Correspondence with the Neufeld lab. University of Waterloo

[dw1305]

Hi all,
Following on from the discussion in <"Does depleted KH stop the nitrogen cycle?"> I said I'd try and contact <"Josh Neufeld's lab">., I did and I'm delighted to say that both Prof. Neufeld and Dr Michelle McKnight have replied. I'll put the correspondence in the subsequent post, as there are several "sections" to it.

The reason for attempting to contact them was that his research group have published some very interesting scientific papers on novel nitrifying organisms and aquarium filters. Before I get onto the correspondence, I'll list the papers we've referenced on UKAPS.

• This one isn't a paper in a peer reviewed Scientific Journal, but it is well worth a read and I might start with it: <"http://cichlidpower.org.au/TCM/PDF/041/TCM041-201203_Apr_wcon.pdf"> (pages 14 - 20) in <"Hello from Kent">.
• McKnight, M & Neufeld, J. (2021) "Microbial community analysis of biofilters reveals a dominance of either comammox Nitrospira or archaea as ammonia oxidizers in freshwater aquaria" <"Microbial community analysis of biofilters reveals a dominance of either comammox Nitrospira or archaea as ammonia oxidizers in freshwater aquaria"> in <"Nitrites never zero"> & <"Does depleted KH stop the nitrogen cycle?">.
• Al-Ajeel, S., Spasov, E., Sauder, L.A., McKnight, M.M. and Neufeld, J.D., (2022). <"Ammonia-oxidizing archaea and complete ammonia-oxidizing bacteria in water treatment systems">. Water Research X, p.100131. in <"Seeding new filter media . . ."> I obviously liked this one as it is linked into multiple threads: <"Dr Timothy Hovanec's comments about Bacterial supplements">, <"Does depleted KH stop the nitrogen cycle?"> & <"The nitrifying microbes in aquariums and cycling">.

cheers Darrel

 

[dw1305]

Hi all,
Prof. Neufeld's initial reply:

Hello Darrell,
So sorry for the delay. Conference travel and holiday.
I’ve cc’d Michelle McKnight, the lead author on the more recent aquarium study. I don’t think we’ve done any specific analyses looking for plant-specific indicator species, but this could be done. One thing I can say is that we have some “new aquarium start-up” data that points to plants as a likely inoculation source for a broader diversity of nitrifiers, but the data in hand are only able to weakly support this hypothesis.
Michelle, anything you’d like to add?
Darrell, if you are ever looking for a guest speaker for a society meeting, please let me know. I’m eager to connect with our most important “stakeholders” for this research 😊
Thanks for reaching out – it’s great to hear from you.
Josh

and my question, which to cut to the chase, the short version is:

Which says:
“Approximately half of the sampled aquaria contained live plants”
Our questions were:
Whether you used this variable in your analysis?
And if you did?
Do aquaria with live plants have a different microbial assemblage in the filter?

and the full email was:

Dear Dr Neufeld,
Apologies for this unsolicited email. I appreciate that academics are busy people.
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 freshwater planted aquariums, 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.
We’ve contacted you partially because your 2012 article “Strangers in your home: Archaea thrive in aquarium biofilters” suggests that you have a long-standing interest in aquaria and the microbial assemblage they contain.
The real genesis of this email were your labs. <"Neufeld Research Group | Neufeld Research Group | University of Waterloo"> papers with Laura Sauder and Michelle McKnight (on AOA and comammox Nitrospira in aquaria filter systems) and particularly:
McKnight MM, Neufeld JD. (2024). "Comammox Nitrospira among dominant ammonia oxidizers within aquarium biofilter microbial communities". Appl Environ Microbiol 90:e00104-24. <"https://doi.org/10.1128/aem.00104-24">
Which says:
“Approximately half of the sampled aquaria contained live plants”
Our questions were:
Whether you used this variable in your analysis?
And if you did?
Do aquaria with live plants have a different microbial assemblage in the filter?
About UKAPS
As a body we are keen on fish keepers keeping planted tanks.
In recent years we’ve had threads (in this ~ subject area) with contributions from Tim Hovanec <"The nitrifying microbes in aquariums and cycling"> and Ryan Newton <"Correspondence with Dr Ryan Newton - School of Freshwater Sciences, University of Wisconsin—Milwaukee">.
I'll "nail my colours to the mast" and say that I was expecting that people would find novel ammonia oxidising microbes before they did, and that “new” nitrifiers would be discovered that occur in acidic, and oligotrophic, conditions.
Yours Darrel

cheers Darrel

 

[dw1305]

Hi all,
And there was some follow up correspondence: My email

Hi Josh,
Thank you ever so much for replying, I wish I was as quick to answer my emails.
Also thank you for adding in Michelle McKnight, it is much appreciated. I've enjoyed your labs. research papers, they are written in terms that the non-specialist can understand.
Is it all right if I share your response (in this thread) on UKAPS? <"Does depleted KH stop the nitrogen cycle?">.

