive. Let me fill in the missing parts and answer your questions.

The primary source of nitrifying bacteria for most people is their tap water. Especially since the start of using chloramines to disinfect tap water the unintended consequence has been the nitrification of drinking water. This happens because the chloramine added to dis-infect the water dissociates along the journey to your house producing ammonia. The ammonia is then nitrified by nitrifying bacteria living in biofilms on the walls of the pipes. This is why water reaching your house can have high nitrate levels. There are several published studies showing how resistant nitrifiers (in their biofilm) are to chloramines -- much more so than free swimming bacteria.

Further, as you noted, research has shown that even in freshwater nitrifying cultures there are some cells that will dominate when the salinity is increased. There are always some 'rogue' bugs in every culture that adapt to changing conditions. As I have written before these bugs are very patient and work on a time scale that we have a hard time imagining.

So why does unassisted saltwater take 3-4 times longer. Two factors are involved in this. 1) Numbers - the bacteria might be circling in the air but the prime source is the water but the number of saltwater tolerant cells in the water is very low so it takes a long time for the numbers to increase to the point where they can take care of the ammonia in the tank, why?; 2) Physiology - there is a nice study predicting the upper salt tolerance of ammonia- and nitrite-oxidizing near 40-50 ppt with nitrite-oxidizing bacteria (NOB) being less tolerant then the ammonia-oxidizing bacteria (AOB). This is exactly what has been found when culturing these bacteria. The saltier the water the slower the AOB and NOB grow. This is due to the cell having to spend more and more energy just maintaining the cell shape (meaning keeping it intact a process called cell turgor). NOB are more affected then AOB which is why nitrite rather than ammonia is the one that takes so long to cycle.

So the practical advice of this - when first setting up a marine tank use a salinity of 20-24 ppt. The bacteria will work faster and once established you can increase the salinity to 30-34 ppt.

Best

@DrTim great info and great to see you join in on Reef2Reef! Welcome!

 

[DrTim]

That's just fantastic information and in the first paragraph alone you covered items we've pondered and chatted about as a great paradox, tap water nitrifiers, in detail I'd never read before. You have given me lots more to read on now much appreciated

The salinity adjustment, wonderful strategy how fun is that for tank tuning!

Thank you much for the reply Dr T it means a lot
B

For those that want to go deeper on this PM me and I can provide references or I can post a reference list here.

 

[RichtheReefer21]

Perhaps my choice of dialogue could have been, well, softer.

I was not implying that it was purely the added bacteria that resulted in my success. But I am quite confident that a cycle in 3 weeks on a brand new tank is not a typical result without some serious help to control spikes.

The first week I dosed 34mL a day. 17mL in the a.m., 17mL p.m.
Week 2 I added hermit, snails, and the crab and dropped to 25mL a day.
Week 3 I had a full cycle and added what I call cockroaches of the sea. (Hearty and tough to kill)
A juvenile damsel. Dosing 10mL twice a day.
Mini cycle successful again.
At this point I went down to 7mL a day and added my 2 clowns.
When that mini cycle took place I was done and stopped treatment. 1 month after starting I had a bio load and no spikes.

Over the next 2 months I simply dropped 30mL of stability each time I added a new fish. 1 at a time of course. And never once got to dangerous nitrogen cycle levels at all.

And I didnt know any better, so I was only doing 10% water changes every 3 weeks thru all of this. I did 1 big (40%) one after my first cycle to stay ahead of nitrates.

I know every situation is different... but I truly believe (though I cannot prove) that I could not have achieved these results as fast as I did without dosing the bacteria.

I chose 2 different brands to keep the bacteria diverse and not overpopulated 1 general strain.

Following this thread. Very nice article and write up. Glad to have this topic up and buzzing.

 

[Mr.Asbury]

Awesome idea. I'm setting up a tank soon, so I'm really excited to see your results.

 

[CherBear811]

Ok, GREAT idea! I haven't read all 7 pages, so please forgive me if someone has already pointed this out, but.....

Both Dr Tim's and Bio Spira WILL knock out that 1.5 ppm ammonia fast, then if the tank doesn't have waste breakdown, the bacteria will starve and die off. I have used both products multiple times. Working in a LFS for 3 years, I sold I don't know how much Bio Spira, but it was a lot - to pretty much every new set up and to many people dealing with crashes and ammonia spikes. It always worked. I have used it myself on multiple new setups and also used it whenever I moved my systems (which as a renter has been more moves than I would have liked) and I have had ZERO losses in fish using this practice.

But my big concern here is the methodology. When we recommend cycling with live bacteria, we also recommend putting a fish or 2 (for larger tanks) in the system so there is something in there peeing and pooping for the bacteria to feed on as it colonizes and grows.

Is the purpose of the test just to see how long each method takes to bring ammonia and nitrite down to zero?

 

[Brew12]

For those that want to go deeper on this PM me and I can provide references or I can post a reference list here.

Not a question, but thank you for putting this on your website! I've read some of your studies but its nice to have them all in the same place!
http://www.drtimsaquatics.com/resources/library-presentations/scientific-papers

 

[User1]

@DrTim Thanks for stopping by and answering some questions. Appreciate it!

 

[Dr. Reef]

Is the purpose of the test just to see how long each method takes to bring ammonia and nitrite down to zero?

Yes by now we know bacteria works just by sheer number of success stories.
How well is what test will prove. Time for it to take ammonia of 2ppm and bring it down to 0.