# Correspondence with Dr Ryan Newton  - School of Freshwater Sciences, University of Wisconsin—Milwaukee



## dw1305 (7 Dec 2022)

Hi all,
Leading on from our discussion about the <"microbes that occur in aquarium filters"> I contacted, Ryan Newton <"Microbiology | Newton Lab Uwm">, one of the authors of  "Bartelme RP, Smith MC, Sepulveda-Villet OJ, Newton RJ. (2019). "Component microenvironments and system biogeography structure microorganism distributions in recirculating aquaculture and aquaponic systems". _mSphere_ *4*:e00143-19. <"https://doi.org/ 10.1128/mSphere.00143-19">.

He has very kindly allowed me to post the correspondence: I'll start with his reply, and then I'll post my original  email in the next post. 


> Hi Darrell,
> Thank you for reaching out with your questions. I am glad Dr. Bartelme’s work is of interest.
> 
> It is a good question to ask where the initial inoculum of nitrifiers comes from & it is a question that I do not have a definitive answer. Nitrifiers are present in many environments because they can live with comparably low external nutrients (carbon particularly). There are a couple of good possibilities, 1) the water - most municipal water systems contain some number of nitrifiers, which then come out of your residence tap; 2) the plants - nitrifiers are also commonly associated with plants. Or, it could be they drift in from the air - seems less likely, but it is not impossible.
> ...


cheers Darrel


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## dw1305 (7 Dec 2022)

Hi all,
And this was the email I sent.  For those who are short of time the two questions asked were:


> If we planted an aquarium and waited for the plants to "grow in" over ~6 weeks, where does the initial inoculum of AOA and comammox _Nitrospira_ come from? and
> If we did need to add them? What would be the best source of initial inoculum?


My email


> Dear Dr Newton,
> Apologies for this unsolicited email. We were initially going to try and contact Ryan Barthelme, but I see he has moved on to different fields.
> 
> My name is Darrel Watts and, when I'm not at work, I'm a science adviser for the <"United Kingdom Aquatic Plant Society (UKAPS)">. This is a forum which deals with planted tanks, and has a mixed membership, mainly, but not exclusively, from the UK, with a fair sprinkling of scientists as members and a generally "informed" level of debate. The genesis of this email were the conclusions in <"Component Microenvironments and System BiogeographyStructure Microorganism Distributions in RecirculatingAquaculture and Aquaponic Systems">
> ...


cheers Darrel


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## Tim Harrison (7 Dec 2022)

This is great, thanks Darrell. It pretty much confirms what you/we all suspected. I'd say this puts you on a firm footing when it comes to sharing with other more fish orientated forums. Still be interesting to know what they make of it.


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## John q (7 Dec 2022)

Fantastic effort Darrel, appreciate the work you've put into this subject over the years.


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## MichaelJ (7 Dec 2022)

John q said:


> Fantastic effort Darrel, appreciate the work you've put into this subject over the years.


hear, hear! 

Cheers,
Michael


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## jaypeecee (7 Dec 2022)

dw1305 said:


> He has very kindly allowed me to post the correspondence: I'll start with his reply, and then I'll post my original email in the next post.


Hi @dw1305 

Looks like I have some serious catching up to do!

Congratulations on pulling together all this information!

JPC


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## Wookii (9 Dec 2022)

Nice one Darrel  👍 really interesting to read his response, and confirmation of what has been discussed on this forum.

His point on commercial microbial tank start bottles is interesting, and could explain why some people appear to have success with them, but others don't.  Is there a reason why these manufacturers don't use the actual microbes commonly found in our filters - presumably they don't survive adequately in a bottle, or are more difficult to farm?

It would be interesting to know how long these microbes can survive outside of their intended environment.


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## dw1305 (12 Dec 2022)

Hi all,


Wookii said:


> Is there a reason why these manufacturers don't use the actual microbes commonly found in our filters - presumably they don't survive adequately in a bottle, or are more difficult to farm?