.... One thing I can say is that we have some “new aquarium start-up” data that points to plants as a likely inoculation source for a broader diversity of nitrifiers, but the data in hand are only able to weakly support this hypothesis......

That is something <"that really interests me">, I don't have any empirical evidence but that is definitely where I think we are.
My main motivation is to get aquarium keepers to keep planted tanks, purely because I think it makes them more likely to enjoy their hobby and that makes them more likely to remain involved with fish keeping.
Yours Darrel

Josh's reply

Thanks Michelle. Perhaps an indicator species analysis could be worthwhile to see if there may be specific taxa that correlate with the planted tanks specifically? But good point that there doesn’t seem to be an obvious trend in established tanks. By the time we sampled them, they had lots and lots of time to be seeded by environmental microbes. But the new tanks – how best to inoculate? The data we have are consistent with intuition, pointing toward live plants being an ideal source.

Darrell, feel free to share my response(s) as you like 😊

Josh

and Michelle McKnight's reply

Hi Darrell and Josh,
So nice to hear that the results are of value/interest to the aquarium community!

The only thing I will add to what Josh said is that I did look for connections between live plants in the aquarium and differences in nitrifier profiles. There wasn’t any obvious pattern between those that did and didn’t contain live plant. As mentioned by Josh thought, it appears to be of benefit to nitrifier establishment when including live plants during new aquarium ’start-up’.

Best,
Michelle

cheers Darrel

 

[dw1305]

Hi all,
and the follow up follow up correspondence:

Hello Darrell,
All good points and I sympathize with your concerns and communication challenges. In some ways, ammonia/nitrite is a solved problem, so we are adding ecological information without gamechanger practical solutions. Although brand new aquarists will always need support and advice (and product-based solutions), experienced aquarists will just take some sponge from an established aquarium and a new aquarium is then fully cycled without fuss.

I think the holy grail has always been nitrate. A microbial solution to eliminate nitrate would be a welcome gamechanger, where ecology and practical solutions would align better in terms of impact on the hobby. For now though, plants (thumbs up to you) and water changes remain the best options, as per always.
Let’s keep in touch Darrell – glad you reached out.

Josh

and my email that preceded this:

Thanks Josh, Much appreciated, I'll post your answer on UKAPS in a new thread.
I'm not entirely surprised that "planted / not planted", isn't a major factor in established tanks. I'd guess that the microbial assemblage is "fine tuned" to the supply of organic (and inorganic) carbon, fixed nitrogen and oxygen. I'd also guess that relatively oligotrophic conditions support a more diverse assemblage of microbes, but it is all conjecture from my part.
As well as the filter media, it would be interesting to see if the substrate has a different microbial assemblage in planted tanks, again I'd guess it does, purely because you have radial oxygen loss from the roots, there are zones of fluctuating REDOX values, the roots are leaking carbohydrates and there are microbial siderophores etc. within the rhizosphere.
Cheers Darrel

Hi Michelle,
Thank you for your reply.
I'd say that the "scientific revolution" in our understanding of the nitrifying microbes that occur in our aquaria hasn't really had as much effect on fish keeping community as I would have hoped.
I think part of the problem is that the WWW has made information a lot more available to aquarists, but the sheer weight of information has made it difficult for people to sort out the "coffee from the froth", and the ammonia based linear view of cycling, with a binary switch from "not fish safe" to "fish safe" and "cycled", is still firmly entrenched. I think some of the reason is that people like "black and white" answers.
I actually started posting about "cycling" about 15 years ago, so really before the discovery of COMAMMOX Nitrospira and the ubiquity of ammonia oxidising archaea (AOA), and it would be, fair to say, that I received pretty short shrift.
My argument was always that it doesn't make any ecological sense that there are only a limited number of nitrifying bacteria (AOB), with a requirement for high carbonate and TAN levels, when fixed nitrogen is a scarce and patchy resource in the environment.
Your research has allowed me to add some science to that "shades of grey ecological" premise, but it is still going to be an uphill struggle.
Cheers Darrel

Cheers Darrel

 

[MichaelJ]

I'll put the correspondence in the subsequent post, as there are several "sections" to it.

This is great stuff Darrel!

Cheers,
Michael

 

[dw1305]

Hi all,

and the follow up follow up correspondence:

There is just a little bit more:
Josh's reply:

All good points and I sympathize with your concerns and communication challenges. In some ways, ammonia/nitrite is a solved problem, so we are adding ecological information without gamechanger practical solutions. Although brand new aquarists will always need support and advice (and product-based solutions), experienced aquarists will just take some sponge from an established aquarium and a new aquarium is then fully cycled without fuss.

I think the holy grail has always been nitrate. A microbial solution to eliminate nitrate would be a welcome gamechanger, where ecology and practical solutions would align better in terms of impact on the hobby. For now though, plants (thumbs up to you) and water changes remain the best options, as per always.

Let’s keep in touch Darrell – glad you reached out.

Josh

and my reply:

Hi Josh and Michelle,
We really can't thank you enough for your time (and your papers).
I can see that for Marine (and Rift Lake Cichlid) aquarists a microbial solution to nitrate would be a game changer.

Josh's reply:

Only for marine and African cichlids?

Josh

and my reply:

Hi Josh,
I'm going to say that for the majority of freshwater aquarium keepers that higher plants (and particularly plants with access to atmospheric CO2) are always the best answer.
They aren't a replacement for water changes, and there may need to be a spatial separation of plants from fish.
Having said that I'm a botanist by training, so I may not be the most objective commentator.
We've actually developed a "plug and play" plant-based method for fixed nitrogen management (derived from phytoremediation and Rice culture) which I've called the "Duckweed Index". It has got a bit of traction, mainly in continental Europe, from keepers of Apistogramma etc.
This method makes use of the visual appearance (leaf colour and growth rate) of a floating aquarium plant, originally Lemna minor and more recently Limnobium (Hydrocharis laevigata) laevigatum.
I'm sure you have much better things to do, but there is a thread at <"What is the “Duckweed Index” all about?">......

And that is where we've ended (at the moment).

cheers Darrel

 

[louis_last]

Really appreciate you taking the time to provide us all with these fascinating insights

 

[dw1305]

Hi all,

Really appreciate you taking the time to provide us all with these fascinating insights

It has been really good of them to reply. I'd guess we are only at the start of the revolution in (finding) microbial diversity.

<"Some results"> are probably what <"you might have predicted">: - <"https://pubs.acs.org/doi/abs/10.1021/acs.est.2c03299">*.

...... Although comammox Nitrospira have been identified in wastewater treatment systems, the conditions for their dominance over canonical ammonia oxidizers remain unclear. Here, we report the dominance of comammox Nitrospira in a moving bed biofilm reactor (MBBR) fed with synthetic mainstream wastewater. Integrated 16S rRNA gene amplicon sequencing, fluorescence in situ hybridization (FISH), and metagenomic sequencing methods demonstrated the selective enrichment of comammox bacteria when the MBBR was operated at a dissolved oxygen (DO) concentration above 6 mg O2/L. The dominance of comammox Nitrospira over canonical ammonia oxidizers (i.e., Nitrosomonas) was attributed to the low residual ammonium concentration (0.02–0.52 mg N/L) formed in the high-DO MBBR......

But I'd also guess there will be some totally unexpected finds, where microbes are utilising novel substrates and new metabolic pathways.

It isn't my field, but I'd guess, be pretty confident that where ever there is a <"niche to exploit">? An organism <"will have found it">.

*Zhao J., Zeng M. et al. (2022) "Selective Enrichment of Comammox Nitrospira in a Moving Bed Biofilm Reactor with Sufficient Oxygen Supply" Environ. Sci. Technol., 56, 18, 13338–13346

cheers Darrel

 

[Bradders]

This is great Darrel. Very interesting.

I am struggling a bit to understand how this equates into the home aquarium, or whether there is enough here to do that? i.e. these new organisms, how long they take to develop etc.

Sorry, if I am missing it. I am not the greatest at understanding chemistry stuff.

 

[dw1305]

Hi all,

I am struggling a bit to understand how this equates into the home aquarium, or whether there is enough here to do that?

Yes, there is. If you have a look at: <"Correspondence with Dr Ryan Newton - School of Freshwater Sciences, University of Wisconsin—Milwaukee">, it covers this subject fairly comprehensively, in a study that looked at aquarium filters. There is also <"Tim Hovanec's "Nitrification in marine aquarium" article">, it is for marine aquariums, but covers the microbial side pretty thoroughly.