I think some of it maybe to do with supply and demand and some to do with less easy to culture. I'm going to presume that the aquarium trade is a very small percentage of the market for microbial supplements, and that most of the demand is for <"sludge buster"> type products. To produce those (for both ponds and septic tanks) you are going to want a high ammonia load bioreactor, which isn't going to be very suitable for low ammonia situations. It maybe that the <"oligotrophic microbes"> are still present at high ammonia levels, which could be the why Dr Nelson found


> ........... We also tested a commercial product of nitrifiers & it did decrease the time to ammonia oxidation start-up. It was slower than our biofilter material transfer, but much quicker than doing nothing. However, the microbes present in the system from the commercial product disappeared over a few weeks and were replaced by those more common to our system. So, it seems some products could help “jump-start” the process,..........


I'm going to guess that they are still present in commercial supplements, but only at low abundance. 


Wookii said:


> It would be interesting to know how long these microbes can survive outside of their intended environment.


That is an interesting question. It looks like COMAMMOX _Nitrospira_  and Ammonia Oxidising Archaea (AOA) are pretty much universal in low ammonia situations, but that could be because,

they are very efficient at finding these resources, or
it could be because they've had a very long time (most of the last 3.5 billion years) to become universal or
a combination of both factors.
I don't know the answer to that one, again I'd guess it is a mixture of both factors, but I have no idea of the relative proportions of "_found it_" to "_sat waiting_".

cheers Darrel


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## dw1305 (12 Dec 2022)

Hi all,


Wookii said:


> Is there a reason why these manufacturers don't use the actual microbes commonly found in our filters - presumably they don't survive adequately in a bottle, or are more difficult to farm?


I've been thinking about this and I think the easiest answer to that would be to sell a plant which comes with its own special microbial cargo. You would know it was Kung-Fu Panda's <"secret ingredient soup">, but no-one else would.


> ........... If you do need to add nitrifiers the best source is from an aquaponics or aquaculture system that is already running and removing ammonia. Some water or sediment/soil or part of the biobilter (if there is one) is an excellent starter. Without this source as an inoculum then you could add some roots from plants from any other tank that is running - these are likely to have nitrifiers associated with them. A small clipping put into the tank would be enough........


Which would suggest that any, non-tissue cultured, plant would do.   If you were to culture a submerged plant, in a leaf mold based medium, I'm 99% certain that would work as an inoculum source.

You would just need a plant that is ~guaranteed to grow. I'm going to suggest _Hygrophila corymbosa_ , but there might be another, even more bullet proof, option.

cheers Darrel


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## Wookii (12 Dec 2022)

dw1305 said:


> Hi all,
> 
> I've been thinking about this and I think the easiest answer to that would be to sell a plant which comes with its own special microbial cargo. You would know it was Kung-Fu Panda's <"secret ingredient soup">, but no-one else would.
> 
> ...



My question was a little loaded - thinking of my new tank, I was wondering - if I were to inoculate a fresh substrate with the mulm from an old filter, sometime in advance of completely filling the tank with water, how long would the microbes be able to survive to populate the substrate (assuming it was kept wet).


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## dw1305 (12 Dec 2022)

Hi all,





Wookii said:


> My question was a little loaded - thinking of my new tank, I was wondering - if I were to inoculate a fresh substrate with the mulm from an old filter, sometime in advance of completely filling the tank with water, how long would the microbes be able to survive to populate the substrate (assuming it was kept wet).


I'm pretty sure that it would be fine. I'd guess that even recently "dry" would still be OK.

Cheers Darrel


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## dw1305 (12 Dec 2022)

Hi all,


dw1305 said:


> that even recently "dry" would still be OK.


This one <"Distinct responses from bacterial, archaeal and fungal streambed communities to severe hydrological disturbances"> suggests that even drying may not be that much of an issue.


> ........ Throughout the experiment, archaeal communities colonizing sediments showed greater phylogenetic distances compared to those of bacteria and fungi, suggesting considerable adaptation to severe hydrological disturbances. The increase in the class abundances, such as those of Thermoplasmata within archaea and of Actinobacteria and Bacilli within bacteria, revealed signs of transitioning to a drought-favoured and soil-like community composition......


Gionchetta G, Romaní AM, Oliva F, Artigas J. (2019) "Distinct responses from bacterial, archaeal and fungal streambed communities to severe hydrological disturbances". _Sci Rep_. 2019 Sep 18;9(1):13506

cheers Darrel


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## Wookii (12 Dec 2022)

dw1305 said:


> Hi all,
> 
> This one <"Distinct responses from bacterial, archaeal and fungal streambed communities to severe hydrological disturbances"> suggests that even drying may not be that much of an issue.
> 
> ...