Basically scientists now have the gene sequences that code for ammonia (NH3) oxidation (AO) to nitrite (NO2-), and also for the COMAMMOX process where bacteria, in the genus Nitrospira, convert ammonia directly to nitrate (NO3-).

Because the gene sequences for COMAMMOX, AOA (Ammonia Oxidising Archaea) and Ammonia Oxidising Bacteria (AOB) are different, researchers can get a gene read from an aquarium filter, aquaculture or waste water treatment etc. and

• find the actual microbes that are present and performing nitrification.

Scientists can then look at (and potentially manipulate) dissolved oxygen and ammonia levels in each system and work out the relationship between the microbial assemblage and those factors. They've done this and it looks like:

• COMAMMOX Nitrospira and AOA are the principle players in our aquariums, and
• that they are favoured by low ammonia levels.

All of this just means that the traditional view of cycling isn't right, and that the AOB that need high levels of ammonia and carbonate hardness don't actually occur in our tanks. Also we can't test for a nitrite "spike" after ammonia addition, because if our principal nitrifiers are COMAMMOX Nitrospira? They are oxidising ammonia directly to nitrate etc. without producing any nitrite spike.

i.e. these new organisms, how long they take to develop etc.

<"They aren't new">, they are probably actually billions of years old, we just have been able to <"find them until recently">.

In my life time Archaea have gone from being "strange bacteria that only occur in extreme conditions", to being "not bacteria", "not all extremophiles" and actually being one of the <"three domains of life"> and pretty much <"universal on the Earth">.

We still know next to nothing about the microbial world, away from the organisms that cause diseases and those that we can culture on agar plates.

cheers Darrel

 

[Bradders]

Awesome, thanks @dw1305.

 

[LMuhlen]

Also we can't test for a nitrite "spike" after ammonia addition, because if our principal nitrifiers are COMAMMOX Nitrospira? They are oxidising ammonia directly to nitrate etc. without producing any nitrite spike.

But we do test and measure a noticeable nitrite spike during the tank cycling. This suggests that these COMAMMOX take their time to establish themselves, and/or that the other simpler microbes are still pumping out enough nitrites for us to measure them. This study that found a large presence of COMAMMOX in aquariums tested mostly well established tanks, right?

 

[ElleDee]

But we do test and measure a noticeable nitrite spike during the tank cycling. This suggests that these COMAMMOX take their time to establish themselves, and/or that the other simpler microbes are still pumping out enough nitrites for us to measure them. This study that found a large presence of COMAMMOX in aquariums tested mostly well established tanks, right?

I've only detected nitrite once in my tanks, ever. When I set up a new filter, it takes longer than it "should", but no nitrite spike except for than one time.

I didn't really have an explanation for it until this information, but since it wasn't causing problems I didn't worry too much. I have no idea if my experience is common, but it happened. 🤷🏼‍♀️

 

[dw1305]

Hi all,

But we do test and measure a noticeable nitrite spike during the tank cycling. This suggests that these COMAMMOX take their time to establish themselves, and/or that the other simpler microbes are still pumping out enough nitrites for us to measure them. This study that found a large presence of COMAMMOX in aquariums tested mostly well established tanks, right?

I think so, basically it takes a while for the <"diverse and flexible microbial assemblage"> to become established. I'm guessing (particularly if you add ammonia) that you will get activity by non-COMAMMOX Nitrospira that are converting ammonia (NH3) to nitrite (NO2-). There is some discussion of COMAMMOX and conventional Nitrospira in <"Dr Timothy Hovanec's comments about Bacterial supplements">.

Dr Ryan Newton offers his thoughts in <"Correspondence with Dr Ryan Newton - School of Freshwater Sciences, University of Wisconsin—Milwaukee">. The Neufeld and Newton Labs. are right at the cutting edge of this research. Dr Newton says:

..... 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.

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.

In some lab tests we found that adding previous material from a running biofilter could reduce ammonia oxidation start-up time from 2-3 weeks to 2-3 days. 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, but it will be a lot less predictable and ultimately may not determine what microbe succeed in the long run. ......

Our experience would suggest that, for a heavily planted tank, six weeks growing is enough to make it "fish safe", but we don't know quite how long the <"full maturation process"> takes, and I'm going to guess that there are a lot of variables and that <"no two tanks are exactly the same">.

cheers Darrel