Thanks Darrel - that’s good news, it means I can inoculate my base layer substrate and not be in a rush to flood the tank.

It also raises the possibility of making our own dry bacteria powder if so inclined - just filter out the mulm every time we clean a filter, and let it air dry before scraping into a pot for future use, stirring it into dry substrate when needed. Would that really work?


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## dw1305 (13 Dec 2022)

Hi all,


Wookii said:


> It also raises the possibility of making our own dry bacteria powder if so inclined - just filter out the mulm every time we clean a filter, and let it air dry before scraping into a pot for future use, stirring it into dry substrate when needed. Would that really work?


That is an interesting one.  I'm going to say you almost certainly could, and it would work, but with some provisos.

I'm going to guess it would take a lot longer to "cycle" than if you had fresh filter mulm, but the difference in that time is just an unknown.  

I also think that a plant and substrate from an existing tank is more likely to provide a more complete microbial assemblage, because it would have the <"commensal organisms from the plant rhizosphere"> and surrounding zone of <"fluctuating REDOX"> values.

cheers Darrel


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## Ria95 (13 Dec 2022)

Wookii said:


> Thanks Darrel - that’s good news, it means I can inoculate my base layer substrate and not be in a rush to flood the tank.
> 
> It also raises the possibility of making our own dry bacteria powder if so inclined - just filter out the mulm every time we clean a filter, and let it air dry before scraping into a pot for future use, stirring it into dry substrate when needed. Would that really work?


If you look at the paper, the „dry treatment”  means that no additional water was added to the experiment.  While I can‘t imagine we would call the environment wet, there would be plenty of microenvironments in the leaves that have enough humidity to enable survival and far from complete desiccation. Even then, spore forming microbes will survive. So if we are talking about microbes in general, the answer would be yes and you can find plenty of commercially available powdery bacteria mixes with numerous claims to fame.
If we reduce the discussion to non-spore forming nitrifying  bacteria then likely a home made dry bacteria powder won‘t prove a great starter.


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## dw1305 (13 Dec 2022)

Hi all, 


Ria95 said:


> Even then, spore forming microbes will survive. So if we are talking about microbes in general, the answer would be yes and you can find plenty of commercially available powdery bacteria mixes with numerous claims to fame.
> If we reduce the discussion to non-spore forming nitrifying bacteria then likely a home made dry bacteria powder won‘t prove a great starter.


That would be my guess, it would always be sub-optimal compared to fresh filter mulm or "_growing plant with substrate_" options. 

Hypothetically, if some-one was serious about this, hydrogels might offer a way forward <"Immobilization of active ammonia-oxidizing archaea in hydrogel beads - npj Clean Water">


> _........ In this study, AOA was embedded in small (~2.5 mm) and large (~4.7 mm) poly(vinyl alcohol) (PVA)—sodium alginate (SA) hydrogel beads ......... Sulfate-bound PVA-SA beads were the most stable, releasing the lowest amount of polymer without shrinking. Diffusion coefficients were found to be 2 to 3 times higher in hydrogels than in granules, with ammonium diffusivity being ca. 35% greater than nitrite and nitrate. Despite a longer lag phase in small beads, embedded AOA sustained a high per volume rate of ammonia oxidation compatible with applications in research and wastewater treatment........._


I think this paper also helps answer @Wookii 's earlier question


Wookii said:


> why these manufacturers don't use the actual microbes commonly found in our filters - presumably they don't survive adequately in a bottle, or are more difficult to farm?





> _Ammonia-oxidizing archaea (AOA) are major players in the nitrogen cycle but their cultivation represents a major challenge due to their slow growth rate and limited tendency to form biofilms......._


Landreau, M., You, H., Stahl, D.A. _et al._ (2021) "Immobilization of active ammonia-oxidizing archaea in hydrogel beads". _npj Clean Water_ *4*, 43. 

cheers Darrel


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## dw1305 (14 Dec 2022)

Hi all, 


dw1305 said:


> Hypothetically, if some-one was serious about this, hydrogels might offer a way forward <"Immobilization of active ammonia-oxidizing archaea in hydrogel beads - npj Clean Water">


I've sort of posted another "hydrogel" paper into the wrong thread, so I'll link it here: <"The nitrifying microbes in aquariums and cycling">. 

cheers Darrel


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